BRONCHOPULMONARY INFECTION WITH PSEUDOMONAS … · c) Psychosocial aspects. B. BRONCHOPULMONARY...

BRONCHOPULMONARY INFECTION WITH PSEUDOMONAS AERUGINOSA

IN CYSTIC FIBROSIS

Andrea Regan Lee Penketh

A thesis submitted for the degree of Doctor of Medicine,

to the University of London, 1983.

Department of Medicine Cardiothoracic Institute Brompton Hospital Fulham RoadLONDON SW3 6HP.

ABSTRACT

Chronic pulmonary d i s e a s e is the major cause of

morbidity and mortal i ty in older pa t ien t s with c y s t i c

f i b r o s i s (CF). In fec t ion with Pseudomonas aeruginosa i s

an important fea ture and, once e s ta b l i s h e d , the organism

usua l ly p e r s i s t s in the lower r esp i r a tory t r a c t .

Although f a i l i n g to e r a d i c a t e pseudomonas, a n t i b i o t i c

chemotherapy was shown to improve re sp i r a t or y function

s i g n i f i c a n t l y , both when given as a short course,

intravenously and when given over a longer period by

ae ro so l . Comparative t r i a l s of intravenous gentamicin

combined with one of three semi-synthet ic p e n i c i l l i n s ,

did not demonstrate an advantage of one p a r t i c u l a r

regimen. In a double-bl ind, p lacebo-control led,

randomised, c ros s-over t r i a l aerosol gentamicin and

c a r b e n i c i l l i n given twice d a i l y for s i x months produced a

s i g n i f i c a n t improvement in r e sp i r a to ry function and a

trend towards reduced frequency of hosp i ta l admiss ions ,

without an increase in a n t i b i o t i c r e s i s t a n t organisms.

Cyst ic f i b r o s i s p a t i e n t s colonised by P. aeruginosa may

remain r e l a t i v e l y well for years , but others d e te r i o r a t e

inexorably and t h i s l a t t e r group cannot be i d e n t i f i e d by

routine sputum b a c te r io l o g y . S t ra ins of P. aeruginosa

i s o l a t e d from f i f t y one p a t i e n t s with a range of se v e r i t y

of d i s ea se were studied and cer ta in c h a r a c t e r i s t i c s were

more frequently a s s o c ia t e d with c y s t i c f i b r o s i s s t r a i n s

when compared with c o n t r o l s . Pa t ients recent ly colonised

h a d o r g a n i s m s s i m i l a r t o c o n t r o l s t r a i n s , w h i l e o a t L o n t s

c o l o n i s e d L o r y e a r s h a d o r g a n i s m s w i t h t h r e e u n u s u a l

f e a t u r e s , n a m e l y 1 ) l o s s o f O - s e r o t y p e r e a c t i o n ,

2 ) e x p r e s s i o n o f a n e w s o m a t i c a n t i g e n a n d 3 ) s e n s i t i v i t y

t o t h e b a c t e r i c i d a l a c t i o n o f n o r m a l h u m a n s e r u m .

P a t i e n t s w i t h o r g a n i s m s e x h i b i t i n g o n e o r t w o o f t h e s e

features were the most severe ly a f fec ted by the i r

d i s e a s e , and may there fore represent a high r i s k group

that might benef i t from more intensive chemotherapy.

A further study of the serum s e n s i t i v i t y of

P. aeruginosa revealed s i x p a t ie n t s with a s e l e c t i v e

de fec t in serum b a c t e r i c i d a l a c t i v i t y towards the ir

autologous s t r a i n . Experimental evidence suggested that

an antibody of the IgG c l a s s blocked the na tu ra l ly

occurring b a c t e r i c i d a l mechanisms.

C O N T E N T S

A b s t r a c t

I n d e x of : t a b l e s

I n d e x o f f i g u r e s

A b b r e v i a t i o n s

A i m o f t h i s t h e s i s .

P a g e M o .

Chapter 1 GENERAL INTRODUCTION 2.1

A. CYSTIC FIBROSIS

1. The d i s e a s e

a) Def ini t ion

b) Incidence

c) Genetics

d) Pathophysiology

2. C l i n i c a l f e a t u re s

a) Respi ratory system

b ) Asperg i l lus fumigatus

c) G a s t r o i n t e s t i n a l system

d) Glucose into lerance

e) Growth and development

f) Other complicat ions

3. Diagnosis

4. Treatment

a) Pulmonary d i s e a s e

i Prevention of infect ion

i i Physiotherapy

i i i Bronchodi lators and c o r t i c o s t e r o i d s

iv Sur fiery

v Ant ib io t i c s

b ) G a s t r o i n te s t i na 1 d i s c a s e

i Pancreat ic enzyme replacements

i i N ut r i t io na l supplementation

i i i Meconium i l e u s equivalent .

c) Psychosocial a s pe ct s .

B. BRONCHOPULMONARY INFECTION IN CYSTIC FIBROSIS 36

1 . B a c t e r i a l infec t ion

2. Infec t ion caused by v i ruses

Mycoplasma and re la te d organisms

Mycobacteria

Chapter 2 PSEUDOMONAS AERUGINOSA 45

A. THE ORGANISM 46

1 . Occurrence

2. I s o l a t i o n

3. Identi f i ca t ion

4. Typing schemes for s t r a i n id e n t i f i c a t i o n

5. Mucoid forms

6 . Virulence f a c t o r s of P.aeruginosa

B. HOST DEFENCE MECHANISMS AGAINST P.AERUGINOSA IN 55

CYSTIC FIBROSIS

1. E p i t h e l i a l b a r r i e r

2. Alveolar macrophages

3. Neutrophil function

4. Cell-mediated immunity

5. Humoral immunity.

Chapter 3 GENERAL METHODS 60

A. CYST EC El EROS IS PATH’] NTS

13. RESPIRATORY FUNCTION TESTS

C. CLINICAL SCORE

D. SERUM SAMPLES

E. IDENTIFICATION OF PSEUDOMONAS STRAINS FROM SPUTUM

1. Primary i s o l a t i o n

2. Colonial form

3. O-se ro lo g ica l typing

a) Methods

b) Result s

4. Po lyagglut inat ion and PA antigen

5. Serum s e n s i t i v i t y t e s t

6. Absorption of sera

F. STORAGE OF STRAINS

G. MEDIA AND SOLUTIONS

Chapter 4. TREATMENT OF P.AERUGINOSA INFECTION IN ACUTE EXACERBATIONS OF PULMONARY DISEASE IN CYSTIC FIBROSIS

BY INTRAVENOUS ANTIBIOTICS. 72

A. INTRODUCTION

B. LITERATURE REVIEW

C. AIMS

D. METHODS

1. Study design

2 P a t i e n t s e l e c t i o n

3 . A s s e s s m e n t

4 . D o s e r o n i mo n s

5 . A s s a y oil a n t i b i o t i c l e v e l s i n s e r u m a n d s p u t u m

a ) G e n t a m i c i n

b ) Penicillins6. Follow-up

7. Other treatment

8. S t a t i s t i c s .

E. RESULTS 87

1. T i c a r c i l l i n versus c a r b e n i c i l l i n

a) Respi ratory function

b) Bacter io logy

c) Ant ib io t ic l e v e l s

d) Res i s t an t organisms

e) Follow-up

f ) Toxici ty

2. A z l o c i l l in versus c a r b e n i c i l l i n 94

a) Respi ratory function

b) Bacter io logy

c) Ant ibio t ic l e v e l s

d) R e s i s t a n t organisms

e) Follow-up

f ) Toxic i ty

g) Sub jec t ive assessment.

F. DISCUSSION 101

G S UMMARY 114

Chapter 5 TREATMENT OF P.AERUGINOSA INFECTION IN

CHRONIC PULMONARY DISEASE IN CYSTIC FIBROSIS BY

AEROSOL ANTIBIOTICS LI5

A. I N T R O D U C T I O N

B . L I 'r E K ATUR E R E V I EW

C . A I M S

METHODS, RESULTS AND DISCUSSION

1. C l i n i c a l t r i a l of aeroso l gentamicin and 119

carbenic i l l i n

a ) Pat ient s and methods

b) Result s

c ) Discuss ion

2. Long -term use of aer oso l a n t i b i o t i c s in adult s

withi c y s t i c f i b r o s i s 132

a ) Pa t ient s and methods

b) Results

c ) Discussion

3. Ant ibiot ic l e v e l s in serum and sputum a f t e r 14 0

administrat ion by aeroso l

a ) Methods

b) Resul t s

c ) Discussion

4. Analysis of ae ro so l p a r t i c l e s i z e d i s t r i b u t i o n 145

a ) Introduction

b) Methods

c ) Resul ts

c) Discussion

E. SUMMARY 151

F . F U T U R E P O L I C Y 152

Chapter 6 SOME PHENOTYPE CHANGES OF P.AERUGINOSA IN RELATION TO THE CLINICAL CONDITION OF

CYSTIC FIBROSIS PATIENTS 153

A . I N T R O D U C T I O N

B . L I T E R A T U R E R E V I E W

C . A I M S

D. METHODS

1. Pat ient s

2. I d e n t i £ica t ion of pseudomonas s t r a i n s

3. Control cu l tu re s

4. C l i n i c a l assessment

5. S t a t i s t i c s .

E. RESULTS 158

1. C h a r a c t e r i s t i c s of P . aeruginosa from c y s t i c

f i b r o s i s sputa .

a) Colonia l form

b) O-serology

c) The PA antigen

d) S e n s i t i v i t y to normal human serum

2. Comparison with contro l cul tures

3. Associat ion between some phenotype c h a r a c t e r i s t i c s

of P .aerug inosa in c y s t i c f i b r o s i s .

4. Relat ionship of some phenotype changes in

P. aeruginosa to the c l i n i c a l condition of c y s t i c

f i b r o s i s p a t i e n t s .

P . D I S C U S S I O N 1 6 7

1 . C h a r g e t o r i s t i c s o f P . a e r u g i n o s a I7 r e i n c y s t i c

f i b r o s i s s p u t a

a ) C o 1 o n i a 1 I o r m

b) O - s o r o t y p o

c) PA antigen

d) S e n s i t i v i t y to normal human serum

2. Some phenotype changes of P. aeruginosa in c y s t i c

f ibros i s

3. Rela t ionship of some phenotype changes of

P. aeruginosa to the c l i n i c a l condition of c y s t i c

f i b r o s i s p a t i e n t s - a hypothesis .

G. SUMMARY 177

Chapter 7 BACTERICIDAL ACTIVITY OF CYSTIC FIBROSISSERUM AGAINST P. AERUGINOSA 178

A. INTRODUCTION AND LITERATURE REVIEW

B. AIMS

C. METHODS

1. Sera

2. Organisms

3. Serum t e s t s

a) S erum- sen s i t iv i ty

b) Blocking f a c t o r a c t i v i t y

c) Absorption of se ra

d) C h a ra c te r i sa t i o n of se lec ted c y s t i c f i b r o s i s

s e r a .

4 . C l i n i c a l a s s e s s m e n t

D . RESULTS 1 4 4

1 . B a c t e r i c i d a l a c t i v i t y o f c y s t i c f i b r o s i s

s e r a a g a i n s t P . a e r u g i n o s a .

2 . I n t e r f e r e n c e b y c y s t i c f i b r o s i s s e r a w i t h n o r m a l

h u m a n s e r u m k i l l i n g o f P . a e r u g i n o s a

3 . S t u d i e s o f s e r a w i t h b l o c k i n g a c t i v i t y a n d t h e

a u t o l o g u s s t r a i n s o f p s e u d o m o n a s

4 . C h a r a c t e r i s a t i o n o f b l o c k i n g a c t i v i t y

a ) S t r a i n - s p e c i f i c i t y

b ) A b s o r p t i o n o f c y s t i c f i b r o s i s s e r u m

c ) S e r u m f r a c t i o n s .

5 . C l i n i c a l c o n d i t i o n o f p a t i e n t s w i t h b l o c k i n g f a c t o r s

i n s e r u m .

E . D I S C U S S I O N 1 9 3

1 . S e r u m - s e n s i t i v i t y o f P . a e r u g i n o s a

2 . B l o c k i n g f a c t o r s i n c y s t i c f i b r o s i s s e r u m

F . SUMMARY 1 9 8

Chapter 8 CONCLUSIONS AND GENERAL DISCUSSION 1 9 9

A . SUMMARY O F P O S I T I V E F I N D I N G S 200

B . D I S C U S S I O N 74ND P R O S P E C T S F O R F U T U R E WORK 2 0 3

a ) S i z e oL: t h e i n n o c u l u m

b ) L e n g t h o f t h e i n c u b a t i o n p e r i o d

c ) A g e o f t h e b r o t h c u l t u r e

d ) D i l u t i o n o f n o r m a l h u m a n s e r u m

e ) D e f i n i t i o n o f s e r u m - s e n s i t i v i t y

Appendix I VALIDATION OF THE SERUM-SENSITIVITY ASSAY 20

Appendix II ASSAYS AND LABORATORY PROCEDURES NOT 213 PERFORMED BY THE AUTHOR

a ) P r e p a r a t i o n o f O - a n t i s e r a f o r s e r o t y p i n g o f

P . a e r u g i n o s a

b ) A n a l y s i s o f a e r o s o l p a r t i c l e s i z e

c ) A n t i b i o t i c a s s a y s

d ) I n i t i a l i s o l a t i o n o f p s e u d o m o n a s f r o m s p u t u m .

e ) M e a s u r e m e n t o f i m m u n o g l o b u l i n l e v e l s i n C F s e r a .

Acknowledgements 218

Bibliography 220

Table Page Me

N u m b e r

I C h a n g e s i n t h e b a c t e r i a l f l o r a i s o l a t e d 3 7

f r o m t h e s p u t u m o f c y s t i c f i b r o s i s p a t i e n t s

s i n c e 1 9 3 8 .

I I I d e n t i f i c a t i o n o f p s e u d o m o n a s . 4 9

I I I S u m m a r y o f s t u d i e s o f i n t r a v e n o u s c h e m o t h e r a p y 7 7

a g a i n s t P . a e r u g i n o s a i n c y s t i c f i b r o s i s

p a t i e n t s .

I V T i c a r c i l l i n / c a r b e n i c i l l i n t r i a l : p a t i e n t d a t a 8 9

o n e n t r y t o t h e t r i a l .

V T i c a r c i l l i n / c a r b e n i c i l l i n t r i a l : r e s p i r a t o r y 9 0

f u n c t i o n d a t a .

V I M e a n s e r u m a n d s p u t u m c o n c e n t r a t i o n s o f 9 3

c a r b e n i c i 1 1 i n a n d t i c a r c i l l i n a f t e r a n

i n t r a v e n o u s d o s e o f f i v e g r a m s .

V I I A z l o c i l l i n / c a r b e n i c i l l i n t r i a l : p a t i e n t d a t a 9 5

o n e n t r y t o t h e t r i a l .

V I I I A z l o c i l l i n / c a r b e n i c i l l i n t r i a l : r e s p i r a t o r y 9 6

f u n c t i o n d a t a .

I X i Mean s e r u m a n d s p u t u m c o n c e n t r a t i o n s o f 9 9

a z l o c i l l i n a n d c a r b e n i c i l l i n a f t e r a n

i n t r a v e n o u s d o s e o f f i v e g r a m s .

X A n t i b i o t i c r e s i s t a n c e o f P . a e r u g i n o s a a f t e r 1 0 0

i n t r a v e n o u s c h e m o t h e r a p y .

X I T i m e b e f o r e t h e n e x t a d m i s s i o n f o r i n t r a v e n o u s 1 0 2

c h e m o t h e r a p y f o r p a t i e n t s w h o c o m p l e t e d t h e

INDEX OF TABLES

t r i a l s .

X I I A z l o c i 1 ! i n / c a r b e n i c i L L i n t r i a l : a i e a n v i s u a l 1 0 3

a n a 1 o q u e s c a 1 e s c o r e s .

X I I I S p u t u m l e v e l s o f p e n i c i l l i n s a f t e r i n t r a v e n o u s , 1 1 3

a d m i n i s t r a t i o n i n c y s t i c f i b r o s i s p a t i e n t s .

X I V A e r o s o l a n t i b i o t i c t r i a l : p r e f e r e n c e o f p a t i e n t 1 2 4

a n d d o c t o r f o r a c t i v e t r e a t m e n t o r p l a c e b o .

XV A e r o s o l a n t i b i o t i c t r i a l : r e s p i r a t o r y f u n c t i o n 1 2 5

d a t a .

X V I R e g i m e n s u s e d i n c y s t i c f i b r o s i s p a t i e n t s a t t h e 1 3 4

B r o m p t o n H o s p i t a l r e c e i v i n g a e r o s o l a n t i b i o t i c s .

X V I I A n t i b i o t i c r e s i s t a n c e o f P , a e r u g i n o s a i s o l a t e d 1 3 6

f r o m p a t i e n t s r e c e i v i n g a e r o s o l a n t i b i o t i c s .

X V I I I T h e f r e q u e n c y o f a n t i b i o t i c r e s i s t a n c e i n 1 3 3

P . a e r u g i n o s a f r o m c y s t i c f i b r o s i s p a t i e n t s

o v e r a s i x m o n t h p e r i o d .

X I X S p u t u m l e v e l s o f a n t i b i o t i c s a f t e r 1 4 2

a d m i n i s t r a t i o n b y i n t r a v e n o u s a n d a e r o s o l r o u t e s .

X X S e r u m l e v e l s o f a n t i b i o t i c s a f t e r a d m i n i s t r a t i o n 1 4 3

b y i n t r a v e n o u s a n d a e r o s o l r o u t e s .

X X I M a l v e r n 2 2 0 0 P a r t i c l e S i z e r a n a l y s i s o f 1 4 8

a n t i b i o t i c a e r o s o l s i z e d i s t r i b u t i o n .

X X I I T h e p a t t e r n o f r e a c t i o n s t o 0 - a n t i s e r a o f 1 6 0

t y p a b l e a n d a b e r r a n t s t r a i n s o f c y s t i c f i b r o s i s

c u l t u r e s o f P . a e r u g i n o s a c o m p a r e d w i t h t h o s e o f

c o n t r o l s t r a i n s .

X X I I I C h a r a c t e r i s t i c s o f s t r a i n s o f P . a e r u g i n o s a f r o m 1 6 1

p a t i e n t s w i t h c y s t i c f i b r o s i s c o m p a r e d w i t h

s t r a i n s f r o m o t h e r s o u r c e s

X X I V A s s o c i a t i o n b e t w e e n t h r o e p h e n o t y p e 1 5 3

c h a r a c t o r i s t i c s of : P . n e r u q i n o s a f r o m c y s t i c

f i b r o s i s n n u t u n n .

XX V C l i n i c a l f e a t u r e s o f c y s t i c f i b r o s i s p a t i e n t s 1 5 ' i

g r o u p e d a c c o r d i n g t o t h e c h a r a c t e r i s t i c s o f

P . a e r u g i n o s a i s o l a t e d f r o m t h e i r s p u t u m .

X X V I B a c t e r i c i d a l a c t i v i t y o f c y s t i c f i b r o s i s s e r u m 1 8 1

a g a i n s t s t r a i n s o f P . a e r u g i n o s a f r o m v a r i o u s

s o u r c e s .

X X V I I T h e e f f e c t o f c y s t i c f i b r o s i s s e r u m f r o m 1 3 7

p a t i e n t B o n t h e b a c t e r i c i d a l a c t i v i t y o f n o r m a l

h u m a n s e r u m t o w a r d s p s e u d o m o n a s s t r a i n 1 3 0 b .

X X V I I I T h e p a t t e r n o f ' b l o c k i n g ' a n d b a c t e r i c i d a l 1 8 9

a c t i v i t y o f s e r a f r o m s i x c y s t i c f i b r o s i s

p a t i e n t s f o r a u t o l o g o u s a n d h e t e r o l o g o u s s t r a i n s

o f P . a e r u g i n o s a

X X I X I n t e r f e r e n c e w i t h t h e b a c t e r i c i d a l a c t i o n o f 1 9 1

n o r m a l h u m a n s e r u m o n a u t o l o g o u s s t r a i n s o f

p s e u d o m o n a s b y t w o c y s t i c f i b r o s i s s e r a , a n d

f r a c t i o n s o f t h o s e s e r a o b t a i n e d b y i o n - e x c h a n g e

c h r o m a t o g r a p h y .

X X X C l i n i c a l d a t a o f s i x c y s t i c f i b r o s i s p a t i e n t s 1 9 2

w i t h b l o c k i n g a c t i v i t y i n s e r u m .

X X X I T h e e f f e c t o f t h e s i z e o f t h e i n o c u l u m o n t h e 2 0 7

v i a b l e c o u n t i n t h e s e r u m - s e n s i t i v i t y t e s t .

X X X I I T h e e f f e c t o f d i l u t i o n o f n o r m a l h u m a n s e r u m 2 1 2

o n i t s b a c t e r i c i d a l a c t i o n t o w a r d s s t r a i n 7 / 1 .

X X X I I I O - s e r o l o g i c a l t y p i n g o f P . a e r u g i n o s a : O - t y p e 2 1 5

s t r a i n s .

Figure Ti 110 Page U(

d u m b e r

1 . T h e p a t h o g e n e s i s o f c y s t i c f i b r o s i s . 2 5

2 . S t r a i n s o f P . a e r u g i n o s a p r o d u c i n g p y o c y a n i n 5 0

a n d m u c o i d o n K i n g ’ s ’ A ’ A g a r .

3 . S h w a c h m a n s y s t e m o f r a t i n g s e v e r i t y o f d i s e a s e . 6 3

4 . T h e s e r u m - s e n s i t i v i t y p l a t e a s s a y . 6 0

5 . S c h e m e f o r i d e n t i f i c a t i o n o f p s e u d o m o n a s 6 9

s t r a i n s .

6 . P E E R b e f o r e a n d a f t e r t r e a t m e n t w i t h 9 1

t i c a r c i l l i n o r c a r b e n i c i l l i n .

7 . P E E R b e f o r e a n d a f t e r t r e a t m e n t w i t h 9 7

a z l o c i l l i n o r c a r b e n i c i l l i n .

8 . V i s u a l a n a l o g u e s c a l e s c o r e s o f c y s t i c f i b r o s i s 1 0 4

p a t i e n t s t r e a t e d w i t h a z l o c i l l i n o r c a r b e n i c i l l i n .

9 . T h e n e b u l i z e r a p p a r a t u s . 1 2 1

1 0 . R e s p i r a t o r y f u n c t i o n o f a c y s t i c f i b r o s i s p a t i e n t 1 2 3

u s i n g a e r o s o l a n t i b i o t i c s a n d p l a c e b o .

1 1 . F o r c e d v i t a l c a p a c i t y r e c o r d i n g s o f s e v e n 1 2 7

p a t i e n t s u s i n g a e r o s o l a n t i b i o t i c s a n d p l a c e b o .

1 2 . R e s p i r a t o r y f u n c t i o n o f a c y s t i c f i b r o s i s p a t i e n t 1 2 9

u s i n g a e r o s o l a n t i b i o t i c s a n d p l a c e b o .

1 3 . T h e r e l a t i o n s h i p o f s o m e p h e n o t y p e c h a n g e s i n 1 7 6

P . a e r u g i n o s a t o t h e c l i n i c a l c o n d i t i o n o f

c y s t i c f i b r o s i s p a t i e n t s .

I N D E X OF F I G U R E S

P h o p M o

1 4 . T h e b a c t e r i c i d a l e f f e c t of : a u t o l o g o u s c y s t i c 1 0 5

f i b r o s i s s c r u m a n d n o r m a l h u m a n s e r u m o n o n e

c y s t i c f i b r o s i s p s e u d o m o n a s s t r a i n .

1 5 . T h e e f f e c t o f t h e l e n g t h o f t h e i n c u b a t i o n p e r i o d 2 0 9

o n t h e v i a b l e c o u n t i n t h e s e r u m - s e n s i t i v i t y t e s t .

ABBREVIATIONS

A A A u t o a q g 1 u t i n a t i o n .

C F C y s t: i. c I’ i b r o s i s .

C F S C y s t i c [ f i b r o s i s . s e r u m ,

c i u C o l o n y f o r m i n g u n i t s .

C P Br.. C e n t r a l P u b l i c H e a l t h L a b o r a t o r y , C o l i n d a l e , L o n d o n .

FEV]_ F o r c e d e x p i r a t o r y v o l u m e i n o n e s e c o n d ( m i s ) .

F V C F o r c e d v i t a l c a p a c i t y ( m i s ) .

L P S L i p o p o l y s a c c h a r i d e .

NA N u t r i e n t a c j a r .

NHS N o r m a l h u m a n s e r u m .

N S N o t s t a t i s t i c a l l y s i g n i f i c a n t ( p > 0 * 0 5 ) .

NT N o t t y p a b l e .

P A P o l y a g g l u t i n a t i o n .

P E F R P e a k e x p i r a t o r y f l o w r a t e ( l i t r e s / m i n u t e ) .

R R e s i s t a n t .

S S e n s i t i v e .

T S B T r y p t o n e s o y a b r o t h .

C y s t i c f i b r o s i s i s t h e m o s t c o m m o n i n h e r i t e d d i s o r d e r

a m o n g t h e C a u c a s i a n p o p u l a t i o n s t h a t i s p o t e n t i a l l y

l e t h a l . O n c e t h o u g h t t o b e i n v a r i a b l y f a t a l i n i n f a n c y ,

t h e m a j o r i t y of : p a t i e n t s n o w s u r v i v e i n t o t h e i r

t w e n t i e s ^ - . I n a r e v i e w o f t h e w o r l d l i t e r a t u r e , c h r o n i c

p u l m o n a r y d i s e a s e w a s f o u n d i n 97% o f a d u l t c a s e s a n d

w a s t h e m a j o r c a u s e o f m o r b i d i t y a n d m o r t a l i t y ^ . T h e

p u l m o n a r y d i s e a s e c o n s i s t s o f t h e s t r u c t u r a l a n d

f u n c t i o n a l c o n s e q u e n c e s o f d i f f u s e b r o n c h o p u l m o n a r y

i n f e c t i o n . I n o l d e r p a t i e n t s t h e d o m i n a n t p a t h o g e n i s

P s e u d o m o n a s a e r u g i n o s a a n d t h e p e r s i s t e n t p r e s e n c e o f

t h i s o r g a n i s m i s a s s o c i a t e d w i t h d e t e r i o r a t i o n i n b o t h

l u n g f u n c t i o n a n d g e n e r a l c l i n i c a l c o n d i t i o n ^ / 4 #

T h e i n i t i a l e v e n t i n t h e c y c l e o f b a c t e r i a l i n f e c t i o n

a n d l u n g d a m a g e i n c y s t i c f i b r o s i s i s n o t k n o w n . N o

s p e c i f i c a b n o r m a l i t y i n t h e l o c a l o r s y s t e m i c i m m u n e

r e s p o n s e s h a s b e e n d e s c r i b e d t h a t e x p l a i n s s a t i s f a c t o r i l y

t h e s u s c e p t i b i l i t y o f t h e s e p a t i e n t s t o b a c t e r i a l

c o l o n i s a t i o n , a n d t h i s i s t h e s u b j e c t o f m u c h c u r r e n t

r e s e a r c h . T h i s t h e s i s i s c o n c e r n e d w i t h t h e c l i n i c a l

p r o b l e m s o f c h e m o t h e r a p y f o r p s e u d o m o n a s i n f e c t i o n i n

c y s t i c f i b r o s i s . I n t h e l i t e r a t u r e t h e r e i s l i t t l e

o b j e c t i v e e v i d e n c e t h a t a n t i b i o t i c t r e a t m e n t i s

b e n e f i c i a l . P . a e r u g i n o s a i s r a r e l y e r a d i c a t e d f r o m t h e

s p u t u m f o r a n y l e n g t h o f t i m e , a n d m a y b e c o n t i n u o u s l y

p r e s e n t f o r y e a r s i n s o m e i n d i v i d u a l s w h i l e , i n c o n t r a s t ,

A I M S O F T H I S T H E S I S

other nationt' j may doe l ine rapidly within months oi:

co l on i sa t ion by pseudomonas. The presence of: this

organism in .sputum cannot therefore bo used alone as an

indicat ion (ior treatment, nor does i t ind icate those

pa t ie n t s at r i s k of sudden d e t e r i o r a t i o n . This t h e s i s

has been designed v;ith the following ob jec t ives

1 . T o e v a l u a t e t h e e f f e c t o f i n t r a v e n o u s c h e m o t h e r a p y

a g a i n s t P . a e r u g i n o s a i n p a t i e n t s w i t h c y s t i c f i b r o s i s

s u f f e r i n g f r o m a n a c u t e e x a c e r b a t i o n o f t h e i r r e s p i r a t o r y

s y m p t o m s ( C h a p t e r 4 ) .

2 . T o a s s e s s t h e l o n g t e r m u s e o f c h e m o t h e r a p y a g a i n s t

P . a e r u g i n o s a g i v e n b y t h e a e r o s o l r o u t e ( C h a p t e r 5 ) .

3 . T o s t u d y t h e c h a r a c t e r i s t i c s o f t h e s t r a i n s o f

P . a e r u g i n o s a i s o l a t e d f r o m c y s t i c f i b r o s i s p a t i e n t s

a t v a r i o u s s t a g e s i n t h e d i s e a s e , a n d t o c o m p a r e

t h e s e f e a t u r e s w i t h s t r a i n s f r o m o t h e r s p u t a a n d f r o m

o t h e r c l i n i c a l s p e c i m e n s . T o c o r r e l a t e a n y

d i f f e r e n c e s f o u n d i n c y s t i c f i b r o s i s s t r a i n s w i t h t h e

c l i n i c a l c o n d i t i o n o f t h e p a t i e n t s , w i t h t h e a i m o f

f i n d i n g m a r k e r s f o r t h o s e p a t i e n t s a t r i s k o f s u d d e n

d e t e r i o r a t i o n i n t h e i r c o n d i t i o n ( C h a p t e r 6 ) .

4 . T o a s s e s s t h e b a c t e r i c i d a l a c t i v i t y o f n o r m a l h u m a n

s e r u m a n d c y s t i c f i b r o s i s s e r u m a g a i n s t P . a e r u g i n o s a

f r o m c y s t i c f i b r o s i s p a t i e n t s a n d f r o m o t h e r s o u r c e s ,

a s a s i m p l e i n d i c a t o r o f h o s t r e s p o n s e t o t h e

o r g a n i s m ( C h a p t e r 7 ) .

Chapter 1

GENERAL INTRODUCTION

C Y S T I C F I B R O S I S

THE DISEASE

Dot ini tion

C y s t i c f i b r o s i s i s t h e m o s t c o m m o n , p o t e n t i a l l y

l e t h a l , i n h e r i t e d c o n d i t i o n s e e n i n C a u c a s i a n s . I t

w a s f i r s t d e s c r i b e d a s a d i s t i n c t e n t i t y i n 1 9 3 3 b y

A n d ersen^ , a l t h o u g h F a n c o n i ^ i n 1 9 3 6 h a d n o t e d

b r o n c h i e c t a s i s i n p a t i e n t s w i t h c o n g e n i t a l c y s t i c

p a n c r e a t i c f i b r o s i s . T h e b a s i c g e n e t i c d e f e c t a n d

i t s s t r u c t u r a l o r b i o c h e m i c a l m a n i f e s t a t i o n s r e m a i n s

u n k n o w n . T h e d i s e a s e i s c l a s s i c a l l y d e f i n e d a s a

c l i n i c a l s y n d r o m e o f p r o g r e s s i v e , o b s t r u c t i v e

p u l m o n a r y d i s e a s e , w i t h e x o c r i n e p a n c r e a t i c

i n s u f f i c i e n c y a n d a h i g h s o d i u m c o n t e n t o f t h e s w e a t .

O n c e t h e p r o v i n c e o f t h e p a e d i a t r i c i a n , i t i s n o w

i n c r e a s i n g l y s e e n b y t h o r a c i c p h y s i c i a n s , d u e t o

i m p r o v e d s u r v i v a l a n d t o t h e r e c o g n i t i o n o f c a s e s

w i t h m i l d e r r e s p i r a t o r y a n d / o r g a s t r o i n t e s t i n a l

i n v o l v e m e n t , w h i c h a r e d i a g n o s e d i n a d o l e s c e n t o r

a d u l t l i f e .

I n c i d e n c e

T h e i n c i d e n c e o f C F i s f a r h i g h e r i n C a u c a s i a n

p o p u l a t i o n s t h a n i n o t h e r e t h n i c g r o u p s . An

i n c i d e n c e o f 1 i n 2 , 0 0 0 l i v e b i r t h s i s g e n e r a l l y

a c c e p t e d , a l t h o u g h t h e r a n g e i s w i d e , f r o m 1 i n 6 2 0

i n S o u t h W e s t A f r i c a n A f r i k a n e r s , t o 1 i n 1 5 , 0 0 0 i n

I t a l y " ^ . S t u d i e s i n t h e U S A h a v e s h o w n a n i n c i d e n c e

o f 1 i n 1 7 , 0 0 0 i n n e g r o s ( a l t h o u g h t h i s i s n o t

r e p r e s e n t a t i v e o f o t h e r n e g r o p o p u l a t i o n s ) , a n d 1 i n

9 0,000 in oiricMitals of Hawaii. Only a Cow cases have

boon reoorted from Afr ica , Japan, Malaysia, the India

subcontinent and the Middle East ,

c ) Genetics

C y s t i c f i b r o s i s i s i n h e r i t e d a s a n a u t o s o m a l

r e c e s s i v e g e n e a n d o b e y s s i m p l e M e n d e l i a n g e n e t i c s .

I n a p o p u l a t i o n w i t h a n o b s e r v e d i n c i d e n c e o C 1 i n

2 , 0 0 0 a f f e c t e d p a t i e n t s a t b i r t h , t h e c a l c u l a t e d

h e t e r o z y g o t e c a r r i e r r a t e i s 4 - 5 % . C F i s t h u s t h e

m o s t c o m m o n , p o t e n t i a l l y l e t h a l g e n e i n t h e U n i t e d

K i n g d o m . T h e c a r r i e r s t a t e i s c o m p l e t e l y

a s y m p t o m a t i c , a n d a t p r e s e n t t h e r e i s n o r e l i a b l e

m e t h o d f o r i t s d e t e c t i o n . U n t i l r e c e n t l y t h e d i s e a s e

w a s l e t h a l b e f o r e r e p r o d u c t i v e a g e , b u t t h e h i g h

c a r r i e r f r e q u e n c y i n t h e p o p u l a t i o n p e r s i s t s . T h i s

g e n e f r e q u e n c y i s p r o b a b l y h i g h e r t h a n c a n b e

m a i n t a i n e d b y s p o n t a n e o u s m u t a t i o n , a n d s u g g e s t s a

h e t e r o z y g o t e a d v a n t a g e ^ . T h e n a t u r e o f t h i s

h e t e r o z y g o t e a d v a n t a g e i s o b s c u r e ^ ,

d ) P a t h o p h y s i o l o g y

T h e r e i s n o s i n g l e b i o c h e m i c a l o r s t r u c t u r a l

d e f e c t t h a t p r o v i d e s a u n i f y i n g h y p o t h e s i s f o r t h e

p a t h o p h y s i o l o g y o f C . F . M a n y a b n o r m a l i t i e s a r e

d e s c r i b e d , a n d i t i s i m p o s s i b l e t o d i s t i n g u i s h t h o s e

d u e t o t h e b a s i c d e f e c t , a n d t h o s e d u e t o s e c o n d a r y

c o m p l i c a t i o n s . T h r o u g h o u t t h e b o d y t h e r e i s a c h a n g e

i n t h e n a t u r e o f m u c o u s a n d s e r o u s s e c r e t i o n s , w i t h

o b s t r u c t i o n t o e x o c r i n e g l a n d d u c t s . S i t e s o f

o b s t r u c t i o n i n c l u d e t h e b r o n c h i a l t r e e , p a n c r e a s a n d

b i l i a r y t r e e , s m a l l i n t o s t i

t h e m u c u s - s c c r e t i n g s a l i v a r

s e c r e t i o n s i s a s s u m e d t o c a

g l a n d s ; d e h y d r a t i o n a n d a l t

e l e c t r o l y t e c o n t e n t o f s e e r

i - m p l i c a t e d . 0 1 p a r t i c u l a r

c o n t e n t o f s o d i u m a n d c h l o r

w h i c h f o r m s t h e b a s i s o f d i

s u m m a r i s e s t h e p a t h o g e n e s i s

n o , p a r a n a s a l s i n u s e s a n d

Y p l a n d s . I n s o i s n a t i o n o f

u s e t h e o b s t r u c t i o n o f

e r e d g l y c o o r o t e i n o r

e t i o n s h a v e a l l b e e n

i m p o r t a n c e i s t h e e l e v a t e d

i d e i n t h e s w e a t i n C F

a g n o s i s 1 0 . F i g u r e 1

o f C F

2 • C L I N I C A L F E A T U R E S

a ) R e s p i r a t o r y s y s t e m

P u l m o n a r y d i s e a s e a p p e a r s a t s o m e s t a g e i n a l m o s t

a l l p a t i e n t s , f r e q u e n t l y d o m i n a t e s t h e c l i n i c a l

p i c t u r e , a n d u s u a l l y d e t e r m i n e s t h e f a t e o f t h e

p a t i e n t . I n a r e v i e w o f t h e w o r l d l i t e r a t u r e o n C F

i n a d u l t s i n 1 9 7 9 , p u l m o n a r y d i s e a s e w a s p r e s e n t i n

9 7 % o f c a s e s a n d w a s t h e m a j o r f a c t o r c o n t r i b u t i n g t o

m o r b i d i t y a n d m o r t a l i t y 2 . T h e l u n g s a r e

m o r p h o l o g i c a l l y n o r m a l a t b i r t h * - - * - . H y p e r t r o p h y o f

b r o n c h i a l g l a n d s a n d g o b l e t c e l l s o c c u r s w i t h m u c o u s

p l u g g i n g o f p e r i p h e r a l a i r w a y s . B r o n c h i t i s a n d

b r o n c h i o l i t i s f o l l o w , s e t t i n g u p a c y c l e o f

o b s t r u c t i o n , c h r o n i c i n f e c t i o n a n d t i s s u e d a m a g e .

B r o n c h i e c t a s i s , p e r i - b r o n c h i a l f i b r o s i s a n d a i r w a y s

o b s t r u c t i o n o c c u r w i t h p r o g r e s s i v e l o s s o f l u n g

f u n c t i o n . T h e e a r l i e s t m a n i f e s t a t i o n s o f f u n c t i o n a l

a b n o r m a l i t i e s i n t h e p e r i p h e r a l a i r w a y s a r e

FIGURE .1

T H E P A T H O G E N E S I S O F C Y S T I C

d e f e c t

A l t e r e d m u c o u s a n d

s e r o u s s e c r e t i o n s

B l o c k a g e o f

e x o c r i n e d u c t s

A l s o : 1

A l t e r e d e l e c t r o l y t e c o n t r o l

M e s o n e p h r i c d u c t s t r u c t u r e s

F I B R O S I S

- B r o n c h i a l t r e e

- P a n c r e a s

- B i l i a r y t r e e

- S m a l l i n t e s t i n e

- S i n u s e s

- S a l i v e r y g l a n d s

o f s w e a t .

, ( e p i d i d y m i s , v a s

d e f e r e n s a n d s e m i n a l v e s i c l e s f a i l t o d e v e l o p )

d e m o n s t r a b l e - } o n c l o w - v o l u m e l o o p s , a n d b y

a l v e o l a r - a r t e r i a l o x y g e n d i f f e r e n c e s d u e t o u n e v e n

v e n t i l a t i o n . A s a i r f l o w o b s t r u c t i o n i n c r e a s e s t h e r e

i s a c a l l i n F E V ^ / F V C r a t i o , w i t h a i r t r a p p i n g a n d

e l e v a t i o n o f r e s i d u a l v o l u m e a n d f u n c t i o n a l r e s i d u a l

c a p a c i t y . F V C i s r e d u c e d a s t h e d i s e a s e p r o g r e s s e s ,

b u t u s u a l l y t o a l e s s e r e x t e n t t h a n F E V ^ . T h e m o s t

p r o m i n e n t s y m p t o m i s a p r o d u c t i v e c o u g h a n d t h i s i s

f o l l o w e d b y d e c r e a s i n g e x e r c i s e t o l e r a n c e a n d

t a e n y p n o e a . A i r t r a p p i n g c a u s e s a n i n c r e a s e i n t h e

a n t e r o p o s t e r i o r d i a m e t e r o f t h e c h e s t w i t h h y p e r

r e s o n a n c e . T h e l o w e r r i b s m a y b e c o m e r e t r a c t e d w i t h

t h e w o r k o f b r e a t h i n g . D i g i t a l c l u b b i n g i s a n e a r l y

a n d p r o m i n e n t s i g n . C r a c k l e s w i t h o r w i t h o u t w h e e z e

a r e h e a r d o n a u s c u l t a t i o n . P u l m o n a r y d i s e a s e i s

u s u a l l y d i f f u s e , b u t o f t e n w o r s e a t t h e a p i c e s

e s p e c i a l l y o n t h e r i g h t - * - . C o m p l i c a t i o n s i n c l u d e

a t e l e c t a s i s , h a e m o p t y s i s , p n e u m o t h o r a x a n d

c a r d i o - r e s p i r a t o r y f a i l u r e . T h e e a r l y r a d i o l o g i c a l

c h a n g e s a r e h y p e r i n f l a t i o n , b r o n c h i a l w a l l t h i c k e n i n g

a n d a t e l e c t a s i s ; r i n g s h a d o w s f o l l o w w i t h l i n e a r a n d

n o d u l a r o p a c i t i e s - ^ # T r i e u p p e r r e s p i r a t o r y t r a c t i s

c o m m o n l y i n v o l v e d w i t h n a s a l p o l y p s a n d s i n u s i t i s .

T h e p r e v a l e n c e a n d s i g n i f i c a n c e o f r e v e r s i b l e a i r w a y s

o b s t r u c t i o n i n C F i s c o n t r o v e r s i a l . R e s t i n g

b r o n c h i a l t o n e i s i n c r e a s e d , a n d . f a l l s d u r i n g

e x e r c i s e * - - * . I n c o n t r a s t t o a s t h m a h o w e v e r t h e r e i s

l i t t l e b r o n c h o c o n s t r i c t i o n a f t e r e x e r c i s e . T h e

i n c i d e n c e o f r e v e r s i b i l i t y v a r i e s f r o m 1 4 - 5 0 %

d e p e n d i n g o n t h e m e t h o d of : c h a l l e n g e i n d i f f e r e n t

s e r i e s . A r e c e n t s t u d y f r o m B i r m i n g h a m s h o w e d t h a t

a l m o s t a t h i r d of : C F c h i l d r e n s t u d i e d h a d a

s i g n i f i c a n t e l e m e n t o f r e v e r s i b l e a i r w a y s

o b s t r u c t i o n , a n d a l l o f t h e s e c h i l d r e n w e r e a t o p i c .

T h e a u t h o r s s u g g e s t e d t h a t C F a n d e x t r i n s i c a s t h m a

c o - e x i s t 1 4

b ) A s p e r g i l l u s f u m i g a t u s

A s p e r g i l l u s f u m i g a t u s T y p e 1 h y p e r s e n s i t i v i t y o n

s k i n t e s t i n g i s c o m m o n , t h e p e r c e n t a g e i n c r e a s i n g

w i t h a g e r e a c h i n g 7 5 - 8 0 % i n a d u l t s - * - ^ . S e r u m

p r e c i p i t i n s t o a s p e r g i l l u s a r e s e e n i n a b o u t a t h i r d

o f c a s e s , b u t b r o n c h o p u l m o n a r y a s p e r g i l l o s i s i s m u c h

r a r e r , a n i n c i d e n c e o f 0 . 5 - 5 % b e i n g r e p o r t e d ^ - 0 .

c ) G a s t r o i n t e s t i n a l s y s t e m

T h e e a r l i e s t g a s t r o i n t e s t i n a l m a n i f e s t a t i o n o f C F

i s m e c o n i u m i l e u s , w h i c h o c c u r s i n 1 0 - 1 5 % o f n e w b o r n s

w i t h t h e d i s e a s e . T h e r e i s f a i l u r e t o p a s s m e c o n i u m

a n d i n t e s t i n a l o b s t r u c t i o n d e v e l o p s b e t w e e n 2 4 a n d 4 8

h o u r s o f b i r t h . M e c o n i u m i l e u s e q u i v a l e n t , a s m a l l

b o w e l o b s t r u c t i o n , o c c u r s i n o l d e r c h i l d r e n a n d

a d u l t s u s u a l l y i n t h e i l e o - c a e c a l r e g i o n . P a n c r e a t i c

i n s u f f i c i e n c y m a n i f e s t s i t s e l f a s s t e a t o r r h o e a , a n d

f a i l u r e t o t h r i v e o r g a i n w e i g h t d e s p i t e a g o o d

a p p e t i t e . O t h e r c o m p l i c a t i o n s i n c l u d e r e c t a l

p r o l a p s e , i n t u s s u s c e p t i o n , v o l v u l u s , d u o d e n a l

u l c e r a t i o n a n d p a n c r e a t i t i s . T h e l i v e r u n d e r g o e s

f a t t y c h a n g e i n m a n y c a s e s , a n d m a n y p a t i e n t s d e v e l o p

a f o c a l b i l i a r y f i b r o s i s w h i c h i s p a t h o g n o m o n i c o f

C P . C i r r h o s i s ; l i v e r f a i l u r e , p o r t a l h y p e r t e n s i o n

a n d v a r i c e s o c c u r i n a s m a l l n u m b e r o f p a t i e n t s w i t h

l i v e r d i s e a s e . G a l l s t o n e s a r e a l s o f o u n d i n a f e w

o l d e r p a t i e n t s .

d ) G l u c o s e i n t o l e r a n c e

G l u c o s e i n t o l e r a n c e i s c o m m o n a n d i n c r e a s e s w i t h

a g e . I t i s r e l a t e d t o p r o g r e s s i v e p a n c r e a t i c

d i s e a s e a n d r e d u c e d c a p a c i t y f o r i n s u l i n r e l e a s e ^ 7 .

K e t o a c i d o s i s i s r a r e a n d t h e m i c r o v a s c u l a r

c o m p l i c a t i o n s o f d i a b e t e s h a v e n o t y e t b e e n d e s c r i b e d

c l i n i c a l l y i n C F . H o w e v e r m u s c l e c a p i l l a r y b a s e m e n t

m e m b r a n e i s t h i c k e n e d i n a l m o s t a l l C F p a t i e n t s , a n d

b e c o m e s a d d i t i o n a l l y t h i c k e n e d w i t h f a s t i n g

1 qh y p e r g l y c a e m i a . T h i c k e n i n g o f c a p i l l a r y b a s e m e n t

m e m b r a n e s i s a d i s t i n c t i v e f e a t u r e o f d i a b e t i c

m i c r o a n g i o p a t h y , a n d i t m a y b e t h a t w i t h i n c r e a s i n g

l i f e e x p e c t a n c y C F p a t i e n t s w i l l d e v e l o p d i a b e t i c

c o m p l i c a t i o n s .

e ) G r o w t h a n d d e v e l o p m e n t

T h e r e i s a h i g h e r m o r b i d i t y a n d m o r t a l i t y i n

f e m a l e s a f t e r p u b e r t y r e s u l t i n g i n a s l i g h t m a l e

p r e d o m i n a n c e i n t h e a d u l t C F p o p u l a t i o n . I n c r e a s e i n

h e i g h t w i t h a g e i s l e s s s e v e r e l y a f f e c t e d b y t h e

d i s e a s e t h a n i n c r e a s e i n w e i g h t . M o s t p a t i e n t s r e a c h

t h e 2 5 t h p e r c e n t i l e f o r h e i g h t , b u t a r e o f t e n b e l o w

t h e 3 r d p e r c e n t i l e f o r w e i g h t . D e l a y e d p u b e r t y

o c c u r s i n m a n y b u t d e l a y e d s k e l e t a l m a t u r i t y i s a

m o r e c o n s i s t e n t f e a t u r e . I t d o e s n o t s e e m t o b e

r e l a t e d t o t h e s e v e r i t y o f l u n g d i s e a s e a s a s s e s s e d

b y s o i r o i a e L r y ^ ^ . M a l e s t e r i l i t y i s d u e t o t h e

d e v e l o p m e n t a l f a i l u r e of : m e s o n e p h r i c d u c t s t r u c t u r e s ,

t h e e p i d i d y m i s , s e m i n a l v e s i c l e s a n d v a s d e f e r e n s .

T h e r e a r e a l e v / r e p o r t s o f a C P m a l e f a t h e r i n g a

c h i l d , b u t m o r e t h a n 9 5 % h a v e o b s t r u c t i v e

a z o o s p e r m i a 2 0_ G y n a e c o m a s t i a m a y a l s o b e a p r o b l e m

i n m a l e s . F e r t i l i t y i n w o m e n i s r e d u c e d , p r o b a b l y

d u e t o t h e t h i c k , v i s c i d c e r v i c a l m u c u s , w h i c h d o e s

n o t e x h i b i t f e r n i n g a t o v u l a t i o n . S e c o n d a r y

a m e n o r r h o e a i s s e e n w i t h s e v e r e p u l m o n a r y d i s e a s e . A

n u m b e r o f C F f e m a l e p a t i e n t s h a v e b o r n e c h i l d r e n , b u t

t h e r e i s a g r e a t r i s k o f d e t e r i o r a t i o n i n l u n g

f u n c t i o n d u r i n g p r e g n a n c y a n d t h e p u e r p e r i u m a n d

m e d i c a l a t t e n t i o n t h r o u g h o u t t h i s p e r i o d w i t h

s c r u p u l o u s p h y s i o t h e r a p y i s m o s t i m p o r t a n t ^ ' ^ 1 #

f . O t h e r c o m p l i c a t i o n s

T h e s e i n c l u d e h e a t p r o s t r a t i o n d u e t o e x c e s s i v e

s a l t l o s s i n t h e s w e a t , h y p e r t r o p h i c p u l m o n a r y

o s t e o a r t h r o p a t h y , i n a p p r o p r i a t e ADH s e c r e t i o n a n d

t r a n s i e n t a r t h r a l g i a , w h i c h i s p r o b a b l y a n i m m u n e

c o m p l e x - m e d i a t e d p h e n o m e n o n . T h r e e c a s e s o f

a m y l o i d o s i s h a v e b e e n described^. P o l y c y t h a e m i a i s

r a r e d e s p i t e t h e f r e q u e n c y o f h y p o x i a - * - .

3 • d i a g n o s i s

A h i g h s w e a t s o d i u m c o n c e n t r a t i o n i s a c a r d i n a l

f e a t u r e o f t h e d i a g n o s i s o f C F . I t i s m o s t r e l i a b l y

p e r f o r m e d b y p i l o c a r p i n e i o n t o p h o r e s i s a n d a s a m p l e

o f a t l e a s t l O O m g s o f s w e a t m u s t b e c o l l e c t e d ^ 3 .

The u p p e r l i m i t o f n o r m a l i n c h i l d r e n i s g e n e r a l l y

a c c e n t e d a s 7 0 r n m o l / l i n t h i s c o u n t r y , a n d t h e

m a j o r i t y of : p a t i e n t s w i t h C F h a v e v a l u e s i n t h e r a n g e

o f 8 G - 1 2 0 m m o l / l . N o r m a l a d o l e s c e n t s a n d a d u l t s t e n d

t o h a v e s l i g h t l y h i g h e r v a l u e s t h a n c h i l d r e n , s o

t h e r e a r e s o m e n o r m a l a d u l t s w i t h a s w e a t s o d i u m

a b o v e 7 0 m m o l / L . A s u p p r e s s i o n t e s t w i t h

f l u d r o c o r t i s o n e h a s r e c e n t l y b e e n s h o w n t o b e u s e f u l

h e r e a s t h e s w e a t s o d i u m c o n c e n t r a t i o n o f n o r m a l

p a t i e n t s i s s u p p r e s s e d b y t a k i n g o r a l

m i n e r a l o c o r t i c o i d , b u t t h a t o f C F p a t i e n t s i s n o t ^ .

F a l s e p o s i t i v e r e s u l t s o c c u r i n c o n d i t i o n s s u c h a s

A d d i s o n ' s d i s e a s e , n e p h r o g e n i c d i a b e t e s i n s i p i d u s a n d

i n m a l n u t r i t i o n . O e d e m a m a y g i v e f a l s e n e g a t i v e

r e s u l t s - * - . P a n c r e a t i c f u n c t i o n t e s t s a r e r a r e l y

p e r f o r m e d r o u t i n e l y , b u t m a y b e u s e f u l w h e n t h e

d i a g n o s i s i s i n d o u b t . S t e a t o r r h o e a i s e s t a b l i s h e d

b y a 3 d a y f a e c a l f a t c o l l e c t i o n o n a d i e t o f k n o w n

f a t c o n t e n t a n d w i t h o u t p a n c r e a t i c r e p l a c e m e n t

t h e r a p y . P a n c r e a t i c f u n c t i o n i s m o s t r e l i a b l y

d e t e r m i n e d b y d u o d e n a l i n t u b a t i o n a n d c o l l e c t i o n o f

s e c r e t i o n s f o l l o w i n g s e c r e t i n a n d p a n c r e o z y m i n

i n j e c t i o n s ^ . i n t h o s e p a t i e n t s w i t h o u t c l i n i c a l

p a n c r e a t i c i n s u f f i c i e n c y , e n z y m e c o n c e n t r a t i o n s m a y

b e n o r m a l b u t t h e r e i s d i m i n i s h e d b i c a r b o n a t e

p r o d u c t i o n . R e c e n t s t u d i e s o n s e r u m i m m u n o r e a c t i v e

t r y p s i n s u g g e s t t h a t t h i s i s a u s e f u l a d j u n c t t o

diagnosis^ # A t b i r t h , p a t i e n t s w i t h C F h a v e a v e r y

h i g h i m m u n o r e a c t i v e t r y p s i n c o m p a r e d t o t h e n o r m a l

p o p u l a t i o n v / h i c h l a t e r C a l l s t o a v e r y l o w l e v e l .

T h i s C a l l i n i m m u n o r e a c t i v e t r y o s i n p r o b a b l y

p a r a l l e l s t h e d e c l i n e i n p a n c r e a t i c C u n c t i o n .

S i m i l a r a b n o r m a l i t i e s i n s e r u m p a n c r e a t i c l i p a s e

a c t i v i t y a r e a l s o r e p o r t e d 2 7 f a n d i t i s s u q q e s t e d

t h a t l e a k a g e o C e n z y m e s C r o m t h e p a n c r e a t i c a c i n i

o c c u r s d i r e c t l y i n t o t h e c i r c u l a t i o n .

4 . T R E A T M E N T

a ) P u l m o n a r y D i s e a s e

i P r e v e n t i o n o f i n f e c t i o n

P a t i e n t s w i t h C F s h o u l d r e c e i v e t h e r o u t i n e

i m m u n i s a t i o n p r o g r a m m e i n c h i l d h o o d , a l t h o u g h

v a c c i n a t i o n d u r i n g a n y f e b r i l e e p i s o d e s h o u l d b e

a v o i d e d . I n f l u e n z a v a c c i n e i s o f t e n g i v e n

a l t h o u g h t h e r e h a s n o t b e e n a s t u d y o f i t s

e f f e c t i v e n e s s i n C F . P a t i e n t s a r e s t r o n g l y

d i s c o u r a g e d f r o m s m o k i n g a n d w h e r e p o s s i b l e t h i s

a d v i c e i s e x t e n d e d t o t h e i r f a m i l y . F a m i l y

m e m b e r s w i t h u p p e r r e s p i r a t o r y i n f e c t i o n s s h o u l d

a v o i d c o n t a c t w i t h t h e C F p a t i e n t a s f a r a s

p o s s i b l e .

i i P h y s i o t h e r a p y

S e g m e n t a l p o s t u r a l d r a i n a g e , w i t h c h e s t

p e r c u s s i o n a n d v i b r a t i o n f o l l o w e d b y c o u g h i n g , i s

p r o b a b l y t h e s i n g l e m o s t i m p o r t a n t f e a t u r e o f

t r e a t m e n t . T h e f o r c e d e x p i r a t i o n t e c h n i q u e ( F E T )

h a s b e e n s h o w n t o b e a t l e a s t a s e f f e c t i v e a s

c o n v e n t i o n a l p h y s i o t h e r a p y , a n d m a y b e p e r f o r m e d

o f t i c i e n t l y b y t h e p a t i e n t w i t h o u t a 53 a i s t a n c e ,

g i v i n g o l d e r p a t i e n t s a d e q r e e o f i n d e p e n d e n c e ^ .

P h y s i c a l e x e r c i s e o f a n y k i n d i s a u s e f u l a d j u n c t

t o p h y s i o t h e r a p y , a n d p a t i e n t s s h o u l d b e

e n c o u r a g e d t o e x e r c i s e a s m u c h a s p o s s i b l e w i t h i n

t h e i r o w n p h y s i c a l l i m i t a t i o n s .

S i m p l e h u m i d i f i c a t i o n o f i n s p i r e d a i r i s v e r y

h e l p f u l i n C F t o r e d u c e t h e v i s c o s i t y o f s p u t u m ,

t o a l l o w i t s e x p e c t o r a t i o n a n d t o m i n i m i s e

d e h y d r a t i o n i n t a c h y p n o e i c p a t i e n t s w h o o f t e n

m o u t h - b r e a t h e . H y p e r t o n i c s a l i n e ( 7 %

w e i g h t / v o l u m e ) g i v e n b y n e b u l i z e r m a y b e h e l p f u l

w h e n s p u t u m i s p a r t i c u l a r l y t e n a c i o u s .

i i i B r o n c h o d i l a t o r s a n d c o r t i c o s t e r o i d s

S a l b u t a m o l i s a d m i n i s t e r e d b y n e b u l i z e r o r

p r e s s u r i s e d a e r o s o l i n p a t i e n t s w i t h d e m o n s t r a b l e

r e v e r s i b i l i t y o f a i r w a y s o b s t r u c t i o n , a n d i t s u s e

b e f o r e p h y s i o t h e r a p y m a y f a c i l i t a t e p o s t u r a l

d r a i n a g e . T e r b u t a l i n e h a s b e e n r e p o r t e d t o

i n c r e a s e t h e r a t e o f m u c o c i l i a r y t r a n s p o r t ^ .

C o r t i c o s t e r o i d s r n a y b e o f b e n e f i t b u t h a v e o n l y

b e e n u s e d i n t h e l a t e r s t a g e s o f t h e d i s e a s e .

i v S u r g e r y

T h e m a i n i n d i c a t i o n f o r s u r g e r y i n C F i s f o r

p n e u m o t h o r a x ^ . P a t i e n t s w i t h s e v e r e h a e m o p t y s i s

h a v e u n d e r g o n e s u r g e r y t o l i g a t e t h e o f f e n d i n g

b r o n c h i a l a r t e r y , b u t e m b o l i s a t i o n o f t h e a r t e r y

v i a a f e m o r a l a r t e r y c a t h e t e r i s a l e s s i n v a s i v e

a p p r o a c h . L o b a r b r o n c h i e c t a s i s r a t h e r t h a n

d i f f u s c l u n q d i s e a s e i s n o t c o m m o n b u t d o c s o c c u r

i n s o m e p a t Lo n t s / a n d l o b e c t o m y i s p e r f o r m e d

( Ol : t o n t h e d i a g n o s i s h a s n o t b e e n m a d e a i: t h e

o f s u r g e r y i n t h e s e c a s e s ) . V i g o r o u s o r e a n d

o o s t - o p e r a ti v e p h y s i o t h e r a p y a n d a n t i b i o t i c c

X s e s s e n t i a 1 f; o r a 11 s u r g i c a l o r o c e d u r e s

F i b r e o p t i c b r o n c h o s c o p y i s u s e f u l i n m a s s i v e

h a e m o p t y s i s t o d e t e r m i n e t h e s i t e o f b l e e d i n g .

S u c t i o n a n d l a v a g e m a y b e a u s e f u l t r e a t m e n t f o r

p e r s i s t e n t a t e l e c t a s i s ,

v A n t i b i o t i c s

A n t i m i c r o b i a l c h e m o t h e r a p y i n g e n e r a l i s

d i s c u s s e d l a t e r i n t h i s c h a p t e r , a n d

a n t i p s e u d o m o n a l c h e m o t h e r a p y i s r e v i e w e d i n

C h a p t e r 4 .

b ) G a s t r o i n t e s t i n a l d i s e a s e

i P a n c r e a t i c e n z y m e r e p l a c e m e n t s

P a n c r e a t i c e n z y m e r e p l a c e m e n t i s g i v e n a s

p r e p a r a t i o n s d e r i v e d f r o m a n i m a l p a n c r e a s a n d

t a k e n w i t h f o o d . T h e d o s e a n d p a r t i c u l a r

p r e p a r a t i o n i s a d j u s t e d t o t h e i n d i v i d u a l , t o

e l i m i n a t e o v e r t s t e a t o r r h o e a a n d m a i n t a i n

n u t r i t i o n . F a t r e s t r i c t i o n i s o f t e n n e c e s s a r y i n

c h i l d r e n , b u t l e s s s o i n a d u l t s , a n d m a n y p a t i e n t s

a r e a b l e t o e a t a n o r m a l d i e t . T y p e 1

h y p e r s e n s i t i v i t y r e a c t i o n s i n c l u d i n g b r o n c h o -

c o n s t r i c t i o n m a y o c c u r w i t h a c c i d e n t a l i n h a l a t i o n

o f p a n c r e a t i n p o w d e r ^ l .

F a t s o l u b l e v i t a m i n s A , D a n d E a r e g i v e n

re gul ar ly . Vitamin K may be given in haemopt ys i s .

B•} 2 de f ic iency i s uncommon, but iron and {folate

d e f i c i e n c i e s are seen, e s p e c i a l l y in women.

r.ipid s o l u b i l i s a t i o n and b i l e acid

p r e c i p i t a t i o n are pH dependent proc es ses , and are

adversely a f f e c t e d by a low je j una l pH in p a t i e n t s

with reduced pancreat ic bicarbonate sec re t ion .

T h e u s e o f c i m e t i d i n e 4 0 m i n u t e s b e f o r e m e a l s t o

r a i s e j e j u n a l p H b y r e d u c i n g g a s t r i c a c i d

s e c r e t i o n f a c i l i t a t e s t h e a c t i o n o f p a n c r e a t i n 3 2 #

i i N u t r i t i o n a l s u p p l e m e n t a t i o n

A c o m p l e t e l y a r t i f i c a l d i e t c o m p r i s i n g m e d i u m

c h a i n t r i g l y c e r i d e , a g l u c o s e p o l y m e r a n d b e e f

s e r u m h y d r o l y s a t e h a s b e e n described^. s o m e

p a t i e n t s s h o w e d a n i m p r o v e m e n t i n g r o w t h a n d i n

w e l l - b e i n g , b u t t h e d i e t i s d i f f i c u l t t o a d h e r e t o

a n d d e f i c i e n c i e s m a y o c c u r , p a r t i c u l a r l y o f

e s s e n t i a l f a t t y a c i d s ^ f 1 . M e d i u m c h a i n

t r i g l y c e r i d e o i l h a s b e e n w i d e l y u s e d a s i t w a s

t h o u g h t t o b e d i r e c t l y a b s o r b e d i n t o l a c t e a l s .

T h e m o s t i m p o r t a n t p a r t o f a n y n u t r i t i o n a l

s u p p l e m e n t s i s t o m a i n t a i n a d e q u a t e c a l o r i e

i n t a k e .

i i i M e c o n i u m i l e u s e q u i v a l e n t

T h i s i s t r e a t e d w i t h N - a c e t y l c y s t e i n e , b o t h

o r a l l y a n d b y e n e m a . E n e m a s o f h y g r o s c o p i c

r a d i o a c t i v e c o n t r a s t m e d i a , s u c h a s g a s t r o g r a p h i n

m a y a l s o b e u s e f u l . I f o b s t r u c t i o n i s n o t

r e l i e v e d s u r g e r y m a y b e n e e d e d , b u t t h i s s h o u l d

not bo the ( i irst l ine of: treatment. Recurrent

col icky pain, 'chronic meconium i l e u s e q u iv a l e n t ' ,

may ind ica te impending obstruct ion and may be

re l ieved by o r a l N-acety lcys te ine^S .

c ) Psychosocial Aspects

T h e i n c r e a s i n g n u m b e r s o f a d o l e s c e n t s a n d a d u l t s

w i t h C F r e s u l t s i n a n u m b e r of : p s y c h o s o c i a l p r o b l e m s .

T h e p a t i e n t s n e e d t o a c q u i r e r e s p o n s i b i l i t y f o r t h e i r

o w n c a r e , a n d t o m a k e t h e t r a n s i t i o n f r o m t h e f a m i l y

h o m e t o i n d e p e n d e n t l i v i n g . T h e c h o i c e o f j o b o r

f u r t h e r e d u c a t i o n i s i m p o r t a n t . P a t i e n t s n e e d t o

d e v e l o p s k i l l s t h a t a r e i n d e m a n d t o c o m p e n s a t e f o r

t h e i r m e d i c a l h a n d i c a p , a n d t h e y m a y n e e d t o w o r k f r o m

h o m e o r i n a n e n v i r o n m e n t s u i t a b l e f o r t h o s e w i t h

c h r o n i c l u n g d i s e a s e .

An i n c r e a s i n g n u m b e r o f a d u l t s w i t h C F a r e

m a r r i e d , a n d t h e r e i s a n e e d f o r c o u n s e l l i n g o f t h e

s p o u s e a b o u t t h e p a t i e n t ' s c o n d i t i o n . I n f e r t i l i t y i n

m a l e s , t h e r i s k o f p r e g n a n c y t o t h e h e a l t h o f t h e

m o t h e r ^ , t h e g e n e t i c s o f C F , a n d t h e e f f e c t s o f

c h r o n i c d i s e a s e o n s e x u a l f u n c t i o n a r e a l l i m p o r t a n t

p r o b 1 e m s .

C F i s a l i f e - t h r e a t e n i n g i l l n e s s w i t h a n a l m o s t

i n e v i t a b l e d o w n w a r d c o u r s e , y e t p a t i e n t s c o p e w i t h

t h i s r e m a r k a b l y w e l l w i t h n o s i g n i f i c a n t i n c r e a s e i n

p s y c h i a t r i c d i s t u r b a n c e s c o m p a r e d w i t h t h e i r p e e r

g r o u p - ^ . C l i n i c a l l y , i t i s o f t e n t h e f a m i l y r a t h e r

t h a n t h e p a t i e n t w h o i s m o s t i n n e e d o f p s y c h o l o g i c a l

s u p p o r t .

T h u q o n o t i c b a s i s of: C F renains to bo o L u c i da tod,

and at present theirs are no proven .methods of: c a r r i e r

detec t ion , or ante-natal , d i agno s i s . If: the deflective

qene or i t s products could be i d e n t i f i e d , then

management may in the future involve the techniques of

molecular b iology.

B. BRONCHOPULMONARY INFECTION IN CYSTIC FIBROSIS1 . B A C T E R I A L I N F E C T I O N

P a t i e n t s w i t h C F h a v e a c h r o n i c p r o d u c t i v e c o u g h ,

a n d a w i d e v a r i e t y o f o r g a n i s m s m a y b e i s o l a t e d f r o m

t h e i r s p u t u m . T h e m o s t c o m m o n o r g a n i s m i s P s e u d o m o n a s

a e r u g i n o s a b u t S t a p h y l o c o c c u s a u r e u s , H a e m o p h i l u s

i n f l u e n z a e a n d K l e b s i e l l a p n e u m o n i a e v a r . a e r o g e n e s

( K . a e r o g e n e s ) a r e f r e q u e n t l y i s o l a t e d . T h e p a t t e r n o f

s p u t u m f l o r a i s r e l a t e d t o a g e a n d s e v e r i t y o f d i s e a s e

a n d h a s u n d e r g o n e s o m e c h a n g e s o v e r t h e d e c a d e s s i n c e

1 9 3 8 . T h e m a i n e v e n t s a r e s u m m a r i s e d i n T a b l e 1 .

I n t h e i n i t i a l s t u d i e s o f C F b y A n d e r s e n ^ a n d

d i S a n t ' A g n e s e ^ a l m o s t a l l p a t i e n t s w e r e c o l o n i s e d b y

S . a u r e u s a n d t h i s o r g a n i s m w a s i s o l a t e d f r o m m o s t

p o s t - m o r t e m s p e c i m e n s . A t t h a t t i m e s t a p h y l o c o c c i

w e r e p r e v a l e n t i n t h e h o s p i t a l e n v i r o n m e n t a n d n a s a l

c a r r i a g e w a s p r e v a l e n t i n t h e g e n e r a l p o p u l a t i o n . T h e

a v e r a g e a g e a t d e a t h i n 5 7 p a t i e n t s s e e n b e t w e e n 1 9 4 0

a n d 1 9 4 9 i n t h e s e s e r i e s w a s j u s t o v e r o n e y e a r - ^ .

F r o m 1 9 3 7 o n w a r d s o r a l s u l p h o n a m i d e s w e r e f o u n d t o b e

u s e f u l i n t h o s e p a t i e n t s w i t h r e c e n t l y a c q u i r e d

i n f e c t i o n s , b u t n o t i n t h o s e w h o w e r e s e v e r e l y i l l o r

CHANGES I N THE BACTERIAL FLORA ISOLATED FROM THE SPUTUM OF

CYSTIC FIBROSIS PATIENTS SINCE 1938.

19631969

| Antibiotic | therapy | available 1

| Sputum pathogens | isolated

Mean | survival of | CF patients | (years) |

Refe renee

1| SuIphonamides 1

| S.aureus only1

1-1 1 1

1! Pencillin 1

1I Tetracycline 1

13-8 |

1| Cloxacillin | Emergence of

| 'prophylaxis' 1

| P.aeruginosa 11I

| Gentamicin | P.aeruginosa 12 I 39| Carbenici11in | (40-90% in 11l

* Brompton Hospital Cystic Fibrosis Unit, Unpublished data.

MB Patients had all survived to 16 years to be referred to this unit.

c y a n o s e d . S m a l l d o s e s of : s u l p h a d i a z i n c w e r e g i v e n t o

e i g h t C r i s e s o v e r t h e w i n t e r o c 1 9 4 4 - 4 5 a n d t h i s w a s

c l a i m e d t o h a v e r e d u c e d t h e n u m b e r a n d s e v e r i t y o f

u p p e r r e s p i r a t o r y t r a c t i n f e c t i o n s . P e n i c i l l i n b e c a m e

a v a i l a b l e i n 1 9 4 4 a n d w a s u s e d b o t h p a r e n t e r a l l y a n d

h y a e r o s o l . T h e u s e o f p e n i c i l l i n b y a e r o s o l i n s o m e

m i l d c a s e s o f C F p r o d u c e d a s u s t a i n e d i m p r o v e m e n t i n

r e s p i r a t o r y f u n c t i o n a n d i n w e i g h t g a i n o v e r a p e r i o d

o f m o n t h s ^ . S h w a c h m a n e t a l ^ / ^ l r e p o r t e d t h e u s e o f

t e t r a c y c l i n e b o t h i n a c u t e r e s p i r a t o r y e x a c e r b a t i o n s

a n d a s l o n g t e r m p r o p h y l a x i s . A v e r a g e a g e a t d e a t h o f

4 2 p a t i e n t s s e e n i n t h e i r c l i n i c b e t w e e n 1 9 4 5 a n d 1 9 5 4

w a s e x t e n d e d t o n e a r l y f o u r y e a r s . I t i s i n t e r e s t i n g

t o n o t e t h a t t h e d i s c u s s i o n i n t h i s p a p e r m e n t i o n s

s e v e r a l c a s e s o f f i t , a c t i v e t e e n a g e r s b o r n b e f o r e t h e

a g e o f a n t i b i o t i c s a n d d i a g n o s e d o n l y i n a d o l e s c e n t

l i f e .

A n t i - s t a p h y l o c o c c a l d r u g s w e r e i n t r o d u c e d i n t h e

1 9 5 0 s , a n d t h e i r u s e w a s f o l l o w e d b y a r i s e i n t h e

i s o l a t i o n r a t e o f P . a e r u g i n o s a . A t t h i s t i m e i t w a s

f e l t t h a t t h e i n i t i a l l u n g d a m a g e i n C F w a s c a u s e d b y

s t a p h y l o c o c c a l i n f e c t i o n , a n d t h a t t h e p r e v e n t i o n o f

s u c h i n f e c t i o n w o u l d p r e v e n t l u n g d a m a g e a n d s e c o n d a r y

i n f e c t i o n b y o t h e r p a t h o g e n s . C o n t i n u o u s

’ p r o p h y l a x i s ' w i t h c l o x a c i l l i n w a s r e c o m m e n d e d b y m a n y

p a e d i a t r i c i a n s . M e a r n s e t a l ^ ® i n t r o d u c e d c o n t i n u o u s

p r o p h y l a x i s w i t h c l o x a c i l l i n f r o m t h e f i r s t y e a r o f

l i f e i n t h e i r c l i n i c i n 1 9 5 7 . T w o c o h o r t s o f p a t i e n t s

w e r e c o m p a r e d , b o t h s e e n i n t h e f i r s t y e a r o f l i f e

w i t h . s i m i l a r r a d i o l o g i c a l , c l i n i c a l a n d

b a c t o r i o 1 o q L c a 1 1! i n d i n g s o n o n t r y t o t h e s t u d y . T h e

g r o u p s o o n b e t o r e 1 9 5 7 h a d a m o r t a l i t y of ; 1 4 1 ; a t t h e

a g o o f f i v e y e a r s . T h e g r o u p s e e n a l t e r 1 9 5 7 h a d a

m o r t a l i t y o f 6 . 5 $ a t t h e s a m e a g e . B a c t e r i a l f l o r a i n

t h e s e c h i l d r e n w e r e s t u d i e d o v e r a p e r i o d o f 20 y e a r s

f r o m 1 9 5 0 t o 1 9 7 1 . T h e i s o l a t i o n r a t e o f S . a u r e u s

f e l l f r o m 4 5 % i n 1 9 5 6 t o 1 2 % i n . 1 9 7 1 , w h i l e t h e r a t e

f o r P . a e r u g i n o s a r o s e f r o m 3% t o 2 8 % . T h e i s o l a t i o n

r a t e o f s t a p h y l o c o c c u s f e l l i n a l l a g e g r o u p s , b u t

p a r t i c u l a r l y i n t h e f i r s t y e a r o f l i f e , a n d t h i s

o c c u r r e d i n p a t i e n t s a t a l l s t a g e s o f t h e d i s e a s e .

T h e i s o l a t i o n r a t e o f p s e u d o m o n a s i n c r e a s e d m o s t

m a r k e d l y i n t h o s e s e v e r e l y a f f e c t e d b y t h e d i s e a s e ^ .

T h e p r e v a l e n c e o f P . a e r u g i n o s a i n C F i s n o w

b e t w e e n 4 0 a n d 9 0 % i n d i f f e r e n t s e r i e s ^ . I t i s

f r e q u e n t l y t h e o n l y p a t h o g e n i s o l a t e d a t t h e t i m e o f

d e a t h / 1 . B a c t e r i a i s o l a t e d i n t h e h o s p i t a l e n v i r o n m e n t

h a v e u n d e r g o n e a s i m i l a r c h a n g e , w i t h P . a e r u g i n o s a

r e p l a c i n g S . a u r e u s . T h e m u c o i d f o r m o f p s e u d o m o n a s

w i t h i t s h i g h l y v i s c o u s s l i m e e n v e l o p e w a s d e s c r i b e d

b y D o g g e t t i n 1 9 5 6 ^ 4 . i t i s v e r y c o m m o n i n C F a n d

a p p e a r s t o d e v e l o p a f t e r a p e r i o d o f t i m e i n t h e C F

h o s t ; t h i s m u c o i d f o r m i s r a r e i n s t r a i n s f r o m o t h e r

s o u r c e s . T h e m u c o i d f o r m h a s b e e n c o n s i d e r e d t o b e

m o r e p a t h o g e n i c t h a n o t h e r f o r m s , a s t h e i n c i d e n c e o f

s e r u m a n t i b o d i e s i s h i g h e r w h e n t h i s f o r m i s

p r e s e n t ^ . & h i g h a n t i b o d y r e s p o n s e a n d t y p e t h r e e

h y p e r s e n s i t i v i t y r e a c t i o n h a s b e e n p o s t u l a t e d a s t h e

m e c h a n i s m d o r t h e i n i t i a l . l u n q d a m a g e . H o w e v e r , a s

m u c o i d s t r a i n s t e n d t o a p p e a r o n l y a l t e r a p e r i o d o l :

c o l o n i s a t i o n b y n o r m a l d o r m s , t h e p r e c i p i t i n l e v e l s

m a y m e r e l y r e d L e c t t h e d u r a t i o n o f e x p o s u r e . M u c o i d

a n d n o r m a l d o r m s m a y c o - e x i s t i n t h e s a m e p a t i e n t a n d

t h e m o r p h o l o g i c a l c h a n g e c a n b e s e e n t o o c c u r i n b o t h

d i r e c t i o n s o n a g a r c u l t u r e ^ 5 .

G e n t a m i c i n , t h e f i r s t a m i n o g l y c o s i d e h i g h l y a c t i v e

a g a i n s t p s e u d o m o n a s , w a s i n t r o d u c e d i n 1 9 6 3 . T h e

f i r s t a n t i - p s e u d o m o n a l p e n i c i l l i n , c a r b e n i c i l l i n ,

f o l l o w e d i n 1 9 6 9 . S i n c e t h e n c o m b i n a t i o n s o f t h e s e

d r u g s , o r t h e i r s u c c e s s o r s , h a v e f o r m e d t h e b a s i s o f

t r e a t m e n t o f a c u t e r e s p i r a t o r y e x a c e r b a t i o n s i n C F

p a t i e n t s c o l o n i s e d b y P . a e r u g i n o s a .

H a e m o p h i l u s i n f l u e n z a e w a s i s o l a t e d i n o n l y a f e w

c a s e s o f C F i n t h e 1 9 6 0 ’ s d e s p i t e t h e p r e s e n c e o f

p r e c i o i t i n s t o t h i s o r g a n i s m i n t h e s e r u m o f u p t o 8 0 %

o f p a t i e n t s w i t h C F ^ ^ . T h i s d i s c r e p a n c y w a s p r o b a b l y

d u e t o t h e d i f f i c u l t y i n i s o l a t i o n o f H . i n f l u e n z a e i n

t h e p r e s e n c e o f P . a e r u g i n o s a . A t e c h n i q u e , u s i n g a

s e l e c t i v e m e d i u m , d e v e l o p e d b y R o b e r t s ^ ® h a s b e e n i n

u s e i n o u r l a b o r a t o r y f o r t h e l a s t t w o y e a r s , a n d t h e

i s o l a t i o n r a t e o f H . i n f l u e n z a e h a s i n c r e a s e d t o n e a r l y

3 0 % . T h i s s y s t e m , i n v o l v i n g a n a e r o b i c c u l t u r e s a n d

t h e u s e o f b a c i t r a c i n i n t h e m e d i u m h a s b e e n u t i l i s e d

b y o t h e r s t o s h o w a h i g h i s o l a t i o n r a t e o f

H . i n f l u e n z a e ^ . T h i s u n m a s k e d p r e v a l e n c e o f

H . i n f l u e n z a e p r o v i d e s a n e x p l a n a t i o n f o r t h e c l i n i c a l

o b s e r v a t i o n s o f t h e b e n e f i t s o f c h l o r a r n o h e n i c o l i n C F

o n t i o n 1 s w i t ii p s o u c l o i n o n a s i n f l e c t i o n . P n o u i n o c o c c a . l

i n f l e c t i o n n a n a s i m i l a r i n c i d e n c e i n C F t o o t h e r

c h r o n i c i n f l e c t i v e l u n g d i s e a s e s a n d a n a e r o b i c

o r g a n i s m s a r e c u l t u r e d f r o m a s m a l l n u m b e r of :

p a t i e n t s . K l e b s i e l l a s p e c i e s a r e r e l a t i v e l y f r e q u e n t

i n C F s p u t a a n d H u a n g e t a l ( 1 9 7 1 ) n o t i c e d a n

i n c r e a s e d i n c i d e n c e o f K l e b s i e l l a i n p a t i e n t s

p r e v i o u s l y t r e a t e d w i t h c a r b e n i c i 1 1 i n ^ O .

T h u s t h e a d v e n t o f a n t i b i o t i c t h e r a p y m a y h a v e

i n f l u e n c e d t h e n a t u r a l h i s t o r y o f b a c t e r i a l p u l m o n a r y

i n f e c t i o n s i n C F . T h e p r e d o m i n a n t o r g a n i s m i s n o w

P . a e r u g i n o s a r a t h e r t h a n S . a u r e u s a n d i t i s p e r s i s t e n t

i n m o s t p a t i e n t s . A m i n o r i t y o f p a t i e n t s c o n t i n u e t o

h a r b o u r s t a p h y l o c o c c i e v e n a f t e r i n t e n s i v e a n t i b i o t i c

t r e a t m e n t . B o t h o f t h e s e o r g a n i s m s , a n d p o s s i b l y

H . i n f l u e n z a e , h a v e a n a s s o c i a t i o n w i t h C F b u t w h e t h e r

t h e s e b a c t e r i a a r e p r i m a r y p a t h o g e n s w h i c h c a u s e

p r o g r e s s i v e l u n g d a m a g e , o r a r e p r e s e n t a s c o m m e n s a l s

i s n o t k n o w n . I t i s w i d e l y a s s u m e d t h a t t h e i m p r o v e d

s u r v i v a l i n C F i s r e l a t e d t o a n t i b i o t i c u s a g e , b u t

t h i s i s c o m p l i c a t e d b y t h e f a c t t h a t o t h e r e f f e c t i v e

t r e a t m e n t w a s d e v e l o p e d i n p a r a l l e l , p a r t i c u l a r l y

p h y s i o t h e r a p y b u t a l s o p a n c r e a t i c e n z y m e r e p l a c e m e n t ,

n u t r i t i o n a l s u p p l e m e n t s a n d t h e i m p r o v e d p a e d i a t r i c

c a r e . T h e n e t r e s u l t h a s b e e n a r a d i c a l c h a n g e i n t h e

s u r v i v a l a n d q u a l i t y o f l i f e o f C F p a t i e n t s ^ .

2 . I N F E C T I O N C A P S 1:1 D BY V I R U S E S

The* r o l e o f v i r u s e s i n a c u t e r e s p i r a t o r y

e x a c e r b a t i o n s of : C F h a v e r e c e i v e d l i t t l e a t t e n t i o n

c o m p a r e d t o b a c t e r i a l i n f e c t i o n . I t i s p o s s i b l e t h a t

v i r a l i n f e c t i o n m a y b e t h e i n i t i a l t r i g g e r i n

p u l m o n a r y d a m a g e . P a t i e n t s d o n o t a p p e a r t o b e m o r e

s u s p e c t i b l e t o v i r a l i n f e c t i o n t h a n n o r m a l c h i l d r e n ,

b u t t h e c o n s e q u e n c e s m a y b e m o r e s e r i o u s . W r i g h t e t

a l ^ 2 s t u d i e d r e s p i r a t o r y d i s e a s e i n c h i l d r e n w i t h C F

o v e r t h e w i n t e r o f 1 9 7 3 . S e r o l o g i c a l e v i d e n c e o f

v i r a l i n f e c t i o n w a s p r e s e n t i n 3 9 % o f m a j o r

e x a c e r b a t i o n s a n d 2 8 % o f t h e m i n o r o n e s . I n f l u e n z a A ,

p a r a - i n f l u e n z a a n d r e s p i r a t o r y s y n c y t i a l v i r u s ( R S V )

w e r e t h e m a i n o r g a n i s m s . T h e r e w a s n o e v i d e n c e o f

m y c o p l a s m a i n f e c t i o n a n d t h e a u t h o r s d i d n o t t e s t f o r

r h i n o v i r u s e s a n d c o r o n a v i r u s e s . P e t e r s o n e t a l ^

s t u d i e d 1 1 6 p a t i e n t s w i t h C F o v e r t h e w i n t e r o f

1 9 7 7 / 7 8 . T h e r e w e r e s e r o l o g i c a l l y p r o v e n v i r a l

i n f e c t i o n s i n 4 9 % o f p a t i e n t s o v e r t h i s p e r i o d ,

a l t h o u g h a s m a l l n u m b e r w e r e n o t a s s o c i a t e d w i t h a n y

a c u t e e x a c e r b a t i o n s . R S V w a s t h e d o m i n a n t a g e n t

a l t h o u g h a d e n o v i r u s e s , i n f l u e n z a A a n d p a r a - i n f l u e n z a

v i r u s e s w e r e c o m m o n ( t h e i n c i d e n c e o f p o s i t i v e

i n f l u e n z a A s e r o l o g y m a y b e a f f e c t e d b y r o u t i n e

i n f l u e n z a v a c c i n a t i o n g i v e n t o C F p a t i e n t s ) . R S V w a s

p a r t i c u l a r l y c o m m o n i n t h e s m a l l n u m b e r o f p a t i e n t s

w h o d e v e l o p e d p s e u d o m o n a s i n f e c t i o n d u r i n g t h e c o u r s e

o f t h e s t u d y . S o m e p a t i e n t s s h o w e d a r i s e i n t i t r e o f

ant ibodies to pseudomonas a f t e r RSV infec t ion an ci a

■1 3

synergism of action was suggested, with RSV damaging

bronchial, rnucosa to render the pat ient s u sc e p t ib le to

pseudomonas c o l o n i s a t ion.

3 . M Y C O P L A S M A AND R E L A T E D O R G A N I S M S

O u t o f 3 3 2 e p i s o d e s o f a c u t e r e s p i r a t o r y

e x a c e r b a t i o n s f o u r w e r e a t t r i b u t e d t o m y c o p l a s m a a n d

c h l a m y d i a i n t h e s t u d y b y P e t e r s e n ^ . O n e e p i s o d e o f

M y c o p l a s m a p n e u m o n i a e i n f e c t i o n a n d o n e o f C o x i e l l a

b u r n e t i w a s n o t e d b y E f t h i m i o u e t a l ^ 4 # a

s u r p r i s i n g l y h i g h i n c i d e n c e ( 2 2 * 3 % ) o f s e r u m a n t i b o d y

t o L e g i o n e l l a p n e u m o p h i l a w a s n o t e d b y Holsclaw^, c o m p a r e d t o 4 . 5 % i n a g e - m a t c h e d c o n t r o l s .

S e r o l o g i c a l s t u d i e s h a v e b e e n p e r f o r m e d i n o u r

i n - p a t i e n t s o v e r t h e f i r s t s i x m o n t h s o f 1 9 8 0 . A

g r o u p o f 2 4 p a t i e n t s w i t h d e t e r i o r a t i n g c l i n i c a l

c o n d i t i o n b e i n g c o m p a r e d w i t h a g r o u p o f a g e a n d

s e x - m a t c h e d n o r m a l c o n t r o l s , a n d a g r o u p o f p a t i e n t s

w h o r e m a i n e d c l i n i c a l l y s t a b l e . 2 9 % o f t h e

d e t e r i o r a t i n g g r o u p h a d s e r o l o g i c a l e v i d e n c e o f v i r a l

i n f e c t i o n d u r i n g t h a t s i x m o n t h p e r i o d , c o m p a r e d t o

o n l y o n e o f t h e s t a b l e p a t i e n t s . E i g h t p a t i e n t s a l s o

s h o w e d h i g h b a s a l t i t r e s t o l e g i o n e l l a ^ .

4 . M Y C O B A C T E R I A

M y c o b a c t e r i a l i n f e c t i o n s a r e s a i d t o b e r a r e i n

C F ^ . T h e m o s t c o m m o n i s M y c o b a c t e r i u m t u b e r c u l o s i s ,

a n d p a t i e n t s r e s p o n d w e l l t o s t a n d a r d t h e r a p y ; f o u r

p a t i e n t s i n o u r u n i t h a v e b e e n t r e a t e d f o r

L u o e r c u l o o i .o . B o x o r b a u m ~> ° r e p o r t e d a c a a o o 1; f a t a 1

p u l m o n a r y i n [ f o o t L o n w i t h t h e f a s t - g r o v / i n q o r g a n i s m

M . c h o L o n c i , a n d n o t e d s e v e n o t h e r s i n t h e l i t e r a t u r e

a s w e l l a s t w o c a s e s w i t h M . f o r t u i t u r n . We h a v e s e e n a

c a s e o f p e r s i s t e n t g r o w t h o f M . f o r t u i t u r n i n t h e s p u t u m

‘l u r i n g a f a t a l i l l n e s s 5 7 . / \ t p o s t - m o r t e m c a s e a t i n g

qranulomata were seen sc a t t er ed throughout both lungs.

C h a p t e r 2

PSEUDOMONAS AERUGINOSA

A . T H E O R G A N I S M

1 . O C C U R R E N C E

P s e u d o m o n a s a e r u g i n o s a h a s b e e n d e s c r i b e d a s ' a

p a t h o g e n o f t h e 2 0 t h c e n t u r y , t h e r e s u l t a n t h a z a r d o f

a n u m b e r o C a d v a n c e s i n m e d i c a l s c i e n c e , s e n s i t i v e t o

o n l y a r e v ; a n t i b i o t i c s a n d r a p i d l y a b l e t o d e v e l o p

r e s i s t a n c e t o t h o s e f e w ' 5 8 . I t i s a G r a m - n e g a t i v e ,

s t r a i g h t o r s l i g h t l y c u r v e d , n o n - s p o r i n g r o d , w i t h

n u m e r o u s p i l i g r o u p e d a t o n e p o l e . N e a r l y a l l s t r a i n s

a r e m o t i l e w i t h a s i n g l e p o l a r f l a g e l l u m .

P . a e r u g i n o s a i s a n e x t r e m e l y v e r s a t i l e o r g a n i s m ,

w i d e l y d i s t r i b u t e d i n s o i l , w a t e r , s e w a g e , t h e

m a m m a l i a n g u t a n d i n p l a n t s . I t i s p a t h o g e n i c t o m a n ,

s o m e ' a n i m a l s , i n s e c t s a n d p l a n t s . I t i s a b l e t o

s u r v i v e i n a l m o s t a n y m o i s t e n v i r o n m e n t o r f l u i d f o u n d

i n h o s p i t a l w a r d s , i n c l u d i n g i n f u s i o n f l u i d s ,

o p h t h a l m i c s o l u t i o n s , a n t i s e p t i c s o l u t i o n s ,

h e x a c h l o r o p h a n e s o a p , d e t t o l , s u c t i o n . a p p a r a t u s ,

n e b u l i z e r s a n d h u m i d i f i e r s , r e s p i r a t o r y v e n t i l a t o r s ,

f l o w e r v a s e s , s i n k s a n d t a p s ; a n d a l s o i n c o l d f o o d .

T h e o r g a n i s m c a n b e r e c o v e r e d f r o m t h e s t o o l i n a b o u t

1 0 % o f t h e n o r m a l p o p u l a t i o n , b u t t h e c o l o n i s a t i o n

r a t e i s h i g h e r i n h o s p i t a l p a t i e n t s w h o a r e e x p o s e d t o

a n t i b i o t i c s . I n f e c t e d w o u n d s , b u r n s o r u r i n e m a y

c o n t a m i n a t e b e d d i n g , h o s p i t a l p e r s o n n e l o r e q u i p m e n t .

T r a n s f e r i s m a i n l y f r o m p e r s o n t o p e r s o n a n d a i r b o r n e

i n f e c t i o n i s r a r e .

N o s o c o m i a l i n f e c t i o n s d u e t o G r a m - n e g a t i v e b a c i l l i

h a v e i n c r e a s e d s t e a d i l y s i n c e t h e 1 9 5 0 s 8 8 .

P. aerug i nosa

at (:ent ion in

natural r e s ls

r e s u l t a n t hiq

a l s o because

i mmu nosup pr e s

proportion of:

P. aeruginosa

has become the locus of increased

the l a s t ten years , pa r t ly because of i t s

tance to many a n t i b i o t i c s and the

h m or ta l i t y lor infected p a t i e n t s , but

of i t s presence in in fec t ion s in the

sed p a t i e n t . However, the ov era l l

Gram-negative in fec t ions due to

has remained r e l a t i v e l y c o n s t a n t ^ .

2 . I S O L A T I O N

P . a e r u g i n o s a h a s s i m p l e n u t r i t i o n a l r e q u i r e m e n t s

a n d w i l l g r o w o n m o s t m e d i a u s e d f o r p r i m a r y i s o l a t i o n

i n a r o u t i n e l a b o r a t o r y . S e l e c t i v e i s o l a t i o n f r o m a

s o u r c e t h a t c o n t a i n s o t h e r b a c t e r i a i s f a c i l i t a t e d b y

m e d i a c o n t a i n i n g c e t y l t r i m e t h y l a m m o n i u m b r o m i d e

( C e t r i m i d e ) , c h l o r o x y l e n o l ( D e t t o l ) , o r 2 , 4 , 4 ,

t r i c h l o r o - 2 - h y d r o x y d i p h e n o l e t h e r ( I r g a s a n ) . I n

a d d i t i o n , n a l i d i x i c a c i d o r n i t r o f u r a n t o i n m a y b e

a d d e d t o c e t r i m i d e a g a r .

T h e o r g a n i s m i s u s u a l l y a s t r i c t a e r o b e b u t w i l l

g r o w a n a e r o b i c a l l y i n a n i t r a t e - c o n t a i n i n g m e d i u m w i t h

t h e p r o d u c t i o n o f n i t r o g e n g a s . A f e a t u r e d i a g n o s t i c

o f t h e s p e c i e s i s t h e f a c t t h a t i t w i l l g r o w a t 4 2 ° C .

3 . I D E N T I F I C A T I O N

M a n y s t r a i n s o f P , a e r u g i n o s a c a n b e i d e n t i f i e d b y

t h e i r o d o u r , ( t h e f r u i t y o d o u r o f t r i m e t h y l a m i n e ) ,

c o l o n i a l m o r p h o l o g y 0 -*- a n d p r o d u c t i o n o f a

c h a r a c t e r i s t i c p i g m e n t p y o c y a n i n . P y o c y a n i n i s a

n o n - f l u o r e s c c - ' i i t: b l . u o p h o n a z i n e p i g m e n t : , w h i c h i s

i n s o l u b l e i n ' w a t e r b u t s o l u b l e i n c h l o r o f o r m , a n d

u n i q u e t o P . a e r u q i n o s a . M a n y s t r a i n s w i l l o n l y

p r o d u c e i t w h e n g r o w n o n s p e c i a l m e d i a c o n t a i n i n g

m a g n e s i u m , p o t a s s i u m a n d i r o n , c o r e x a m p l e K i n g ' s " A"

a g a r 6 2 w h i c h a l s o c o n t a i n s g l y c e r o l t o e n h a n c e g r o w t h

o f t h e o r g a n i s m . P y o r u b r i n i s a r e d , w a t e r - s o l u b l e

p i g m e n t a l s o u n i q u e t o t h i s o r g a n i s m , p y o m e l a n i n a

b r o w n / b l a c k p i g m e n t , a n d p y o v e r d i n , a f l u o r e s c e n t

y e l l o w / g r e e n p i g m e n t a r e a l s o p r o d u c e d . T h e p i g m e n t s

a r e a n t i b a c t e r i a l , a n d a l s o p r o t e c t t h e o r g a n i s m f r o m

p r o t o z o a i n t h e n a t u r a l e n v i r o n m e n t . I d e n t i f i c a t i o n

o f s t r a i n s i s n o t u s u a l l y c a r r i e d b e y o n d t h e s e

c h a r a c t e r i s t i c s . M e t h o d s f o r t h e c o n f o r m a t i o n o f

i d e n t i t y o f u n u s u a l i s o l a t e s , o r s t r a i n s t h a t d o n o t

p r o d u c e p y o c y a n i n , a r e s u m m a r i s e d i n T a b l e 1 1 ^ ^ .

P . a e r u g i n o s a p r o d u c e s c y t o c h r o m e o x i d a s e , c a t a l a s e ,

a r g i n i n e d i h y d r o l a s e , a n d o x i d a t i v e l y m e t a b o l i s e s

g l u c o s e . M o s t s t r a i n s p r o d u c e t h e f l u o r e s c e n t p i g m e n t

f l u o r e s c e i n a n d w i l l g r o w a n a e r o b i c a l l y i n

n i t r a t e - c o n t a i n i n g m e d i a . A n t i b a c t e r i a l s u b s t a n c e s o r

b a c t e r i o c i n s t e r m e d " p y o c i n e s " a r e a l s o p r o d u c e d .

F i g u r e 2 s h o w s s t r a i n s o f P . a e r u g i n o s a p r o d u c i n g

m u c o i d s u b s t a n c e a n d p y o c y a n i n g r o w i n g o n K i n g ' s " A "

a g a r .

4 . T Y P I N G S C H E M E S F O R S T R A I N I D E N T I F I C A T I O N

T h e r e a r e f o u r m e t h o d s c u r r e n t l y i n u s e f o r t y p i n g

P . a e r u g i n o s a : p y o c i n e t y p i n g , p h a g e t y p i n g , 0 ( s o m a t i c

T A B L E I I

I D E N T I F I C A T I O N OF P S E U D O M O N A S

K i n g ' s m e d i urn " A "

G r e e n , r e d o r N o p i g m e n t

b r o w n p i g m e n t

P . a e r u g i n o s a K i n g ' s m e d i u m

F l u o r e s c e i n N o p i g m e n t

i G r o w t h a t 4 2 ° C o t h e r s i A r g i n i n e d i h y d r o l a s e

i i G r o w t h i i O x i d a t i o n p a t t e r n

a n a e r o b i c a l l y i n f o r c a r b o h y d r a t e s

n i t r a t e m e d i a

o t h e r s

P . a e r u g i n o s a P . a e r u g i n o s a

FIGURE 2

STRAINS OF P.AERUGINOSA PRODUCING PYOCYANIN AND MUCOID

SUBSTANCE ON KING'S "A" AGAR

King and Phillips 1978 63

a n t i g e n ) a n d H ( l l a g e l l a r a n t i g e n ) s e r o l o g i c a l t y p i n g .

P y o c i n c t y o i n g i s a c o m m o n m e t h o d , w h i c h i s p e r f o r m e d

b y c r o s s - s t r e a k i . ng o f i n d i c a t o r s t r a i n s o n a n a r e a oL :

m e d i u m t h a t h a s p r e v i o u s l y s u p p o r t e d t h e g r o w t h of : a

t e s t s t r a i n o f p s e u d o m o n a s . I f t h e l a t t e r h a s

p r o d u c e d a p y o c i n e w h i c h h a s d i f f u s e d i n t o t h e m e d i u m ,

t h e g r o w t h o f i n d i c a t o r s t r a i n s i s i n h i b i t e d i n a

s p e c i f i c p a t t e r n . T h i s m e t h o d h a s b e e n r e v i e w e d b y

G o v a n ^ , a n d i s b o t h r e p r o d u c i b l e a n d r e l a t i v e l y

d i s c r i m i n a t i n g . H o w e v e r i t r e q u i r e s t h r e e d a y s t o

c o m p l e t e , a n d a l t h o u g h 1 0 5 t y p e s h a v e b e e n d e s c r i b e d ,

f o u r o f t h e m a c c o u n t f o r t w o t h i r d s o f t h e s t r a i n s

t y p e d t h r o u g h o u t t h e w o r l d .

S e r a r a i s e d a g a i n s t w h o l e u n h e a t e d c e l l s m a y b e

u s e d t o d e t e c t t h e h e a t - l a b i l e H a n t i g e n o f

p s e u d o m o n a s , b u t t h e s p e c i f i c i t y i s q u e s t i o n a b l e a n d

d i s c r i m i n a t o r y p o w e r i s p o o r , a n d a l m o s t h a l f t h e

s t r a i n s s h a r e t h e s a m e H a n t i g e n * ^ . B a c t e r i o p h a g e

t y p i n g i s n o t r e p r o d u c i b l e a n d c a n n o t b e u s e d a l o n e

f o r e p i d e m i o l o g i c a l s t u d i e s . I t i s u s e d t o

d i s t i n g u i s h b e t w e e n s t r a i n s o f t h e s a m e O - s e r o g r o u p ^ .

S t r a i n s o f t h e s a m e O - g r o u p a r e c o n s i d e r e d d i s t i n c t i f

t h r e e o r m o r e s t r o n g r e a c t i o n d i f f e r e n c e s i n p h a g e

t y p i n g p a t t e r n a r e o b s e r v e d .

T h e d e t e c t i o n o f g r o u p - s p e c i f i c , h e a t - s t a b l e

l i p o p o l y s a c c h a r i d e a n t i g e n s b y a g g l u t i n a t i o n w i t h

m o n o s p e c i f i c r a b b i t a n t i s e r a f o r m s t h e b a s i s o f

O - s e r o l o g i c a l t y p i n g . T h i s m e t h o d i s r e p r o d u c i b l e a n d

di scr i minat ing , and now has a proposed Internat iona l

Antigenic Tyoing Scheme1̂ . I t i s easy to per form in

batches using the m i cr o t i t r e method and i s the method

in use at the Centra l Public Health Laboratory. This

method was therefore the system chosen for typing C'7

cu l tu res in these s t u d i e s .

M U C O I D F O R M S

S o m e s t r a i n s o f P . a e r u g i n o s a f r o m c h r o n i c

i n f e c t i o n s , p a r t i c u l a r l y C F , m a y p r o d u c e s i g n i f i c a n t

a m o u n t s o f e x o p o l y s a c c h a r i d e a n d f o r m m u c o i d

c o l o n i e s 0 0 . T h i s p h e n o m e n o n i s r e v e r s i b l e o n

s u b c u l t u r e i n t h e l a b o r a t o r y ' * 0 a n d m a y b e i n d u c e d b y

t h e a c t i o n o f p h a g e 0 . T h e m u c o i d s u b s t a n c e i s a n

a n i o n i c p o l y m e r o f D - m a n n u r o n i c a c i d a n d L - g u l u r o n i c

a c i d , a n d c l o s e l y r e s e m b l e s t h e s e a w e e d p o l y s a c c h a r i d e

a l g i n i c a c i d ' . E x a m i n a t i o n o f p o s t - m o r t e m l u n g

m a t e r i a l f r o m C F p a t i e n t s u n d e r t h e e l e c t r o n

m i c r o s c o p e h a s s h o w n t h a t P , a e r u g i n o s a f o r m s

m i c r o c o l o n i e s i n t h e a l v e o l i w h i c h a r e s u r r o u n d e d b y a

f i b r o u s e x t r a c e l l u l a r m a t r i x w i t h s i m i l a r s t a i n i n g

p r o p e r t i e s t o m u c o i d ^ l . T h i s s u g g e s t s t h a t m u c o i d

s t r a i n s m a y e x i s t a s s u c h w i t h i n t h e C F l u n g , a n d t h i s

i s s u p p o r t e d b y t h e w o r k o f C a s h e t a l ^ 2 V7 h o c r e a t e d

c h r o n i c p s e u d o m o n a s p u l m o n a r y i n f e c t i o n i n r a t s b y

i n t r o d u c i n g t h e o r g a n i s m i n t o t h e t r a c h e a i n a n

a r t i f i c i a l a n i o n i c f i b r o u s m a t r i x o f a g a r .

T h e c o n s i s t a n t i s o l a t i o n o f m u c o i d s t r a i n s f r o m C F

s p u t a d e s p i t e t h e t e n d e n c y f o r n o n - m u c o i d s t r a i n s t o

o v e r q r o w o n s u b c u l t u r e , s u g g e s t s t h a t t h e e n v i r o n m e n t

of ; t h e C F l u n g e x e r t s a s e l e c t i v e d i r e c t i n ( f a v o u r o f

m u c o i d s t r a i n s . M u c o i d v a r i a n t s of : o t h e r

G r a m - n o q a t i. v e b a c i l l i , i n p a r t i c u l a r F s c h e r i c h i a c o l i ,

h a v e b e e n i s o l a t e d f r o m C F s p u t a , s o t h e p h e n o m e n o n i s

n o t u n i q u e t o P . a c r u q i n o s a ^ . M u c o i d c u l t u r e s c a n b e

m a i n t a i n e d i n v i t r o w i t h t h e a d d i t i o n o f a s u r f a c t a n t

( d e o x y c h o l a t e ) , o r b y t h e a d d i t i o n o f l o w

c o n c e n t r a t i o n s o f c a r b e n i c i l l i n , f l u c l o x a c i l l i n a n d

t o b r a m y c i n ^ ^ / 7 5 b T h u s s u r f a c t a n t w i t h i n t h e l u n g ,

a n d / o r t h e u s e o f a n t i b i o t i c s , m a y f a v o u r t h e

s e l e c t i o n o f m u c o i d v a r i a n t s .

T h e a n t i b i o t i c s e n s i t i v i t i e s o f m u c o i d s t r a i n s o f

P . a e r u g i n o s a a r e v e r y v a r i a b l e . G o v a n a n d F y f e ^

f o u n d t h a t m u c o i d s t r a i n s w e r e m o r e r e s i s t a n t t o

c a r b e n i c i l l i n , f l u c l o x a c i l l i n a n d t o b r a m y c i n , w h i l e

o t h e r s h a v e f o u n d t h e r e v e r s e ^ . P s e u d o m o n a s s t r a i n s

f r o m t h e s a m e s p u t u m s p e c i m e n m a y h a v e d i f f e r e n t

a n t i b i o t i c s u s c e p t i b i l i t i e s ^ . F u r t h e r m o r e , m u c o i d

m a t e r i a l h a s r e c e n t l y b e e n s h o w n t o e f f e c t t h e

d i f f u s i o n r a t e s o f p o s i t i v e l y - c h a r g e d a n t i b i o t i c s s u c h

a s a m i n o g l y c o s i d e s a n d n e o m y c i n ^ .

S o m e o f t h e e f f e c t o f m u c o i d o n h o s t d e f e n c e

m e c h a n i s m s a r e d i s c u s s e d l a t e r i n t h i s c h a p t e r . T h e

p r e s e n c e o f m u c o i d s t r a i n s i s a s s o c i a t e d w i t h t h e

p r e s e n c e o f m o r e p r e c i p i t i n s t o p s e u d o m o n a s i n

p a t i e n t ' s s e r u m , a n d t h u s m u c o i d s t r a i n s h a v e b e e n

c o n s i d e r e d t o b e t h e m o r e p a t h o g e n i c f o r m o f t h e

o r g a n i s m 1̂ , 7 9 . i t i s e q u a l l y p o s s i b l e t h a t b o t h

p h e n o m e n a a r e m e r e l y f e a t u r e s o f p r o l o n g e d

c o 1 o n i. s a t i o n b y p 55 o u cl 01 n o n a 3 .

6 . V I R U L E N C E F A C T O R S O F P S E U D O M O N A S A E R U G I N O S A

T h e k n o w n v i r u l e n c e f a c t o r s o f P . a e r u g i n o s a f a l l

i n t o t w o g r o u p s , t h e c e l l s u r f a c e s t r u c t u r e s a n d

e x t r a c e l l u l a r p r o d u c t s . T h e l a t t e r i n c l u d e

h a e m o l y s i n s , p h o s p h o l i p a s e s , p r o t e a s e s , a n e n t e r o t o x i n

a n d a h e a t - l a b i l e e x o t o x i n t e r m e d e x o t o x i n A . T h e s e

s u b s t a n c e s a r e i n v o l v e d i n t i s s u e i n v a s i o n a n d

c e l l u l a r d a m a g e , a n d e x o t o x i n A h a s b e e n s h o w n t o

i m p a i r t h e a c t i o n o f m o n o c y t e s i n p e r i p h e r a l b l o o d ^ 0 .

T h e r o l e o f p r o t e a s e s i n t i s s u e d a m a g e a n d t h e e f f e c t

o f p h o s p h o l i p a s e s o n s u r f a c t a n t h a v e n o t b e e n s t u d i e d

i n C F . C e l l s u r f a c e c o m p o n e n t s o f i m m u n o l o g i c a l

i m p o r t a n c e i n c l u d e p i l i , e x t r a c e l l u l a r s l i m e

p o l y s a c c h a r i d e , m u c o i d p o l y s a c c h a r i d e a n d c e l l - w a l l

l i p o p o l y s a c c h a r i d e ( L P S ) . P i l i a l l o w a d h e r e n c e t o

e p i t h e l i a l c e l l s a n d c o l o n i s a t i o n o f s u r f a c e s a n d t h e

s l i m e p o l y s a c c h a r i d e h a s b e e n s h o w n t o b e t o x i c t o

n e u t r o p h i l s . T h e l i p i d A c o m p o n e n t o f P . a e r u g i n o s a

L P S i s r e s p o n s i b l e f o r t h e t o x i c p r o p e r t i e s o f t h e

e n d o t o x i n b u t i s g e n e r a l l y t h o u g h t t o b e l e s s p o t e n t

t h a n t h e e n d o t o x i n o f o t h e r g r o u p s o f G r a m - n e g a t i v e

o r g a n i s m s .

T h e m u c o i d m a t e r i a l i s v e r y v i s c o u s a n d m a y

c o n t r i b u t e t o a i r w a y s o b s t r u c t i o n . M u c o i d f o r m s a r e

m o r e r e s i s t a n t t o p h a g o c y t o s i s a n d k i l l i n g b y

p o l y m o r p h o n u c l e a r l e u c o c y t e s ^ . I n n o n - c y s t i c

oat Louts with urinary in fec t ions the mucoid layer

i n t e r e f e r e s with oosoni sa t ion by antibody^^.

13. H O S T D E F E N C E M E C H A N I S M S A G A I N S T P S E U D O M O N A S A E R U G I N O S A

I N C Y S T I C F I B R O S I S

A d u l t s w i t h C F h a v e c h r o n i c b a c t e r i a l

b r o n c h o p u l m o n a r y i n f e c t i o n , b u t b a c t e r a e m i a i s r a r e ^ ,

, f o /a s a r e t h e c o m p l i c a t i o n s o f e m p y e m a 0 ^ a n d

amyloidosis^. T h e r e d o e s n o t s e e m t o b e a n y i n c r e a s e

i n i n f e c t i o n a t o t h e r s i t e s , s u c h a s t h e s k i n o r

u r i n a r y t r a c t , c o m p a r e d w i t h n o r m a l s u b j e c t s o f t h e

s a m e a g e . T h i s w o u l d s u g g e s t t h a t t h e r e i s a d e f e c t

i n t h e l o c a l d e f e n c e m e c h a n i s m s o f t h e r e s p i r a t o r y

t r a c t , r a t h e r t h a n i n s y s t e m i c d e f e n c e s , w h i c h r e n d e r s

C F p a t i e n t s s u s c e p t i b l e t o i n f e c t i o n b y b a c t e r i a .

1 . E P I T H E L I A L B A R R I E R

T h e e p i t h e l i a l c e l l s a n d m u c o u s m e m b r a n e s t o g e t h e r

w i t h t h e m u c o c i l i a r y a p p a r a t u s f o r m s t h e f i r s t l i n e o f

d e f e n c e a g a i n s t p s e u d o m o n a s i n t h e l u n g . I t h a s b e e n

s h o w n t h a t s e c r e t o r y i m m u n o g l o b u l i n A i n h i b i t s t h e

a d h e r e n c e o f s t r e p t o c o c c i t o e p i t h e l i a l c e l l s a n d

Reynolds*^ h a s s u g g e s t e d a s i m i l a r r o l e f o r s e c r e t o r y

I g A i n p s e u d o m o n a s c o l o n i s a t i o n . T h e a b i l i t y o f

P . a e r u g i n o s a t o p e r s i s t i n t h e l o w e r r e s p i r a t o r y t r a c t

i s r e l a t e d t o i t s a b i l i t y t o a d h e r e i n i t i a l l y t o

b u c c a l e p i t h e l i u m . C F p a t i e n t s h a v e h i g h l e v e l s o f

s a l i v a r y p r o t e a s e s a n d l o s e f i b r o n e c t i n f r o m b u c c a l

e p i t h e l i a l c e l l cur (races which allows the organism to

adhere more e a s i l y to the epithelium®®.

E f ' t e c t i v e e m u c o c i l i a r y c l e a r a n c e d e p e n d s o n t h e

v i s c o - e l a s t i c p r o p e r t i e s o f m u c u s a n d i n t a c t c i l i a r y

f u n c t i o n , w i t h c i l i a b e a t i n g a t a n o r m a l r a t e , i n a

c o n s i s t e n t d i r e c t i o n a n d i n a c o - o r d i n a t e d m a n n e r .

P u l m o n a r y m u c o c i l i a r y c l e a r a n c e i n p a t i e n t s w i t h C F

h a s b e e n s h o w n t o b e d e c r e a s e d ® ^ , a s jl a s n a s a l

m u c o c i l i a r y c l e a r a n c e ^ . T h i s m a y b e d u e t o a b n o r m a l

m u c u s , w i t h a c h a n g e o f v i s c o s i t y d u e t o t h e p r e s e n c e

o f a b n o r m a l g l y c o p r o t e i n s ® ® . W o r k o n a n i m a l m o d e l s

u s i n g t h e o y s t e r g i l l a n d r a b b i t t r a c h e a h a s

d e m o n s t r a t e d t h a t t h e c i l i a r y b e a t p a t t e r n o f t h e s e

p r e p a r a t i o n s i s r a p i d l y d i s t o r t e d b y t h e a d d i t i o n o f

C F s e r u m ® ® ' ® - * - . A s e r u m c i l i a r y d y s k i n e t i c f a c t o r h a s

b e e n p r o p o s e d v / h i c h m a y d i s o r g a n i s e c i l i a b y a d i r e c t

e f f e c t o r b y r e l e a s i n g a b n o r m a l m u c u s . R e c e n t w o r k

s u g g e s t s t h a t t h i s m a y b e d u e t o a n I g M i n h u m a n

s e r u m , b u t i s n o t n e c e s s a r i l y s p e c i f i c t o C F ® ® . O t h e r

w o r k o n c i l i a r y b e a t f r e q u e n c y u s i n g s a m p l e s o f c i l i a

f r o m n a s a l b r u s h i n g s o f C F p a t i e n t s h a v e s h o w n t h a t

t h i s a s p e c t o f c i l i a r y f u n c t i o n i s n o r m a l i n p a t i e n t s

w i t h C F ® ® a n d i s u n a f f e c t e d b y s e r u m f r o m t h e p a t i e n t ,

f r o m o t h e r c y s t i c s o r f r o m n o r m a l s u b j e c t s ® ® . S e r u m

f r o m C F p a t i e n t s d o e s n o t e f f e c t t h e c i l i a r y b e a t

f r e q u e n c y o f c i l i a r y s a m p l e s f r o m n o r m a l h u m a n

s u b j e c t s b u t d o e s a r r e s t c i l i a r y b e a t i n g i f p l a c e d o n

r a b b i t t r a c h e a ® ® .

2 . A L V E O L A R MACR OPHAGES

Theiro lg sonic evidence to suggest that the

function of a l v eo la r m a c r o p h a g e s i. s abnormal in CF.

C P s e r u m d o e s n o t p r o m o t e p h a g o c y t o s i s o t p s e u d o m o n a s

b y r a b b i t a l v e o l a r m a c r o p h a g e s , a l t h o u g h t h i s m a y b e

c o r r e c t e d b y i n c r e a s i n g t h e a m o u n t o t s e r u m i n t h e

a s s a y . A s i m i l a r e f f e c t i s s e e n w i t h I g A d e f i c i e n t

s e r u m , b u t a l l s e r a s u p p o r t p h a g o c y t o s i s b y

p o l y m o r p h o n u c l e a r leucocytes'^. C F s e r u m a l s o e f f e c t s

h u m a n a l v e o l a r m a c r o p h a g e f u n c t i o n b y i n h i b i t i n g t h e

p h a g o c y t o s i s o f p s e u d o m o n a s , b u t n o t s t a p h y l o c o c c u s ,

b y m a c r o p h a g e s f r o m b o t h C F a n d n o r m a l s u b j e c t s . T h e

m a c r o p h a g e s f r o m C F p a t i e n t s w e r e s t r u c t u r a l l y n o r m a l

a n d a b l e t o p h a g o c y t o s e p s e u d o m o n a s i n t h e p r e s e n c e o f

n o r m a l s e r u m ^ .

3 . N E U T R O P H I L F U N C T I O N

M o n o c y t e a n d n e u t r o p h i l c h e m o t a x i s s e e m s n o r m a l i n

C F , a s i s n e u t r o p h i l p h a g o c y t o s i s ^ . I n t r a c e l l u l a r

k i l l i n g b y n e u t r o p h i l s i n v o l v i n g t h e m y e l o p e r o x i d a s e

s y s t e m m a y b e d e f e c t i v e ^ . M i g r a t i o n o f C F l e u k o c y t e s

m a y b e i n h i b i t e d b y e x t r a c t s o f n o r m a l o r C F l u n g ,

s u g g e s t i n g s e n s i t i s a t i o n t o t i s s u e a n t i g e n s ^ .

4 . C E L L - M E D I A T E D I M M U N I T Y

S t u d i e s u s i n g s h e e p e r y t h r o c y t e r o s e t t e t e c h n i q u e s

h a v e s h o w n t h a t t h e t o t a l n u m b e r a n d t h e p e r c e n t a g e o f

T c e l l s i s n o r m a l , a l t h o u g h a b s o l u t e n u m b e r s i n c r e a s e

w i t h infection^. P r o l i f e r a t i o n o f T c e l l s i n

t r e s o o n s o t o m i t o g e n s i s n o r m a l ’o u t m a y ’n o r e d u c e r ) i n

t h e p r e s e n c e o c C F s e r u m ^ - ^ . 3 ! : i n t e s t s o f d e l a y e d

11y o e r s e n s i t i v i h y a r e n o r m a 1 a n d p a t i e n t s r e s p o n d

a p p r o p r i a t e l y t o B C G . S t u d i e s o n T c e l l

s u b - p o p u l a t i o n s , m e d i a t o r - r e l e a s e a n d c y t o t o x i c i t y

h a v e n o t b e e n r e p o r t e d . T h e r e i s o n e r e p o r t o f

r e d u c e d r e s p o n s i v e n e s s o f l y m p h o c y t e s t o p s e u d o m o n a s

a n t i g e n b u t r i o t t o o t h e r m i t o g e n s i n a d v a n c e d

p u l m o n a r y d i s e a s e ^ ! - .

5 . HUMORAL I M M U N I T Y

T h e p e r c e n t a g e o f B c e l l s i s n o r m a l b u t a b s o l u t e

n u m b e r s a r e e l e v a t e d i n t h e p r e s e n c e o f c h r o n i c

p u l m o n a r y i n f e c t i o n ^ . H y p e r g a m m a g l o b u l i n a e m i a w i t h

r a i s e d l e v e l s o f I g G , I g M a n d I g A i s c o m m o n a n d

r e l a t e s t o t h e i n c r e a s i n g s e v e r i t y o f t h e d i s e a s e .

Y o u n g c h i l d r e n w i t h l e s s s e v e r e d i s e a s e m a y h a v e

a b n o r m a l l y l o w l e v e l s o f I g G ^ ® 2 f w h i c h m a y i n d i c a t e

f a i l u r e t o r e c o g n i s e f o r e i g n a n t i g e n s i n e a r l y l i f e .

T h e s e a u t h o r s s u g g e s t e d t h a t a b n o r m a l m u c u s m a y

p r e v e n t f o r e i g n a n t i g e n r e c o g n i t i o n a c r o s s m u c o s a l

b a r r i e r s i n e a r l y l i f e , b u t s u b s e q u e n t l u n g d a m a g e

a l l o w s s e n s i t i s a t i o n t o t h e s e a n t i g e n s a n d p r o d u c e s

t h e e v e n t u a l h y p e r g a m m a g l o b u l i n a e m i a s e e n i n o l d e r

c h i l d r e n .

P o s t - m o r t e m s t u d i e s o n C F b r o n c h i a l t i s s u e

d e m o n s t r a t e n o r m a l n u m b e r s o f c e l l s c o n t a i n i n g I g G ,

I g M a n d I g A . T h e n u m b e r o f I g A c o n t a i n i n g c e l l s i s

p r o p o r t i o n a l t o t h e s e v e r i t y o f b r o n c h i a l

[ n f oc t i.on ^^ ̂ . A d e f e c t i n t h e s y n t h e s i s of; s e c r e t o r y

IgA h a s boon s u g g e s t e d by t h e f i n d i n g o f t r e e

s e c r e t o r y n i e c e s i n C F s e r u m l ' ^ . Wor m a 1. h u m a n s e r u m

c o n t a i n s a l o w l i t r e o f I g M a g a i n s t t h e p r o t e a s e s and

e l a s t a s e s o f p s e u d o m o n a s . H i g h t i t r e s a g a i n s t

p r o t e a s e s a n d e x o t o x i n A a r e f o u n d i n C F s e r a ^ - ^ .

A n t i b o d i e s t o t h e e x t r a c e l l u l a r s l i m e a r e o f t h e I g M

c l a s s . N o r m a l h u m a n s e r u m c o n t a i n s l o w l e v e l s o f

n a t u r a l n o n - s p e c i f i c o p s o n i n s , m a i n l y I g M , w h i c h a r e

w e a k , b u t c o n v a l e s c e n t p a t i e n t s h a v e s p e c i f i c I g G

o p s o n i n s d i r e c t e d t h e a g a i n s t t h e O - a n t i g e n c o m p o n e n t

o f L P S 1 0 6 ^ C F p a t i e n t s h a v e h i g h t i t r e s o f s u c h

a n t i b o d i e s t o L P S a n d o t h e r s u r f a c e a n t i g e n s - * - 1- ^ . A

c e l l e n v e l o p e p r o t e i n o f m o l e c u l a r w e i g h t 5 8 , 5 0 0

d a l t o n s h a s b e e n s h o w n t o b e i m m u n o g e n i c i n r a b b i t s ,

a n d a n t i b o d i e s t o i t a r e f o u n d i n C F s e r a ^ - ^ . S u c h

p r o t e i n s a r e c o m m o n t o a l l s e r o t y p e s o f P . a e r u g i n o s a

a n d m a y o f f e r t h e b e s t h o p e f o r a f u t u r e v a c c i n e .

B o t h t h e c l a s s i c a n d a l t e r n a t e p a t h w a y s o f t h e

c o m p l e m e n t s y s t e m m a y b e i n v o l v e d i n o p s o n i s a t i o n a n d

p h a g o c y t o s i s o f p s e u d o m o n a s ^ - ^ . N o d e f i n i t i v e

a b n o r m a l i t y o f c o m p l e m e n t f u n c t i o n h a s b e e n r e p o r t e d

i n C F , a l t h o u g h a b n o r m a l l e v e l s o f C 3 , C 4 a n d C 5 h a v e

b e e n o b s e r v e d - ^ - ^ .

C h a p t e r 3

GENERAL METHODS

A. C Y S T I C F I B R O S I S P A T I E N T S

T h e n a t i o n t a a t u cl i e d w e r 0 a d o l g s c e n t s a n d a d u 1 1: s

a t t e n d i n g t h e C y s t i c F i b r o s i s U n i t a t t h e B r o m p t o n

H o s p i t a l u n d e r t h e c a r e o f D r J C B a t t e n a n d D r

M a r g a r e t E f i o d s o n . E a c h p a t i e n t h a d t h e c l i n i c a l

f e a t u r e s o f r e c u r r e n t b r o n c h o p u l m o n a r y i n f e c t i o n a n d

b r o n c h i e c t a s i s , a n d m o s t h a d p a n c r e a t i c m a l a b s o r p t i o n .

A l l h a d a s v / e a t s o d i u m c o n c e n t r a t i o n o f > 7 0 m m o l s / l b v

p i l o c a r p i n e i o n t o p h o r e s i s ^ . T h o s e t a k i n g p a r t i n

t r i a l s o f c h e m o t h e r a p y g a v e i n f o r m e d w r i t t e n c o n s e n t .

A l l t r i a l p r o t o c o l s w e r e a p p r o v e d b y t h e B r o m p t o n

H o s p i t a l E t h i c s C o m m i t t e e .

B. RESPIRATORY FUNCTION TESTSP e a k e x p i r a t o r y f l o w r a t e ( P E E R ) w a s r e c o r d e d a s

t h e b e s t o f t h r e e a t t e m p t s o n a W r i g h t ' s P e a k F l o w

M e t e r . F o r c e d e x p i r a t o r y v o l u m e i n o n e s e c o n d ( F E V g )

a n d f o r c e d v i t a l c a p a c i t y ( F V C ) w e r e r e c o r d e d o n a

V i t a l o g r a p h s p i r o m e t e r , r e a d i n g f r o m t h e A T P S ( a m b i e n t

t e m p e r a t u r e a n d p r e s s u r e ) s c a l e . R e s u l t s w e r e

e x p r e s s e d a s a p e r c e n t a g e o f p r e d i c t e d n o r m a l v a l u e s

u s i n g t a b l e s p u b l i s h e d b y C o t e s - ^ ^ - .

C. CLINICAL SCOREAn o v e r a l l c l i n i c a l a s s e s s m e n t w a s s c o r e d b y t h e

m e t h o d o f S h w a c h m a n ^ - ^ # j n b r i e f t h i s m e t h o d

c o n s i d e r s f o u r v a r i a b l e s , r a d i o l o g i c a l a p p e a r a n c e ,

n u t r i t i o n a l s t a t u s , c l i n i c a l s i g n s o n e x a m i n a t i o n o f

t h e r e s p i r a t o r y s y s t e m , a n d t h e p a t i e n t ' s e x e r c i s e

t o l e r a n c e . Point : . -- ; a r e a w a r d e d i n i n c r e m e n t s of ; i l v o

u n t o a m a x i i i u i i n o f 2 5 p e r f e a t u r e , t o g i v e a m a x i m u m

p o s s i b l e s c o r c o n 1 0 0 . F i g u r e 3 s umm a r i s e s t h i s

s c o r i n g s y s t e m . P a t i e n t s w e r e e x a m i n e d a n d a w a r d e d a

s c o r e b y t h e a u t h o r o n t h e s a m e d a y a s s a m p l e s o f

s e r u m a n d s p u t u m w e r e c o l l e c t e d , b e f o r e a n y r e s u l t s o f

t h e s u b s e a u e n t s t u d i e s w e r e k n o w n .

D . SERUM SAMPLESB l o o d w a s t a k e n b y s t e r i l e v e n e p u n c t u r e i n t o g l a s s

t u b e s a n d a l l o w e d t o c l o t a t r o o m t e m p e r a t u r e f o r 2 - 3

h o u r s . T u b e s w e r e s p u n a t 2 0 0 0 g f o r 1 0 m i n u t e s ,

s e r u m w a s t a k e n o f f i n t o g l a s s b o t t l e s a n d s t o r e d i n

1 m l a l i q u o t s a t - 2 0 ° C . S e r a w e r e t h a w e d o n l y o n c e a n d

s t u d i e d t h e s a m e d a y , a n d a l l s e r a v / e r e s t u d i e d w i t h i n

2 m o n t h s .

E . IDENTIFICATION OF PSEUDOMONAS STRAINS FROM SPUTUM1 . P R I M A R Y I S O L A T I O N

T h e s p u t a w e r e d i g e s t e d b y p a n c r e a t i n , p l a t e d o n t o

c e t r i m i d e - n a l i d i x i c a c i d a g a r ( O x o i d ) a n d i n c u b a t e d a t

3 7 ° C f o r 1 8 h o u r s . T h i s m e d i u m i s s e l e c t i v e f o r

p s e u d o m o n a s ( C h a p t e r 2 ) . A s w e e p o f t h e p r i m a r y

g r o w t h w a s s u b c u l t u r e d o n t o K i n g ' s " A " A g a r , a m e d i u m

d e s i g n e d t o e n h a n c e p y o c y a n i n p r o d u c t i o n , a n d d i s c r e e t

c o l o n i e s v / e r e p i c k e d f o r i d e n t i f i c a t i o n .

SYSTEM OF RATING SEVERITY OF DISEASE

Grading Points General Activity

Physical Nutrition X-RayExamination Findings

Maintains we’ght and Clear lung fieldsheight above 25thpercentile well formedstools, almost normal.fair muscle tone andmass

Excellent 25 (86-100)

Good 20(71-85)

Mild 15(56-70)

Full normal activity: plays bail, goes to school regularly and soon

Lacks endurance and tires at end ol day: good school attendance

May rest voluntarily during the day; tires easily after exertion; fan school attendance

Normal: no cough pulse and respirations normal clear lungs, good posture

Resting pulse and respirations normal: rare coughing or deanngofthioat.no clubbing: clear lungs; minimal emphysema

Weight ana height at approximately 15th to 20th percentile, stools slightly abnormal fair muscle tone and mass

Minimal accentuation of brcnchovascular markings, early emphysema

Occasional cough, perhaps in morning upon rising; respirat-ons slightly elevated; mild emphysema; coarse breath sounds; rarely localized rales, early clubbing

Mild emphysema with patchy atelectasis.

Weight and height above 3rd percentile stools usua'iy abnormal, increased large and poo'iy bronchovascuiarformed, very l.ttie. if markingsany abdominal distention; poor muscle tone with reduced muscle mass

Moderate 10 (41-55)

Home teacher; dyspnoeic after short walk; rests a great deal

Frequent cough usually productive; chest retraction; moderate emphysema, may have chest deformity, rales usually present; clubbing 2 to 3+

Weight and height below 3rd percentile poorly formed, bulky, fatty, offensive stools; flabby muscles and reduced mass: abdominal detention, mild to moderate

Moderate empt ysema widespread areas ol atelectasis with superimposed areas of infection; minimal bronchial extasia

Severe 5 (40 or below)

Orthopnoeic, confined to bed or chair

Severe coughing spells; tachypnoeawithtachycardia and extensive pulmonary changes; may show signs of right-sided cardiac failure; clubbing 3 to 4+

Malnutrition ma'ked: large proude-ant abdomen, rectal prolapse.'large, foul, frequent, fatty- movements

Extensive changes with pulmonary obstructive phenomena and infection; lobar atelectasis and bronchiectasis

(Shwachman and Kulczycki, 1 9 5 8 1 1 2 )

King's "a " Agar^- formula - Bacto peptoneBacto agarG l y c e r o l

K2SO4 (anhydous)MqC ] .2 ( a n h y d o u s )

pH 7 . 2 a u t o c l a v e d a t 1 2 1 ° C

f o r 1 5 m i n s .

2 . C O L O N I A L FORM

C o l o n i a l f o r m w a s r e c o r d e d f o l l o w i n g t h e c r i t e r i a

o f P h i l l i p s 1 9 6 9 6 1 .

C o l o n y t y p e 1 - t h e m o s t c o m m o n l y o c c u r r i n g f o r m .

L a r g e c o l o n i e s t e n d i n g t o b e f l a t w i t h i r r e g u l a r s h a p e

a n d f e a t h e r y e d g e , o c c a s i o n a l l y c o a r s e a n d g r a n u l a r i n

a p p e a r a n c e , s h i n y a n d m o i s t .

C o l o n y t y p e 2 - s m a l l , r o u n d , r a i s e d , d i s c r e t e , s h i n y ,

s m o o t h c o l o n i e s .

C o l o n y t y p e 3 - s m a l l , r a i s e d , d r y , c r e n a t e d a n d

i r r e g u l a r l y r o u n d c o l o n i e s .

C o l o n y t y p e 4 - M u c o i d . R a i s e d , s l i m y a n d v i s c o u s ,

e i t h e r f o r m i n g d i s c r e t e c o l o n i e s o r t e n d i n g t o f l o w

i n t o e a c h o t h e r . On s t a n d i n g a t r o o m t e m p e r a t u r e t h e

s l i m e a p p e a r s t o g e l i n m o u n d s .

C o l o n y t y p e 5 - r a r e l y s e e n , c o m m o n l y g e l a t i n o u s i n

t e x t u r e a n d s i m i l a r t o t y p e 3 b u t n o t a s d r y .

C o l o n y t y p e 6 - d w a r f f o r m , v e r y s m a l l , i . e . < 0 . 5 m m s

a f t e r o v e r n i g h t i n c u b a t i o n . A f t e r 4 8 h o u r s i n c u b a t i o n

t h e s i z e i s a p p r o x i m a t e l y 1 - 2 m m ) ,

t o c o l o n y t y p e 2 .

O t h e r w i s e s i m i l a r

3 . O - S V, RO LOG I C A L T Y P 1 N G

T h e h e a t - s t a b l e O - a n t i g e n s of : t h e c e l l w a l l

1 i f ) o p o 1 y s a c c h a r i d e o f P s e u d o m o n a s a e r u g i n o s a p t r o u i d o

t h e b a s i s f o r s e r o l o g i c a l t y p i n g o f t h i s o r g a n i s m .

T h e a n t i g e n s a r e d e t e c t e d b y a g g l u t i n a t i o n w i t h r a b b i t

a n t i s e r a r a i s e d a g a i n s t a h e a t - k i l l e d s u s p e n s i o n o f a

s t r a i n ( p r e p a r a t i o n a n d c h a r a c t e r i s a t i o n o f

O - a n t i s e r a , s e e A p p e n d i x I I ) . T h e s y s t e m u s e d f o r

t h e s e s t u d i e s w a s t h a t e m p l o y e d b y t h e C e n t r a l P u b l i c

H e a l t h L a b o r a t o r y f o r r o u t i n e e p i d e m i o l o g i c a l t y p i n g

o f p s e u d o m o n a s . I t i s b a s e d o n t h e s y s t e m o f H a b s - * - - ^

w i t h s o m e a d d i t o n s ^ - - ^ * a n d c o r r e s p o n d s w i t h t h e

I n t e r n a t i o n a l A n t i g e n i c T y p i n g S c h e m e ( I A T S ) ^ .

1 7 O - s e r o g r o u p s a r e s u b d i v i d e d b y m i n o r a n t i g e n s t o

d e f i n e a t o t a l o f 21 s e r o t y p e s .

a ) M e t h o d s

T h e s t r a i n w a s c u l t u r e d o n t o K i n g ' s " A " A g a r a n d a

s i n g l e c o l o n y s u b c u l t u r e d i n t o 3 m l s o f T r y p t o n e S o y a

B r o t h ( T S B , O x o i d ) i n a 5 x t e s t t u b e . A f t e r

i n c u b a t i o n a t 3 7 ° C o v e r n i g h t t h e t u b e w a s p l a c e d i n

b o i l i n g w a t e r f o r t w o h o u r s . A 0 . 0 2 5 m l a l i q u o t o f t h e

s u s p e n s i o n w a s a d d e d t o a n e q u a l v o l u m e o f s e r u m

d i l u t i o n s i n t h e w e l l s o f a m i c r o t i t r e t r a y a n d a

s a l i n e c o n t r o l w a s i n c l u d e d . T h e t r a y s w e r e i n c u b a t e d

a t 3 7 ° C f o r f o u r h o u r s , a n d a t 4 ° C o v e r n i g h t . T h e

t r a y s w e r e l e f t a t r o o m t e m p e r a t u r e f o r 1 5 m i n u t e s a n d

t h e n r e a d u s i n g a m i r r o r .

b ) K o s u ]. t o

P o s i t i v e - aqt] l u t i n a t i o n s e e n a s a ' f l a k e ' 1 i n inn

t h e w e l l .

N e g a t i v e = b u t t o n o f c o l l s w i t h a c l e a r

s u p e r n a t a n t .

A l l n o n - t y p a b l e c u l t u r e s w e r e c h e c k e d b y s l i d e

a g g l u t i n a t i o n w i t h g r o w t h t a k e n d i r e c t l y f r o m a g a r

o 1 a 1 0 s •

4 . P O L Y A G G L U T I N A T I O N AMD P A A N T I G E N

O - s e r o l o g i c a l t y p i n g i s r e p r o d u c i b l e a n d 9 0 - 9 5 % o f

c l i n i c a l s t r a i n s m a y b e t y p e d b y t h i s m e t h o d ^ A f e w

s t r a i n s g i v e u n s t a b l e s u s p e n s i o n s i n s a l i n e a n d a r e

t e r m e d a u t o a g g l u t i n a b l e ( A A ) , a n d a f e w g i v e n o

r e a c t i o n s w h a t s o e v e r a n d a r e t e r m i n e d n o n - t y p a b l e

( N T ) . A b o u t 5% o f s t r a i n s a r e p o l y a g g l u t i n a t a b l e ( P A )

a n d r e a c t w i t h m o r e t h a n o n e s e r u m o u t s i d e t h e

e s t a b l i s h e d c r o s s - r e a c t i o n s o f t h e s e r o t y p e s t r a i n s

( s e e A p p e n d i x I ) . T h i s i s d u e t o t h e p r e s e n c e o f a

h e a t - s t a b l e s u r f a c e a n t i g e n , d i s t i n c t f r o m t h e

O - a n t i g e n ( P A a n t i g e n ) , a n d a n t i b o d y t o t h i s i s

d i s t i n c t f r o m t h e O - a n t i b o d y a n d o f t h e I g M c l a s s

p r e d o m i n a n t l y - ^ - - ^ . A n t i s e r u m t o P A a n t i g e n w a s

i n c l u d e d i n t h e s e r o t y p i n g s c h e m e .

5 . S E R U M ^ S B N S I T I V I T Y T E S T S

An 1 8 h o u r b r o t h c u l t u r e w a s d i l u t e d 1 i n 1 0 0 i n

H a n k s ' b a l a n c e d s a l t s o l u t i o n ( O x o i d ) a n d a l i q u o t s o f

1 0 0 p i w e r e a d d e d t o e q u a l v o l u m e s o f N H S a n d t o

c o n t r o l t u b e s of ; H a n k s s o l u t i o n . T h e m i x t u r e s w o r e

i n c u b a t e d w i t h g e n t l e a g i t a t i o n a t 3 7 ° C t o r 1 h o u r ,

a n d 3 0 p i v o l u m e s w e r e s o o t t e d o n t o n u t r i e n t a g a r

( MA ) . A f u r t t i e r 2 0 p i w a s a d d e d t o 2 m l s o f f l a n k s

s o l u t i o n a n d 2 0 p i o f t h i s w a s p l a t e d . A g a r p l a t e s

w o r e i n c u b a t e d a t 37oq f o r 1 3 h o u r s a n d t h e c o l o n i e s

w e r e c o u n t e d . S e r u m - s e n s i t i v e c u l t u r e s w e r e d e f i n e d

b o t h a s t h o s e t h a t y i e l d e d n o m o r e t h a n 5 0 c o l o n i e s

f r o m 20 j t i l o f t h e u n d i l u t e d s e r u m s a m p l e a n d a l s o a s

t h o s e w h e r e t h e v i a b l e c o u n t w a s r e d u c e d b y a t l e a s t

1 0 0 0 f o l d c o m p a r e d w i t h t h e c o n t r o l ( A p p e n d i x I ) .

P a r t i a l l y s e n s i t i v e c u l t u r e s g a v e c o u n t s o f 5 1 t o 2 0 0

c o l o n i e s f r o m t h e u n d i l u t e d s a m p l e , a n d o f t e n

r e c o v e r e d w h e n d i l u t e d , b u t i n c o n t r a s t t o t h e c o n t r o l

s a m p l e s g r o w t h w a s n e v e r c o n f l u e n t . R e s i s t a n t

c u l t u r e s a l w a y s g a v e c o n f l u e n t g r o w t h f r o m b o t h

d i l u t i o n s o f t h e t e s t s a m p l e . F i g u r e 4 s h o w s t h e

n u t r i e n t a g a r p l a t e i n o n e t e s t o n a s e n s i t i v e s t r a i n .

F o u r c o l o n i e s g r e w f r o m t h e u n d i l u t e d t e s t s a m p l e s e e n

a t t h e b o t t o m o f t h e p l a t e , w i t h r e c o v e r y i n t h e

d i l u t e d s a m p l e t o a p p r o x i m a t e l y 1 0 0 c o l o n i e s . I n t h e

c o n t r o l s a m p l e a p p r o x i m a t e l y 100 c o l o n i e s g r e w f r o m

t h e u n d i l u t e d a l i q u o t , a n d c o n f l u e n t g r o w t h r e s u l t e d

f r o m t h e d i l u t e d a l i q u o t . P r e l i m i n a r y e x p e r i m e n t s t o

e s t a b l i s h t h e c o n d i t i o n s f o r t h e s e r u m - s e n s i t i v i t y

t e s t a r e d e s c r i b e d i n A p p e n d i x 1 .

A s u m m a r y o f t h e p r o c e d u r e f o r t h e i d e n t i f i c a t i o n

o f p s e u d o m o n a s s t r a i n s f r o m s p u t u m i s s h o w n i n

F i g u r e 5 .

FIGURE 4

THE SERUM-SENSITIVITY PLATE ASSAY

Nutriant agar plate after 18 hours incubation

NORMAL HUMAN SERUM

20 fil fromundiluted samp 1e

20 j i l from sample

di luted 1 in 100

CONTROL(Hanks' solution)

F I G U R E 5

SCHEME FOR IDENTIFICATION OF PSEUDOMONAS STRAINS

3 puturn

^--------------- p a n c r e a t i n d i g e s t i o n

C e t r i m i d e - n a l i d i x i c a c i d a g a r

P r i m a r y g r o w t h

K i n g ' s " A " a g a r

C o l o n y f o r m O - s e r o t y p e P A a n t i g e n S e r u m s e n s i t i v i t y

6 . A B S O R P T I O N 0 0 S E R A

■ The n t r a i n of : n s e u d o i n o n a s t o b e u s e d t o r t h e

a b s o r p t i o n oi s e r a w a s g r o w n i n T r y n t o n c S o y a B r o t h

(Oxoid) a t 3 7 ° C tor 4 h o u r s . F i v e NA p l a t e s v j e r e

i n o c c u l a t e d u s i n g a s t e r i l e s w a b a n d i n c u b a t e d

o v e r n i g h t a t 3 7 ° C . T h e g r o w t h v ; a s w a s h e d o f t w i t h a

0 . 5 % s o l u t i o n o r n h e n o l i n s a l i n e a n d c e n t r i f u g e d a t

1 0 , 0 0 0 r p m f o r 2 0 m i n u t e s . T h e c e l l s w e r e r e s u s p e n d e d

i n 1 m l o f s e r u m a n d i n c u b a t e d a t 3 7 ° C f o r 2 h o u r s a n d

t h e n 4 ^ C o v e r n i g h t . T h e s u s p e n s i o n w a s c e n t r i f u g e d

f o r 10 m i n u t e s i n a n E p p e n d o r f c e n t r i f u g e , t h e s e r u m

t a k e n o f f a n d p a s s e d t h r o u g h a 0 . 4 5 p s t e r i l i s i n g

g r a d e m e m b r a n e f i l t e r .

F. STORAGE OF STRAINSO n c e i d e n t i f i e d , e a c h d i s t i n c t s t r a i n o f

p s e u d o m o n a s w a s a l l o c a t e d a r e f e r e n c e n u m b e r a n d

s t o r e d i n t h r e e w a y s

- o n a s l o p e o f D o r s e t E g g a t r o o m t e m p e r a t u r e

- i n 1% s k i m m e d m i l k a t - 2 0 ° C

- i n 1 6 % ( w / v ) g l y c e r o l i n B r a i n H e a r t I n f u s i o n

B r o t h a t - 7 0 ° C .

T h e D o r s e t E g g c u l t u r e s w e r e u s e d f o r a l l t h e

s t u d i e s , t h e o t h e r s b e i n g s t o r e d f o r r e f e r e n c e .

C u l t u r e s w e r e t r a n s f e r r e d t o 3 m l o f T S B a n d i n c u b a t e d

a t 3 7 ° C f o r 1 8 h o u r s , a n d t h e n s u b c u l t u r e d o n t o K i n g ' s

" A " A g a r b e f o r e u s e i n e x p e r i m e n t s .

M E D I A AND S O L U T I O N S

T r y p t o n o S o y a B r o t h ( O x o i d ) w a s u s e d t h r o u g h o u t

!: o r l i q u i d c u l t u r e , a n d t h e s o l i d m e d i u m u s e d w a s

0 >:o i d i' iu t r i e n t B r o t h ( n o . 2 ) s o 1 i d i l i e d w i t h 1 % ( v ; / v )

a g a r . P h o s p h a t e - b u f f e r e d s a l i n e p h 7 * 4 ( P B S ; 0 * 0 0 2 M )

w a s p r e p a r e d b y d i s s o l v i n g r e a g e n t t a b l e t s ( O x o i d ) i n

d i s t i l l e d w a t e r . h a n k s b a l a n c e d s a l t s o l u t i o n w a s

p r e p a r e d f r o m H a n k s s t o c k s o l u t i o n ( O x o i d ) . S a l i n e

w a s a q u e o u s s o d i u m c h l o r i d e 0 * 3 5 % ( w / v ) , a n d p h e n o l

s a l i n e c o n t a i n e d 0 * 5 % ( w / v ) o f s o l i d p h e n o l .

C h a p t e r 4

TREATMENT OF P.AERUGINOSA INFECTION IN ACUTE EXACERBATIONS OF PULMONARY DISEASE IN CYSTIC FIBROSIS BY

INTRAVENOUS ANTIBIOTICS

/ \ . I N T R O D U C T I O N

T r e a t m e n t o f a c u t e e x a c e r b a t i o n s o f r e s p i r a t o r y

s y m p t o m s i n C F p a t i e n t s w i t h p s e u d o m o n a s i s o l a t e d f r o m

t h e i r s p u t u m i s b a s e d o n t w o p r e m i s e s . T h e f i r s t i s

t h a t c h r o n i c P . a e r u g i n o s a i n f e c t i o n i s r e l a t e d t o t h e

s e v e r i t y o f p u l m o n a r y d i s e a s e , a n d t h a t p s e u d o m o n a s

h a s a r o l e i n t h e p a t h o g e n e s i s o f t h i s p u l m o n a r y

d i s e a s e . T h e s e c o n d p r e m i s e i s t h a t c h e m o t h e r a p y

d i r e c t e d a g a i n s t P . a e r u q i n o s a p r o d u c e s c l i n i c a l

i m p r o v e m e n t e v e n t h o u g h t h e o r g a n i s m m a y n o t b e

e r a d i c a t e d f r o m t h e s p u t u m . T h e f o l l o w i n g i s a b r i e f

s u m m a r y o f t h e e v i d e n c e t o s u p p o r t t h e s e a s s u m p t i o n s ,

a n d a r e v i e w o f t h e l i t e r a t u r e o n t r i a l s o f a n t i b i o t i c

c h e m o t h e r a p y i n t h i s c o n d i t i o n .

T h e p r e v a l e n c e o f P . a e r u g i n o s a i s o l a t e d f r o m t h e

s p u t u m o f C F p a t i e n t s i s r e p o r t e d t o b e b e t w e e n

4 0 - 9 0 % ^ , 4 3 , 1 1 5 b T h e p r e s e n c e o f t h e o r g a n i s m i n

s p u t u m i s i n t e r p r e t e d a s i n f e c t i o n r a t h e r t h a n m e r e

b a c t e r i a l c a r r i a g e , s i n c e m o s t p a t i e n t s s h o w c h a n g e s

i n s y s t e m i c i m m u n e r e s p o n s e s t o p s e u d o m o n a s

a n t i g e n s - * - 1̂ ' ( a l s o r e v i e w e d i n C h a p t e r 2 ) . I t i s

t h o u g h t t h a t t h e o r g a n i s m , o r i t s i m m u n o g e n i c

p r o d u c t s , p e n e t r a t e t h e e p i t h e l i a l b a r r i e r t o i n i t i a t e

t h e s e r e s p o n s e s , w h i c h w o u l d n o t o c c u r w i t h s i m p l e

b a c t e r i a l c o l o n i s a t i o n . K u l c z y c k i e t a l ^ r e v i e w e d 1 6 0

C F p a t i e n t s o v e r a f i v e y e a r p e r i o d a n d s h o w e d a

p o s i t i v e c o r r e l a t i o n b e t w e e n s e v e r i t y o f p u l m o n a r y

d i s e a s e a n d c h r o n i c P . a e r u g i n o s a i n f e c t i o n . A s i m i l a r

p o s i t i v e c o r r e l a t i o n b e t w e e n c h r o n i c p s e u d o m o n a s

i s o l a t i o n f r o m s p u t u m a n d d e c l i n e i n r e s p i r a t o r y

f u n c t i o n a n d c h e s t x - r a y s c o r e w a s s h o w n b y H o d s o n ' - .

I n b o t h t h e s e s t u d i e s t h e o r g a n i s m w a s i s o l a t e d f r o m

p o s t - m o r t e m s p e c i m e n s i n m o s t c a s e s . I n a d e t a i l e d

s t u d y o f 1 0 4 C F c h i l d r e n f r o m t h e H o s p i t a l f o r S i c k

C h i l d r e n , G r e a t O r m o n d S t r e e t , p u l m o n a r y f u n c t i o n

t e s t s , c h e s t x - r a y s c o r e s , h o s p i t a l a d m i s s i o n s ,

h y p e r s e n s i t i v i t y r e s p o n s e s a n d b a c t e r i a l c o l o n i s a t i o n

w e r e s u b j e c t e d t o a n a n a l y s i s o f v a r i a n c e . T h e o n l y

s t a t i s t i c a l l y s i g n i f i c a n t f a c t o r a s s o c i a t e d w i t h

d e t e r i o r a t i o n i n t h e p a t i e n t ' s c o n d i t i o n w a s

c o l o n i s a t i o n b y P . a e r u g i n o s a - * - ^ .

I n s u m m a r y , t h e r e i s r e a s o n a b l e e v i d e n c e t h a t

w i t h i n a g r o u p o f C F p a t i e n t s , t h o s e w i t h o u t

p s e u d o m o n a s a r e f i t t e r t h a n t h o s e i n f e c t e d b y t h e

o r g a n i s m , a n d t h a t t h e r e i s a r e l a t i o n s h i p b e t w e e n

p s e u d o m o n a s i n f e c t i o n a n d t h e s e v e r i t y o f p u l m o n a r y

d i s e a s e . T h i s r e l a t i o n s h i p i s n o t n e c e s s a r i l y a

c a u s a l o n e , a n d t h e r e i s l i t t l e e v i d e n c e t h a t t h e

o r g a n i s m i s i m p o r t a n t t o t h e s e v e r i t y o f d i s e a s e

w i t h i n t h e i n d i v i d u a l .

B . LITERATURE REVIEW

T h e o b j e c t o f a n y c l i n i c a l t r i a l i s t o o b t a i n a n

a n s w e r t o a p a r t i c u l a r c l i n i c a l q u e s t i o n . I n t h i s

c a s e t h e q u e s t i o n i s ' d o e s a n t i b i o t i c t h e r a p y a g a i n s t

P . a e r u g i n o s a i m p r o v e t h e c l i n i c a l c o n d i t i o n o f C F

p a t i e n t s c h r o n i c a l l y i n f e c t e d b y t h i s o r a a n i s o w h o a r c

s u f f e r i n g f r o m a n a c u t e e x a c e r b a t i o n o f t h e i r

r e s p i r a t o r y s y m p t o m s ? ' . S c i e n t i f i c a l l y v a l i d d a t a i s

b e s t o b t a i n e d i f t h e p a t i e n t s a r e r a n d o m l y a l l o c a t e d

t o t r e a t m e n t a n d c o n t r o l g r o u p s , b o t h g r o u p s o f

p a t i e n t s a r e c o m p a r a b l e i n t e r m s o f s e v e r i t y o f

d i s e a s e , a n d t h e o u t c o m e i s a s s e s s e d b y s t a t i s t i c a l

a n a l y s i s o f o b j e c t i v e n u m e r i c a l d a t a . T h e s e b a s i c

r u l e s h a v e r a r e l y b e e n a p p l i e d t o t r i a l s o f

c h e m o t h e r a p y i n C F , l a r g e l y b e c a u s e o f t h e f e a t u r e s o f

p s e u d o m o n a s i n f e c t i o n i n t h i s c o n d i t i o n . T h e o r g a n i s m

r e m a i n s l o c a l i s e d w i t h i n t h e r e s p i r a t o r y t r a c t , a n d

b a c t e r a e m i a i s e x t r e m e l y r a r e ^ . A n t i b i o t i c s m u s t

t h e r e f o r e p e n e t r a t e t h e r e s p i r a t o r y e p i t h e l i a l b a r r i e r

t o b e e f f e c t i v e , b u t t h e y a r e a l m o s t a l w a y s g i v e n i n

t h e r o u t i n e r e g i m e n s f o r s y s t e m i c i n f e c t i o n s , w i t h o u t

r e g a r d f o r s p u t u m l e v e l s o b t a i n e d . O n c e t h e o r g a n i s m

i s d e t e c t e d i n t h e s p u t u m o f a p a r t i c u l a r p a t i e n t i t

i s r a r e l y e r a d i c a t e d f o r a n y l e n g t h o f t i m e , s o t h a t

t h e b a c t e r i o l o g i c a l o b j e c t i v e o f s t e r i l i s a t i o n o f t h e

s p u t u m s p e c i m e n i n m o s t t r i a l s o f c h e m o t h e r a p y , i s n o t

a p p l i c a b l e t o C F T h e c a r d i n a l s y m p t o m s a n d s i g n s

o f i n f e c t i o n , s u c h a s p y r e x i a , r a i s e d v z h i t e c e l l

c o u n t , r a i s e d s e d i m e n t a t i o n r a t e a n d a c u t e p h a s e

p r o t e i n s , m a y n o t b e p r e s e n t i n t h e a c u t e o n c h r o n i c

e x a c e r b a t i o n s o f C F p u l m o n a r y d i s e a s e . S i m i l a r l y i t

m a y b e e x t r e m e l y d i f f i c u l t t o s e e n e w r a d i o l o g i c a l

c h a n g e s i n a n a l r e a d y s e v e r e l y a b n o r m a l c h e s t x - r a y .

T.n t h e 1 9 7 0 ' s , o p e n t r i a l s o f a n t i p s e u d o m o n n 1

a n t i b i o t i c s w e r e p e r f o r m e d a s t h e s e d r u q s b e c a m e

a v a i l a b l e ^ 0 , 1 1 8 - 1 2 1 # T h e _in_ v i t r o a n d i n v i v o

s y n e r g i s m o f a m i n o g l y c o s i d e s a n d p e n i c i l l i n s i s w e l l

k n o w n , a n d b y t h e e n d o f t h e d e c a d e t h e c o m b i n a t i o n o f

g e n t a m i c i n a n d c a r b e n i c i l l . i n f o r 1 0 - 1 4 d a y s h a d b e c o m e

s t a n d a r d c h e m o t h e r a p y f o r a c u t e e x a c e r b a t i o n s o f C F

p u l i r f o n a r y d i s e a s e w h e r e p s e u d o m o n a s w a s p r e s e n t .

• S u b s e q u e n t t r i a l s o f o t h e r a m i n o g l y c o s i d e s a n d

p e n i c i l l i n s h a v e e i t h e r b e e n o p e n o r c o m p a r a t i v e .

T a b l e I I I s u m m a r i s e s t h e m a j o r s t u d i e s t o d a t e .

T h e g e n e r a l c o n c l u s i o n s t h a t m a y b e d r a w n a r e : -

1 . T h e h a r d c l i n i c a l d a t a i s s p a r s e . I n m o s t s t u d i e s

c l i n i c a l s c o r e s , x - r a y s c o r e s a n d p u l m o n a r y f u n c t i o n

t e s t s w e r e m e a s u r e d , b u t f e w d e t a i l e d r e s u l t s a r e

g i v e n .

2 . P a t i e n t s w i t h " s e v e r e d i s e a s e " , d o n o t r e s p o n d t o a n y

1 2 0 1 2 2 1 2 3 r e g u n e n 1 , i U , #

3 . P s e u d o m o n a s i s o n l y ' c l e a r e d * f r o m s p u t u m f o r a f e w

w e e k s a t t h e m o s t , w i t h v e r y f e w e x c e p t i o n s . W h e r e

t h i s h a s b e e n i n v e s t i g a t e d , p a t i e n t s a p p e a r t o b e

' r e c o l o n i s e d ' b y o r g a n i s m s o f t h e s a m e s e r o t y p e a n d

p y o c i n e t y p e .

4 . P a t i e n t S e l e c t i o n

M o s t s t u d i e s h a v e d e f i n e d p a t i e n t s w i t h c h r o n i c

p s e u d o m o n a s i n f e c t i o n a s t h o s e w h e r e t h e o r g a n i s m h a s

b e e n i s o l a t e d f r o m e v e r y s p u t u m s a m p l e f o r a t l e a s t

TABLE IIISUMMARY OF STUDIES OF INTRAVENOUS CHEMOTHERAPY AGAINST P.AERUGINOSA IN CF PATIENTS

I Study | 1 1 1 1 1 1

Antibiotic regimen | (dose in mg/kg/day) |

No. of courses evaluated

Design | of study |

Assessment | of |

response | 1

No. of positive responses ' a) clinical I b) transient ! improvement ! eradication j

1 of pseudomonasBoxerbaum( Hd) ) Carbenici1lin | 41 Open | Clinical features | 33 j 0 !let a 1, 1968. | (300 - 350) | 1 CXR, RFT. | i j| Phair et al | Carbenici1lin | 13 Open | Clinical features | 7 1 0 i| 1970 (119) | (2 - 300) | (4 with 1 1 1 11 1 gentamicin 1 1 1 I| Huang(^u) | Carbenici1lin | 85 Open | Clinical features | 54 1 28 iI et al, 1971 | (150 - 500) for | 1 CXR, RFT, | 1 I11 1 14 days | 1 bacteriology. | ! l| Marks | Carbenicillin | 14 Open | Clinical features | 2 j i || et al, 1971 | (600 - 2400) | 1 CXR, RFT, blood gases, | i 11 1 1 bacteriology. | i 1| McCrae(-^z -̂) | Tobramycin (5 - 10) for | 17 Open | White cell count, ESR, | 17 i 4 (3 for |1 1976 | 14 days | 1 RFT, blood gases. | iunstated period)| Parry(^zz) | Ticarcillin (300) | 42 Comparative I Shwachman score | 28 ! Not stated II et al, 1977 | Gentamicin (4-7) | Alternate | CXR, RFT, | 1 11 1 for 11 days | allocation I blood gases. | 1 1| Beaudry(^J) | Carbenicillin (200) | 22 Comparative | Clinical score | 22 | 3 1| et al, 1980 | + gentamicin (5) | Random | CXR, RFT, j ii 1i 1 or cloxacillin (200) | allocation | blood gases | 1 1| Rabin(iZ )̂ I Ticarcillin (600) I 38 Open | Colony count reduced I Not | 21 11 et al, 1980 I + tobramycin (12-16) I 1 by 10^/ml. | stated 1 || Martin(^z )̂ I Carbenicillin (800) | 20 Comparative | Clinical score | 19 1 15 I| et al, 1980 | + gentamicin or I Alternate | CXR 1 1 (1 for 15/12) 11 1tobramycin (9) for 14 days | allocation | 1 I 1I MjzjI ler ( ̂ z^) | Tobramycin (10) | 54 Comparative I Bacteriology | 21 1 21 1| et al, 1982 I+ azloci 1lin/piperaci1lin/ | Allocation on | PEFR | 1 11 1 carbenicillin | sensitivity |Precipitating antibodies | 1 11 1 results | 1 i 1| Hod son(^z )̂ | Carbenicillin (400) I 18 Cross over I Clinical features ! 9 1 5 1I et al, 1982 | + gentamicin or | study | PEFR | 1 |1 1 tobramycin 1 1 1 1 I

RFT = r e s p i r a t o r y f u n c t i o n t e s t s . = ne as u r e m e n t s c o r r e l a t e d w i t h clinical outcome and results given in paper.RFT

s i x m o n t h s . A c u t o e x a c a r b a t i o n s o f t h e d i s e a s e h a v e

b e e n l o o s e l y d e f i n e d i n t e r m s o f i n c r e a s e d c o u g h ,

s p u t u m p r o d u c t i o n a n d s h o r t n e s s ol : b r e a t h o n

e x e r t i o n .

Des Lqn

I n m o s t c o m p a r a t i v e t r i a l s p a t i e n t s w e r e a l l o c a t e d

a l t e r n a t e l y t o t r e a t m e n t g r o u p s . I n o n l y o n e s t u d y

w a s t h e a l l o c a t i o n r a n d o m i s e d ! 2 3 f a n t i j_n o n e f u r t h e r

s t u d y p a t i e n t s w e r e u s e d a s t h e i r o w n c o n t r o l i n a

c r o s s - o v e r d e s i g n - ^ ^ .

V a r i a b l e s o f r e s p o n s e

a ) C l i n i c a l a s s e s s m e n t o r s c o r e ; t h i s w a s d o n e e i t h e r

119o n t h e s y s t e m o r S h w a c h m a n o r o n t h e a u t h o r ' s

o w n s y s t e m , b u t o n l y s h o w n t o i m p r o v e w i t h

t r e a t m e n t b y M a r t i n e t a l ^ - * a n d B e a u d r y e t a l - ^ 3 ^

b ) W h i t e c e l l c o u n t a n d E S R ; t h e s e v a r i a b l e s w e r e n o t

c o n s i s t e n t l y a b n o r m a l a n d d i d n o t r e l a t e t o

c l i n i c a l o u t c o i n e ^ ^ ^ ^ ' ^ ^ .

c ) C h e s t x - r a y ; s t u d i e s o n o l d e r p a t i e n t s a n d t h e m o r e

s e v e r e l y i l l d i d n o t f i n d m e a s u r a b l e r a d i o l o g i c a l

c h a n g e s , b u t s t u d i e s i n c h i l d r e n h a v e f o u n d t h e

c h a n g e d x - r a y s c o r e t o b e a u s e f u l m e a s u r e o f

r e s p o n s e x ^ J .

d ) B l o o d g a s e s ; t h e s e a r e m e n t i o n e d a s b e i n g m e a s u r e d

i n m a n y s t u d i e s b u t n o r e s u l t s a r e g i v e n .

e ) S p u t u m b a c t e r i o l o g y ; s i m p l e b a c t e r i o l o g y d i d n o t

c o r r e l a t e w i t h c l i n i c a l r e s u l t s . T h e o r g a n i s m w a s

n o t e r a d i c a t e d , b u t d e s p i t e t h i s p a t i e n t s w e r e

c o n s i d e r e d t o h a v e i m p r o v e d . Q u a n t i t a t i v e

b a c t e r i o l o g y o n y o u t u r n i s n o t o r i o u s l y d i f f i c u l t t o

i n t e r p r e t , a n d t h e o n e s t u d y b y R a b i n e t n l - ^ * ^ t h a t

u s e s i t t o a s s e s s r e s p o n s e d o e s n o t q i v e a n y

c l i n i c a l r e s u l t s . M o s t p a p e r s d i s c u s s d e t a i l e d

b a c t e r i o l o g y o f t h e i s o l a t e d o r g a n i s m s , a n d M I C s

i o r t h e a n t i b i o t i c s u s e d b u t p h a i r e t a . U ^ n o t e d

t h a t c l i n i c a l r e s p o n s e d i d n o t c o r r e l a t e w i t h i n

v i t r o s e n s i t i v i t y , a n d s o m e p a t i e n t s w i t h r e s i s t a n t

o r g a n i s m s r e s p o n d e d .

f ) R e s p i r a t o r y f u n c t i o n t e s t s ; m o s t s t u d i e s p e r f o r m e d

r e s p i r a t o r y f u n c t i o n t e s t s b u t f e w q u o t e d e t a i l e d

r e s u l t s . P e a k e x p i r a t o r y f l o w r a t e ^ ^ ' ^ ^ a n d

f o r c e d e x p i r a t o r y f l o w r a t e b e t w e e n 2 5 a n d 7 5 % o f

F V C X “ J w e r e s h o w n t o i m p r o v e s i g n i f i c a n t l y w i t h

c h e m o t h e r a p y , t h e d e g r e e o f i m p r o v e m e n t b e i n g

b e t w e e n 3 0 a n d 8 5 % o f b a s e l i n e v a l u e s ,

g ) P a t i e n t a s s e s s m e n t ; m a l a i s e w a s o f t e n q u o t e d a s a

m a j o r s y m p t o m , b u t n o s t u d i e s h a v e m a d e a n y

m e a s u r e m e n t o f p a t i e n t ' s s u b j e c t i v e r e s p o n s e s t o

t r e a t m e n t .

C . A I M S

T o e v a l u a t e t h e c l i n i c a l b e n e f i t o f i n t r a v e n o u s

c h e m o t h e r a p y a g a i n s t P . a e r u g i n o s a i n a c u t e

e x a c e r b a t i o n s o f r e s p i r a t o r y s y m p t o m s i n c h r o n i c a l l y

i n f e c t e d C F p a t i e n t s a n d t o c o m p a r e s o m e o f t h e

p e n i c i l l i n s a v a i l a b l e f o r t h i s p u r p o s e .

D . M E T H O D S

1 . S T U D Y DFS T. GU

T w o p r e v i o u s t r L a I s h a v e c o m p a r e d g e n t a i n i c i n a n

t o b r a m c y i n ( i n c o m b i n a t i o n w i t h a p e n i c i l l i n ) a n d

f o u n d n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e m 1 - 2 6 , 1 2 7 #

T h e r e i s n o e v i d e n c e o f t o x i c i t y i n t h e s e o r o t h e r

s t u d i e s o f e i t h e r d r u g i n C F , a n d t h e r e f o r e g e n t a m i c i n

w a s u s e d a s t h e a m i n o g l y c o s i d e i n t h e p r e s e n t s t u d i e s .

T h e r e a r e t w o c l a s s e s o f p e n i c i l l i n s w i t h

a n t i o s e u d o m o n a l a c t i v i t y , t h e a l p h a c a r b o x y l

p e n i c i l l i n s , c a r b e n i c i l l i n a n d t i c a r c i l l i n a n d t h e

a c y l a m i n o p e n i c i l l i n d e r i v a t i v e s o f a m p i c i l l i n w h i c h

i n c l u d e a z l o c i l l i n , m e z l o c i l l i n a n d p i p e r a c i l l i n . T h e

l a t t e r g r o u p a r e c o n s i d e r a b l y m o r e a c t i v e ini v i t r o

a g a i n s t P . a e r u g i n o s a , a n d a l l a r e s u s c e p t i b l e t o

b e t a - l a c t a m a s e s . T i c a r c i l l i n a n d a z l o c i l l i n w e r e

s e l e c t e d a s r e p r e s e n t a t i v e s o f t h e t w o g r o u p s t h a t a r e

a v a i l a b l e i n m o s t h o s p i t a l s . I n s e p a r a t e r a n d o m i s e d

p r o s p e c t i v e t r i a l s t i c a r c i l l i n o r a z l o c i l l i n p l u s

g e n t a m i c i n w a s c o m p a r e d w i t h c a r b e n i c i l l i n p l u s

g e n t a m i c i n , t h e l a t t e r b e i n g s t a n d a r d c h e m o t h e r a p y a t

t h e B r o m p t o n H o s p i t a l .

P a t i e n t s w h o w e r e s e v e r e l y i l l ( F EV] _ l e s s t h a n

2 0 % p r e d i c t e d n o r m a l v a l u e ) w e r e e x c l u d e d . A

c o m p a r a t i v e t r i a l w a s p e r f o r m e d , r a t h e r t h a n a

c r o s s - o v e r s t u d y , b e c a u s e w e w i s h e d t o i n c l u d e a

r e l a t i v e l y l a r g e n u m b e r o f p a t i e n t s . M o s t p a t i e n t s d o

n o t r e q u i r e f u r t h e r t r e a t m e n t f o r s i x m o n t h s o r

l o n g e r , a n d t o u s e e a c h p a t i e n t a s t h e i r o wn c o n t r o l

w o u l d m e a n t h a t t h e s t u d y w o u l d h a v e t a i con s e v e r a l

y e a r s . R e s p i r a t o r y f u n c t i o n t e s t s ' w e r e u s e d a s t h e

p r i m a r y m e a s u r e m e n t s o f r e s p o n s e . S i m p l e m e a s u r e m e n t s

oi : d y n a m i c l u n g v o l u m e s , FFV] _ a n d F V C w e r e m a d e u s i n g

a s p i r o m e t e r a n d p e a k f l o w m e a s u r e d o n a p e a k f l o w

m e t e r . S u c h t e s t s a r e e a s i l y p e r f o r m e d a t t h e b e d s i d e

a n d l e s s d i s t r e s s i n g t o t h e s i c k p a t i e n t t h a n b e i n g

t r a n s p o r t e d t o a r e s p i r a t o r y f u n c t i o n l a b o r a t o r y t o

p e r f o r m t h e m a n o e u v r e s f o r m e a s u r i n g s t a t i c l u n g

v o l u m e s a n d g a s t r a n s f e r . L u n g f u n c t i o n t e s t s w e r e

m o n i t o r e d s e r i a l l y i n C F c h i l d r e n a d m i t t e d t o h o s p i t a l

b y R e d d i n g e t a l x . T h e y s h o w e d t h a t v i t a l c a p a c i t y

w a s t h e f i r s t v a r i a b l e t o i m p r o v e , a c c o m p a n i e d b y a

r i s e i n P E F R a n d F E V ^ . C h a n g e s i n o t h e r l u n g v o l u m e s

a n d i n f l o w r a t e s a t v a r i o u s p e r c e n t a g e s o f v i t a l

c a p a c i t y o c c u r r e d l a t e r a n d w e r e s u b j e c t t o

p e r f o r m a n c e v a r i a b i l i t y e s p e c i a l l y i n t h e m o r e

s e v e r e l y i l l p a t i e n t s . I n t h e s e c o n d s t u d y o n

a z l o c i l l i n , a s u b j e c t i v e a s s e s s m e n t o f t h e p a t i e n t ' s

r e s p o n s e w a s m e a s u r e d u s i n g a v i s u a l a n a l o g u e s c a l e .

P a t i e n t s w e r e r a n d o m l y a l l o c a t e d t o t h e t w o

t r e a t m e n t g r o u p s u s i n g i n s t r u c t i o n s i n s e a l e d a n d

s h u f f l e d e n v e l o p e s . T h e t r i a l s w e r e s i n g l e b l i n d ;

p a t i e n t s g a v e i n f o r m e d c o n s e n t f o r b l o o d a n d s p u t u m

s a m p l e s f o r a n t i b i o t i c a s s a y s a n d w e r e t o l d t h e y w e r e

r e c e i v i n g ’ s t a n d a r d ’ c h e m o t h e r a p y . A l l d r u g s u s e d i n

t h e s e s t u d i e s h a v e f u l l p r o d u c t l i c e n c e s a n d a l l

e x c e p t a z l o c i l l i n h a v e b e e n o n t h e m a r k e t f o r m a n y

y e a r s . T h e t r i a l s w e r e s i n g l e b l i n d a s t h e d o s e

I n t e r v a l s a n d I n I: u s i o n v o l u m e of : t h e p e n i c i l l i n s

v a r i e d , s o t h a t n u r s i n g s t a f f ! : w e r e a w a r e w h i c h

p r e p a r a t i o n w a s b e i n g g i v e n . A l l c l i n i c a l a s s e s s m e n t s

w e r e m a d e b y t h e a u t h o r . R e s p i r a t o r y f u n c t i o n t e s t s

w e r e r e c o r d e d b y t h e h o u s e p h y s i c i a n w h o w a s n o t

o t h e r - w i s e i n v o l v e d i n t h e s t u d i e s i n a n y o t h e r w a y , a s

p a t i e n t s m a y p e r f o r m t h e s e t e s t s b e t t e r w i t h

e n c o u r a g e m e n t .

2 . P A T I E N T S E L E C T I O N

A l l C F p a t i e n t s a d m i t t e d t o h o s p i t a l o v e r a p e r i o d

o f 2 0 m o n t h s f r o m J u l y 1 9 3 0 t o A p r i l 1 9 8 2 , w e r e

c o n s i d e r e d f o r i n c l u s i o n i n t h e t r i a l s . T h e c r i t e r i a

f o r i n c l u s i o n w e r e : -

a ) C o n f i r m e d d i a g n o s i s o f C F b a s e d o n c r i t e r i a i n

C h a p t e r 3 .

b ) C h r o n i c p s e u d o m o n a s i n f e c t i o n , i . e . P . a e r u g i n o s a

i s o l a t e d f r o m e v e r y s p u t u m c u l t u r e f o r a p e r i o d o f

a t l e a s t s i x m o n t h s .

c ) N o h i s t o r y o f p e n i c i l l i n a l l e r g y .

d ) N o b i o c h e m i c a l e v i d e n c e o f l i v e r o r r e n a l

d i s e a s e .

e ) N o s y m p t o m s o r s i g n s o f c o r p u l m o n a l e .

f ) N o p o s s i b i l i t y o f p r e g n a n c y i n t h e c a s e o f f e m a l e

p a t i e n t s .

g ) F E V g > 2 0 % p r e d i c t e d n o r m a l v a l u e s o n e n t r y t o t h e

trial-**̂ .h ) P a t i e n t s w e r e c o n s i d e r e d t o b e s u f f e r i n g f r o m a n

a c u t e e x a c e r b a t i o n o f t h e i r r e s p i r a t o r y d i s e a s e

a n d t o r e q u i r e i n t r a v e n o u s c h e m o t h e r a p y i f t h e i r

a d m i s s i o n r e s p i r a t o r y f u n c t i o n t e s t s h a d s h o w n a

f a l l o f a t l e a s t 2 0 3 c o m p a r e d t o p r e v i o u s

o u t - p a t i e n t m e a s u r e m e n t s . T h e m a j o r s y m p t o m s w e r e

o f i n c r e a s e d c o u g h , s p u t u m p r o d u c t i o n a n d

s h o r t n e s s o f b r e a t h o n e x e r t i o n .

v S i x t e e n p a t i e n t s w e r e i n c l u d e d i n t h e t i c a r c i l l i n

v e r s u s c a r b e n i c i l l i n t r i a l , a n d t w e n t y i n t h e

a z l o c i l l i n v e r s u s c a r b e n i c i l l i n t r i a l .

3 . A S S E S S M E N T

T h e f o l l o w i n g m e a s u r e m e n t s w e r e m a d e o n d a y 0 a n d

d a y 1 0 : -

a ) R e s p i r a t o r y f u n c t i o n t e s t s

P E F R , FEV- j ^ , F V C w e r e r e c o r d e d a s d e s c r i b e d i n

C h a p t e r 3 .

b ) P a t i e n t a s s e s s m e n t

A s u b j e c t i v e a s s e s s m e n t o f p a t i e n t w e l l - b e i n g w a s

r e c o r d e d o n a 1 0 c m v i s u a l a n a l o g u e s c a l e ,

( a z l o c i l l i n t r i a l o n l y ) w i t h z e r o r e p r e s e n t i n g t h e

w o r s t t h a t t h e i n d i v i d u a l h a d e v e r f e l t , a n d 10

r e p r e s e n t i n g t h e b e s t t h a t t h e y h a d f e l t i n t h e

p r e v i o u s t w o y e a r s .

c ) S p u t u m c u l t u r e

S p u t u m s a m p l e s w e r e p r o c e s s e d t o i s o l a t e

p s e u d o m o n a s a s d e s c r i b e d i n C h a p t e r 3 . O t h e r

o r g a n i s m s w e r e i s o l a t e d b y s t a n d a r d m e t h o d s . A l l

o r g a n i s m s w e r e t e s t e d f o r a n t i b i o t i c s e n s i t i v i t y

b y t h e S t o k e s c o m p a r a t i v e d i s c m e E h o d ^ - ^ .

d ) T o x i c i t y m o n i t o r i n g

b u l l b l o o d c o u n t , E S 8 , l i v e r f u n c t i o n t e s t s , u r e a

a n d e l e c t r o l y t e s w e r e p e r f o r m e d o n a l l p a t i e n t s .

D O S E R E G I M E N S

A l l p a t i e n t s r e c e i v e d g e n t a m i c i n i n t r a v e n o u s l y 8

h o u r l y , t h e d o s e b e i n g a d j u s t e d t o g i v e a s e r u m l e v e l

o f 8 - 1 0 m i c r o g r a m s / m l a t 1 5 m i n u t e s a f t e r a b o l u s

i n j e c t i o n . T h e d o s e o f a z l o c i l l i n w a s 5 g r a m s 8

h o u r l y , a n d o f c a r b e n i c i l l i n a n d t i c a r c i l l i n 5 g r a i n s 6

h o u r l y . A l l p a t i e n t s w e r e g i v e n t h e p e n i c i l l i n s b y

s l o w i n t r a v e n o u s i n f u s i o n o v e r 2 0 m i n u t e s . A l l

t r e a t m e n t s w e r e a r b i t r a r i l y g i v e n f o r t e n d a y s .

A S S A Y O F A N T I B I O T I C L E V E L S I N S E R U M AMD S P U T U M

A l l s a m p l e s w e r e t a k e n o n t h e t h i r d d a y o f

t r e a t m e n t , t o e s t i m a t e ' s t e a d y s t a t e ' l e v e l s o f

a n t i b i o t i c s . L e v e l s o f t h e p e n i c i l l i n s i n s e r u m w e r e

m e a s u r e d a t 1 5 m i n u t e s a f t e r t h e e n d o f t h e i n f u s i o n ,

a n d a l s o j u s t b e f o r e t h e n e x t d o s e ( s i x h o u r s f o r

c a r b e n i c i 11 i n o r t i c a r c i l l i n , 8 h o u r s f o r a z l o c i l l i n ) .

S p u t u m c o n c e n t r a t i o n s w e r e m e a s u r e d a t o n e a n d t w o

h o u r s a f t e r t h e e n d o f t h e i n f u s i o n , a n d t h e s a m p l e

j u s t b e f o r e t h e n e x t d o s e w a s d e s i g n a t e d t i m e 0 .

A d d i t i o n a l s a m p l e s w e r e a s s a y e d i n t h e t i c a r c i l l i n

t r i a l . S e r u m g e n t a m i c i n l e v e l s w e r e p e r f o r m e d a s a

r o u t i n e p r o c e d u r e b e f o r e a n d 1 5 m i n u t e s a f t e r a n

i n t r a v e n o u s b o l u s d o s e S p u t u m l e v e l s o f g e n t a m i c i n

w o r e m e a s u r e d i n t h e s a m e s a m p l e s t a k e n t o t p e n i c i l l i n

o s t i m a t L o n s .

a ) G e n t a m i c i n a s s a y

i S 0 r u m s a i n n 1 e s

■ S e r u m g e n t a m i c i n a s s a y s were p e r f o r m e d b y

h o m o g e n e o u s e n z y m e i m m u n o a s s a y ( E M I T , S y v a C o . )

u s i n g a S t a s a r I I I s p e c t r o p h o t o m e t e r c o u p l e d t o a

C P - 1 0 0 0 c l i n i c a l p r o c e s s o r a c c o r d i n g t o t h e

m a n u f a c t u r e r ' s i n s t r u c t i o n s ,

i i S p u t u m s a m p l e s

S p u t u m w a s h o m o g e n i s e d b y v i g o r o u s a g i t a t i o n o n

a v o r t e x m i x e r i n a b o t t l e c o n t a i n i n g s i x 5 • 5mm

g l a s s b e a d s . B i o a s s a y w a s u s e d t o d e t e r m i n e t h e

g e n t a m i c i n c o n c e n t r a t i o n s u s i n g K l e b s i e l l a

N . C . T . C . 1 0 8 9 6 a s t h e i n d i c a t o r o r g a n i s m o n D . S . T .

a g a r ( O x o i d ) i n 2 4 c m s q u a r e a s s a y d i s h e s ( N u n c . ) .

S t a n d a r d s o f g e n t a m i c i n f r o m 1 * 0 m g / L t o 1 6 * 0 m g / L

w e r e p r e p a r e d i n s t e r i l e h o r s e s e r u m ( N o . 3

W e l l c o m e ) . S p u t u m h o m o g e n a t e s a n d s t a n d a r d

s o l u t i o n s w e r e t e s t e d i n t r i p l i c a t e . F o l l o w i n g

i n c u b a t i o n f o r 1 8 h o u r s a t 3 7 ^ C , i n h i b i t i o n z o n e

d i a m e t e r s w e r e m e a s u r e d u s i n g a z o n e p r o j e c t o r

( L e e b r o o k I n s t r u m e n t s ) . D e - r a n d o m i s a t i o n a n d

c a l c u l a t i o n o f t h e r e s u l t s w a s p e r f o r m e d a c c o r d i n g

t o t h e m e t h o d o f P e r k i n s - ^ 0 .

b ) A z l o c i l l i n / c a r b e n i c i l l i n / t i c a r c i l l i n a s s a y s

i S e r u m s a m p l e s

S e r u r n p e n i c i l l i n a s s a y s w e r e p e r f o r m e d b y

b i o a s s a y f o l l o w i n g p r e - t r e a t m e n t o f t h e s e r u m w i t h

a r e s i n ! : o r e m o v e a n y q e n t a m i c i n p r e r e n t . 1m l o r

s e r u m " / a s a d r i e d t o 1 0 0 mq of : ' D o w e : c ' 5 0 - X 8 r e s i n

( R . n . O . ) a n d m i x e d o n a r o t a t o r t o r 1 5 m i n u t e s .

T h e r e s i n w a s a l l o w e d t o s e t t l e b y s t a n d i n g t o r a

f u r t h e r f i v e m i n u t e s a n d t h e s u p e r n a t a n t s e r u m

r e m o v e d f o r p e n i c i l l i n a s s a y ( i n o r d e r t o e n s u r e

r e m o v a l o f t h e g e n t a m i c i n h a d b e e n e f f e c t e d , t h e

s u p e r n a t a n t w a s a s s a y e d f o r g e n t a m i c i n b y t h e E M I T

p r o c e d u r e p r e v i o u s l y d e s c r i b e d ) .

B i o a s s a y f o r p e n i c i l l i n s w a s p e r f o r m e d o n

D . S . T . a g a r ( O x o i d ) u s i n g P . a e r u g i n o s a N . C . T . C .

1 0 7 0 1 a s t h e i n d i c a t o r o r g a n i s m . S t a n d a r d s o f 2 * 5

m g / L t o 4 0 m g / L w e r e p r e p a r e d f o r t h e a n t i b i o t i c s

i n h o r s e s e r u m ( M o . 3 W e l l c o m e ) a n d r e s i n a b s o r b e d

t e s t s e r a w e r e a p p r o p r i a t e l y d i l u t e d i n s e r u m i n

o r d e r t o b r i n g t h e m w i t h i n t h e c o n c e n t r a t i o n s o f

t h e s t a n d a r d c u r v e r a n g e . I n c u b a t i o n , r e a d i n g a n d

c a l c u l a t i o n o f r e s u l t s w a s p e r f o r m e d a s d e s c r i b e d

f o r s p u t u m g e n t a m i c i n a s s a y ,

i i S p u t u m s a m p l e s

S p u t u m w a s h o m o g e n i s e d a s a b o v e a n d

h o m o g e n a t e s w e r e t e s t e d a s f o r s e r u m s a m p l e s .

6 . F O L L O W - U P

P a t i e n t s w e r e s e e n i n o u t - p a t i e n t s a t o n e a n d

t h r e e m o n t h s a f t e r d i s c h a r g e , w h e n r e s p i r a t o r y

f u n c t i o n t e s t s a n d s p u t u m c u l t u r e s w e r e p e r f o r m e d .

T h e d a t e o f t h e p a t i e n t s s u b s e q u e n t h o s p i t a l a d m i s s i o n

f o r c h e m o t h e r a p y ( i f a n y ) w a s n o t e d .

7. OTHER TREATMENT

A l l . p a t i e n t s r e c e i v e d p h y s i o t h e r a p y w i t h p o s t u r a l

d r a i n a g e f o u r t i m e s a d a y . T h o s e w i t h a p r o v e n

r e v e r s i b l e e l e m e n t o £ a i r G l o w o b s t r u c t i o n r e c e i v e d

b r o n c h o d i l a t o r s . T h o s e o a t i e n t s w i t h o t h e r p a t h o g e n s

i n t h e i n i t i a l s p u t u m c u l t u r e r e c e i v e d a p p r o p r i a t e

o r a l c h e m o t h e r a p y . M u c o l y t i c a g e n t s w e r e n o t u s e d .

3 . S T A T I S T I C S

B a s e l i n e d a t a o n p a t i e n t g r o u p s a t e n t r y t o e a c h

t r i a l w a s c o m p a r e d u s i n g W i l c o x o n ' s r a n k s u m t e s t .

R e s p i r a t o r y f u n c t i o n b e f o r e a n d a f t e r t r e a t m e n t w i t h i n

e a c h g r o u p w a s c o m p a r e d u s i n g a W i l c o x o n ' s m a t c h e d - p a i r s

s i g n e d - r a n k t e s t . T h e i m p r o v e m e n t i n r e s p i r a t o r y

f u n c t i o n o n e a c h t r e a t m e n t w a s c o m p a r e d u s i n g a n

a n a l y s i s o f c o v a r i a n c e .

E . RESULTS1 . T I C A R C I L L I N V E R S U S C A R B E N I C I L L I N

a ) R e s p i r a t o r y f u n c t i o n

T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e

t w o g r o u p s i n a g e , s e x o r r e s p i r a t o r y f u n c t i o n o n

e n t r y t o t h e t r i a l ( T a b l e I V ) . B o t h a n t i b i o t i c

r e g i m e n s p r o d u c e d a s i g n i f i c a n t i m p r o v e m e n t i n a l l

t h r e e v a r i a b l e s o f r e s p i r a t o r y f u n c t i o n , P E F R , F E V g

a n d F V C ( T a b l e V) v a r y i n g f r o m 2 7 * 0 % t o 4 6 * 2 % o f

b a s e l i . n o v a l u e s . T h e r e w a s n o s i g n i f . i c o n t d i i: T e r e n c e

b e tv; c o n t r e a t m e n t s . F i g u r e 6 s h o w s t h e i m p r o v e m e n t i n

p e a t e x p i r a t o r y f l o w r a t e f o r b o t h t r e a t m e n t s .

b ) B a c t e r i o l o g y

A l l p a t i e n t s h a d P . a e r u g i n o s a i s o l a t e d f r o m t h e

s p u t u m o f a t t h e b e g i n n i n g a n d e n d o f t r e a t m e n t .

H . i n f l u e n z a e a n d 5 . a u r e u s w e r e i s o l a t e d f r o m t h e c o n t r o l

c u l t u r e f r o m o n e p a t i e n t i n t h e t i c a r c i l l i n g r o u p , b u t

t h e s e p a t h o g e n s w e r e n o t p r e s e n t o n t h e c u l t u r e o n d a y

1 0 . H , i n f l u e n z a e v ; a s i s o l a t e d f r o m t w o p a t i e n t s i n t h e

c a r b e n i c i l l i n g r o u p o n d a y 0 , b u t n o t f r o m t h e c u l t u r e s

o n d a y 1 0 .

TABLE IV

T ICARCTLLIN v CARBENICILLIN TRIAL: PATIENT DATA ON ENTRY TO THE TRIAL

111111

TICARCILLIN+

GENTAMICIN

1CARBENICILLIN |

+ | GENTAMICIN |

Statist ica1 comparison

(WiLcoxon's rank sum test)

1Number of patients |

Sex | 1

5 M 3 F1

4 M 4 F | 1

1Age range (years) |

116 - 26

12 1 - 3 3 |

1Range of PEFR (l/min)|

1105 - 300

1140 - 370 |

1Range of FEV^ (l/min)|

1600 - 1850

1550 - 1750 |

1Range of FVC (mis) |

11350 - 2400

11250 - 2800 |

1Number of patients |

with organisms | 1in the original | Isputum culture:- |

1 111P.aeruginosa |

18 I 11

H.influenzae |1

2 1 11S. aureus |

* same individual NS = not significant

TABLE V

TICARCILLIN/CARBENICILLIN TRIAL: RESPIRATORY FUNCTION DATA

Mean values of each group _+ standard deviationn = 8

Treatmentgroup

Respiratory function vari able

DAY 0 DAY 10

Improvement | Difference with treatment between the

(Wilcoxon signed rank

two treatments (analysis of covariance)

Ticarc i11in

Carbenici1lin

PEER (1/min)

196*3 (+ 61*8)

247*5 (+ 66*4)

PEFR (1/min)

286.9 (+ 69*4)

314*4 (+ 92*8)

46.2% p<0.01

27.0% p<0.02NS

Ticarcillin

Carbenicillin

FEV1 mis

986-3 (+ 454)

962-5 (+ 447)

FVC mis

FEV] mis

1381.3 (+ 578)

1318.8 (+ 602)

FVC mis

40.1% p<0.001

37.0% p<0.001NS

T icarcillin

Carbenicillin

1881-3 (+ 723)

1675 (+ 794)

2530 (+ 892)

2368.8 (+1079)

34.5% p<0.001

41.4% p<0.001NS

FIGURE 6

PEFR BEFORE AND AFTER TREATMENT WITH TICARCILLIN AND CARBENICILLIN

c) Ant ibiot ic l e v e l s

Mean a n t i b i o t i c l e v e l s in scrum and sputum in f iv e

pa t ien t s on each treatment arc shown in Table VI.

Mean serum l e v e l s a t 15 minutes were s im i la r tor the

two drugs, but the 1 hour level of t i c a r c i l l i n (319*5

jicj/ml) was higher than for carbenici 11 in (199 j ig/ml).

Serum l e v e l s of p e n i c i l l i n s j u s t before the next dose

were very low. Sputum l e v e l s of both a n t i b i o t i c s were

low, the highest mean values obtained were 4*72 jug/ml

for t i c a r c i l l i n (range 2*38-4*72) and 6*6 pg/ml for

c a r b e n c i l l i n , (range 2*06 - 6* 6) . This represents 1*5

and 2*4% r e s p e c t i v e l y of the highest serum values .

Gentamicin was not detected in any sputum sample, the

lower l i m i t of the b i o a s s a y being l'Qjig/ml.

d ) Res i s ta nt organisms

Transient r e s i s t a n c e of pseudomonas to gentamicin

was seen in one p a t i e n t , but the cul ture had become

s e n s i t i v e at one month fol low-up. Organisms from one

pat ie nt in each group developed r e s i s t a n c e to

c a r b e n i c i l l i n , which p e r s i s t e d for up to 4 months

(Table X).

e > Follow up (Table XI)

Six p a t ie n t s in the c a r b e n i c i l l i n group have been

readmitted for fur ther treatment at an average of 5*7

months between t reatments : the remaining two p a t ie n t s

are well 16 months l a t e r . Five pa t ie n t s from the

t i c a r c i l l i n group have subsequently been readmitted,

at an average of 4*7 months between admissions: the

MEAN SERUM AND SPUTUM CONCENTRATIONS OF CARBENIC ILLIN (n=5),

AND TICARC1LLIN (n-5) AFTER AN INTRAVENOUS DOSE OF FIVE GRAMS

TABLE VI

Ant ibi ot ic and sample

after end of i.v. infusion

1 hr | 2 hr | 4 hr | 6 hr 1 1 1 1 1 1

TICARCILLIN

(fig/ml) 11- serum 7.7 | 296.6 | 319.6 |

- sputum 3.1 | 3.9 | 4.7 |1

3.2 | 1

2.4 3.6

CARBENICELLIN

(jig/ml) 11

- serum 2.1 | 278.6 | 199.0 |111

- sputum 2.1 | 6.6 | 3.6 I1

4.5 | 1 1

6.1 4.3

r e m a i n i n g t h r o e a r e w e l l 1 7 t o 2 6 m o n t h s l a t e r . T h e

i n t e r v a l b e t w e e n h o s p i t a l a d m i s s i o n s i s n o t

s t a t i s t i c a l l y d i f f e r e n t ( W i . l c o x o n ' s r a n k s u m t e s t ) .

£ ) Tox ic i ty

There were no s i d e - e f f e c t s of treatment and no

evidence of t o x i c i t y in th i s small t r i a l .

2. AZLOCILLIN VERSUS CARBENICILLIN

a ) Respi ratory function

There was no s i g n i f i c a n t d i f fer enc e between the

two groups of p a t ie n t s on entry to the t r i a l (Table

VII ) . Both a n t i b i o t i c regimens produced a s i g n i f i c a n t

improvement in PEFR, FEV^ and FVC, between 41% and 46%

of ba se l ine va lues , and there was no d i f fer enc e

between the two treatments (Table VIII ) . Figure 7

shows the improvement in PEFR for both treatments.

b ) Bacteriology

P, aeruginosa was i s o l a t e d from the i n i t i a l sputum

cul ture in a l l p a t i e n t s but was not present in the

cu l ture at day 10 in two pat ien t s in the a z l o c i l l i n

group and one in the c a r b e n i c i l l i n group. Follow-up

cu l tu res at one month showed that the organism was

present in a l l ca se s .

TABLE VII

AZLOCILLIN v CARBENIC ILL IN TRIAL : PATIENT DATA ON ENTRY TO THE TRIAL

1I AZLOCILLIN

1| CARBEN1CILLIN

1| Statistical

I + gentamicin 1 1

1 + gentamicin 1 1

I comparison I (Wilcoxon's I rank sum test

Number of patients11 10 1

I1 10 1

Sex11 6 M 4 F 1

1| 9 M I F1

Age range (years)11 18-25 1

1I 17-29 1

11 MS 1

Range of PEFR (1/min)11 125-490 1

1I 165-410 1

II NS 1

Range of FEV^ (mis)1| 660-2200 1

1I 400-2200 1

11 NS 1

Range of FVC (mis)11 700-3750 1

1I 950-2600 1

11 NSI

Number of patients with organisms in initial sputum culture:-

P. aeruginosa ( 10 1 1011

S. aureus 1 5 1 4 1H. influenzae I 6

1 2 1 1

TABLE VIII

AZLOCILLIN v CARBENICILLIN TRIAL: RESPIRATORY FUNCTION DATA

Mean values of each group +_ standard deviation n = 10

T reatment Group

Respiratory function variable

DAY 0 DAY 10

Imp rovement on treatment (Wilcoxon

signed rank test)

Di f ference between two

treatment s (analysis of covariance)

Azloci11 in

Carbenici11 in

PEFR 1/min

243-0 (+ 108)

266*6 (+ 77.7)

PEFR 1/min

343*5 (+ 127)

376*2 (+ 94.4)

41*4% p<0.001

41*1% p<0.001

Azloci1lin

Carbenici1lin

FEVt mis

1206-0 (+ 419)

1116*0 (+ 514)

FEV^ mis

1760-0 (+ 862)

1619*0 (+ 871)

45*9% p<0.001

45*1% p<0.001

Azloci11 in

Carbenici1lin

FVC mis

2045*0 (+ 824)

1810-0 (+ 503)

FVC mis

2897*5 (+ 1228)

2588-0 (+ 929)

41*7% pCO.OOl

43*0% p<0.02

FIGURE 7

PEFR BEFORE AND AFTER TREATMENT WITH AZLOCILLIN AND CARBENICILLIN

c ) A n t i b i o t i c 1 c. v e l s

•Moan a n t i b i o t i c l e v e l s i n s e r u m a n d s p u t u m a r e

s h o w n i n T a b l e I X . A z l o c i l l i n w a s d e t e c t e d i n t h e

s p u t u m i n l i v e o u t o f : t e n p a t i e n t s , r a n g e 2*5 - 13*8 fiq/m l , a n d c a r b e n i c i l l i n w a s d e t e c t e d i n t h e

s p u t u m o f : a l l s e v e n p a t i e n t s t e s t e d , r a n g e 2*9 -

1 2 * 2 j u g / m l . T h e s e s p u t u m l e v e l s a r e e x t r e m e l y l o w .

A z l o c i l l i n g a v e s o m e w h a t h i g h e r s e r u m l e v e l s t h a n

c a r b e n i c i l l i n d e s p i t e t h e l o n g e r d o s e i n t e r v a l .

G e n t a m i c i n w a s o n l y d e t e c t a b l e i n t h e s p u t u m o f t o u r

i n d i v i d u a l s , t h e m e a n v a l u e a t 1 h o u r b e i n g 1 * 2 3

ji g/ml.d ) R e s i s t a n t O r g a n i s m s

I n t h e c a r b e n i c i l l i n t r e a t e d g r o u p , c u l t u r e s f r o m

t w o p a t i e n t s d e v e l o p e d t r a n s i e n t r e s i s t a n c e t o

c a r b e n i c i l l i n d u r i n g t h e c o u r s e o f t r e a t m e n t , w h i c h

w a s l o s t b y o n e m o n t h . C u l t u r e s f r o m t h r e e p a t i e n t s

d e v e l o p e d r e s i s t a n c e t o e i t h e r c a r b e n i c i l l i n ( 2 ) o r

a z l o c i l l i n ( 1 ) w h i c h w a s s t i l l p r e s e n t a t o n e m o n t h

b u t l o s t b y t h r e e m o n t h s . I n t h e a z l o c i l l i n - t r e a t e d

g r o u p o n e c u l t u r e b e c a m e r e s i s t a n t t o b o t h a z l o c i l l i n

a n d c a r b e n i c i l l i n f o r t h r e e m o n t h s a n d t w o c u l t u r e s

w e r e t r a n s i e n t l y r e s i s t a n t t o p e n c i l l i n s , ( o n e t o e a c h

o f t h e d r u g s u s e d , T a b l e X ) .

e ) F o l l o w - u p

S i n c e t h e t r i a l w a s c o m p l e t e d , t e n p a t i e n t s h a v e

b e e n r e a d m i t t e d , f i v e i n t h e a z l o c i l l i n g r o u p a t a n

a v e r a g e o f f o u r m o n t h s a f t e r t r e a t m e n t , a n d f i v e i n

t h e c a r b e n i c i l l i n g r o u p w i t h a n a v e r a g e o f 3*6 m o n t h s

MEAN SERUM AND SPUTUM CONCENTRATIONS OF CARBENICILLIN (n=7)

AND AZLOCILLIN (n=10), AFTER AN INTRAVENOUS DOSE OF FIVE GRAMS

TABLE IX

Ant ibiot ic

AZLOCILLIN(pg/ml)

Sample

Sputum

Time after end of i.v. infusion

0 iy_ 1 hr 2 hrs

44.3 628.6

2.2 - 3.2 3.1

CARBENICILLIN(jig/ml)

Sputum

12.8 308.9

4.6 - 8.9 7.5

ANTIBIOTIC RESISTANCE OF P.AERUGINOSA AFTER INTRAVENOUS CHEMOTHERAPY

TABLE X

Az loc i11 in v Carbenicil1 in j Ticarcillin v Carbenici11 in

Treatment group (n=10)1| Treatment group (n=8) |

a)Azloci11 in I b )Carbenici11 in1| a)Ticarcillin | b)Carbenici1lin

Cultures | resistant | on disc | test to:- |

1Gentamicin |

. . . 1Carbenici11 in |1

ITicarcillin |

1Azlocillin |

* Resistant for four months. All other cultures reverted to full sensitivity by three months.

- = Mot applicable

s i n c e t h e p r e v i o u s c o u r s e o f t r e a t m e n t . T h i s

d i f f e r e n c e i s n o t s t a t i s t i c a l l y s i g n i f i c a n t . One

p a t i e n t in t h e a z l o c i l l i n g r o u p a i d n o t i m p r o v e

s u f f i c i e n t l y t o b e d i s c h a r g e d f rom h o s p i t a l and l a t e r

d i e d . The r e m a i n i n g n i n e p a t i e n t s a r e s t i l l w e l l , 5 -

12 m o n t h s a f t e r t r e a t m e n t ( T a b l e X I ) .

f ) T o x i c i t y

B o t h r e g i m e n s w e r e w e l l t o l e r a t e d w i t h no e v i d e n c e

o f t o x i c i t y .

g ) S u b j e c t i v e a s s e s s m e n t

A l l p a t i e n t s c o n s i d e r e d t h e m s e l v e s t o h a v e

i m p r o v e d o n t r e a t m e n t ( T a b l e X I I ) a n d t h e i m p r o v e m e n t

i n v i s u a l a n a l o g u e s c a l e s c o r e s w a s s i g n i f i c a n t i n

b o t h g r o u p s . T h e r e w a s n o d i f f e r e n c e b e t w e e n t h e m e a n

v a l u e s o f b a s e l i n e s c o r e o r f i n a l s c o r e o n t h e

a n a l o g u e s c a l e i n t h e t w o t r e a t m e n t g r o u p s . F i g u r e 8

s h o w s t h e v i s u a l a n a l o g u e s c a l e s c o r e s o n d a y 0 a n d

d a y 10 o f t r e a t m e n t f o r e a c h p a t i e n t , w i t h t h e g r o u p

m e a n v a l u e s .

F . DISCUSSION

T i c a r c i l l i n i s a s e m i - s y n t h e t i c p e n i c i l l i n f i r s t

d e v e l o p e d i n 1 9 6 4 b u t o n l y r e c e n t l y i n t r o d u c e d t o t h e

U K . I t h a s a v e r y s i m i l a r s t r u c t u r e t o c a r b e n i c i l l i n

w i t h a t h e o p h e n e r i n g i n p l a c e o f t h e b e n z e n e r i n g o f

c a r b e n i c i l l i n . I t s a n t i b a c t e r i a l a c t i v i t y i s s i m i l a r

t o i t s p a r e n t c o m p o u n d a n d i t i s a c t i v e a g a i n s t

G r a m - n e g a t i v e r o d s , a n a e r o b e s a n d n o n - p - l a c t a m a s e

TIME BEFORE THE NEXT ADMISSION FOR INTRAVENOUS CHEMOTHERAPY FOR PATIENTS WHO COMPLETED THE TRIALS

TABLE XI

T reatment group

Mean time | before readmissionl

(months) |

Time since trial treatment for patient s

not yet readmitted (months)

Carbenici11 in n = 8

1 1 1 6 | 1 1 1 1

5*7(range 2 - 9 ) |

T icarc i 1 lin n = 8

1 1 1 5 | 1 1 1 1

4*7 | (range 1*5-7) |

17 - 26

Carbenici1lin n - 10

1 1 1 1 ! 5 | 1 1 1 1

3*6 | (range 2 - 6 ) |

5 - 1 2

Azloci1lin n = 10

1 1 1 5 | 1 1 1 1 1 !

4*0(range 3 - 6 ) |

table xii

AZLOCILLIN v CARBENICILLIN: MEAN VISUAL ANALOGUE SCALE SCORES

n = 10

111 Ant ibiot ic Treatment1

Time of 1 Assessment | Azloc il1 in

11 Carbenic i11 in

| Difference | between

| treatments

Mean + SD11111

Mean + SD | (analysis of | covariance)

Day 1 |1I

4*28 + 1*19

3*54 + 1*78 I NS

Day 10 I 1 8-67 + 1•42

11I1 8*23 + 1*08 I NS1

Significance of | improvement |

(Paired t test) |I1

p<0*00111111

p<0*001

. 1 0 4

FIGURE 8

AZLOCILLIN/CARBENICILLIN TRIALVisual analogue scale scores

on day 0 and day 10 of treatment------ Mean value of group

o r o r l u c i n g s t a n h v l o c o c c i . I n v i t r o i t i s m o r e a c t i v e

a g a i n s t P . a e r u g i n o s a t b a n c a r b e n i c i L l i n . I n a s t u d y

b y S u t h e r l a n d e t a l ( 1 9 7 0 ) 1 3 1 , 3 0 g Q r s t r a i n s t e s t e d

w o r e i n h i b i t e d b y 2 5 m i c r o g r a m s / m l o i t i c a r e i l l i n ,

w h i l e o n l y 202 w e r e i n h i b i t e d b y t h a t l e v e l o f

c a r b e n i c i 1 t i n . O t h e r s t u d i e s h a v e s h o w n t h a t

t i c a r c i l l i n i s 1 * 4 - 3 * 9 t i m e s m o r e p o t e n t i n v i t r o

t h a n c a r b e n i c i l l i n 1 2 2 , 1 3 2 , 1 3 3 a B o t h d r u g s a r e

a v a i l a b l e a s a d i s o d i u m s a l t c o n t a i n i n g 5 * 3 5

m m o l s / g r a m of : s o d i u m . T i c a r c i l l i n e x h i b i t s i n v i t r o

s y n e r g y w i t h a m i n o g l y c o s i d e s , a s d o e s

carbenicillinl^l'-^^.A z l o c i l l i n i s a r e l a t i v e l y n e w a c e y l - u r e i d o

p e n i c i l l i n , w i t h a h i g h i n v i t r o a c t i v i t y a g a i n s t

G r a m - n e g a t i v e o r g a n i s m s , i n c l u d i n g p s e u d o m o n a s ,

p r o t e u s , c o l i l o r m s , k l e b s i e l l a a n d h a e m o p h i l u s . I t i s

a l s o a c t i v e a g a i n s t s o m e G r a m - p o s i t i v e o r g a n i s m s , b u t

i s r e l a t i v e l y s e n s i t i v e t o t h e a c t i o n o f p • l a c t a m a s e s .

S t r a i n s o f p s e u d o m o n a s s e n s i t i v e t o c a r b e n i c i l l i n a n d

g e n t a m i c i n a r e s e n s i t i v e t o c o n c e n t r a t i o n s o f

a z l o c i l l i n o f e i g h t u g / m l o r l e s s . T h e m a j o r i t y o f

s t r a i n s r e s i s t a n t t o c a r b e n i c i l l i n a n d g e n t a m i c i n a r e

s u s c e p t i b l e t o 3 2 u g / m l o f a z l o c i l l i n . T h u s a g a i n s t

p s e u d o m o n a s , a z l o c i l l i n h a s a p p r o x i m a t e l y e i g h t t i m e s

m o r e i n v i t r o a c t i v i t y t h a n c a r b e n i c i l l i n - ^ 4 .

C o m p a r a t i v e k i l l i n g c u r v e s o f c a r b e n i c i l l i n a n d

a z l o c i l l i n s h o w t h a t t h e l a t t e r i s b a c t e r i c i d a l a t

a p p r o x i m a t e l y e i g h t f o l d l o w e r s e r u m l e v e l s - ^ 5 .

T h e i m p r o v e m e n t i n r e s p i r a t o r y f u n c t i o n t e s t s i n

n i l q 1: 0110s o i ' p a t i e n t s i n t h e s e s t u d i o n w a s b o t h

s t a t i s t i . c n i l y s i q n i. f i c a n t n n d s u b s t a n t i n i , v n r y i n g

f r o m 2 7 t o 4 G b of : b a s e l i n e v a l u e s . T r i o t h r o e

v a r i a b l e s m e a s u r e d , P E F R , FEV-j_ a n d F V C , a l l i mo r o v e d

t o a s i m i l a r d e g r e e . I n t h e a z l o c i l l i n s t u d y p a t i e n t s

o n b o t h t r e a t m e n t s r e c o r d e d s i g n i f i c a n t s u b j e c t i v e

i m p r o v e m e n t i n t h e i r w e l l b e i n g . I t i s c o n c l u d e d t h a t

a n t i b i o t i c t h e r a p y a g a i n s t P . a e r u g i n o s a d o e s h a v e

c l i n i c a l b e n e f i t t o C F p a t i e n t s c h r o n i c a l l y i n f e c t e d

w i t h t h i s o r g a n i s m . A s s u g g e s t e d i n p r e v i o u s s t u d i e s ,

e r a d i c a t i o n o f p s e u d o m o n a s f r o m t h e s p u t u m w a s

u n c o m m o n . I t w a s s e e n i n o n l y t h r e e p a t i e n t s , a l l i n

t h e s e c o n d s t u d y , a n d w a s t r a n s i e n t .

T h e i m p r o v e m e n t i n r e s p i r a t o r y f u n c t i o n d a t a m a y

b e d u e t o h o s p i t a l c a r e a s a w h o l e a n d n o t a r e s u l t o f

c h e m o t h e r a p y ; t h i s i s d i s c u s s e d b e l o w . P a t i e n t

r e c o r d i n g s o n a v i s u a l a n a l o g u e s c a l e a r e s u b j e c t i v e

a n d t h e r e w i l l b e a s t r o n g b i a s t o w a r d s a p o s i t i v e

r e s u l t , a s p a t i e n t s f e e l a n o b l i g a t i o n t o t h e i r

d o c t o r s t o c o m m e n t f a v o u r a b l y o n t h e i r t r e a t m e n t . T h e

i m p r o v e m e n t i n s c o r e s i s n o t s u r p r i s i n g a n d m e a n s

l i t t l e e x c e p t s o m e s a t i s f a c t i o n f o r t h e p h y s i c i a n t h a t

h i s p a t i e n t s f e e l b e t t e r . T h e l a c k o f d i f f e r e n c e

b e t w e e n r e g i m e n s i n t h i s r e s p e c t a s w e l l a s t h e

o b j e c t i v e r e s p i r a t o r y f u n c t i o n d a t a s t r e n g t h e n s t h e

c a s e f o r t h e r e g i m e n s t o b e c o n s i d e r e d e q u a l l y

e f f e c t i v e . T h e s i m i l a r i t y o f t h e m e a n t i m e b e f o r e t h e

n e x t a d m i s s i o n f o r a l l t r e a t m e n t g r o u p s a l s o a d d s

w e i g h t t o t h i s c o n c l u s i o n . H o w e v e r t h i s i s q u i t e

u n c o n t r o l l e d d a t a ; p a t i e n t s w e r e a d m i t t e d t o t h e

t r i a l s o n s p e c i f i c c r i t e r i a , b u t t h e s e c r i t e r i a w e r e

n o l . a p p l i e d t o l a t e r a d m i s s i o n s .

I n b o t h t h e p r e s e n t s t u d i e s t h e r e v ; a s n o o b v i o u s

a d v a n t a g e o f o n e a n t i b i o t i c o v e r a n o t h e r , d e s p i t e t h e

• w i d e l y d i f f e r i n g M i e s in_ v i t r o . S i m i l a r r e s u l t s w e r e

s e e n c o m p a r i n g g e n t a m i c i n a n d t o b r a m y c i n i n c f - ^ 5 , 1 2 7 ^

I n v i e w o f t h e l a c k o f c l i n i c a l a d v a n t a g e o f t h e m o r e

o o w e r f u l i n v i t r o a n t i b i o t i c , i t i s p o s s i b l e t h a t t h e

b e n e f i t s e e n w a s n o t d u e t o t h e a n t i b i o t i c s t h e m s e l v e s

b u t d u e t o h o s p i t a l c a r e i n c l u d i n g h y d r a t i o n ,

n u t r i t i o n , r e s t a n d i n t e n s i v e p h y s i o t h e r a p y . A n y

i n t e r c u r r e n t v i r a l i n f e c t i o n w o u l d a l s o r e s o l v e w i t h

t i m e . B e a u d r y e t a l - ^ 3 r a n d o m i s e d . C F p a t i e n t s t o

t r e a t m e n t w i t h c l o x a c i l l i n o r a l l y , ( a c t i v e a g a i n s t

S . a u r e u s b u t i n a c t i v e a g a i n s t P . a e r u g i n o s a ) , o r

g e n t a m i c i n a n d c a r b e n i c i 1 1 i n , a n d c o n c l u d e d t h a t

p a t i e n t s w h o w e r e h o s p i t a l i s e d a n d t r e a t e d i n t e n s i v e l y

i m p r o v e , a n d t h a t s p e c i f i c a n t i - p s e u d o m o n a l

a n t i b i o t i c s a r e l e s s i m p o r t a n t . H o w e v e r , t h e p a t i e n t s

i n B e a u d r y ' s s t u d y w e r e s e v e r e l y i l l , 1 6 o u t o f 2 2

h a v i n g b a s e l i n e r e s p i r a t o r y f u n c t i o n o f l e s s t h a n 2 0 %

p r e d i c t e d , w i t h o n l y 10 p a t i e n t s s h o w i n g a n y r e s p o n s e

t o t r e a t m e n t . H y a t t e t a l ^ - 3 6 c o n d u c t e d a s i m i l a r

c o n t r o l l e d t r i a l o f o x a c i l l i n p l u s s i s o m i c i n a n d

c a r b e n i c i l l i n , v e r s u s o x a c i l l i n a l o n e , i n 2 4

e x a c e r b a t i o n s o f C F p u l m o n a r y d i s e a s e i n p a t i e n t s

c h r o n i c a l l y i n f e c t e d w i t h P . a e r u g i n o s a . T h r e e o u t o f

1 5 p a t i e n t s i n t h e ' t r e a t m e n t ' g r o u p ( 2 0 % ) h a d t o b e

r e m o v e d f r o m t h e s t u d y o r r e q u i r e d f u r t h e r t r e a t m e n t ,

s i . q n i f i c o n t l y l e s s t h a n t h e s e v e n o u t o f n i n e ( 7 8 % ) i n

t h e o x n c i 1 L i n - o n l y g r o u p . T h e d i f f e r e n c e i n

i m o r o v e m e n t i n F E Vg w a s a i s o s i q n i f i c a n t . T h e y

c o n c l u d e d t h a t a n t i p s e u d o m o n a l c h e m o t h e r a p y w a s o f

b e n e f i t i n t h e s e c i r c u m s t a n c e s . T h i s t r i a l w a s

c o n d u c t e d d o u b l e - b l i n d s o t h a t t h e a u t h o r s c o u l d u s e

' f a i l u r e ' t o r e s p o n d a s a n e n d - p o i n t , o t h e r w i s e t h e

r e s u l t s w o u l d h a v e b e e n s i m i l a r t o t h a t o f B e a u d r y e t

a l ^ - 2 3 , a s n o n e o f t h e c l i n i c a l v a r i a b l e s e x c e p t F E V p

s h o w e d a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e t w o

g r o u p s . T h e q u e s t i o n s r a i s e d b y t h e s e t r i a l s c a n o n l y

d e f i n i t e l y b e a n s w e r e d w i t h a n p l a c e b o - c o n t r o l l e d

t r i a l , b u t m a n y g r o u p s h a v e c o n s i d e r e d t h i s u n e t h i c a l .

T h e u n i q u e d o u b l e - b l i n d , p l a c e b o - c o n t r o l l e d t r i a l

p e r f o r m e d b y W i e n t z e n a n d c o - a u t h o r s i s t h e r e f o r e

w o r t h y o f d e t a i l e d d i s c u s s i o n ^ 7 . o v e r a p e r i o d o f 1 4

m o n t h s t h e y a l l o c a t e d C F p a t i e n t s a d m i t t e d t o h o s p i t a l

t o t r e a t m e n t w i t h e i t h e r t o b r a m y c i n o r p l a c e b o .

P a t i e n t s w i t h ' l i f e - t h r e a t e n i n g ' i l l n e s s w e r e e x c l u d e d

b u t i t i s n o t s t a t e d h o w t h i s c a t e g o r y w a s d e f i n e d .

C l i n i c a l r e s p o n s e w a s d e s c r i b e d l o o s e l y i n t e r m s o f

t h e p a t i e n t s a c t i v i t y , m o o d , a p p e t i t e a n d s p u t u m

p r o d u c t i o n . S p e c i f i c m e a s u r e m e n t s o f P E E R , F E V q a n d

F V C w e r e m a d e a t f i v e d a y i n t e r v a l s w i t h a c h a n g e o f

1 5 % o r g r e a t e r b e i n g c o n s i d e r e d a p o s i t i v e r e s u l t .

T h e t w o g r o u p s o f 1 2 p a t i e n t s w e r e c o m p a r a b l e a t e n t r y

t o t h e s t u d y , a l t h o u g h t h e p l a c e b o g r o u p w e r e y o u n g e r ,

h a d m o r e s e v e r e l y a b n o r m a l c h e s t x - r a y s , a g r e a t e r

1. 0 9

n u m b e r o f p r e v i o u s h o s p i t a l a d m i s s i o n : . ' , a n d h a d a

h i g h e r ; i n i t i a l s p u t u m c o l o n y c o u n t o f P . a e r u q i n o s a .

( A l l b u t t h r o e p a t i e n t s w e r e c o l o n i s e d b y

p s e u d o m o n a s ) . T h e s e f a c t o r s w e r e n o t s t a t i s t i c a l l y

s i g n i f i c a n t l y d i f f e r e n t w h e n c o m p a r e d w i t h t o b r a m y c i n

t r e a t e d g r o u p h o w e v e r , a n d t h e r e s u l t s i n t h e t w o

g r o u p s w e r e v e r y d i f f e r e n t . E l e v e n p a t i e n t s i n e a c h

g r o u p c o m p l e t e d t h e s t u d y a n d a l l e l e v e n i n t h e

t o b r a m y c i n g r o u p ' r e s p o n d e d 1 c l i n i c a l l y , w i t h

r e s p i r a t o r y f u n c t i o n t e s t s i m p r o v i n g s i g n i f i c a n t l y i n

t w o t h i r d s o f t h o s e a b l e t o c o - o p e r a t e . O n l y s e v e n o f

t h e p l a c e b o g r o u p ' r e s p o n d e d ' , n o n e h a d s i g n i f i c a n t

i m p r o v e m e n t i n r e s p i r a t o r y f u n c t i o n a n d t w o d i e d ,

d e s p i t e t h e i n i t i a l e x c l u s i o n o f s e v e r e l y i l l

p a t i e n t s . T w o o f t h e p l a c e b o ' f a i l u r e s ' w e r e

s u c c e s s f u l l y t r e a t e d w i t h a n t i b i o t i c s s u b s e q u e n t l y .

T h e g r e a t e r o v e r a l l c l i n i c a l ' r e s p o n s e ' , a n d r e d u c t i o n

i n s p u t u m P . a e r u g i n o s a c o l o n y c o u n t i n t h e t o b r a m y c i n

g r o u p d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e , b u t t h e

i m p r o v e m e n t i n r e s p i r a t o r y f u n c t i o n t e s t s d i d r e a c h

s i g n i f i c a n c e d e s p i t e t h e s m a l l n u m b e r o f p a t i e n t s .

T h e a u t h o r s c o n c l u d e d t h a t u n t i l a b i g g e r t r i a l

p r o v i d e d b e t t e r d a t a , i t w a s a d v i s a b l e t o t r e a t

r e s p i r a t o r y e x a c e r b a t i o n s o f C F w i t h a n t i b i o t i c s .

T h e r e s u l t s o f t h e p r e s e n t s t u d y s u g g e s t t h a t a l l

o f t h e r e g i m e n s u s e d , a r e e f f e c t i v e . T h e o n e w i t h t h e

l o w e s t i n c i d e n c e o f s i d e e f f e c t s a n d l o w e s t c o s t

s h o u l d t h e r e f o r e b e u s e d . P e r n a g s t h e t i m e h a s c o m e

f o r a l a r g e p l a c e b o c o n t r o l l e d t r i a l t o a s s e s s t h e

a n dv a l u e of : h o s p i t a l c a r e a l o n e c o m p a r e d w i t h t h i n

a n t i p s e u d o i n o n a 1 c h o m o t h e r a p y .

a i d e e f f e c t s r e p o r t e d w i t h c a r b o n i c i l . l i n a n d i t s

d e r i v a t i v e t i c a r c i l l i n i n c l u d e a l l e r g i c r e a c t i o n s ,

h a e m o r r h a g i c c y s t i t i s , e o s i n o n h i l i a , d e f e c t i v e

p l a t e l e t a g g r e g a t i o n a n d a l t e r a t i o n s i n l i v e r f u n c t i o n

t e s t s ^ ^ “ 1 2 0 e T h e i n f u s i o n i s v e r y i r r i t a n t t o v e i n s

a n d t h i s m a y b e a p r o b l e m i n y o u n g e r p a t i e n t s . B e r g a n

a n d M i c h a l s e n ^ - ® ® r e p o r t e d t h a t f i v e o u t o f n i n e C F

p a t i e n t s t r e a t e d w i t h a z l o c i l l i n d e v e l o p e d a r a s h ,

w h i l e M i l l e r e t n o t i c e d t h a t s e v e n o u t o f 2 0

p a t i e n t s o n a z l o c i l l i n d e v e l o p e d s y s t e m i c s i d e e f f e c t s

w i t h r a s h e s , p y r e x i a , a r t h r a l g i a a n d l y n v p h a d e n o p a t b y .

I n t h e p r e s e n t s t u d i e s n o s i d e e f f e c t s w e r e o b s e r v e d

a n d a l l p e n i c i l l i n s w e r e w e l l t o l e r a t e d . A z l o c i l l i n

h a s t h e p r a c t i c a l a d v a n t a g e s o f a n e i g h t h o u r d o s e

i n t e r v a l a n d c a n b e g i v e n a t t h e s a m e o c c a s i o n a s t h e

a m i n o g l y c o s i d e b y t h e n u r s i n g s t a f f . T h e l o w s o d i u m

c o n t e n t ( 2 * 1 7 m i l l i e q u i v a l e n t s / g r a m ) , a p p r o x i m a t e l y

h a l f t h a t o f t i c a r c i l l i n o r c a r b e n i c i l l i n , m a y b e

u s e f u l i n p a t i e n t s w i t h c a r d i o r e s p i r a t o r y f a i l u r e a n d

f l u i d r e t e n t i o n .

T h e s e r u m l e v e l s o f a z l o c i l l i n , t i c a r c i l l i n a n d

c a r b e n i c i l l i n a c h i e v e d a f t e r i n t r a v e n o u s i n f u s i o n s o f

f i v e g r a m s o f e a c h d r u g w e r e h i g h , a n d c o m p a r a b l e w i t h

v a l u e s f r o m o t h e r s t u d i e s o n C F p a t i e n t s , a n d o t h e r

i n d i v i d u a l s ^ - ^ ® r122 , 1 3 8 - 1 4 0 ̂ j n a r e v i e w o f a n t i b i o t i c

t h e r a p y i n C F , S m i t h - ^ l r e v i e w e d p h a r m a c o k i n e t i c

s t u d i e s o f p e n c i l l i n s a n d a m i n o g l y c o s i d e s i n C F

c h i l d r e n . Peak si^rum c or i c e n t r a i i i o n : . ; w e r e lowc-r f o r C f’

c h i l d r e n khan c o n c r o l c , o r i m a r . i l y rlnc t o i n c r e a s e d

r e n a l , e x c r e t i o n . A s t u d y on a z l o c i 1 1 in in CP c h i l d r e n

'3howod a s i m i l a r r e r u . l t ^ ' - ® . In t h e p r e s e n t s t u d i e s in

a d u l t s , s e r u m l e v e l s o f a l l t h r o e p e n i c i l l i n s w e r e

c o m p a r a b l e w i t h t h o s e f o u n d in n o r m a l a d u l t s .

T h e k i n e t i c s o f a n t i b i o t i c d i f f u s i o n f r o m t h e

c i r c u l a t i o n i n t o t h e r e s p i r a t o r y t r a c t c a n n o t b e

s t u d i e d w i t h o u t i n v a s i v e p r o c e d u r e s , s o m o s t h u m a n

s t u d i e s u s e s p u t u m l e v e l s o f d r u g s a s a n a p p r o x i m a t i o n

t o l e v e l s i n b r o n c h i a l s e c r e t i o n s . T h e p e n e t r a t i o n o f

a n a n t i b i o t i c i n t o s p u t u m d e p e n d s o n m a n y f a c t o r s : t h e

p h y s i c o c h e m i c a l p r o p e r t i e s o f t h e d r u g , i t s b i n d i n g t o

s e r u m a n d t i s s u e p r o t e i n s , i n f l a m m a t i o n o f t h e m u c o s a l

e p i t h e l i u m i n t h e c a s e o f p e n i c i l l i n s , a n d t h e n a t u r e

o f s p u t u m i t s e l f . S a g g e r s a n d L a w s o n s h o w e d t h a t

t h e p e n e t r a t i o n o f a n t i b i o t i c s i n t o s p u t u m i n v i v o w a s

d e p e n d e n t o n m o l e c u l a r s i z e a n d w a s n o t d i r e c t l y

r e l a t e d t o s e r u m l e v e l s . I t h a s b e e n s h o w n t h a t

l i p o p h i l i c m o l e c u l e s p e n e t r a t e s p u t u m m o r e e f f i c i e n t l y

t h a n l y p o p h o b . i c o n e s * “̂ . O n c e p r e s e n t i n s p u t u m ,

l e v e l s o f a n t i b i o t i c s m e a s u r e d i n b i o a s s a y m a y b e a n

u n d e r e s t i m a t e f o r s e v e r a l r e a s o n s . P u r u l e n t s p u t u m

r e d u c e s t h e i n v i t r o a c t i v i t y o f g e n t a m i c i n a n d

p o l y m i x i n s b u t n o t c a r b e n i c i l l i n ^ ^ ^ . I n v i v o a

p e n i c i l l i n - i n a c t i v a t i n g a g e n t d e r i v e d f r o m h o s t

l e u c o t y e s m a y b e p r e s e n t i n p u s ^ ^ . A m i n o g l y c o s i d e s

a r e a f f e c t e d b y t h e p H , c a l c i u m a n d DMA c o n t e n t o f

s p u t u m - ^ l , a n d b a c t e r i a l b e t a - l a c t a m a s e s m a y

i n a c t i v a t a s a n s i t i u e p e n i c i 1 1 i n s e n d c e p h a l o s o o r i n :•>.

T h e hi e a . s u r o d p e n e t r a t i o n o f p o n e i l l . i n s i n t o C F s p u t u m

i n t h o s e s t u d i e s w a s v e r y l e v ; . T a b l e X I I I s h o w s t h e

s p u t u m . l e v e l s a c h i e v e d , t o g e t h e r w i t h t h e r e s u l t s L: r o m

o t h e r a u t h o r s . I n a l l c a s e s s p u t u m l e v e l s w e r e l e s s

t h a n 1 0 % o f p e a t s e r u m l e v e l s , u s u a l l y i n t h e r a n g e

0 - 4 % . F a i l u r e t o e r a d i c a t e p s e u d o m o n a s f r o m t h e

s p u t u m o f C F p a t i e n t s h a s b e e n a t t r i b u t e d t o p o o r

s p u t u m p e n e t r a t i o n o f a n t i b i o t i c , a n d t h i s m a y i n d e e d

b e a f a c t o r . H o w e v e r , i t s e e m s u n l i k e l y t h a t

s i g n i f i c a n t l y h i g h e r s p u t u m l e v e l s c a n b e a c h i e v e d

' whe n t h e s e d r u g s a r e g i v e n b y t h e i n t r a v e n o u s r o u t e ,

a s e v e n l a r g e i n t r a v e n o u s d o s e s p r o d u c e l o w s p u t u m

l e v e l s . H i g h e r a n t i b i o t i c l e v e l s i n s p u t u m m i g h t b e

o b t a i n e d b y u s i n g a d i f f e r e n t r o u t e o f a d m i n i s t r a t i o n ,

t h a t i s b y i n h a l a t i o n d i r e c t l y i n t o t h e a i r w a y s .

S e v e r a l o f t h e s t u d i e s r e v i e w e d i n T a b l e I I I

c o m m e n t o n t h e d e v e l o p m e n t o f a n t i b i o t i c - r e s i s t a n t

s t r a i n s o f p s e u d o m o n a s d u r i n g t h e c o u r s e o f

c h e m o t h e r a p y . H u a n g e t a l ^ Q a l s o c o m m e n t e d o n t h e

i n c r e a s e i n r e s i s t a n t k l e b s i e l l a i s o l a t e d . I n t h e

p r e s e n t s t u d i e s t h e i n c i d e n c e o f r e s i s t a n c e w a s l o w ,

a n d m o s t c u l t u r e s r e v e r t e d t o s e n s i t i v i t y b y t h e t h r e e

m o n t h f o l l o w - u p .

TABLE XIII

SPUTUM LEVELS OF PENICILLINS AFTER INTRAVENOUS ADMINISTRATION INCYSTIC FIBROSIS PATIENTS

Study 111

IV antibiotic and dose

Mean sputum level (fig/ml)

Sputum/ [leak serum level

Huang 501I Carbonic i11 in Only detectable in

e t a 1, 1971 11

90 mg/kg/dose 2 of 8 patients

Marks 120 11 Carbenic i11 in 78 3*6

e t al, 1971 11

60 tng/kg/dose

Parry 122 11 Ticarci1 lin 4*9 2*7

et al, 1977 11

50 mg/kg/dose

Malmborg11 Azloci 11 in 1-5 - 38 0*4 to 9*5

et ai, 1981 11

200 mg/kg

Levy 147 11 Azloci 11 in 0*5 - 4 0*4 to 2*8

e t a 1, 1981 1 40 mg/kg Only detectable in11

34 of 111 samples.

Present11 Carbenici1lin

studies 1 5 gm/dose1 a) v | ticarcillin trial 2* 1 - 6*6 0*7 to 2*4

1 b) vi azlocillin trial 4*6 - 8*9 1*5 to 2*9iI Ticarcillin 5 gm/dose 1 2*4 - 4*7 0*8 to 1-51| Azlocillin 5 gin/doseI1

2 * 2 - 3 * 2 0 * 4 t o 0 * 5

SUMMARY

C y s t i c f i b r o s l s p a t i e n t s w i t h P . a e r u a i n o s a

i. n f e c t i. o n w 11 o e >: n e r i e n c e d an a c u t !a e x a c e r b a t i o n o

r e s o i r a t o r y s ym p t oms wer e t r e a t e d f o r t e n d a y s wi

i n t r a v e n o u s g e n t a m i c i n , and one o f t h r e e p e n i c i l l i n s ,

a z . l o c L l l . i n , t i c a r c i l l i n o r c a r b e n i c i l l i n . S i g n i f i c a n t

i m p r o v e m e n t s i n mean PEFR, F.EV| and FVC w e r e s e e n w i t h

a l l r e g i m e n s . T h e r e V7as l i t t l e d i f f e r e n c e b e t w e e n t h e

t r e a t m e n t s , d e s p i t e t h e w i d e l y d i f f e r i n g M I C ' s f o r

P . a e r u g i n o s a o f t h e t h r e e p e n i c i l l i n s . Se r um l e v e l s

o f a n t i b i o t i c s w e r e a d e q u a t e , b u t s p u t u m p e n e t r a t i o n

was p o o r . T h e r e wa s no t o x i c i t y o r s i d e - e f f e c t f r o m

t r e a t m e n t . A n t i b i o t i c r e s i s t a n t s t r a i n s o f

p s e u d o m o n a s e m e r g e d i n a s m a l l number o f p a t i e n t s b u t

a l l c u l t u r e s r e v e r t e d t o f u l l s e n s i t i v i t y by t h r e e

m o n t h s .

Mo d i f f e r e n c e i n t h e d e g r e e o f i m p r o v e m e n t i n

r e s p i r a t o r y f u n c t i o n c o u l d be d e m o n s t r a t e d b e t w e e n t h e

d i f f e r e n t t r e a t m e n t r e g i m e n s . F a c t o r s s u c h a s

t o x i c i t y , s o d i u m l e v e l , c o s t and c o n v e n i e n c e o f

a d m i n i s t r a t i o n c a n t h e r e f o r e be c o n s i d e r e d i n

s e l e c t i n g t h e a n t i b i o t i c r e g i m e n f o r f u t u r e u s e .

Chapter 5

TREATMENT OF PSEUDOMONAS AERUGINOSA INFECTION IN CHRONICPULMONARY DISEASE IN CYSTIC FIBROSIS BY

AEROSOL ANTIBIOTICS

T h e i: a t i o n a t o f o r L h e i: o 1 1 ow i n q w o r k w a a 13 a a < ̂ d o n

t h o t w o p r e m i s e r , d i s c u s s e d i n C h a p t e r 4 . P i 1: s t: 1. y f

t h a t p s e u d o m o n a s c o l o n i s a t i o n of : t h e r e s p i r a t o r y t r a c t

i s c a u s a l l y r e l a t e d t o t h e d e t e r i o r a t i o n i n t h e

r e s p i r a t o r y f u n c t i o n o f C F p a t i e n t s , a n d s e c o n d l y ,

t h a t a l t h o u g h s h o r t i n t r a v e n o u s c o u r s e s of :

c h e m o t h e r a p y d o n o t e r a d i c a t e t h e p a t h o g e n , t h e y d o

h a v e a t h e r a p e u t i c e f f e c t . C h e m o t h e r a p y a g a i n s t

P . a e r u g i n o s a g i v e n o v e r a l o n g e r p e r i o d o f t i m e m i g h t

t h e r e f o r e b e b e n e f i c i a l t o C F p a t i e n t s . T h e r e a r e n o

o r a l a n t i b i o t i c s e f f e c t i v e a g a i n s t P . a e r u g i n o s a a p a r t

f r o m t h e c a r b e n i c i l l i n e s t e r , c a r f e c i l l i n , w h i c h i s

h y d r o l y s e d i n t h e u r i n e t o r e l e a s e c a r b e n i c i 1.1 i n a n d

m a y b e u s e d t o t r e a t u r i n a r y t r a c t i n f e c t i o n s o n l y .

T o s t u d y t h e e f f e c t o f l o n g - t e r m o u t - p a t i e n t u s e o f

c h e m o t h e r a p y a g a i n s t p s e u d o m o n a s , i t w a s t h e r e f o r e

d e c i d e d t o g i v e t h e a n t i b i o t i c s b y a e r o s o l .

B . LITERATURE REVIEW

P r o m i n g i v e n b y a e r o s o l t o g u i n e a p i g s w a s

d e m o n s t r a t e d t o p r e v e n t t h e d e v e l o p m e n t o f

t u b e r c u l o s i s i n e x p e r i m e n t a l a n i m a l s b y B a r a c h e t

ail48. P e n i c i l l i n a e r o s o l s w e r e u s e d i n t h e l a t e

1 9 4 0 s o n p a t i e n t s w i t h C F a n d a s h o r t t e r m b e n e f i t w a s

n o t e d / ' - ® • A e r o s o l s o f n e o m y c i n , g e n t a m i c i n ,

c a r b e n i c i l l i n , c o l i s t i n a n d o t h e r a n t i b i o t i c s h a v e

b o o n u s e d i n CP on an ' a d h o c ' b a s i n , but : L h e r o hnvr>

boon no i": o r ,• a a 1. t: r L a 1. s r e p o r t : ed on t hc ua o o i: a a r oa o 1

a n l: i. blot" L o s in t h i s c o n d i t i o n .

I n h a l e d a n t i b i o t i c s h a v e be en a i. von t o a nun b e t o f

q n o u n s o l p a t i e n t s w i t h a t r a c h e o s t o m y t o c o m b a t

G r a m - n e q a t i v e i n f e c t i o n . b e e p e r e t a l - 1- 4 ^ s h o w e d t h a t

a n a e r o s o l o f p o l y m i x i n p r e v e n t e d c o l o n i s a t i o n o f t h e

r e s p i r a t o r y t r e e b y p s e u d o m o n a s i n p o l i o m y e l i t i s

p a t i e n t s w i t h a t r a c h e o s t o m y . K l a t e r s k y a n d

c o - w o r k e r s t u d i e d t h e e n d o t r a c h e a l a d m i n i s t r a t i o n

o f g e n t a m i c i n t o u n c o n s c i o u s p a t i e n t s w i t h a

t r a c h e o s t o m y a d m i t t e d t o a n e u r o s u r g i c a l u n i t .

G e n t a m i c i n s i g n i f i c a n t l y r e d u c e d t h e i n c i d e n c e o f

c o l o n i s a t i o n o f t r a c h e a l s e c r e t i o n s b y G r a m - n e g a t i v e

r o d s , a n d s i g n i f i c a n t l y d e c r e a s e d t h e f r e q u e n c y o f

b a c t e r i o l o g i c a l l y p r o v e n e p i s o d e s o f r e s p i r a t o r y t r a c t

i n f e c t i o n w h e n c o m p a r e d w i t h p l a c e b o i n s t i l l a t i o n o f

s a l i n e . T h e e m e r g e n c e o f g e n t a m i c i n r e s i s t a n t s t r a i n s

o f p s e u d o m o n a s a n d k l e b s i e l l a , p r o m p t e d t h e s a m e g r o u p

t o c o m p a r e g e n t a m i c i n w i t h a n e n d o t r a c h e a l r e g i m e n o f

k a n a m y c i n a n d p o l y m i x i n B , a n d t h i s w a s a l s o f o u n d t o

b e e f f e c t i v e i n r e d u c i n g c o l o n i s a t i o n o f t h e t r a c h e a l

s e c r e t i o n s b y G r a m - n e g a t i v e b a c t e r i a ^ i . G e n t a m i c i n

a n d c o l i s t i n w e r e g i v e n b y d i r e c t e n d o t r a c h e a l

i n s t i l l a t i o n t o p a t i e n t s w i t h c h r o n i c p u r u l e n t

b r o n c h i a l i n f e c t i o n b y P i n e s e t a l l 5 2 V/ i t h s o m e

s u c c e s s .

I n c o n t r a s t t o d i r e c t e n d o t r a c h e a l i n s t i l l a t i o n ,

p u b l i s h e d s t u d i e s o n t h e a d m i n i s t r a t i o n o f a n t i b i o t i c s

b y n o b u I. L/; : :) r a r c - s c a r c e . R o s e e t a 1., .1.f! 7 0 1 5 3 t r e a t e d

20 a d u l t m a l e s w i t h b r o n c h o n u . l m o n a r v d i s e a s e a n d

G r a n - n e q a t i v e o r g a n i s m s i n t h e s p u t u m w i t h c o l i s t i n

t h r e e t i m e s d a i l y b y a e r o s o l . T h e s e p a t i e n t s v/o r c

m a i n l y s u f l i e r i n g f r o m c h r o n i c , o b s t r u c t i v e p u l m o n a r y

d i s e a s e o r b r o n c h i e c t a s i s , b u t t h r e e h a d a s p i r a t i o n

p n e u m o n i a s f o l l o w i n g n e u r o s u r g e r y . T h e y r e p o r t e d a

s i g n i f i c a n t r e d u c t i o n o f s p u t u m b a c t e r i a l c o l o n y

c o u n t s . i n 1 8 o u t o f 2 0 p a t i e n t s , b u t i n s e v e n p a t i e n t

t h e r e w a s r e c u r r e n c e o f p r e t r e a t m e n t o r g a n i s m s

i m m e d i a t e l y a f t e r t r e a t m e n t w a s s t o p p e d , a n d i n t h r e e

p a t i e n t s s t r a i n s r e s i s t a n t t o c o l i s t i n e m e r g e d d u r i n g

t h e c o u r s e o f t r e a t m e n t .

C . A I M S

T h e a i m s o f t h i s s t u d y w e r e : - t o e v a l u a t e t h e

l o n g - t e r m u s e o f a n t i p s e u d o m o n a l a n t i b i o t i c s g i v e n b y

t h e a e r o s o l r o u t e t o C F p a t i e n t s c h r o n i c a l l y i n f e c t e d

w i t h P . a e r u g i n o s a ; t o m e a s u r e a n t i b i o t i c l e v e l s i n

s e r u m a n d s p u t u m a f t e r a d m i n i s t r a t i o n b y a e r o s o l ; t o

a s s e s s t h e d e v e l o p m e n t o f a n t i b i o t i c - r e s i s t a n c e i n

P . a e r u g i n o s a d u r i n g t h e u s e o f a e r o s o l a n t i b i o t i c s ,

a n d t o i n v e s t i g a t e t h e d i s t r i b u t i o n o f p a r t i c l e s i z e

i n t h e a e r o s o l s g e n e r a t e d .

13. M E T H O D S A N D R E S U L T S

l. CLINICAL TRIAL OK AEROSOL GENTAMICIN AND

CAR PENICILLIN

n ) P a t i e n t s a n d M e t h o d s

T w e n t y p a t i e n t s , 1 1 m a l e s a n i l 9 f e m a l e s , a q e r a n g e

1 5 t o 4 2 y e a r s , e n t e r e d t h e s t u d y . A l l h a d C F b y t h e

c r i t e r i a d e f i n e d i n C h a p t e r 3 , a n d a l l h a d

P . a e r u g i n o s a i s o l a t e d f r o m t h e s p u t u m f o r a t l e a s t s i r

m o n t h s b e f o r e t h e t r i a l . Mo p a t i e n t h a d r e c e i v e d

i n t r a v e n o u s c h e m o t h e r a p y d u r i n g t h e f o r t n i g h t b e f o r e

t h e t r i a l .

T h e d e s i g n o f t h e t r i a l w a s a d o u b l e - b l i n d ,

p l a c e b o - c o n t r o l l e d , r a n d o m i s e d , c r o s s - o v e r s t u d y ,

p a t i e n t s a c t i n g a s t h e i r o w n c o n t r o l . E a c h t r e a t m e n t

p e r i o d w a s f o r s i x m o n t h s a n d p a t i e n t s w e r e r a n d o m l y

a l l o c a t e d t o a c t i v e o r p l a c e b o t r e a t m e n t s f i r s t .

A c t i v e t r e a t m e n t c o n s i s t e d o f g e n t a m i c i n 8 0 m g , a n d

c a r b e n i c i l l i n 1 g r a m , b o t h g i v e n t w i c e d a i l y . T h e

d o s e o f c a r b e n i c i l l i n c h o s e n w a s t h e m a x i m u m a m o u n t

t h a t c o u l d b e d i s s o l v e d i n 4 m i s o f w a t e r f o r

n e b u l i s a t i o n . T h e d o s e o f g e n t a m i c i n c h o s e n w a s t h e

s t a n d a r d i n t r a v e n o u s d o s e . T r e a t m e n t w a s g i v e n t w i c e

d a i l y ; m o r e f r e q u e n t l y w o u l d h a v e b e e n t o o g r e a t a n

i m p o s i t i o n o n t h e p a t i e n t ' s d a i l y l i f e . T w o p l a c e b o s

m a t c h i n g t h e a c t i v e p r e p a r a t i o n s i n a p p e a r a n c e w e r e

u s e d a n d c a r e w a s t a k e n t o a d j u s t t h e t a s t e a n d

t o n i c i t y o f t h e c a r b e n i c i l l i n p l a c e b o t o a p p r o x i m a t e l y

t h a t o f t h e a c t i v e d r u g .

T hc) nebs1. ier aopa ra fc ua con s i a i;ed of an a i r

co:nnr(]",soi;, 'Bird1 m icr onc’nu 1 l x r-j c ( 3M UK Ltd), Ambu

valve, and length of corrupatcd tubing so that: exhaled

antibiotics could be di a charged out ol: a window.

F i g u r e 9 s h o w s t h e n e b u l i z e r a p p a r a t u s . T h r e e a i r

c o m p r e s s o r s w e r e u s e d , M e d i x M i n o r I I , a n d WOBL p u m p

( M e d i x L i m i t e d ) a n d t h e C F l A ( A e r o s o l P r o d u c t s

L i m i t e d ) . P a t i e n t s w e r e i n s t r u c t e d f i r s t t o p e r f o r m

t h e i r p h y s i o t h e r a p y a s u s u a l , a n d t h e n t o i n h a l e e a c h

a n t i b i o t i c s e p a r a t e l y , w a s h i n g t h e n e b u l i s e r a f t e r

u s e . G e n t a m i c i n o r i t s p l a c e b o w a s i n h a l e d f i r s t ,

f o l l o w e d b y c a r b e n i c i l l i n o r i t s p l a c e b o . T h e

e q u i p m e n t w a s s t e r i l i s e d i n M i l t o n s o l u t i o n o n c e a

w e e k . U s e o f t h e A m b u - v a l v e a n d t u b i n g r e d u c e d t h e

r i s k o f s e n s i t i s a t i o n o f o t h e r m e m b e r s o f t h e f a m i l y

t o p e n i c i l l i n s , a n d r e d u c e d t h e s m e l l i n t h e p a t i e n t ' s

r o o m .

An o u t - p a t i e n t a s s e s s m e n t w a s m a d e a t t h e

b e g i n n i n g o f t h e t r i a l a n d m o n t h l y t h r o u g h o u t i t s

c o u r s e . A t e a c h v i s i t a h i s t o r y w a s t a k e n a n d t h e

p a t i e n t e x a m i n e d . P e a k e x p i r a t o r y f l o w r a t e ( P E F R )

w a s r e c o r d e d a s t h e b e s t o f t h r e e a t t e m p t s o n a W r i g h t

P e a k F l o w M e t e r . F o r c e d e x p i r a t o r y v o l u m e i n o n e

s e c o n d ( F E V g ) a n d f o r c e d v i t a l c a p a c i t y ( F V C ) w e r e

r e c o r d e d o n a b e l l o w s s p i r o m e t e r ( V i t a l o g r a p h ) .

S p u t u m s a m p l e s o b t a i n e d a t e a c h a t t e n d a n c e w e r e s e n t

f o r c u l t u r e , a n d o r g a n i s m s i s o l a t e d w e r e t e s t e d f o r

a n t i b i o t i c s e n s i t i v i t y b y t h e S t o k e s c o m p a r a t i v e d i s c

m e t h o d 1 2 9 .

F I G U R E 9

I f , d u r i n g t h e c o u r s e o f t h e s t u d y , t h e p a t i e n t

w a s a d m i t t e d t o h o s p i t a l w i t h a n a c u t e e x a c e r b a t i o n o f

s y m p t o m s , a 1 0 d a y c o u r s e o f i n t r a v e n o u s g e n t a m i c i n

a n d c a r b o n i c ! l l i n w a s g i v e n , a n d t h e t r i a l a e r o s o l

c o n t i n u e d b l i n d l y . A t t h e e n d o f t h e t r i a l b o t h

d o c t o r a n d p a t i e n t g a v e s u b j e c t i v e a s s e s s m e n t s o f t h e

p r e f e r r e d t r e a t m e n t b e f o r e t h e c o d e w a s b r o k e n . An

a u d i o g r a m was p e r f o r m e d o n e a c h p a t i e n t u s i n g a

A m p l i v o x a u d i o m e t e r ( A m p l i v o x , M i d d l e s e x ) .

b ) R e s u l t s

T h r e e p a t i e n t s w e r e w i t h d r a w n ; o n e d u r i n g t h e

f i r s t m o n t h w i t h a r a s h ( o n p l a c e b o l i m b ) , o n e w i t h

r e s p i r a t o r y f a i l u r e r e q u i r i n g m a x i m u m t h e r a p y ( o n

p l a c e b o l i m b ) , a n d o n e w i t h b i l a t e r a l p n e u m o t h o r a c e s

t h a t r e q u i r e d s u r g i c a l i n t e r v e n t i o n ( a c t i v e l i m b ) . ' I n

t h e c a s e o f a f o u r t h p a t i e n t , t h e c o d e w a s b r o k e n a t

m o n t h 1 0 b e c a u s e h e r c o n d i t i o n h a d d r a m a t i c a l l y

i m p r o v e d d u r i n g t h e f i r s t 6 m o n t h s o f t r e a t m e n t a n d

d e t e r i o r a t e d r a p i d l y d u r i n g t h e s e c o n d 4 m o n t h s w i t h

t w o h o s p i t a l a d m i s s i o n s . T h i s p a t i e n t r e f u s e d t o

c o n t i n u e w i t h t h e s e c o n d p r e p a r a t i o n ( F i g u r e 1 0 ) . T h e

s u b j e c t i v e a s s e s s m e n t s a t t h e e n d o f t h e t r i a l a r e

s h o w n i n T a b l e X I V . N o p a t i e n t p r e f e r r e d t h e p l a c e b o .

T a b l e X V s h o w s t h e r e s p i r a t o r y f u n c t i o n r e s u l t s . M e a n

P E F R , FEV]_ a n d F V C w e r e a l l s i g n i f i c a n t l y h i g h e r

d u r i n g t h e a c t i v e t r e a t m e n t p e r i o d . S o m e p a t i e n t s h a d

a g r e a t e r r e s p o n s e t h a n o t h e r s ; i n 1 5 o u t o f 1 7

p a t i e n t s m e a n F E V i w a s h i g h e r d u r i n g t h e a c t i v e

t r e a t m e n t p e r i o d t h a n d u r i n g t h e p l a c e b o , a n d t h i s

FIGURE 10

CF PATIENT USING AEROSOL ANTIBIOTIC

AEROSOL ANTIBIOTIC TRIAL: PREFERENCE OF PATIENT AND DOCTORFOR ACTIVE TREATMENT OR PLACEBO

n = 17

I PATIENT | DOCTOR

ACTIVE 1 14 | 12

PLACEBO 1 o | 1

UNCERTAIN 1 3 | 4

TABLE XV

AEROSOL ANTIBIOTIC TRIAL: RESPIRATORY FUNCTION DATA

ACTIVE' PLACEBO+ Statistical analysis Student's unpaired T test

Mean FEVx ml 1566 1300 P < 0-001

Mean FVC ml 2656 2314 p <0*02

Mean PEFR 1/min 323 283 p < 0-001

* 17 patients had monthly recordings on six occasions- total 102 assessments.

+ 16 patients had monthly recordings on six occasions and one had monthly recordings on four occasions- total 100 assessments.

r e a c h e d . s t a t i s t i c a l s i g n i f i c a n c e i n n i n e c a s e s . M e a n

F V C w a s h i g h e r i n t h e a c t i v e t r e a t m e n t p e r i o d i n .16

p a t i e n t s , s i g n i f : i c a n t l y s o i n s e v e n , a n d t h e s e r e s u l t s

a r e s h o w n i n F i g u r e 1 1 . F o u r t e e n p a t i e n t s h a d a

h i g h e r m e a n P E F R o n a c t i v e a e r o s o l , a n d t h i s r e a c h e d

s i g n i f i c a n c e i n s e v e n . N o p a t i e n t h a d a s i g n i f i c a n t

i m p r o v e m e n t i n F E V ^ , F V C o r P E F R w h i l e r e c e i v i n g

p l a c e b o .

T h r e e p a t i e n t s w e r e a d m i t t e d t o h o s p i t a l f o r

i n t r a v e n o u s c h e m o t h e r a p y w h i l e r e c e i v i n g a n t i b i o t i c

a e r o s o l a n d s e v e n w h i l e r e c e i v i n g p l a c e b o . A u d i o g r a m s

w e r e n o r m a l i n a l l c a s e s . P . a e r u g i n o s a w a s i s o l a t e d

f r o m a l l s p u t u m s a m p l e s f r o m a l l p a t i e n t s t h r o u g h o u t

t h e s t u d y . I n s a m p l e s f r o m o n e i n d i v i d u a l t h e r e w e r e

g e n t a m i c i n - r e s i s t a n t o r g a n i s m s b e f o r e , d u r i n g a n d

a f t e r t h e s t u d y . S h e w a s i n c l u d e d i n t h e t r i a l a s h e r

t w i n s i s t e r t o w h o m s h e w a s v e r y a t t a c h e d w a s a l r e a d y

p a r t i c i p a t i n g i n t h e s t u d y , a n d t h i s p a t i e n t i n f a c t

h a d a g o o d r e s p o n s e d e s p i t e t h e r e s i s t a n c e o f h e r

o r g a n i s m . ( T h e s e t w i n s w e r e a l s o u n d e r t h e c a r e o f D r

A G C h a p p e l l o f B r i g e n d H o s p i t a l ) N o p a t i e n t d e v e l o p e d

g e n t a m i c i n r e s i s t a n t o r g a n i s m s . P s e u d o m o n a s r e s i s t a n t

t o c a r b e n i c i l l i n w a s i s o l a t e d t r a n s i e n t l y i n t w o

p a t i e n t s r e c e i v i n g a c t i v e t r e a t m e n t , t w o d u r i n g t h e

p l a c e b o p e r i o d a n d i n t w o d u r i n g b o t h p e r i o d s . A l l

c u l t u r e s r e v e r t e d t o f u l l s e n s i t i v i t y d u r i n g t h e

c o u r s e o f t h e t r i a l .

F O R C E D V I T A L C A P A C I T Y R E C O R D I N G S O F S E V E N C F P A T I E N T ' S

U S I N G A E R O S O L A N T I B I O T I C S AND P L A C E B O

F I G U R E 1 1

— M ean±SD • Active o Placebo ▼ 0 .0 1 > P > 0 .0 0 1 * P < 0 .001

F i g u r e 1 2 s h o w s t h e r e s p i r a t o r y f u n c t i o n

r e c o r d i n g s t o r o n e p a t i e n t w h o w a s a d m i t t e d t o

h o s p i t a l t w i c e f o r i n t r a v e n o u s c h e m o t h e r a p y s h o r t l y

b e f o r e t h e t r i a l . H e d r e w a c t i v e t r e a t m e n t f i r s t w i t h

a g o o d c l i n i c a l r e s p o n s e , b u t d e t e r i o r a t e d d u r i n g t h e

p l a c e b o p e r i o d a n d w a s r e a d m i t t e d . A t t h e e n d o f t h e

s t u d y h e w a s r e - s t a r t e d o n a c t i v e t r e a t m e n t a n d h i s

c o n d i t i o n i m p r o v e d ,

c ) D i s c u s s i o n

A e r o s o l t r e a t m e n t w i t h c a r b e n i c i l l i n a n d

g e n t a m i c i n o v e r a s i x m o n t h p e r i o d p r o d u c e d

s i g n i f i c a n t o b j e c t i v e i m p r o v e m e n t i n l u n g f u n c t i o n i n

a g r o u p o f 1 7 C F p a t i e n t s w i t h m o d e r a t e l y s e v e r e

d i s e a s e . T h e r a n d o m i s e d , c r o s s - o v e r d e s i g n m i n i m i s e s

v a r i a t i o n i n r e s p i r a t o r y f u n c t i o n d u e t o t h e s e a s o n ,

o r t o n a t u r a l p r o g r e s s i o n o f t h e d i s e a s e i t s e l f .

S o m e b e n e f i t w a s o b t a i n e d t h r o u g h o u t t h e t r i a l b y

t h e d i s c i p l i n e o f t h e r e g i m e n w h i c h c o m p e l l e d p a t i e n t s

t o p a y a t t e n t i o n t o r e g u l a r p h y s i o t h e r a p y . T h e u s e o f

a n i s o s m o t i c p l a c e b o w i t h a n i d e n t i c a l s o d i u m c o n t e n t

e l i m i n a t e s t h e e f f e c t o f h u m i d i f i c a t i o n a n d t h e

r e d u c t i o n o f v i s c o s i t y o f b r o n c h i a l s e c r e t i o n s b y

s o d i u m c h l o r i d e .

T h e c o n t e n t o f a n a e r o s o l m a y b e i r r i t a n t t o t h e

l u n g , D a l l y a n d c o - w o r k e r s l 5 4 d e m o n s t r a t e d a f a l l i n

F E V g o f 2 0 % , o r m o r e i n s e v e n o u t o f 1 8 a s t h m a t i c

s u b j e c t s g i v e n 8 0 m g o f g e n t a m i c i n i n 2 m l s o f a v e h i c l e

b y n e b u l i z e r . T h e s e r e a c t i o n s a l s o o c c u r r e d w i t h

i n h a l a t i o n o f t h e v e h i c l e a l o n e a n d w e r e n o t p r e v e n t e d

FIGURE 12

CF PATIENT USING AEROSOL ANTIBIOTIC

. 1979 1980▼ = iv. Carbenicillin & Gentamicin

b y s o d i u m c r o m o g l y c a t e , s u g g e s t i n g a d i r e c t i r r i t a n t

e l : I f e c t o l r t h e v e h i c l e i n s u s c e p t i b l e p a t i e n t s . N o

s u c h a c u t e r e a c t i o n s o c c u r r e d i n t h e p r e s e n t s t u d y

d e s p i t e t h e f a c t t h a t m a n y C F p a t i e n t s h a v e b r o n c h i a l

l a b i l i t y ^ . S u c h r e a c t i o n s w o u l d h a v e t e n d e d t o

r e d u c e t h e i m p r o v e m e n t i n P E F R a n d FEV-^ s e e n o n t h e

a c t i v e t r e a t m e n t . T h e g e n t a m i c i n s u l p h a t e u s e d i n t h e

p r e s e n t s t u d y w a s p r o v i d e d b y R o u s s e l L a b o r a t o r i e s

w h i l e t h a t u s e d b y D a l l y e t a l w a s f r o m t h e S o b e r i n g

C o r p o r a t i o n ; t h e v e h i c l e s u s e d b y t h e s e c o m p a n i e s m a y

b e d i f f e r e n t . A l s o , i n t h e s t u d y b y D a l l y e t a l

p a t i e n t s h a d s t o p p e d a l l b r o n c h o d i l a t o r s f o r 7 2 h o u r s ,

w h e r e a s a n y p a t i e n t i n t h e p r e s e n t s t u d y w h o h a d

r e v e r s i b l e a i r w a y s o b s t r u c t i o n w o u l d h a v e b e e n u s i n g

b r o n c h o d i l a t o r s r e g u l a r l y .

T h e r e w a s a t r e n d t o w a r d s a r e d u c e d f r e q u e n c y o f

h o s p i t a l a d m i s s i o n s o n t h e a c t i v e t r e a t m e n t , b u t

n u m b e r s w e r e s m a l l . T h e s u b j e c t i v e d a t a o n p a t i e n t

a n d d o c t o r p r e f e r e n c e f o r a c t i v e o r p l a c e b o t r e a t m e n t

s h o w s a c l e a r r e j e c t i o n o f t h e p l a c e b o . H o w e v e r

a l t h o u g h t h e s t u d y w a s p e r f o r m e d b l i n d , b o t h p a t i e n t

a n d d o c t o r w e r e w e l l a w a r e o f t h e p a t i e n t ' s

p e r f o r m a n c e s o n s p i r o m e t e r a n d p e a k f l o w m e t e r , w h i c h

i n t r o d u c e s a s t r o n g b i a s f o r c o r r e l a t i o n o f p r e f e r e n c e

w i t h t h e p e r i o d o f h i g h e r r e s p i r a t o r y f u n c t i o n .

S i x p a t i e n t s d e v e l o p e d c a r b e n i c i l l i n - r e s i s t a n t

o r g a n i s m s d u r i n g t h e s t u d y , i n t w o c a s e s t h i s o c c u r r e d

d u r i n g t h e a c t i v e p e r i o d a n d o n e c a s e i n t h e p l a c e b o

p e r i o d f o l l o w i n g a c t i v e t r e a t m e n t . T h i s r e s i s t a n c e

m a y b e a t t r i b u t a b l e t o a e r o s o l u s e , b u t i n t h e o t h e r

Ccises r e s i s t a n c e o c c u r r e d d u r i n g t h e i n i t i a l p l a c e b o

p e r i o d .

T h i s s t u d y i s t h e f i r s t c o n t r o l l e d t r i a l oLr

a e r o s o l c h e m o t h e r a p y f o r p s e u d o m o n a s i n f e c t i o n i n C F ,

a n d t h e o n l y s t u d y o f a e r o s o l t h e r a p y t o s h o w

o b j e c t i v e i m p r o v e m e n t i n t h e p a t i e n t ' s c o n d i t i o n . T h e

o n l y o t h e r p l a c e b o - c o n t r o l l e d t r i a l o f a e r o s o l

c h e m o t h e r a p y i n C F c o n c e r n e d t h e r o l e o f S . a u r e u s

i n f e c t i o n i n t h e p a t h o g e n e s i s o f t h e d i s e a s e . T h e

H o s p i t a l f o r S i c k C h i l d r e n i n T o r o n t o h a s f o r s o m e

y e a r s u s e d a e r o s o l c e p h a l o r i d i n e , c o m b i n e d w i t h o r a l

c l o x a c i l l i n , a s c o n t i n u o u s a n t i s t a p h y l o c o c c a l

p r o p h y l a x i s . I n a t w o y e a r p r o s p e c t i v e s t u d y t h i s w a s

c o m p a r e d w i t h o r a l c l o x a c i l l i n a l o n e i n 4 7 p a t i e n t s

w i t h m i l d t o m o d e r a t e d i s e a s e . T h e r e w e r e n o

s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e t w o g r o u p s i n

c l i n i c a l s c o r e , c h e s t x - r a y s c o r e , p u l m o n a r y f u n c t i o n

t e s t s o r h o s p i t a l a d m i s s i o n s , a n d t h e p r o g r e s s i o n o f

t h e d i s e a s e s e e m e d u n e f f e c t e d . C a r r i a g e r a t e s o f

S . a u r e u s a n d P . a e r u g i n o s a w e r e s i m i l a r , w h i l e t h a t o f

H . i n f l u e n z a e w a s l o w e r i n t h e g r o u p r e c e i v i n g

a e r o s o l l 5 5 # A e r o s o l c e p h a l o r i d i n e h a s t h e r e f o r e b e e n

d i s c o n t i n u e d i n T o r o n t o , w i t h t h e s u g g e s t i o n t h a t

p r o p h y l a x i s f o r s t a p h y l o c o c c a l i n f e c t i o n b y w h a t e v e r

r o u t e s h o u l d p e r h a p s b e l o o k e d a t a g a i n .

T h e d i s a d v a n t a g e s o f a e r o s o l t h e r a p y f o r t h e

p a t i e n t a r e g r e a t . I t i s t i m e - c o n s u m i n g t a k i n g a t

l e a s t 20 m i n u t e s t w i c e d a i l y , a n d r e q u i r i n g t h e

c l e a n i n g a n d m a i n t e n a n c e o f t h e e q u i p m e n t . I t i s

e x p e n s i v e i n t e r m s o f t h e c o s t o f t h e a n t i b i o t i c s o v e r

a p r o l o n g e d p e r i o d , a l t h o u g h t h e c a p i t a l c o s t o f t h e

e q u i p m e n t i s o n l y a b o u t £ 1 0 0 ( 1 9 8 2 ) . T h e m a j o r

b e n e f i t i s i n t h e r e d u c t i o n o f h o s p i t a l a d m i s s i o n s , a

s o c i a l g a i n t o t h e p a t i e n t a n d a f i n a n c i a l g a i n t o t h e

H e a l t h S e r v i c e . T h e g a i n f o r t h e p a t i e n t i n t e r m s o f

i m p r o v e d w e l l - b e i n g o v e r a l o n g p e r i o d i s i m p o s s i b l e

t o q u a n t i f y . H o w e v e r , a l l t h o s e t a k i n g p a r t i n t h e

t r i a l e l e c t e d t o c o n t i n u e t h e a c t i v e p r e p a r a t i o n a t

t h e e n d o f t h e s t u d y .

2 . L O N G - T E R M U S E O F A E R O S O L A N T I B I O T I C S I N A D U L T S WI T H

C Y S T I C F I B R O S I S

T h e r e i s a r i s k o f i n d u c i n g a n t i b i o t i c - r e s i s t a n t

o r g a n i s m s w i t h a n y l o n g - t e r m c h e m o t h e r a p y . T h i s w a s

n o t a p r o b l e m i n t h e r e l a t i v e l y s h o r t c l i n i c a l t r i a l

j u s t d e s c r i b e d , b u t i t w a s d e c i d e d t o l o o k

r e t r o s p e c t i v e l y a t a l l p a t i e n t s w h o h a d r e c e i v e d s u c h

t r e a t m e n t t o a s s e s s t h e e m e r g e n c e o f r e s i s t a n t

o r g a n i s m s a n d t h e o c c u r r e n c e o f a n y o t h e r

s i d e - e f f e c t s . T h e m a i n a d v a n t a g e o f l o n g - t e r m a e r o s o l

t h e r a p y w o u l d b e a r e d u c t i o n i n t h e f r e q u e n c y o f

h o s p i t a l a d m i s s i o n s , s o t h i s w a s a l s o a s s e s s e d

r e t r o s p e c t i v e l y ,

a ) P a t i e n t s a n d M e t h o d s

O n 3 1 . 1 2 . 8 1 . t h e r e w e r e 1 6 2 p a t i e n t s a t t e n d i n g

t h e B r o m p t o n H o s p i t a l C F U n i t . On r e v i e w o f a l l

p a t i e n t s ' n o t e s , 4 3 w e r e f o u n d t o b e r e c e i v i n g

a n t i b i o t i c s b y a e r o s o l . T h e r e w e r e 1 5 f e m a l e s a n d 2 3

m a l e s , a g e r a n g e 1 6 - 4 3 y e a r s , w h o h a d r e c e i v e d a e r o s o l

a n t i b i o t i c s f o r a n a v e r a g e o f 2 1 m o n t h s . T h e

a n t i b i o t i c r e g i m e n s a r e s h o w n i n T a b l e X V I . F r o m t h e

h o s p i t a l n o t e s t h e f o l l o w i n g i n f o r m a t i o n w a s

o b t a i n e d : -

i T h e n u m b e r o f h o s p i t a l a d m i s s i o n s p e r y e a r f o r t h e

y e a r b e f o r e t r e a t m e n t , a n d f o r t h e y e a r f o l l o w i n g

t h e s t a r t o f a e r o s o l t h e r a p y .

ii Any side effects of treatment.i i i A l l s p u t u m c u l t u r e s o b t a i n e d d u r i n g a e r o s o l

t r e a t m e n t w e r e r e v i e w e d a n d a n t i b i o t i c r e s i s t a n c e

p a t t e r n s o f p s e u d o m o n a s n o t e d . A n t i b i o t i c

s e n s i t i v i t y w a s p e r f o r m e d b y t h e S t o k e s

c o m p a r a t i v e d i s c m e t h o d - * - 2 9 ^ u s i n g d i s c s o f

1 0 u g / m l o f g e n t a m i c i n , 1 00 u g / m l c a r b e n i c i l l i n

a n d 7 5 u g / m l a z l o c i l l i n . ' T r a n s i e n t ' r e s i s t a n c e

w a s d e f i n e d a s o n e o r t w o i s o l a t e s r e p o r t e d a s

r e s i s t a n t p e r y e a r , ' i n t e r m i t t e n t ' r e s i s t a n c e a s

m o r e t h a n t w o i s o l a t e s r e p o r t e d a s r e s i s t a n t p e r

y e a r b u t n o t c o n s e c u t i v e c u l t u r e s , a n d

’ p e r s i s t e n t ' r e s i s t a n c e a s t h r e e o r m o r e

c o n s e c u t i v e c u l t u r e s .

REGIMENS USED IN CYSTIC FIBROSIS PATIENTS AT THE BROMPTON HOSPITAL

TABLE XVI

RECEIVING AEROSOL ANTIBIOTICS

Number of patients

I Antibiotic regimen receiving treatment

on 31.12.81.

| All given twice daily

1 1. Gentamicin 80mg + carbenicillin lgm 35

I 2. Gentamicin 80mg 5

1 3. Carbenicillin lgm 1

I 4. Colomycin 100,000 units 1

1 5. Neomycin 500mg 1

1 Total 43

I Average length of treatment 2 1 * 2 months

I Range 2 to 61 months

i v I n o r d e r t o c o m p a r e t h e a n t i b i o t i c r e s i s t a n c e o f

P . a e r u g i n o s a f r o m t h e s p u t u m o f C F p a t i e n t s

r e c e i v i n g a e r o s o l t r e a t m e n t , w i t h t h a t o f

o r g a n i s m s f r o m p a t i e n t s w h o w e r e n o t r e c e i v i n g

t h i s t r e a t m e n t , a f u r t h e r r e t r o s p e c t i v e s t u d y w a s

d o n e . T h e f i r s t 3 0 p a t i e n t s i n e i t h e r g r o u p t o b e

s e e n i n o u t p a t i e n t s a f t e r 1 . 7 . 8 1 . w e r e s e l e c t e d ,

a n d a l l c u l t u r e s o b t a i n e d f r o m t h e s e p a t i e n t s o v e r

s i x m o n t h s w e r e a n a l y s e d . I n t h e a e r o s o l g r o u p

t h e r e w e r e 21 m a l e s a n d n i n e f e m a l e s , a g e r a n g e

1 6 - 4 2 y e a r s , a n d i n t h e c o n t r o l g r o u p 1 6 m a l e s a n d

1 4 f e m a l e s , a g e r a n g e 1 5 - 3 4 y e a r s ,

b ) R e s u l t s

T h e a v e r a g e n u m b e r o f h o s p i t a l a d m i s s i o n s p e r

p a t i e n t i n t h e y e a r b e f o r e t r e a t m e n t w a s 1 - 8 , w h i l e

t h e a v e r a g e n u m b e r o n t r e a t m e n t w a s 1 * 0 3 ( p < 0 * 0 5

t e s t ) . S i d e e f f e c t s w e r e m i n o r . O n e p a t i e n t

c o m p l a i n e d o f a r a s h , a n d o n e o f a s e v e r e s o r e t h r o a t .

T h r e e r e f u s e d t o u s e c a r b e n i c i l l i n b e c a u s e o f t h e

t a s t e a n d s m e l l . I n p a r t i c u l a r , t h e r e w a s n o o r a l

c a n d i d i a s i s a n d n o b r o n c h o c o n s t r i c t i o n . T a b l e X V I I

s h o w s t h e a n t i b i o t i c r e s i s t a n c e o f P . a e r u g i n o s a t o

g e n t a m i c i n , c a r b e n i c i l l i n a n d a z l o c i l l i n , f r o m a l l

p a t i e n t s o n a e r o s o l a n t i b i o t i c s . T r a n s i e n t r e s i s t a n c e

w a s c o m m o n , e s p e c i a l l y w i t h t h e p e n i c i l l i n s , b u t

TABLE XVII

ANTIBIOTIC RESISTANCE OF PSEUDOMONAS AERUGINOSA

ISOLATED FROM PATIENTS RECEIVING AEROSOL ANTIBIOTICS

Cultures of P.aeruginosa from sputa of 43 patients on continuous aerosol antibiotics tested for antibiotic sensitivity by the

disc method.

Antibiotic resistance of P♦aeruginosa

Antibiotic | 1 1 1 I 1 1

1' T R AN S I E NT ' | ' I NTERMI TTENT' j ' P E R S I S T E N T'*

1 - 2 cultures ! per year | (number of | patients) |

> 2 cultures | per year | (number of 1 patients) |

>3 consecutive cultures (number of patient s)

Gentamicin | 1 1

9 1 1 1

Carbenici11 in |11

10 I11

Azlocillin !111I

16 | 1 1 1 1

* All cultures reverted to full sensitivity without cessation of aerosol treatment.

persistant resistance much less so. Resistance to azlocillin, a semisynthetic penicillin not used by aerosol was more common than to either of the two drugs used by aerosol. All cultures reverted to full sensitivity without cessation of aerosol treatment.

T h e f r e q u e n c y o f a n t i b i o t i c r e s i s t a n c e i n

P . a e r u g i n o s a i n 3 0 C F p a t i e n t s o n a e r o s o l t h e r a p y ,

c o m p a r e d t o t h e 3 0 c o n t r o l s i s s h o w n i n T a b l e X V I I I .

T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e i n a m i n o g l y c o s i d e

r e s i s t a n c e i n t h e a e r o s o l g r o u p . R e s i s t a n c e t o

p e n i c i l l i n s w a s s i g n i f i c a n t l y i n c r e a s e d i n t h e a e r o s o l

g r o u p .

c) DiscussionL o n g - t e r m u s e o f a n t i b i o t i c s i n a n y f o r m

e n c o u r a g e s t h e e m e r g e n c e o f d r u g - r e s i s t a n t o r g a n i s m s .

P r i n c e a n d N e u n o t e d t h a t t h e n u m b e r o f s t r a i n s o f

P . a e r u g i n o s a w i t h a c a r b e n i c i l l i n M I C o f g r e a t e r t h a n

1 2 5 u g / m l f r o m t h e i r C F p a t i e n t s r o s e f r o m 7% t o m o r e

t h a n 5 0 % i n 1 9 8 1 - ^ 6 . D r u g r e s i s t a n c e w a s n o t a

p r o b l e m i n t h e a e r o s o l c l i n i c a l t r i a l , b u t t h e

f o l l o w - u p d a t a s h o w s a h i g h f r e q u e n c y o f g e n t a m i c i n

a n d c a r b e n i c i l l i n r e s i s t a n t s t r a i n s o f p s e u d o m o n a s i n

a l l p a t i e n t s o n a e r o s o l s , a n d a g r e a t e r f r e q u e n c y o f

r e s i s t a n c e w h e n c o m p a r e d t o C F p a t i e n t s n o t r e c e i v i n g

a e r o s o l t r e a t m e n t . R e s i s t a n c e t o c a r b e n i c i l l i n w a s

m o r e o f a p r o b l e m t h a n t o g e n t a m i c i n , a l t h o u g h t o d a t e

c u l t u r e s t e n d e d t o r e v e r t b a c k t o s e n s i t i v i t y w i t h o u t

s t o p p i n g t h e t r e a t m e n t . T h e e m e r g e n c e o f r e s i s t a n t

o r g a n i s m s w a s a l s o n o t e d i n t h r e e o f 20 p a t i e n t s

TABLE XVIII

THE FREQUENCY OF ANTIBIOTIC RESISTANCE IN P.AERUGINOSA FROM

CYSTIC FIBROSIS PATIENTS OVER A SIX MONTH PERIOD

Antibiotic30 CF patients on aerosol antibiotics

( 1 0 0 cultures)

Gentamicin

No. of resistsnt

30 control CF patients (87 cultures

cultures

| Significance ) I (x^ test)

Tobramycin 13 5 NS

Carbenici1lin 36 3 p<0.001

Azloci 1lin 36 9 p<0 . 0 0 1

t r e a t e d w i t h c o l . i s t i n b y R o s e e t a l - ^ 3 f a n d a l s o i n

p a t i e n t s g i v e n e n d o t r a c h e a l g e n t a m i c i n ^ ^ 9 .

R e s i s t a n c e of : p s e u d o m o n a s t o c a r b e n i c . i l . l i n i s u s u a l l y

d u e t o t h e p l a s m i d - m e d i a t e d p r o d u c t i o n o f

b e t a - l a c t a m a s e s ^ S ? , a n d t h i s w o u l d a c c o u n t f o r t h e

o b s e r v e d i n c r e a s e i n r e s i s t a n c e a z l o c i l l i n , a

p e n i c i l l i n w h i c h h a d n o t b e e n g i v e n b y a e r o s o l .

P l a s m i d - m e d i a t e d c h a n g e s i n p e r m e a b i l i t y t o

p e n i c i l l i n s a n d c h a n g e s i n t h e e f f i c a c y o f

p e n i c i l l i n - b i n d i n g p r o t e i n s i n t h e c y t o p l a s m i c

m e m b r a n e m a y a l s o c a u s e r e s i s t a n c e . R e s i s t a n c e t o

a m i n o g l y c o s i d e s i s d u e t o t h e p r o d u c t i o n o f

a m i n o g l y c o s i d e m o d i f y i n g e n z y m e s o f v a r i o u s k i n d s - ^ - 5 8 ^

A n t i b i o t i c r e s i s t a n c e m u s t b e c o n t i n u a l l y m o n i t o r e d i n

p a t i e n t s o n a e r o s o l a n t i b i o t i c t h e r a p y , a n d i f

p e r s i s t e n t r e s i s t a n c e p a t t e r n s o f p s e u d o m o n a s d e v e l o p

i n a n u m b e r o f p a t i e n t s , t h e w h o l e p r i n c i p l e o f t h i s

t y p e o f t h e r a p y m u s t b e r e v i e w e d . A t p r e s e n t , i n t h e

B r o m p t o n H o s p i t a l C y s t i c F i b r o s i s U n i t , a e r o s o l

t h e r a p y i s u s e d o n l y f o r t h o s e p a t i e n t s w h o s e

c o n d i t i o n i s d e t e r i o r a t i n g a n d w h o r e q u i r e f r e q u e n t

h o s p i t a l a d m i s s i o n . T h e p a u c i t y o f o t h e r s i d e e f f e c t s

i s e n c o u r a g i n g , b u t i t m u s t b e s e t a g a i n s t t h e f a c t

t h a t s e v e r e l y i l l p a t i e n t s w i t h a t e r m i n a l i l l n e s s a r e

v e r y t o l e r a n t o f a n y t r e a t m e n t t h a t o f f e r s t h e m a

l i t t l e h o p e . T h e r e d u c t i o n i n f r e q u e n c y o f h o s p i t a l

a d m i s s i o n s i s a l s o e n c o u r a g i n g b u t t h e d a t a i s

r e t r o s p e c t i v e a n d b i a s e d . P a t i e n t s w e r e s e l e c t e d f o r

a e r o s o l t h e r a p y p a r t l y o n t h e b a s i s o f n e e d i n g

frequent admission, and other treatment such as physiotherapy may well have been intensified at the same time.

3 . A N T I B I O T I C L E V E L S I N S E R U M AMD S P U T U M A F T E R

A D M I N I S T R A T I O N B Y A E R O S O L

a ) M e t h o d s

A n t i b i o t i c l e v e l s i n s e r u m a n d s p u t u m w e r e

m e a s u r e d i n f i v e p a t i e n t s a f t e r i n h a l a t i o n o f 8 0 m g o f

g e n t a m i c i n a n d 1 g r a m o f c a r b e n i c i l l i n . A f t e r r o u t i n e

p h y s i o t h e r a p y t h e s p u t u m p r o d u c e d w a s d i s c a r d e d ,

b a s e l i n e s a m p l e s o f s e r u m a n d s p u t u m w e r e t a k e n a n d

a n t i b i o t i c s n e b u l i z e d i n t h e u s u a l w a y . S e r u m s a m p l e s

w e r e c o l l e c t e d a t 1 5 m i n u t e s a n d a t o n e a n d f o u r h o u r s

a f t e r t h e e n d o f n e b u l i z a t i o n . S p u t u m s a m p l e s w e r e

c o l l e c t e d a t 1 5 , 3 0 , 6 0 m i n u t e s a n d t h e n e v e r y t w o

h o u r s f o r a p e r i o d o f e i g h t h o u r s a f t e r t h e e n d o f

n e b u l i z a t i o n .

i G e n t a m i c i n a s s a y

S e r u m l e v e l s w e r e m e a s u r e d b y t h e E M I T m e t h o d

( e n z y m e m u l t i p l i e d i m m u n o - a s s a y t e c h n i q u e ) .

S p u t u m s a m p l e s w e r e h o m o g e n i s e d u s i n g g l a s s b e a d s

a n d a b i o a s s a y w i t h K l e b s i e l l a N C T C 1 0 8 9 6

p e r f o r m e d . T h e c o r r e l a t i o n b e t w e e n t h e s e t w o

m e t h o d s i n o u r l a b o r a t o r y i s 0 - 9 8 .

i i C a r b e n i c i l l i n a s s a y

A l a r g e p l a t e b i o a s s a y u s i n g p s e u d o m o n a s

s t r a i n " E l l s w o r t h " w a s u s e d f o r b o t h s e r u m a n d

s p u t u m s a m p l e s . C a l c i u m c h l o r i d e 25 m m o l w a s

a d d e d t o t h e p l a t e s t o i n a c t i v a t e g e n t a m i c i n .

b ) R e s u l t s

T h e r e w e r e n o d e t e c t a b l e a m o u n t s of : g e n t a m i c i n o r

c a r b e n i c i l l i n i n s e r u m i n a n y s a m p l e s f o l l o w i n g t h e

a e r o s o l a d m i n i s t r a t i o n o f a n t i b i o t i c s . T h e p e a k l e v e l

o f g e n t a m i c i n i n s p u t u m o f > 1 6 p g / m l w a s s e e n

i m m e d i a t e l y a f t e r i n h a l a t i o n , f a l l i n g t o 6*4 p g / m l a t

2 h o u r s , 3 * 4 p g / m l a t 8 h o u r s . T h e p e a k l e v e l o f

c a r b e n i c i l l i n w a s a l s o s e e n i m m e d i a t e l y a t 1 0 3 4 p g / m l

f a l l i n g r a p i d l y t o 3 3 2 p g / m l a t 1 h o u r , 7 2 p g / m l a t 2

h o u r s a n d 1 8 * 4 p g / m l a t 6 h o u r s .

T h e s e r e s u l t s a r e s h o w n i n T a b l e s X I X a n d X X , a n d

a r e c o m p a r e d w i t h s e r u m a n d s p u t u m a n t i b i o t i c l e v e l s

o b t a i n e d a f t e r i n t r a v e n o u s i n f u s i o n o f g e n t a m i c i n a n d

c a r b e n i c i l l i n i n t h e f i v e p a t i e n t s i n t h e

c a r b e n i c i l l i n / t i c a r c i l l i n t r i a l ( C h a p t e r 4 ) i n w h o m

l e v e l s w e r e m e a s u r e d .

c ) D i s c u s s i o n

I t i s p o s s i b l e t h a t t h e r e g u l a r u s e o f a e r o s o l

a n t i b i o t i c s , w h i l e f a i l i n g t o e l i m i n a t e s p u t u m

p a t h o g e n s , d o e s r e d u c e t h e b a c t e r i a l l o a d i n t h e s e

p a t i e n t s . P r i n c e a n d N e u - ^ 6 r e c e n t l y r e v i e w e d t h e i n

v i t r o a c t i v i t y o f a v a r i e t y o f a n t i b i o t i c s a g a i n s t 6 2

i s o l a t e s o f P . a e r u g i n o s a f r o m C F p a t i e n t s . T h e M I C 5 Q

( m e a n i n h i b i t o r y c o n c e n t r a t i o n r e q u i r e d t o i n h i b i t 5 0 %

o f t h e i s o l a t e s ) w a s 2 5 0 p g / m l f o r c a r b e n i c i l l i n , a n d

2 * 5 p g / m l f o r g e n t a m i c i n . T h e s p u t u m l e v e l s o f

SPUTUM LEVELS OF ANTIBIOTICS AFTER ADMINISTRATION BY INTRAVENOUS AND AEROSOL ROUTES.

TABLE XIX

1Antibiotics / route |

Peak (jig/ml)Level before next dose (pg/ml)

1 1 CARBENICILLIN I |

i iii 5 g i .v. |

|6 * 6 2 * 1

1ii 1 g aerosol |

1 0 3 4 1 8 * 4

1 11 1

GENTAMICIN | |i iii 80 mg i.v . |

1< 1 < 1

1ii 80 g aerosol |

> 16 3 * 4

* i = five patients from IV antibiotic trial,

ii = five patients from aerosol trial.

SERUM LEVELS OF ANTIBIOTICS AFTER ADMINISTRATION BY INTRAVENOUS AND AEROSOL ROUTES

n = 5 *

TABLE XX

Antibiotics/route |1

Peak(jig/ml)

1 Level before | next dose 1 (fig/ml)1

CARBENICILLIN |1

i 5 g i .v. |I

278*6 | 2 * 1

11ii 1 g aerosol |

< 1 1 < 1

GENTAMICIN |I

i 80 mg i.v. |I

7-38 1 < 1 I1

ii 80 g aerosol |1

< 1 1 < 1

* i = five patients from IV antibiotic trial,

ii = five patients from aerosol trial.

c a r b e n i c i l l i n and g e n t a m i c i n a c h i e v e d by i n t r a v e n o u s

inf: lift i o n a r e much l e s s t h a n t h o s e MIC50 v a l u e s ,

a l t h o u g h p e a k s p u t u m l e v e l s a f t e r a e r o s o l e x c e e d e d

t he m. P e a k s p u t u m l e v e l s r e p r e s e n t o n l y d e p o s i t i o n in

s e c r e t i o n s o f t h e l a r g e r a i r w a y s h o w e v e r , and t h e

s m a l l a mo u nt o f a n t i b i o t i c a e r o s o l whi ch p e n e t r a t e s

t h e p e r i p h e r y o f t h e l u n g s i s l i k e l y t o r e s u l t i n v e r y

low l e v e l s i n d e e d . L o r i a n l 5 ^ h a s c o i n e d t h e t e r m

'minimum a n t i b i o t i c c o n c e n t r a t i o n ' (MAC) f o r t h e

l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c t h a t e f f e c t s t h e

m o r p h o l o g y , b i o c h e m i s t r y , o r r a t e o f g r o w t h o f an

o r g a n i s m . G r a m - n e g a t i v e b a c i l l i e x p o s e d t o s u b- MI C

c o n c e n t r a t i o n s o f / 3 - l a c t a m a n t i b i o t i c s become

e l o n g a t e d w i t h o u t s e p a r a t i n g , t o for m l o n g f i l a m e n t o u s

c e l l s w h i c h a r e o s m o t i c a l l y u n s t a b l e and l y z e . The

r a t i o o f MIC o v e r MAC t h e r e f o r e d e f i n e s t h e r a n g e o f

w h i c h t h e o r g a n i s m i s s e n s i t i v e . F o r 16 s t r a i n s o f

p s e u d o m o n a s , t h e MIC/MAC r a t i o wa s 2 * 1 f o r

c a r b e n i c i l l i n a nd 4 * 8 f o r g e n t a m i c i n , a n d t h e r e f o r e

t h e a u t h o r s u g g e s t e d t h a t l e v e l s o f l e s s t h a n h a l f MIC

f o r c a r b e n i c i l l i n , o r o n e f i f t h MIC f o r g e n t a m i c i n

w o u l d be i n e f f e c t i v e . I t i s u n l i k e l y t h a t l e v e l s o f

g e n t a m i c i n o r c a r b e n i c i l l i n i n t h e p e r i p h e r a l a i r w a y s

w e r e r e m o t e l y c l o s e t o MAC f o r p s e u d o m o n a s i n t h e

c l i n i c a l s t u d y . To e x p l a i n t h e o b s e r v e d c l i n i c a l

b e n e f i t o f a e r o s o l a n t i b i o t i c s i n CF, i t may be t h a t

t h e d r u g s a r e a c t i n g i n an unknown ma nner w h i c h i s

u n r e l a t e d t o t h e i r c o n v e n t i o n a l b a c t e r i c i d a l and

b a c t e r i o s t a t i c e f f e c t s . T h e o r e t i c a l e x a m p l e s m i g h t be

some a c t i o n on m u c o c i l i a r y c l e a r a n c e , o r on t h e

a d h e r e n c e of; b a c t e r i a t o t h e r e s p i r a t o r y e p i t h e l i u m .

S t u d i e s o f g e n t a m i c i n and c o l i s t i n by a e r o s o l h a v e

shown t h a t s e r u m l e v e l s , f o l l o w i n g i n h a l a t i o n , a r e

u n d e t e c t a b l e ^ 3 , 1 6 0 # i n t h e p r e s e n t s t u d y n e i t h e r

g e n t a m i c i n n o r c a r b e n i c . i 11 i n w e r e d e t e c t e d i n s e r u m

a f t e r a d m i n i s t r a t i o n by a e r o s o l . T h i s may be d u e t o

s l o w a b s o r p t i o n o f a n t i b i o t i c s f rom t h e r e s p i r a t o r y

t r a c t , y e t s i g n i f i c a n t s e r u m l e v e l s a r e a c h i e v e d when

g e n t a m i c i n i s g i v e n by d i r e c t t r a c h e a l

i n s t i l l a t i o n - * - ^ r 160 . N e g l i g i b l e s e r u m l e v e l s a r e

t h e r e f o r e f u r t h e r e v i d e n c e t h a t t h e amount o f

a n t i b i o t i c r e a c h i n g t h e p e r i p h e r a l a i r w a y s i s

e x t r e m e l y s m a l l .

4 . ANALYSIS OF AEROSOL PARTICLE S I Z E DISTRIBUTION

a ) I n t r o d u c t i o n

As a p r e l i m i n a r y i n v e s t i g a t i o n o f t h e m e c h a n i s m s

by w h i c h a e r o s o l a n t i b i o t i c s e x e r t a t h e r a p e u t i c

e f f e c t , i t wa s d e c i d e d t o s t u d y t h e c h a r a c t e r i s t i c s o f

t h e a e r o s o l s p r o d u c e d . The s i t e o f d e p o s i t i o n o f an

i n h a l e d a e r o s o l w i t h i n t h e human l u n g d e p e n d s on t h e

p h y s i c a l p r o p e r t i e s o f t h e a e r o s o l p a r t i c l e s ( s i z e ,

s h a p e and d e n s i t y ) , t h e mode o f i n h a l a t i o n , and t h e

a i r w a y c a l i b r e l ^ l / i n g e n e r a l , t h e l a r g e r t h e

a e r o d y n a m i c d i a m e t e r o f a p a r t i c l e , t h e c l o s e r t o t h e

mo u t h i t w i l l b e d e p o s i t e d . P a r t i c l e s o f g r e a t e r t h a n

10 pm i n d i a m e t e r d e p o s i t i n t h e u p p e r r e s p i r a t o r y

t r a c t a n d do n o t e n t e r t h e l o w e r a i r w a y s i n

P a r t i c l e s o f 5 - 1 0 pm d e p o s i t i ns i g n i f i c a n t , a m o u n t s ,

t h e u p p e r a i r w a y s and t h e f i r s t 5 - 6 b r o n c h i a l

b i f u r c a t i o n s ; o n l y s m a l l e r p a r t i c l e s o f l e s s t h a n 5 pm

p e n e t r a t e be yo n d t h e 5 t h b r o n c h i a l d i v i s i o n . M o s t

t h e r a p e u t i c a e r o s o l s a r e h e t e r o d i s p e r s e and f i t a

l o g - n o r m a l d i s t r i b u t i o n i n whi ch t h e p l o t o f t h e

number o f p a r t i c l e s p e r g i v e n s i z e band v e r s u s t h e

l o g a r i t h m o f s i z e , p r o d u c e s a b e l l - s h a p e d n o r m a l

d i s t r i b u t i o n c u r v e . S u c h a e r o s o l s may be d e s c r i b e d by

t h e m a s s m e d i a n d i a m e t e r (MMD), t h e c o u n t m e d i a n

d i a m e t e r and t h e g e o m e t r i c s t a n d a r d o f d e v i a t i o n

( G S D ) 1 ^ 3 . g SD i s t h e r a t i o o f t h e d i a m e t e r o f t h e

p a r t i c l e t h a t i s l a r g e r t h a n 84*2% o f a l l o t h e r s , t o

t h e c o u n t m e d i a n d i a m e t e r . GSD i s e q u a l t o one f o r a

m o n o d i s p e r s e a e r o s o l , a n d t h e g r e a t e r t h e GSD t h e more

d i s p e r s e t h e a e r o s o l .

A e r o d y n a m i c m a s s m e d i a n d i a m e t e r (AMMD) i s a

m e a s u r e o f t h e s e t t l i n g v e l o c i t y o f a g i v e n a e r o s o l

d u e t o g r a v i t a t i o n a l f o r c e s e x p r e s s e d i n t e r m s o f a

p a r t i c l e o f u n i t d e n s i t y h a v i n g t h e s ame s e t t l i n g

v e l o c i t y i n a i r . T h i s c o n c e p t a l l o w s a c o m p a r i s o n o f

a e r o s o l s and a p r e d i c t i o n o f d e p o s i t i o n s i t e s w i t h i n

t h e a i r w a y s .

AMMD = MMD x s q u a r e r o u t e o f d e n s i t y ,

b) M a t e r i a l s a n d M e t h o d s

The a p p a r a t u s u s e d t o d e l i v e r a e r o s o l s t o p a t i e n t s

i n t h e p r e c e e d i n g c l i n i c a l t r i a l w a s s e t up i n an

i d e n t i c a l m a n ne r t o a n a l y s e t h e p a r t i c l e s i z e o f t h e

a e r o s o l g e n e r a t e d . T h r e e a i r c o m p r e s s o r s w e r e u s e d ,

t h e M e d i x M i n o r I I , t h e WOBL pump ( Me d i x UK L t d ) and

t h e CF.1A ( A e r o s o l P r o d u c t s L t d ) . A ' B i r d '

m i c r o n e b u l i z e r ( 3M UK L t d ) was u s e d t h r o u g h o u t . The

f l o w r a t e o f e a c h a i r c o m p r e s s o r wa s m e a s u r e d u s i n g a

r o t a m e t e r ( G a p m e t e r L a b - K i t , F l o w b i t s L t d ) .

The a n t i b i o t i c s o l u t i o n s w e r e made up a c c o r d i n g t o

t h e i n s t r u c t i o n s f o r p a t i e n t s i n t h e t r i a l .

G e n t a m i c i n s u l p h a t e 30mg i n 2 m l s ( R o u s e l l , UK, L t d )

wa s u s e d u n d i l u t e d , a n d c a r b e n i c i l l i n 1 g ram ( B e e c h a m )

w a s made up i n 4 m l s o f w a t e r f o r i n j e c t i o n . The

s o l u t i o n d e n s i t i e s w e r e 1 - 0 7 9 g r a m s / m l and , 1 * 2 5

g r a m s / m l r e s p e c t i v e l y . The p a r t i c l e s i z e d i s t r i b u t i o n

o f e a c h a e r o s o l w a s a n a l y s e d u s i n g a M a l v e r n 2200

P a r t i c l e s i z e r . T h i s m e a s u r e s t h e s c a t t e r i n g e f f e c t

o f t h e a e r o s o l on a l a s e r beam and c o m p u t e s t h e

d i s t r i b u t i o n o f p a r t i c l e s i n s i z e b a n d s ,

c ) R e s u l t s

The f l o w r a t e s f r o m t h e a i r c o m p r e s s o r s w e r e a s

f o l l o w s : -

The WOBL pump 23 L / m i n

M e d i x M i n o r I I . 11*5 L / m i n

CFIA 35 L / m i n .

The p a r t i c l e s i z e a n a l y s i s i s shown i n T a b l e X XI .

C a r b e n i c i l l i n n e b u l i z e d t o g i v e a f i n e a e r o s o l o f l e s s

t h a n 5 pm AMMD w i t h a l l t h r e e a i r c o m p r e s s o r s .

G e n t a m i c i n n e b u l i z e d t o g i v e l a r g e r p a r t i c l e s w i t h

AMMD o f 6 - 1 3 jura u s i n g t h e h i g h e r f l o w r a t e

c o m p r e s s o r s , a nd an AMMD o f 9 * 9 7 pm w i t h t h e M e d i x

M i n o r .

MALVERN 2200 PARTICLE SIZER ANALYSIS OF ANTIBIOTIC AEROSOL

SIZE DISTRIBUTION

TABLE XXI

Antibiotic

Solution

GENTAMICIN

CARBENICILLIN

TYPE OF AIR COMPRESSOR

MMD GSD AMMD

MEDIX MINOR

MMD GSD | AMMD

MMD = Mass median diameter (jam).

GSD = Geometric standard deviation.

AMMD = Aerodynamic mass median diameter (pm).

d ) D i s c u s s i o n

Morrow 61 s h o w e d t h a t t h e s i t e o f d e p o s i t i o n o f an

i n h a l e d a e r o s o l w i t h i n t h e human l u n g d e p e n d s on t h e

p h y s i c a l p r o p e r t i e s o f t h e a e r o s o l , t h e mode o f

i n h a l a t i o n and t h e a i r w a y c a l i b r e . P a v i a e t a l - * - ° J

s howe d t h a t p e n e t r a t i o n o f p a r t i c l e s i n t o t h e human

l u n g i s r e l a t e d t o t h e v o l u m e i n s p i r e d p e r b r e a t h , t h e

f o r c e d e x p i r a t o r y v o l u m e i n one s e c o n d , and i n v e r s e l y

r e l a t e d t o t h e f l o w r a t e d u r i n g i n h a l a t i o n . A

b r e a t h - h o l d i n g p a u s e a f t e r i n h a l a t i o n f a v o u r s t h e

s e t t l e m e n t by g r a v i t y o f p a r t i c l e s o f < 2 pm whi ch w o u l d

o t h e r w i s e be e x h a l e d . T h u s , f u l l b r e a t h s , i n h a l e d

s l o w l y , f o l l o w e d by an i n s p i r a t o r y p a u s e e n h a n c e t h e

d e p o s i t i o n o f t h e r a p e u t i c a e r o s o l s .

D e p o s i t i o n o f a e r o s o l s i s a f f e c t e d by p a t h o l o g y

w i t h i n t h e l u n g a nd a i r w a y s . P a v i a e t a l l 6 4 s h o we d

t h a t t h e d e p t h o f d e p o s i t i o n c o r r e l a t e s w i t h FEV^ , and

p e r i p h e r a l d e p o s i t i o n i s r e d u c e d i n p a t i e n t s w i t h

a i r f l o w o b s t r u c t i o n , s u c h a s CF p a t i e n t s . A s t u d y o f

a e r o s o l d e p o s i t i o n i n 22 CF c h i l d r e n showed t h a t

d e p o s i t i o n w a s p a t c h y i n t h o s e w i t h a i r f l o w

o b s t r u c t i o n - * - ^ . L o u r e n c o e t a l ^-66 s howe d t h a t t h e

d e p o s i t i o n o f 2 pm p a r t i c l e s i n p a t i e n t s w i t h

b r o n c h i e c t a s i s w a s m o r e c e n t r a l t h a n i n n o r m a l

s u b j e c t s . The p u l m o n a r y p a t h o l o g y o f CF w o u l d

t h e r e f o r e t e n d t o r e d u c e p e r i p h e r a l d e p o s i t i o n .

The f a c t o r s a f f e c t i n g t h e t y p e o f a e r o s o l p r o d u c e d

by c o m m e r c i a l l y a v a i l a b l e n e b u l i z e r s and a i r

c o m p r e s s o r s h a v e o n l y r e c e n t l y b e en i n v e s t i g a t e d .

C l a y e t n l - ^ ^ h a v e shown t h a t t h e MMD i s s m a l l e r and

t h e o u t p u t g r e a t e r a t h i g h f l o w r a t e s t h r o u g h t h e

n e b u l i z e r . A ' d e a d v o l u m e ' o f n e b u l i z e d f l u i d o f 1 - 2

m.ls o c c u r s , w h i c h i s r e l a t e d t o t h e i n i t i a l v o l u m e b u t

i n d e p e n d e n t o f f l o w r a t e , and t h e s e d r o p l e t s r e m a i n

w i t h i n t h e a p p a r a t u s . I n t h e p r e s e n t s t u d y t h e f l o w

r a t e s o f a l l t h r e e a i r c o m p r e s s o r s w e r e a d e q u a t e .

G e n t a m i c i n w a s u s e d i n 2ml a l i q u o t s and t h e r e f o r e t h e

d e a d v o l u m e w a s a s u b s t a n t i a l p a r t o f t h e o r i g i n a l

a n t i b i o t i c f l u i d ; t h e d e a d v o l u m e was l e s s i m p o r t a n t

f o r c a r b e n i c . i l l i n w h i c h was n e b u l i z e d i n 4 m l s . The

r e s u l t s o f t h e p r e s e n t s t u d y e m p h a s i z e t h e i m p o r t a n c e

o f f l o w r a t e on s i z e d i s t r i b u t i o n o f t h e a e r o s o l ; t h e

h i g h e r t h e f l o w r a t e t h e s m a l l e r t h e AMMD o b t a i n e d f o r

b o t h a n t i b i o t i c s . A l l a e r o s o l s w e r e p o l y d i s p e r s e w i t h

a GSD o f b e t w e e n 1 * 7 - 2 * 0 f o r g e n t a m i c i n , and 2 - 6 -

3 * 5 f o r c a r b e n i c i l l i n i n t h e d i f f e r e n t a p p a r a t u s .

G e n t a m i c i n a e r o s o l s w e r e c o m p o s e d o f q u i t e l a r g e

p a r t i c l e s , p a r t i c u l a r l y w i t h t h e low f l o w - r a t e M e d i x

M i n o r pump, a n d an AMMD o f 9 * 9 7 pm s u g g e s t s t h a t v e r y

l i t t l e o f t h e a e r o s o l p e n e t r a t e d b e y o n d t h e u p p e r

a i r w a y s . The AMMD o f c a r b e n i c i l l i n a e r o s o l s w a s

s m a l l e r , s u g g e s t i n g g r e a t e r p e r i p h e r a l d e p o s i t i o n .

I t i s g e n e r a l l y a c c e p t e d t h a t o n l y a s m a l l

p r o p o r t i o n o f t h e d o s e p l a c e d i n a n e b u l i z e r w i l l

a c t u a l l y p e n e t r a t e t h e a i r w a y s . Newman e t a l ^-68 when

s t u d y i n g p r e s s u r i z e d a e r o s o l s , n o t e d t h a t o n l y 8 * 8 % o f

t h e d o s e w a s d e p o s i t e d i n t h e l u n g s , 80% i n t h e mouth

a nd p h a r y n x , 9 * 8 % i n t h e a e r o s o l a c t u a t o r and 1 % w a s

e x h a l e d . E x t r a p o l a t i n g t h i s t o a n e b u l i z e r s y s t e m ,

f rom an i n i t i a l d o s e o f 8 0 mg i n 2 m l s o f g e n t a m i c i n , a t

l e a s t h a l f w i l l r e m a i n i n t h e n e b u l i z e r , and o n l y a

f r a c t i o n o f t h a t n e b u l i z e d w i l l p e n e t r a t e b e yond t h e

o r o p h a r y n x . The l a r g e p a r t i c l e s i z e m e a s u r e d i n t h i s

s t u d y m u s t mean t h a t t h e r e was c o n s i d e r a b l e d e p o s i t i o n

i n t h e c e n t r a l a i r w a y s . C a r b e n i c i l l i n was g i v e n a s 1

g r a m i n 4 m l s w i t h t h e a d v a n t a g e o f a p r o p o r t i o n a l l y

s m a l l e r d e a d v o l u m e a n d a s m a l l e r p a r t i c l e s i z e . Even

s o , o n l y a f r a c t i o n o f t h e d o s e p l a c e d i n t h e

n e b u l i z e r w i l l r e a c h t h e p e r i p h e r a l a i r w a y s , t o be

d i s p e r s e d t h r o u g h o u t t h e w h o l e o f t h e b r o n c h i a l t r e e .

E. SUMMARY

A e r o s o l g e n t a m i c i n and c a r b e n i c i l l i n g i v e n t w i c e

d a i l y f o r s i x m o n t h s s i g n i f i c a n t l y i m p r o v e d mean PEFR,

FEVg a nd FVC, i n 1 7 CF p a t i e n t s c o m p a r e d w i t h s i x

m o n t h s o f p l a c e b o t r e a t m e n t . T h e r e w a s a l s o a t r e n d

t o w a r d s a r e d u c e d f r e q u e n c y o f h o s p i t a l a d m i s s i o n s ,

t h i s t r e n d w a s a l s o e v i d e n t i n u n c o n t r o l l e d d a t a on

a l l 41 p a t i e n t s r e c e i v i n g t h i s t r e a t m e n t . A h i g h e r

i n c i d e n c e o f t e m p o r a r y r e s i s t a n c e o f p s e u d o m o n a s t o

p e n i c i l l i n s w a s n o t e d i n 41 p a t i e n t s r e c e i v i n g a e r o s o l

t r e a t m e n t on a r e t r o s p e c t i v e a n a l y s i s .

An a i r c o m p r e s s o r w i t h a h i g h f l o w r a t e i s n e e d e d

t o g e n e r a t e a f i n e a e r o s o l o f a e r o d y n a m i c m a s s m e d i a n

d i a m e t e r o f t h r e e m i c r o n s o r l e s s , f rom a n t i b i o t i c

s o l u t i o n s .

F. FUTURE POLICY

To d e s i g n a p o l i c y o f a e r o s o l t h e r a p y f o r t h e

f u t u r e , i n f o r m a t i o n i s n e e d e d on d o s a g e , d o s e

i n t e r v a l , d u r a t i o n o f t r e a t m e n t and w h i c h d r u g , o r

c o m b i n a t i o n o f d r u g s , t o u s e . An a i r c o m p r e s s o r t h a t

i s l i g h t t o c a r r y , b u t h a s a h i g h f l o w r a t e i s

n e c e s s a r y . F u r t h e r w o r k i s n e e d e d on t h e t y p e o f

n e b u l i z e r t o b e u s e d , a n d e q u i p m e n t t o r e - c y c l e o r

r e mo ve e x h a l e d a n t i b i o t i c f r o m t h e s y s t e m . A e r o s o l

a n t i b i o t i c s i n h i g h e r d o s e s , and g i v e n mor e

f r e q u e n t l y , m i g h t be a u s e f u l f o r m o f t r e a t m e n t f o r

a c u t e e x a c e r b a t i o n s w h i c h c o u l d be g i v e n a t home

r a t h e r t h a n i n h o s p i t a l . The u s e o f a e r o s o l s a t an

e a r l i e r s t a g e i n t h e n a t u r a l h i s t o r y o f t h e d i s e a s e ,

b e f o r e o r j u s t a f t e r p s e u d o m o n a s i s a c q u i r e d , may

p r o d u c e a g r e a t e r c l i n i c a l r e s p o n s e and p r o l o n g l i f e .

Chapter 6

SOME PHENOTYPE CHANGES OF PSEUDOMONAS AERUGINOSA IN

RELATION TO THE CLINICAL CONDITION OFCYSTIC FIBROSIS PATIENTS

T h e r e s p i r a t o r y t r a c t of : m o s t p a t i e n t s v ; i t h C P

b e c o m e s p e r m a n e n t l y c o l o n i s e d b y P . a e r u g i n o s a a n d o n c e

a c q u i r e d t h i s o r g a n i s m i s r a r e l y e r a d i c a t e d , a l t h o u g h

i t m a y t e m p o r a r i l y d i s a p p e a r f r o m t h e s p u t u m a f t e r

a n t i b i o t i c t r e a t m e n t . T h e p r e s e n c e o f t h e o r g a n i s m i s

a s s o c i a t e d w i t h m o r b i d i t y a n d m o r t a l i t y i n C F , a n d t h e

e v i d e n c e f o r i t ' s r o l e i n t h e p a t h o g e n e s i s o f t h e

d i s e a s e h a s b e e n d i s c u s s e d e a r l i e r . T h e c o u r s e o f t h e

d i s e a s e i n p a t i e n t s c o l o n i s e d b y P . a e r u g i n o s a v a r i e s

g r e a t l y . S o m e p a t i e n t s r e m a i n r e l a t i v e l y w e l l f o r

i n a n y y e a r s , o t h e r s d e c l i n e r a p i d l y w i t h i n a f e w m o n t h s

o f a c q u i r i n g t h e o r g a n i s m , a n d o t h e r s a r e w e l l f o r a

p e r i o d o f t i m e a n d t h e n s u d d e n t l y d e t e r i o r a t e . T h i s

l a t t e r g r o u p o f p a t i e n t s c a n n o t b e i d e n t i f i e d b y

r o u t i n e s p u t u m b a c t e r i o l o g y . I n c h a p t e r s 4 a n d 5 t h e

d a t a o n a n t i b i o t i c t r e a t m e n t o f c h r o n i c p s e u d o m o n a s

i n f e c t i o n i n C F p a t i e n t s w a s d i s c u s s e d . W h e t h e r g i v e n

i n t r a v e n o u s l y o r o n a m o r e l o n g - t e r m b a s i s b y a e r o s o l ,

a n t i b i o t i c t r e a t m e n t c a n r e s u l t i n c o n s i d e r a b l e

c l i n i c a l i m p r o v e m e n t , a l t h o u g h t h i s i s p a l l i a t i v e n o t

c u r a t i v e . I t i s p o s s i b l e t h a t m o r e a g g r e s i v e a n d

i n t e n s i v e t r e a t m e n t m a y p r e v e n t t h e s u d d e n a n d o f t e n

f a t a l d e t e r i o r a t i o n i n c o n d i t i o n s e e n i n s o m e p a t i e n t s

w i t h p s e u d o m o n a s i n f e c t i o n , b u t t h i s g r o u p o f p a t i e n t s

m u s t f i r s t b e i d e n t i f i e d .

1 5 . r5

D. LITERATURE REVIEW

P r e v i o u s w o r k o n t h e c h a r a c t e r i s t i c s o f

P . a e r u g i n o s a i s o l a t e d f r o m C F p a t i e n t s h a s s u g g e s t e d

t h a t t h e o r g a n i s m i n C F h a s f e a t u r e s w h i c h a r e r a r e i n

o t h e r c o n d i t i o n s . D o g g e t t e t a l ^ ® f i r s t d e s c r i b e d t h e

a s s o c i a t i o n o f t h e m u c o i d c o l o n i a l f o r m w i t h C F .

H d i b y a n d O l l i n g - ^ 9 s h o w e d t h a t 61% o f s t r a i n s o f

p s e u d o m o n a s f r o m C F p a t i e n t s w e r e s e n s i t i v e t o t h e

b a c t e r i c i d a l e f f e c t o f n o r m a l h u m a n s e r u m , i n c o n t r a s t

t o t h e s e r u m - r e s i s t a n t s t r a i n s f r o m n o n - C F p a t i e n t s ,

f r o m o t h e r s o u r c e s a n d f r o m t h e e n v i r o n m e n t .

C . A I M S

A s t u d y o f t h e p h e n o t y p e c h a r a c t e r i s t i c s o f

P , a e r u g i n o s a f r o m C F p a t i e n t s w a s m a d e i n t w o p a r t s .

F i r s t a c o m p a r i s o n w a s m a d e o f C F s t r a i n s w i t h

o r g a n i s m s f r o m o t h e r s o u r c e s i n o r d e r t o d e f i n e a n y

d i f f e r e n c e s b e t w e e n t h e m . S e c o n d l y t h e n a t u r e o f t h e

o r g a n i s m i s o l a t e d f r o m e a c h p a t i e n t w a s c o r r e l a t e d

w i t h t h e c l i n i c a l c o n d i t i o n o f t h a t i n d i v i d u a l , w i t h

t h e a i m o f i d e n t i f y i n g p a t i e n t s a t r i s k o f s u d d e n

d e t e r i o r a t i o n i n t h e i r c o n d i t i o n b y c h a r a c t e r i s t i c s o f

t h e i r o r g a n i s m s .

D. METHODS1 . P A T I E N T S

F o r t y - n i n e a d u l t s w i t h CF, 2 8 m a l e s a n d 2 1

f e m a l e s , a g e r a n g e 1 6 - 4 3 y e a r s w e r e s t u d i e d a n d s p u t u m

a n d s e r u m s a m p l e s w e r e o b t a i n e d a t o u t - p a t i e n t

a t t e n d a n c e s o v e r a p e r i o d o f s i x m o n t h s . N o r m a l h u m a n

s e r u m ( N H S ) w a s o b t a i n e d b y p o o l i n g e q u a l v o l u m e s o f

s e r u m f r o m f i v e h e a l t h y v o l u n t e e r s . S e r a w e r e s t o r e d

i n a l i q u o t s a t - 2 0 ° C a n d u s e d w i t h i n t w o m o n t h s .

2 . I D E N T I F I C A T I O N O F P S E U D O M O N A S S T R A I N S

( D e t a i l s o f m e t h o d s a r e d e s c r i b e d i n C h a p t e r 3 )

T h e s p u t a w e r e d i g e s t e d b y p a n c r e a t i n , p l a t e d o n t o

c e t r i m i d e - n a l i d i x i c a c i d a g a r ( O x o i d ) a n d i n c u b a t e d a t

3 7 ° C f o r 1 8 h o u r s . A s w e e p o f t h e p r i m a r y g r o w t h w a s

s u b c u l t u r e d o n t o K i n g ' s " A " a g a r ^ 2 a n d d i s c r e e t

c o l o n i e s w e r e p i c k e d f o r i d e n t i f i c a t i o n . C o l o n i a l

f o r m s w e r e c l a s s i f i e d a c c o r d i n g t o P h i l l i p s ^ . o

s e r o l o g i c a l t y p i n g w a s p e r f o r m e d b y a g g l u t i n a t i o n

t e s t s w i t h a n t i s e r a c o r r e s p o n d i n g t o t h e I n t e r n a t i o n a l

A n t i g e n i c T y p i n g S c h e m e ^ . An a n t i s e r u m t o t h e

a n t i g e n a s s o c i a t e d w i t h t h e p o l y a g g l u t i n a t i o n . ( P A ) o f

f i e l d s t r a i n s o f O - t y p i n g s e r a w a s i n c l u d e d - * - - ^ . E a c h

s t r a i n w a s t e s t e d f o r s e n s i t i v i t y t o n o r m a l h u m a n

s e r u m b y t h e m e t h o d d e s c r i b e d i n C h a p t e r 3 .

P s e u d o m o n a s s t r a i n s f r o m e a c h p a t i e n t w e r e i d e n t i f i e d

a n d s t u d i e d b l i n d . O n l y a t t h e e n d o f t h e s t u d y w e r e

o r g a n i s m s r e l a t e d b a c k t o t h e i n d i v i d u a l p a t i e n t a n d

t o h i s o r h e r c l i n i c a l s c o r e .

3 . C O N T R O L C U L T U R E S

2 0 5 d i s t i n c t s t r a i n s o f P . a e r u g i n o s a r e c e i v e d C o t

s e r o t y n i n g o v e r t h e s a m e p e r i o d o f t i m e b y t h e

D i v i s i o n o f H o s p i t a l I n f e c t i o n , P u b l i c H e a l t h

L a b o r a t o r y S e r v i c e , C o l i n d a l e , w e r e u s e d a s c o n t r o l s .

S t r a i n s w e r e d i v i d e d i n t o t w o g r o u p s , t h e f i r s t

c o n s i s t e d o f 8 7 s t r a i n s f r o m s p u t a a n d t h e s e c o n d o f

1 . 1 8 s t r a i n s f r o m o t h e r c l i n i c a l s p e c i m e n s , i n c l u d i n g

e n d o t r a c h e a l t u b e s a n d t r a c h e o s t o m y s i t e s ( a n d f r o m

e n v i r o n m e n t a l s o u r c e s ) . T h e c o l o n i a l f o r m ,

0 - s e r o t y p e , r e a c t i o n t o P A a n t i s e r u m a n d s e n s i t i v i t y

t o n o r m a l h u m a n s e r u m w e r e n o t e d f o r e a c h s t r a i n .

4 . C L I N I C A L A S S E S S M E N T

P a t i e n t s w e r e a s s e s s e d b y t h e a u t h o r a t t h e t i m e

o f s a m p l e c o l l e c t i o n , b e f o r e a n y r e s u l t s o f

b a c t e r i o l o g i c a l s t u d i e s w e r e k n o w n . T h e h i s t o r y o f

c h e s t d i s e a s e , t h e n u m b e r o f d a y s i n h o s p i t a l d u r i n g

t h e p e r i o d o f t h e s t u d y , a n d t h e d u r a t i o n o f

P . a e r u g i n o s a i n f e c t i o n w e r e n o t e d . F o r c e d e x p i r a t o r y

v o l u m e i n o n e s e c o n d ( F E V ^ ) a n d f o r c e d v i t a l c a p a c i t y

( F V C ) w e r e r e c o r d e d o n a V i t a l o g r a p h d r y s p i r o m e t e r

a n d e x p r e s s e d a s a p e r c e n t a g e o f p r e d i c t e d n o r m a l

v a l u e s f r o m t a b l e s p u b l i s h e d b y C o t e s - ^ ^ . An o v e r a l l

a s s e s s m e n t w a s s c o r e d b y t h e s y s t e m o f S h w a c h m a n l ^ - 2 .

5 . S T A T I S T I C S

T h e c h a r a c t e r i s t i c s o f s t r a i n s f r o m e a c h g r o u p o f

c u l t u r e s w e r e c o m p a r e d u s i n g t h e C h i - S q u a r e t e s t .

L i n e a r c o r r e l a t i o n c o e f f i c i e n t s w e r e c a l c u l a t e d f o r

e a c h p a i r o f c h a r a c t e r i s t i c s . T h e c l i n i c a l d a t a o f

p a t i e n t s w i t h e a c h c a t e g o r y o f o r g a n i s m w e r e c o m p a r e d

u s i n g W i l c o x o n ' s r a n k s u m t e s t . T h e d u r a t i o n o f

h o s p i t a l a d m i s s i o n s w a s c o m p a r e d u s i n g t h e C h i - S q u a r e

t e s t .

E . R E S U L T S

1 . C H A R A C T E R I S A T I O N O F S T R A I N S O F P . A E R U G I N O S A I S O L A T E D

F ROM C Y S T I C F I B R O S I S S P U T A

1 0 9 d i s t i n c t s t r a i n s o f P . a e r u g i n o s a w e r e

i d e n t i f i e d f r o m 7 3 s p u t u m c u l t u r e s .

a ) C o l o n i a l f o r m

T h e m a j o r i t y o f C F s t r a i n s w e r e c o l o n y t y p e 2

( 5 3 * 2 % ) , 3 7 s t r a i n s ( 3 4 % ) w e r e m u c o i d o r c o l o n y

t y p e 4 , t h e r e m a i n d e r w e r e c o l o n y t y p e 1 .

b ) O - s e r o l o g y

O n l y 3 5 c u l t u r e s ( 3 2 * 1 % ) r e a c t e d w i t h a s i n g l e

0 - s e r u m a n d w e r e t y p a b l e . A l m o s t h a l f o f t h e t v p a b l e

s t r a i n s w e r e 0 g r o u p 6 , t h e r e m a i n d e r b e i n g

d i s t r i b u t e d a m o n g t h e c o m m o n s e r o g r o u p s .

T a b l e X X I I c o m p a r e s t h e p a t t e r n o f r e a c t i o n s w i t h

0 - t y p i n g s e r a o f t h e 3 5 t y p a b l e C F c u l t u r e s , t h e 5 5

p o l y a g g l u t i n a b l e C F c u l t u r e s , a n d t h e 1 1 2 c u l t u r e s

f r o m t h e c o n t r o l g r o u p o f g e n e r a l h o s p i t a l i s o l a t e s

w h i c h w e r e t y p a b l e . 0 - t y p e 6 w a s t h e c o m m o n e s t i n a l l

g r o u p s . A s i m i l a r p a t t e r n i s s e e n i n t h e d i s t r i b u t i o n

o f O - g r o u p s i n t h e t y p a b l e C F a n d c o n t r o l s t r a i n s ,

G r o u p s 1 , 2 , 3 , 4 , 6 , 1 1 , 1 6 b e i n g w e l l r e p r e s e n t e d , b u t

9 , 1 0 , 1 1 b e i n g l e s s w e l l r e p r e s e n t e d i n t h e C F s t r a i n s .

T e n C F c u l t u r e s ( 9 * 2 % ) d i d n o t r e a c t w i t h a n y o f

t h e t y p i n g s e r a a n d w e r e d e n o t e d a s ' n o t t y p a b l e ' o r

N T . N i n e c u l t u r e s w e r e a u t o a g g l u t i n a b l e i n s a l i n e .

5 5 c u l t u r e s ( 5 5 - 5 % ) w e r e a g g l u t i n a t e d b y m o r e t h a n o n e

a n t i s e r u m a n d w e r e c l a s s i f i e d a s ' p o l y a g g l u t i n a t i n g '

o r P A . T h e O - a n t i s e r a t h a t m o s t f r e q u e n t l y

a g g l u t i n a t e d P A s t r a i n s w e r e 5 d , 6 , 9 , 1 0 a n d 1 3 ,

a l t h o u g h s i x o t h e r s c o n t r i b u t e d t o t h e p a t t e r n o f

p o l y a g g l u t i n a t i o n .

c ) T h e P A a n t i g e n

T h i s w a s d e t e c t e d i n a l l 5 5 p o l y a g g l u t i n a t i n g

s t r a i n s a n d i n t w o O - t y p a b l e s t r a i n s .

d ) S e n s i t i v i t y t o n o r m a l h u m a n s e r u m

T h e m a j o r i t y o f C F s t r a i n s w e r e s e r u m s e n s i t i v e ,

5 8 s t r a i n s ( 5 3 * 2 % ) w e r e f u l l y s e n s i t i v e , a n d 1 5

s t r a i n s ( 1 3 * 8 % ) p a r t i a l l y s e n s i t i v e . T h e r e m a i n i n g 3 6

( 3 3 % ) w e r e r e s i s t a n t t o N H S .

2 . C O M P A R I S O N WI T H C O N T R O L C U L T U R E S

T h e c h a r a c t e r i s t i c s o f t h e s t r a i n s o f P . a e r u g i n o s a

i s o l a t e d f r o m t h e C F s p u t a w e r e c o m p a r e d t o t h o s e o f

s t r a i n s f r o m t h e t w o c o n t r o l g r o u p s , ( T a b l e X X I I I ) .

T h e m u c o i d f o r m w a s f o u n d i n o n l y o n e c o n t r o l s p u t u m

s t r a i n f r o m a p a t i e n t w i t h b r o n c h i e c t a s i s , ( n o n - C F ) .

T h e m a j o r i t y o f c o n t r o l c u l t u r e s w e r e t y p a b l e b y 0 -

s e r o l o g y , ( 8 7 - 4 % o f s p u t u m i s o l a t e s a n d 9 4 - 9 % ) o f

TABLE XXII

T H E P A T T E R N O F R E A C T I O N S TO 0 - A N T I S E R A O F T Y P A B L E AMD

A B E R R A N T S T R A I N S O F C Y S T I C F I B R O S I S C U L T U R E S OF

P . A E R U G I N O S A , C O M P A R E D WI T H T H O S E O F C O N T R O L S T R A I N S .

0 t y p e

s e r u m

H o s p i t a l

i s o l a t e s

n = 1 1 2

N o . o f C F c

w e r e a g g l u t i

a n d c

T y p a b l e ( 3 5 )

u l t u r e s e x a m i n e d t h a t

n a t e d b y s t a t e d s e r u m

l a s s i f r i e d a s s -

P o l y a q q l u t i n a b l e ( 5 5 )

1 6 ( 5 - 4 % ) 1 ( 2 - 9 % ) 4 ( 7 * 3 % )

2 1 3 ( 1 1 - 6 % ) 3 ( 8 - 6 % ) 2

3 6 ( 5 * 4 % ) 6 ( 1 7 * 1 % ) 4

4 5 ( 4 * 5 % ) 3 ( 8 * 6 % ) 3 ( 5 * 5 % )

5 ( d ) 1 ( 0 - 9 % ) 0 2 6 ( 4 7 * 3 % )

6 2 7 ( 2 4 - 1 % ) 1 7 ( 4 8 * 6 % ) 4 0 ( 7 2 * 7 % )

7 4 ( 3 * 6 % ) 0 7 ( 1 2 * 7 % )

8 1 ( 0 - 9 % ) 0 3

9 6 ( 5 * 4 % ) 0 3 7 ( 6 7 * 3 % )

1 0 9 ( 8 * 0 % ) 1 4 0 ( 7 2 * 7 % )

1 1 2 4 ( 2 1 * 4 % ) 2 ( 5 * 7 % ) 0

1 2 1 ( 0 * 9 % ) 0 0

1 3 0 0 2 1 ( 3 8 * 2 % )

1 4 0 0 0

1 5 0 0 0

1 6 6 ( 5 - 4 % ) 2 ( 5 * 7 % ) 1

1 7 0 0 0

P A s e r u m 3 ( 2 * 7 % ) 2 ( 5 * 7 % ) 5 5 ( 1 0 0 % )

TABLE XXIII

CHARACTERISTICS OF STRAINS OF PSEUDOMONAS AERUGINOSA FROM PATIENTS WITH

CYSTIC FIBROSIS COMPARED WITH STRAINS FROM OTHER SOURCES

Straincharacteristic

Source of strainsDi fference between CF other groups

x^ test

II CF 1 n=

sput a 109 (%)

Other | sputa | n=87 GO 1

Other hospital specimens n=l18 (%)

1. Mucoid colony

111 37 1

1 (I'D 1 1

0 p<0*001

2. 0 serotype

111 35 (32*1)

76 (87*4) I 112 (94*9) p<0*001ident i fied 1

3. PA antigen

111 55 (50*5)

15 (17*2) I 12 (10*2) p<0*001expressed 1

4. Sensitive to

normal human 1 58 (53*2) 12 (13*8) | 13 (11*1) p<0*001serum 1

o t h e r i s o l a t e s , ( 1 1 2 o f 118 c u l t u r e s ) and t h i s i s

s i g n i f i c a n t l y d i f f e r e n t f rom t h e low p e r c e n t a g e o f CP

s t r a i n s w h ic h w e r e t y p a b l e ( p < 0 * 0 0 1 ) . 50*5% o f CF

s t r a i n s e x p r e s s e d PA a n t i g e n , c o m p a r e d t o 17*2% o f

sp u tu m i s o l a t e s ( p < 0 * 0 0 1 ) and 1 0 * 2% o f o t h e r s t r a i n s

( p < 0 * 0 0 1 ) . The i n c i d e n c e o f se r u m s e n s i t i v i t y among

CF s t r a i n s w a s s i g n i f i c a n t l y h i g h e r t h a n i n e i t h e r

c o n t r o l g r o u p s ( p < 0 * 0 0 1 ) . A l t h o u g h t h e c o n t r o l

c u l t u r e s f r o m s p u t u m t e n d e d t o h a v e m o re v a r i a n t

s t r a i n s o f p s e u d o m o n a s t h a n t h e o t h e r c o n t r o l g r o u p ,

t h e d i f f e r e n c e b e t w e e n s p u t u m c o n t r o l s and CF s t r a i n s

w a s s t i l l h i g h l y s i g n i f i c a n t .

3 . ASSOCIATION BETWEEN SOME PHENOTYPE CHARACTERISTICS OF

P.AERUGINOSA IN CYSTIC F I B R O S I S

T h e r e w a s no c o r r e l a t i o n b e t w e e n c o l o n i a l f o r m and t h e

o t h e r c h a r a c t e r i s t i c s o f t h e o r g a n i s m o r w i t h t h e c l i n i c a l

c o n d i t i o n o f t h e p a t i e n t s i n t h i s s t u d y . The m a j o r i t y o f

t h e CF s t r a i n s sh o w e d t h e t h r e e p h e n o t y p e c h a r a c t e r i s t i c s

o f l o s s o f 0 - t y p e r e a c t i o n , e x p r e s s i o n o f PA a n t i g e n and

se r u m s e n s i t i v i t y , a l l o f w h ic h w e r e i n f r e q u e n t i n t h e

c o n t r o l g r o u p s . T h e r e w a s a s i g n i f i c a n t a s s o c i a t i o n

b e t w e e n t h e s e t h r e e f e a t u r e s ( T a b l e X X I V ) . T h e s e t h r e e

v a r i a b l e s t e n d e d t o g r o u p t h e m s e l v e s , s u c h t h a t o f t h e 1 0 9

s t r a i n s o f p s e u d o m o n a s i s o l a t e d 59 h ad a l l t h r e e f e a t u r e s ,

26 h a d n o n e , and 24 h a d o ne o r two f e a t u r e s .

TABLE XXIV

ASSOCIATION BETWEEN THREE PHENOTYPE CHARACTERISTICS OF P.AERUGINOSA

FROM CF SPUTUM

Pair ofCharacteristics

1I Association | between them

1| Correlation I coefficient

1| Signi ficance 1

Loss of 0 type11 74*3% of untypable strains 1

v PA antigeniI express PA antigen. 1

11 0*720 I

1I P<0-001 1

1 96*5% of PA strains are |i11

I untypable by 0 serology. 1

Loss of 0 type1| 83*8% of untypable strains 1

v serumi1 are serum sensitive. 1

l1 0-704

1I p<0-001 1

sensit ivity1| 93*1% of SS strains have 1

I lost 0 type. 1

PA antigen 1| 76% of PA strains are SS.I111

v serum1| 72% of SS strains have 1

1I 0-627 1

1I p<0*001 1

sensitivity1I PA antigen 1

4 . RELATIONSHIP OF SOME PHENOTYPE CHANGES IN P . AERUGINOSA

TO THE CLINICAL CONDITION OF CYSTIC F I B R O S IS PATIENTS

P a t i e n t s w e r e d i v i d e d r e t r o s p e c t i v e l y i n t o t h r e e

g r o u p s a c c o r d i n g t o t h e c h a r a c t e r i s t i c s o f t h e

p r e d o m i n a n t s t r a i n o f p s e u d o m o n a s i s o l a t e d f rom t h e i r

s p u t u m ( T a b l e X X V ) . O r g a n i s m s w e r e c o n s i d e r e d t o be

'com mon' i f t h e y w e r e O - t v p a b l e , r e s i s t a n t t o n o r m a l

human s e r u m an d d i d n o t h a v e PA a n t i g e n . O r g a n i s m s

w i t h t h e s e t h r e e f e a t u r e s o f l o s s o f O - t y p e r e a c t i o n ,

p r e s e n c e o f PA a n t i g e n and s e n s i t i v i t y t o NHS w e r e

c o n s i d e r e d c l a s s i c a l l y ' c y s t i c ' . O r g a n i s m s w i t h some

b u t n o t a l l o f t h e s e f e a t u r e s w e r e c l a s s i f i e d a s

' i n t e r m e d i a t e ' . S i x p a t i e n t s , mean a g e 2 1 * 3 y e a r s ,

h a d o n l y 'com m on' s t r a i n s o f p s e u d o m o n a s i n t h e i r

s p u t u m . The a v e r a g e d u r a t i o n o f c o l o n i s a t i o n by t h e

o r g a n i s m w as 3 * 7 y e a r s . T h e s e p a t i e n t s w e r e i n a g o o d

c l i n i c a l c o n d i t i o n w i t h s i g n i f i c a n t l y h i g h e r c l i n i c a l

s c o r e , s p e n t l e s s t i m e i n h o s p i t a l and h ad b e t t e r

r e s p i r a t o r y f u n c t i o n t h a n t h e p a t i e n t s i n t h e o t h e r

two g r o u p s .

T w e n t y - o n e p a t i e n t s , mean a g e 2 4 * 2 y e a r s , h a d o n l y

' c y s t i c ' s t r a i n s i n t h e i r s p u t u m . The a v e r a g e

d u r a t i o n o f c o l o n i s a t i o n w as 7 * 1 y e a r s . R e s p i r a t o r y

f u n c t i o n w a s n o t a s g o o d a s t h e f i r s t g r o u p o f

p a t i e n t s , b u t i t w a s s i g n i f i c a n t l y b e t t e r t h a n t h e

p a t i e n t s w i t h ' i n t e r m e d i a t e ' o r g a n i s m s .

T w e n t y - t w o p a t i e n t s , mean a g e 2 2 - 1 y e a r s , h ad

o r g a n i s m s w i t h ' i n t e r m e d i a t e ' c h a r a c t e r i s t i c s and t h e

a v e r a g e d u r a t i o n o f c o l o n i s a t i o n w a s 4 * 3 y e a r s . T h e i r

TABLE XXV

CLINICAL FEATURES OF CF PATIENTS GROUPED ACCORDING TO THE

CHARACTERISTICS OF P.AERUGINOSA ISOLATED FROM THEIR SPUTUM.

Clinical features Category of P.aeruginosa'Common' 'Intermediate' 'Cystic'

No. of patients Mean age (years)

2222*1

2124*2

Mean duration of colonisation and range (yrs)

3-7(0*3-7)

4*3(0*5- 12)

p<0*001

7*1(0*3-20)

% predicted FEV^ Mean + SD

50*8+21*9p<0*01

31*1+16*4p<0 *05

35*02+13*3

% predicted FVC Mean + SD

63*3+19*8p<0* 05

45*02+19*4p=0*05

54*8+12*5

Average no. of days in hospitalin six months+ SD

6•2+5 * 3

P<0*001

13*0+19*0

p<0*001

10*1+18*9

Clinical score + SD

71+12*0P<0-01

58*3+9*9NS-

65*5+10*9

Number of deaths 0 5 (22*7%) 1 (4*7%)

c l i n i c a l s c o r e a n d r e s p i r a t o r y f u n c t i o n w e r e

s i g n i f i c a n t l y w o r s e t h a n t h a t o f t h e f i r s t (common)

g r o u p o f p a t i e n t s , and t h e i r r e s p i r a t o r y f u n c t i o n

s i g n i f i c a n t l y w o r s e t h a n t h a t o f p a t i e n t s w i t h

' c y s t i c ' s t r a i n s . T h e s e i n d i v i d u a l s s p e n t m ore t i m e

i n h o s p i t a l d u r i n g t h e p e r i o d o f t h e s t u d y t h a n t h e

o t h e r two g r o u p s . F i v e o f t h e s e p a t i e n t s h a v e

s u b s e q u e n t l y d i e d ( 2 3 % ) , w h e r e a s t h e r e h a s been o n l y

o n e d e a t h i n t h e ' c y s t i c ' g r o u p (5%) and none i n t h e

common g r o u p .

The v a r i a t i o n i n s t r a i n s o f p s e u d o m o n a s f rom e a c h

i n d i v i d u a l p a t i e n t w a s r e v i e w e d t o t e s t t h e v a l i d i t y

o f d i v i d i n g p a t i e n t s i n t o g r o u p s a c c o r d i n g t o t h e

n a t u r e o f t h e o r g a n i s m . E a c h o f t h e s i x p a t i e n t s i n

t h e 'com mon' g r o u p h a d o n l y one s t r a i n o f p s e u d o m o n a s

w h ic h w a s i s o l a t e d f r o m s p u t u m a t e v e r y a t t e n d a n c e ,

a l t h o u g h some d i s s o c i a t i o n o f c o l o n i a l fo rm was s e e n .

O - s e r o g r o u p s 1 , 3 , 6 , a n d 11 w e r e f o u n d . I n t h e

' c y s t i c ' g r o u p o f 2 1 p a t i e n t s , a p p a r e n t l y i d e n t i c a l

' c y s t i c ' s t r a i n s w e r e i s o l a t e d f r o m e v e r y s p e c i m e n

f r o m e a c h i n d i v i d u a l i n 20 c a s e s . In o n e s a m p l e f ro m

a s i n g l e i n d i v i d u a l t h e r e w a s an ' i n t e r m e d i a t e '

v a r i a n t . Of t h e 22 p a t i e n t s w i t h ' i n t e r m e d i a t e '

o r g a n i s m s , s e v e n h a d two o r m o re d i s t i n c t s t r a i n s o f

p s e u d o m o n a s i n a s i n g l e s p u tu m s p e c i m e n . However i n

o n l y two o f t h e s e w e r e d i f f e r e n t O - t y p a b l e s t r a i n s

f o u n d . The o t h e r f i v e p a t i e n t s h ad d i f f e r e n t

' i n t e r m e d i a t e ' s t r a i n s v a r y i n g i n e i t h e r

s e r u m - s e n s i t i v i t y , o r t h e p r e s e n c e o f PA a n t i g e n , b u t

w e r e of: o ne O - t y o e w h e r e t h e O - r e a c t i o n w a s o b t a i n e d .

F . DISCUSSION

1 . CHARACTERISTICS OF P.AERUGINOSA FROM CYSTIC F I B R O S I S

a ) C o l o n i a l Form

A s i n g l e s t r a i n o f P . a e r u g i n o s a may d i s s o c i a t e

i n t o s e v e r a l c o l o n i a l f o r m s - * - ^ . The m u c o i d form i s

r a r e l y i s o l a t e d f r o m c l i n i c a l s p e c i m e n s , w i t h t h e

e x c e p t i o n o f s p u t u m f r o m CF p a t i e n t s l ^ l f and i s o f t e n

c o n s i d e r e d a p a t h o g n o m o n i c f e a t u r e o f CF. M u c o id

f o r m s r a p i d l y . b e c o m e n o n - m u c o i d by s e r i a l t r a n s f e r on

s o l i d medium^-*7®, a n d t h e r e l a t i v e l y low i n c i d e n c e o f

m u c o i d f o r m s i n t h i s CF s e r i e s may be d u e t o t h e f a c t

t h a t o r g a n i s m s w e r e s t u d i e d a f t e r t w o o r t h r e e

s u b c u l t u r e s . M u c o i d a n d n o n - m u c o i d f o r m s f r o m t h e

s a m e s p e c i m e n h a v e b e e n shown t o be i d e n t i c a l s t r a i n s ,

t h a t i s o f t h e s a m e s e r o t y p e a n d p y o c i n type-*-7 2 - 1 7 4 0

C o l o n i a l f o r m i s t h e r e f o r e an i n c o n s i s t e n t and

c u l t u r e - d e p e n d e n t f e a t u r e o f a s t r a i n o f p s e u d o m o n a s ,

a n d i n t h i s s t u d y d i d n o t c o r r e l a t e w i t h t h e o t h e r

c h a r a c t e r i s t i c s o f t h e o r g a n i s m , o r w i t h t h e c o n d i t i o n

o f t h e p a t i e n t . T h e r e l a t i o n s h i p o f t h e m u c o i d form

t o t h e p a t h o g e n e s i s o f CF w as d i s c u s s e d i n C h a p t e r 2,

b u t c o l o n i a l fo rm i s o f no i m p o r t a n c e i n i n v i t r o

s t u d i e s o f t h e o r g a n i s m .

b ) O - s e r o t y p e

The h e a t s t a b l e O - a n t i g e n s o f P,a e r u g i n o s a - t h e

c e l l w a l l l i p o p o l y s a c c h a r i d e - p r o v i d e t h e b a s i s t o r

s e r o l o g i c a l t y p i n g o f t h i s o r g a n i s m . 90-95% o f

s t r a i n s f ro m c l i n i c a l s p e c i m e n s c a n b e a l l o c a t e d t o a

s p e c i f i c s e r o t y p e by t h i s m e t h o d s 6 5 . A s m a l l number

o f s t r a i n s a r e a g g l u t i n a t e d by s e v e r a l a n t i s e r a

a g a i n s t u n r e l a t e d O - s e r o g r o u p s , an d s u c h s t r a i n s may

e x p r e s s a new s o m a t i c a n t i g e n , t o w h ic h a s p e c i f i c

r a b b i t a n t i s e r u m may be r a i s e d . T h i s h a s been t e r m e d

t h e p o l y a g g l u t i n a t i n g o r PA a n t i g e n i c .

T y p a b l e CF c u l t u r e s w e r e m a i n l y O - g r o u p s 1 , 2 b , 3 ,

4 , 6 , 1 0 , 11 and 1 6 , w i t h t y p e 6 b e i n g t h e m o s t common

( 4 8 * 6 % ) . T h i s p a t t e r n i s s i m i l a r i n g e n e r a l t o t h a t

s e e n i n o t h e r h o s p i t a l i s o l a t e s , ( T a b l e X X I I ) . Z i e r d t

a n d Williamsl73 t y p e d 173 CF s t r a i n s by t h e Homma

s y s t e m , and f o u n d t h a t t h e t y p e 8 ( c o r r e s p o n d i n g t o

IATS 0 - 6 ) a c c o u n t e d f o r 60% o f s t r a i n s . They a l s o

n o t e d t h a t CF s t r a i n s d i d n o t g i v e c o n s i s t e n t r e s u l t s

on r e p e t i t i v e t y p i n g t e s t s and 2 1 % w e r e n o t t y p a b l e .

I n t h e s t u d y o f D i a z e t a l - ^ 5 on c u l t u r e s f ro m 40 CF

p a t i e n t s , 45% o f s t r a i n s c o u l d n o t be t y p e d . H ^ i b y

a n d R o s e n d a l - -̂^6 t y p e d 4 8 4 CF i s o l a t e s by t h e Habs

m e t h o d and f o u n d t h a t t h e e q u i v a l e n t o f IATS 1 , 3 , 4

a n d 6 w e r e t h e m o s t common, b u t t h a t a l m o s t h a l f t h e

s t r a i n s w e r e p o l y a g g l u t i n a t i n g . In t h e m i n o r i t y o f CF

s t r a i n s o f p s e u d o m o n a s t h a t a r e O - t y p a b l e , i t s e e m s

t h a t t h e d i s t r i b u t i o n o f O - g r o u p s i s s i m i l a r t o a n y

o t h e r c o l l e c t i o n o f i s o l a t e s o f p s e u d o m o n a s . A l t h o u g h

IATS t y p e 6 may b e t h e m o s t common in CF i s o l a t e s , i t

i s a l s o q e n e r a l l v t h e m o s t common, and n o t a 'CF

s e r o t y p e ' a s d e s c r i b e d by Z i e r d t and W i l l i a m s ^ 3 %

c ) PA a n t i g e n

P o l y a g g l u t i n a t i o n o f p s e u d o m o n a s by m u l t i p l e

O - a n t i s e r a f r o m CF p a t i e n t s was f i r s t d e s c r i b e d by

M a r t i n ^ f n 1 9 7 1 . H ^ i b y and R o s e n d a l l ^ G f o u n d many

p o l y a g g l u t i n a t i n g CF s t r a i n s u s i n g H a b s a n t i s e r a , and

a s s i g n e d 36*9% o f s t r a i n s s t u d i e d t o an ' 0 - 3 / 9

c o m p l e x ' w h ic h i n c l u d e d c r o s s - r e a c t i o n s b e tw e e n

0 - g r o u p s 1 , 2 , 3 , 5 , 6 , 7 , and 9 . S e r o g r o u p s 02 a n d

05 a r e w e l l known t o c r o s s - r e a c t , and s u b - t y p e

s p e c i f i c s e r a w e r e u s e d i n t h e p r e s e n t s t u d y .

A l l o w i n g f o r t h i s t h e p a t t e r n o f p o l y a g g l u t i n a t i n g

r e a c t i o n s o b s e r v e d i n t h e p r e s e n t s t u d y , w hich

i n v o l v e d O - g r o u p s 5 , 6 , 9 , 10 and 1 3 , i s d i f f e r e n t

f ro m t h e p a t t e r n d e s c r i b e d by H o ib y and R o s e n d a l . The

p henom enon o f p o l y a g g l u t i n a t i o n w as v e r y common among

CF s t r a i n s o f t h i s s t u d y , and w a s a s s o c i a t e d w i t h t h e

e x p r e s s i o n o f PA a n t i g e n , a s d e t e c t e d by a s p e c i f i c

r a b b i t a n t i s e r u m . A l l s t r a i n s w h ic h p o l y a g g l u t i n a t e d

0 - t y p i n g s e r a r e a c t e d w i t h t h e PA s e r u m .

d ) S e n s i t i v i t y t o n o r m a l human s e r u m

The m a j o r i t y o f s t r a i n s o f P . a e r u g i n o s a i s o l a t e d

f ro m b l o o d c u l t u r e s a n d s a p r o p h y t i c s o u r c e s a r e

r e s i s t a n t t o t h e b a c t e r i c i d a l a c t i o n o f n o r m a l human

serum^-77^ a l t h o u g h t h e a b s o l u t e p r e v a l e n c e o f s e r u m

s e n s i t i v i t y may d e p e n d on t h e i j i v i t r o c o n d i t i o n s o f

t h e t e s t l 7 8 . S e r u m s e n s i t i v e s t r a i n s a r e f r e q u e n t l y

f o u n d in CF and t h e p r e v a l e n c e o f 531 in t h i s s t u d y i.

s i m i l a r t o f i g u r e s p r e v i o u s l y r e p o r t e d - * - ^

S e n s i t i v i t y t o s e r u m w as h i g h l y s i g n i f i c a n t l y

a s s o c i a t e d w i t h t h e l o s s o f O - t y p e r e a c t i o n and t h e

p r e s e n c e o f PA a n t i g e n .

2 . SOME PHENOTYPE CHANGES OF P.AERUGINOSA IN CYSTIC

F I B R O S IS

T h r e e p h e n o t y p e c h a r a c t e r i s t i c s , l o s s o f O - t y p e

r e a c t i o n , e x p r e s s i o n o f PA a n t i g e n and s e n s i t i v i t y

t o n o r m a l human s e r u m w e r e f o u n d t o be common i n

p s e u d o m o n a s s t r a i n s f r o m CF p a t i e n t s and t o be

s i g n i f i c a n t l y a s s o c i a t e d w i t h e a c h o t h e r . O r g a n i s m s

w e r e c l a s s i f i e d a s ' c o m m o n ' , ' i n t e r m e d i a t e ' o r

' c y s t i c ' on t h e b a s i s o f t h e s e f e a t u r e s . T h e s e t h r e e

c h a r a c t e r i s t i c s w e r e a l s o s e e n i n a s m a l l num ber o f

c o n t r o l s t r a i n s o f p s e u d o m o n a s , s u g g e s t i n g t h a t t h e y

a r e n o t s p e c i f i c t o CF b u t p e r h a p s r e l a t e t o c h r o n i c

c o l o n i s a t i o n o f a n y s i t e by t h e o r g a n i s m . I t i s

t h e r e f o r e u n l i k e l y t h a t t h i s i s a phenomenon w h i c h i s

o n l y s e e n i n a d u l t CF p a t i e n t s h o w e v e r i t w o u ld b e o f

g r e a t i n t e r e s t t o p e r f o r m a s i m i l a r s t u d y i n CF

c h i l d r e n .

In o n l y two c a s e s w e r e p a t i e n t s f rom t h e

' i n t e r m e d i a t e ' g r o u p i n f e c t e d by m o re t h a n o n e

d i s t i n g u i s h a b l e s t r a i n o f p s e u d o m o n a s , s o i t i s

u n l i k e l y t h a t m u l t i p l e s t r a i n s w e r e a c o n t r i b u t o r y

f a c t o r t o t h e i r p o o r c l i n i c a l s t a t u s . I f p s e u d o m o n a s

u n d e r g o e s c h a n g e s i n t h r e e p h e n o t y p e c h a r a c t e r i s t i c s

in p a r a l l e l in CP, s e v e r a l i n t e r m e d i a t e v a r i a n t s w i t h

c h a n g e s i n o ne o f : t h e t h r e e f e a t u r e s m i g h t be e x p e c t e d

t o o c c u r i n t h e s a m e s p e c i m e n . T h i s was s e e n i n f i v e

p a t i e n t s in t h e ' i n t e r m e d i a t e ' g r o u p , p r o v i d i n g some

e v i d e n c e f o r a d a p t a t i o n o f t h e o r g a n i s m r a t h e r t h a n

a c q u i s i t i o n o f o t h e r o r g a n i s m s f rom t h e e n v i r o n m e n t .

(Any r e c e n t l y a c q u i r e d o r g a n i s m w o u l d be m o s t l i k e l y

t o be o f t h e ' com m o n ' t y p e ) . F u r t h e r e v i d e n c e w a s

p r o v i d e d when t h e v a r i a n t s f ro m t h e s e f i v e p a t i e n t s

w e r e s t u d i e d i n m o r e d e t a i l . S e r u m - r e s i s t a n t v a r i a n t s

s e l e c t e d f r o m s u r v i v o r s o f t h e s e r u m - s e n s i t i v i t y t e s t

w e r e f o u n d t o be o f t h e sam e O - s e r o t y p e a s t h e

'common' s t r a i n s f r o m ' t h e sam e s p e c i m e n . R e s i s t a n t

v a r i a n t s b e c a m e s e n s i t i v e t o s e r u m on r e t e s t i n g , b u t

c o u l d be r e s e l e c t e d by t h e a c t i o n o f a b a c t e r i o p h a g e .

'Common' and ' c y s t i c ' s t r a i n s f ro m t h e sam e s p e c i m e n

o f o n e p a t i e n t w e r e i d e n t i c a l i n p y o c i n t y p e a n d

v a r i e d by o n l y o n e p h a g e o f t h e p h a g e t y p i n g s e t .

A c q u i s i t i o n o f p s e u d o m o n a s f r o m o t h e r p a t i e n t s ,

t h a t i s c r o s s - i n f e c t i o n b e tw e e n CF p a t i e n t s , i s t h e

s u b j e c t o f c o n t r o v e r s y ; e p i d e m i o l o g i c a l s t u d i e s a r e

h a m p e r e d by t h e d i f f i c u l t i e s i n s e r o t y p i n g p s e u d o m o n a s

f r o m CF s p u t u m , h i g h l i g h t e d i n t h i s s t u d y . Hj&iby and

R o s e n d a l l 7 6 d e f i n e d c l u s t e r s o f o r g a n i s m s on s e r o l o g y

an d i n f e r r e d t h a t c r o s s - i n f e c t i o n h ad o c c u r r e d w i t h o u t

d i s c u s s i n g p a t i e n t c o n t a c t . D i a z e t a l - ^ 5 f o u n d t h r e e

s e r o t y p e s t o p r e d o m i n a n t among CF p a t i e n t s , b u t t h e

o v e r a l l p a t t e r n o f i s o l a t e s e r o t y p e s was s i m i l a r t o

t h o s e f r o m o t h e r p a t i e n t s an d t h e e n v i r o n m e n t . K e l l y

e t a l l ^ O a i S o Hound t h a t t h e d i s t r i b u t i o n o f s e r o t y p e s

f rom CP p a t i e n t s d i d n o t d i f f e r s u b s t a n t i a l l y from

t h a t o f s t r a i n s f r o m a l l s o u r c e s in t h e i r l a b o r a t o r y ,

and w h e r e p a t i e n t s h a r b o u r e d t h e sam e s t r a i n t h e r e w as

no e v i d e n c e o f c o n t a c t b e t w e e n them . They c o n c l u d e d

t h a t p s e u d o m o n a s s t r a i n s w e r e a c q u i r e d by CF p a t i e n t s

f ro m t h e e n v i r o n m e n t . T he s i m i l a r i t y i n p a t t e r n o f

t y p a b l e CF c u l t u r e s i n t h e p r e s e n t s t u d y c o m p a r e d t o

a l l i s o l a t e s s e n t t o t h e r e f e r e n c e l a b o r a t o r y f o r

t y p i n g ( T a b l e X X I I ) s u p p o r t s t h i s c o n c l u s i o n .

T he p h e n o t y p e v a r i a n t s o f p s e u d o m o n a s d e s c r i b e d i n

t h i s s t u d y a r e s e r o l o g i c a l l y d i s t i n c t , and t h e r e f o r e

a r e a s s u m e d t o h a v e a d i s t i n c t a n t i g e n i c s t r u c t u r e .

I n many r e s p e c t s , t h e c h r o n i c c o l o n i s a t i o n o f t h e CF

l u n g by P . a e r u g i n o s a r e p r e s e n t s a h o s t - p a r a s i t e

r e l a t i o n s h i p . CF p a t i e n t s d e m o n s t r a t e s p e c i f i c

a c q u i r e d im m u n i ty t o t h e o r g a n i s m ( C h a p t e r 2 ) , a n d t h e

o r g a n i s m c a n c o m b a t t h i s o n l y by a n t i g e n i c v a r i a t i o n .

A n t i g e n i c v a r i a t i o n may b e d e f i n e d a s t h e p r o c e s s by

w h i c h an i n f e c t i n g o r g a n i s m c h a n g e s i t s a n t i g e n i c

s t r u c t u r e s u f f i c i e n t l y t o a v o i d e l i m i n a t i o n by t h e

h o s t s ' immune s y s t e m , a n d t h u s g a i n s a s e l e c t i v e

a d v a n t a g e . I n f e c t i o n s c a u s e d i n i t i a l l y by an

a p p a r a n t l y h o m o g e n o u s p o p u l a t i o n o f an o r g a n i s m c a n

g i v e r i s e t o a p o p u l a t i o n i m m u n o l o g i c a l l y d i s t i n c t

f r o m t h e p a r e n t s t r a i n . A n t i g e n i c v a r i a t i o n c a n b e

i n d u c e d i n m i c r o - o r g a n i s m s by t h e d e v e l o p m e n t o f

s p e c i f i c i m m u n i t y t o them w i t h i n t h e h o s t p o p u l a t i o n ,

a s i n t h e c a s e o f t h e i n f l u e n z a v i r u s , o r w i t h i n t h e

M a j o ri n d i v i r t u a l h o s t a s in t r e p a n o s o m i a s i s .

a n t i g e n i c v a r i a t i o n s in t h e I n f l u e n z a A v i r u s r e s u l t

in p a n d e m i c s e v e r y 10 t o 30 y e a r s , and a r e c a u s e d by

r e c o m b i n a t i o n s b e t w e e n v i r u s e s i n f e c t i n g man and o t h e r

a n i m a l s . M in o r c h a n g e s t h a t c a u s e e p i d e m i c s a r e

p r o b a b l y t h e r e s u l t o f p o i n t m u t a t i o n s , known a s

a n t i g e n i c d r i f t . C h a n g e s i n t h e s t r u c t u r e o f two

g l y c o p r o t e i n s , h a e m a g g l u t i n i n and n e u r a m i n i d a s e o c c u r ,

a n d m u t a n t s a r e s e l e c t e d o u t w h ich h a v e a g r o w t h

a d v a n t a g e i n t h e p r e s e n c e o f l i m i t i n g c o n c e n t r a t i o n s

o f t h e h o s t a n t i b o d y t o t h e p a r e n t s t r a i n . A n t i g e n i c

d r i f t w o u l d b e t h e m o s t l i k e l y t y p e o f a n t i g e n i c

v a r i a t i o n o c c u r r i n g i n p s e u d o m o n a s i n f e c t i o n i n C F .

A n t i g e n i c v a r i a t i o n o c c u r r i n g w i t h i n t h e

i n d i v i d u a l h o s t h a s b e e n m o s t s t u d i e d i n t h e p r o t o z o a ,

t h e t r e p a n o s o m e s , p l a s m o d i a and b a b a s i a g e n e r a . M o s t

l a b o r a t o r y w o r k h a s b e e n d o n e i n t h o s e g e n e r a w h ic h

i n f e c t a n i m a l s h o w e v e r , and t h e e v i d e n c e t h a t t h e sam e

phenomen o c c u r s i n man i s c i r c u m s t a n t i a l - * - ^ - * - . D o e s

a n t i g e n i c v a r i a t i o n o c c u r i n b a c t e r i a l d i s e a s e i n man?

T h e r e i s e v i d e n c e t h a t c h a n g e s i n t h e a n t i g e n i c

s t r u c t u r e o f c e l l w a l l LPS r e s u l t s i n s e r u m - r e s i s t a n t

s t r a i n s o f N e i s s e r i a g o n o r r h o e a , w h ic h i n t u r n a r e

r e s p o n s i b l e f o r d i s s e m i n a t e d disease-"*-The c h a n g e s

o b s e r v e d i n P . a e r u g i n o s a i n CF i n t h i s s t u d y may be

t h e r e s u l t o f a n t i g e n i c v a r i a t i o n , w h ich o c c u r r e d

d u r i n g t h e c o u r s e o f a c h r o n i c b a c t e r i a l i n f e c t i o n in

an i n d i v i d u a l human h o s t . P s e u d o m o n a s i s one o f t h e

m o s t v e r s i t i l e b a c t e r i a , a d a p t a b l e t o a w i d e r a n g e o f

e n v i r o n m e n t s , and a l i k e l y c a n d i d a t e t o r t h i s

p h e n o m e n o n . T e j e d o r e t a l - ^ 3 r e p o r t e d t h a t t h e s p u t a

o f CF p a t i e n t s c o l o n i s e d w i t h P . a e r u g i n o s a c o n t a i n e d

l a r g e n u m b e r s o f b a c t e r i o p h a g e s . I t w o u l d be o f

i n t e r e s t t o s t u d y t h e r o l e o f p h a g e s i n t h e m e c h a n i s m

o f p h e n o t y p e c h a n g e a n d a n t i g e n i c d r i f t i n p s e u d o m o n a s

i n CF.

RELATIONSHIP OF SOME PHENOTYPE CHANGES OF P . AERUGINOSA

TO THE CLINICAL CONDITION OF CF PATIENTS:

A HYPOTHESIS

P a t i e n t s who h a d b e e n c o l o n i s e d by p s e u d o m o n a s

r e l a t i v e l y r e c e n t l y a n d who w e r e i n a g o o d c l i n i c a l

c o n d i t i o n h a d s t r a i n s o f t h e o r g a n i s m s i m i l a r i n

c h a r a c t e r t o t h o s e f r o m o t h e r c l i n i c a l s o u r c e s . T he

c h a n g e s o f p h e n o t y p e o b s e r v e d i n t h i s s t u d y s e e m e d t o

o c c u r i n p a r a l l e l , s o t h a t a l m o s t h a l f t h e p a t i e n t s

h a d o r g a n i s m s w i t h a l l t h e f e a t u r e s o f ' c y s t i c '

s t r a i n s . P a t i e n t s w i t h ' c y s t i c ' s t r a i n s w e r e o l d e r

a n d h a d b e e n c o l o n i s e d by t h e o r g a n i s m f o r a l o n g e r

p e r i o d o f t i m e , a n d a l s o had a g r e a t e r i m p a i r m e n t o f

r e s p i r a t o r y f u n c t i o n t h a n p a t i e n t s w i t h common

s t r a i n s . P a t i e n t s c o l o n i s e d by o r g a n i s m s o f an

' i n t e r m e d i a t e ' t y p e w e r e m o re s e v e r e l y a f f e c t e d by CF

t h a n e i t h e r o f t h e o t h e r two g r o u p s , t h e i r r e s p i r a t o r y

f u n c t i o n w a s w o r s e a n d f i v e p a t i e n t s f ro m t h i s g r o u p

h a v e s u b s e q u e n t l y d i e d . On t h e b a s i s o f t h e f i n d i n g s

i n t h i s s t u d y t h e f o l l o w i n g h y p o t h e s i s i s p r o p o s e d

I n c h r o n i c b r o n c h o p u l m o n a r y i n f e c t i o n w i t h

P . a e r u g i n o s a in p a t i e n t s w i t h CF, t h e o r g a n i s m

u n d e r g o e s an e n v i r o n m e n t a l a d a p t a t i o n m ar k e d by

c h a n g e s i n s o m a t i c a n t i g e n s and s e n s i t i v i t y t o n o r m a l

human s e r u m . The p r o t e i n - l i p o p o l y s a c c h a r i d e c o m p l e x

o f t h e o u t e r m e m b r an e o f p s e u d o m o n a s i s

i m m u n o g e n i c l 8 4 , s o c h a n g e s i n i t s s t r u c t u r e may r e n d e r

h o s t d e f e n c e m e c h a n i s m s t e m p o r a r i l y l e s s e f f e c t i v e ,

and u p s e t t h e h o s t - p a r a s i t e r e l a t i o n s h i p t h a t e x i s t e d

b e t w e e n t h e CF p a t i e n t a n d p s e u d o m o n a s . P a t i e n t s

w h o s e h o s t d e f e n c e s a r e n o t a b l e t o a d j u s t t o t h e new

v a r i a n t s o f p s e u d o m o n a s d e t e r i o r a t e r a p i d l y and d i e .

P a t i e n t s who a r e a b l e t o a d a p t and s u r v i v e t h i s

i n t e r m e d i a t e p e r i o d may r e m a i n r e l a t i v e l y w e l l f o r

some y e a r s ( F i g u r e 1 3 ) . The a p p e a r a n c e o f t h e s e

v a r i a n t s t r a i n s o f p s e u d o m o n a s may r e p r e s e n t a

c r i t i c a l s t a g e i n t h e p r o g r e s s i o n o f CF. A g g r e s s i v e

c h e m o t h e r a p y t o r e d u c e b a c t e r i a l l o a d d u r i n g t h i s

p e r i o d o f c h a n g e i n t h e o r g a n i s m m i g h t a l l o w t h e h o s t

d e f e n c e s y s t e m t i m e t o f i n d a new e q u i l i b r i u m . A

p r o s p e c t i v e c o n t r o l l e d t r i a l i s o f c o u r s e n e c e s s a r y t o

t e s t t h i s h y p o t h e s i s .

T he p h e n o t y p e c h a r a c t e r i s t i c s o f t h e s t r a i n s w e r e

s t u d i e d f i r s t i n a b l i n d m a n n e r , b u t a l l o c a t i o n o f

p a t i e n t s t o g r o u p s on t h e b a s i s o f t h e i r sp u tu m

o r g a n i s m s w as r e t r o s p e c t i v e and o pen t o b i a s . I t was

a s s u m e d t h a t t h e p r o g r e s s i o n o f t h e d i s e a s e w o u ld be

p a r a l l e l e d by c h a n g e s i n t h e o r g a n i s m s h o w e v e r , an d

t h e f i n d i n g t h a t p a t i e n t s w e r e w o r s e i n t h e

i n t e r m e d i a t e g r o u p w a s u n e x p e c t e d . I t i s p o s s i b l e

FIGURE 13

THE RELATIONSHIP OF SOME PHENOTYPE CHANGES IN

P.AERUGINOSA TO THE CLINICAL CONDITION OF

C Y STIC F I B R O S I S PATIENTS

ORGANISM L o s s o f O t y p e r e a c t i o n .

E x p r e s s i o n o f PA a n t i g e n .

D e v e l o p m e n t o f s e r u m - s e n s i t i v i t y .

'com m on' ' i n t e r m e d i a t e '-------------->

s t r a i n s t r a i n

' c y s t i c '*

s t r a i n

S u d d e n d e t e r i o r a t i o n .

PATIENT

G ood c o n d i t i o n A d a p t t o c h a n g e R e l a t i v e l y w e l l .

i n p s e u d o m o n a s

a n t i g e n s E v e n t u a l s low

d e t e r i o r a t i o n .

that: the organism can progress in one steo from common to cystic, and we have no data on the genetic basis or linkage between the characteristics we have studied.The cystic form does seem to be the ’end-stage', in only- one sample from one patient in this group was an intermediate variant found. The fact that five patients in the intermediate group had several variant forms in their sputum at the same time suggests that the three characteristics are not closely linked genetically.

G. SUMMARY1. The majority of strains of P.aeruginosa isolated

from CF patients had three distinctive features when compared to controls; loss of the O-serotype reaction, presence of a new somatic antigen, and sensitivity to normal human serum.

2. These three phenotype characteristics were significantly associated with each other.

3. Patients who had recently been colonised by the organism and who were relatively fit, had strains with similar characteristics to control strains.

4. Patients who had been colonised for some years and were in a poorer condition had typically 'cystic' organisms, with all three of the above features.

5. Patients with 'intermediate' strains, having one or two of these features were the most severely affected by CF. The appearance of these variants may represent a critical stage in the progression of CF.

Chapter 7

BACTERICIDAL ACTIVITY OF CYSTIC FIBROSIS SERUM AGAINST

PSEUDOMONAS AERUGINOSA

A. INTRODUCTION AND LITERATURE REVIEW

In contrast to strains oE P. aeruginosa from the environment or routine clinical specimens, strains isolated Erom CF sputa are usually sensitive to the bactericidal action of normal human serum^-69,179 (Chapter 6). This bactericidal activity is complement-mediated and initiated by natural opsonic antibodies of the IgM classic. Hjzfiby & 011ingl69 noted that sera from three CF patients failed to kill the strains of pseudomonas from the patient's own sputum. These strains were.sensitive to normal human serum. Thomassen & Demko^^^ found a similar selective defect in five CF sera, and noted that in two cases the CF sera 'blocked' the bactericidal activity of normal human serum against the autologous strain of pseudomonas.

B. AIMS

The aim of this study was to investigate further the bactericidal activity of CF serum against autologous and heterologous strains of pseudomonas, and to study the effect of CF sera on the bactericidal activity of normal human serum.

C. METHODS1. SERA

Normal human serum (NHS) was obtained by pooling equal volumes of serum from five healthy volunteers. Cystic

Fibrosis serum (CFS) was obtained From 2 groups oF CP patients. The first group was composed of 51 patients, 30 males and 21 Females, age range 16-43 years, all of whom were chronically infected by P,aeruginosa, which had been isolated in every sputum sample For at least 6 months before the study. The second group of 10 patients, 4 males and 6 females, age range 16-49 years, had never had P.aeruginosa isolated from their sputum, and were considered to be free of this organism. Sera were stored in aliquots at -20^C and used within 2 months.

2. ORGANISMSSix strains of P.aeruginosa of known sensitivity to

NHS were used as a reference panel for comparing bactericidal activity of patients' sera (Table XXVI).An environmental strain (0985), a strain from a patient with pneumonia secondary to carcinoma of the bronchus (2361) and 2 CF strains (002 and 003) were selected from strains sent for serotyping to the Division of Hospital Infection, Central Public Health Laboratory . Strain 7/1 was the serum-sensitive variant of laboratory reference strain 7. Sputum from CF patients was processed by methods described in Chapter 3, and isolates of P.aeruginosa from each patient were identified.

T A B L E X X V I

BACTERICIDAL ACTIVITY OF CYSTIC F I B R O S IS SERUM AGAINST STRAINS OF PSEUDOMONAS AERUGINOSA FROM VARIOUS SOURCES.

TestStrainNumber

Scourceor

diagnosisSensitivity

to NHSNumber of sera with ba a) CF patients

colonised by P.aeruqinosa

ctericidal activity from b) CF patients free of

P.aeruqinosa n=10

0985 Intensive Care Unit

sink+ 20 10

2361 Pneumonia + 19 107/1 Laboratory + 20 10003 CF + 20 10002 CF - 1 07 Laboratory — 0 0

+ = S e n s i t i v e - = R e s i s t a n t 181

3. SERUM T E S T S

a ) Serum-sensitivitySerum sensitivity tests were performed as described

in Chapter 3. Each CF serum was tested for bactericidal activity against the reference panel of pseudomonas strains, and against the strains isolated from thatpatient's sputum. All strains from CF patients' sputa were tested for sensitivity to NHS.

b) Blocking factor activityEqual volumes (50 ul) of CFS and NHS were

pre-incubated for 1 hour at 37^C, and an aliquot of an 18 hour culture of pseudomonas diluted 1 in 100 in Hank's solution was added. After incubation for a further 1 hour, 20 ul of the mixture was spotted onto an agar plate. A further 20 ul was diluted logarthrimically in Hank's solution to 1 in 10,000, and 20 ul of each dilution plated to give a viable count.The same procedure was followed for control tubes in which the test organism was exposed to 1) NHS and Hank's solution to confirm the serum-sensitivity of the organism, 2) CFS and Hank's solution to monitor the bactericidal action of CFS alone, and 3) Hank’s solution alone to confirm the viability of the test organism.Sera from CF patients containing blocking factors were defined as those that did not kill the organism, but blocked the bactericidal action of NHS on that organism, such that the viable count from tubes containing both NHS and CFS was similar to that from the control tube in which the organism was held in Hanks's solution alone.

c ) A b s o r p t i o n o f s e r a

The absorbing strain of pseudomonas was grown in Tryptone Soya Broth (Oxoid) at 37(̂C for 4 hours. five nutrient agar plates were inoculated using a sterile swab and incubated overnight at 37^C. The growth was washed off with a 0*5% solution of phenol saline and centrifuged at 10,000 G for 20 minutes. The cells were resuspended in 1ml of serum and incubated at 37^C for 2 hours, then at 4^C overnight. The suspension was centrifuged for 10 minutes in an Eppendorf centrifuge at10,000 G, the serum taken off, and passed through a 0*45 um membrane filter (Millipore). Absorbed sera were tested for residual bactericidal and blocking activity.

d) Characterisation of selected cystic fibrosis seraTotal haemolytic complement was measured by the

method of Mayer^-^6. Immunoglobulin was measured by radial immunodiffusion. Ion-exchange chromatography was performed using DEAE cellulose, equilibrated with 0*1M phosphate buffer ph8. The serum was dialysed against this buffer for 24 hours and 1ml was processed through a 1cm by 15cm column with 0*02M phosphate buffer.Fractions (1ml) were collected and tested with rabbit antisera to human immunoglobulin IgG and IgM (Seward) by double-diffusion in agar gel^^.

4. CLINICAL ASSESSMENTRespiratory function was assessed in an out-patient clinic on the day of sample collection. Forced expiratory volume in one second (FEV-j_) , and forced vital

capacity (FVC) were measured on a Vita log rap’n spirometer, and were expressed as a percentage oi predicted normal values using tables published by Cotes-̂ -1-. Overall assessment was scored according to the method of Shwachman^ 2.

D. RESULTS

1. BACTERICIDAL ACTIVITY OF CYSTIC FIBROSIS SERA AGAINST P.AERUGINOSA

Sera from 20 CF patients with pseudomonas infection and 10 patients without it were tested for bactericidal activity against the reference panel of strains (Table XXVI). Sera from both groups of patients were bactericidal for all strains sensitive to NHS. Strains resistant to NHS were also resistant to all but 1 CF serum.

119 cultures of P.aeruginosa were isolated from the sputa of the of 51 CF patients, and 64 (53*8%) were sensitive to NHS. Sera from all 51 patients were tested for activity against autologous cultures, and all strains resistant to NHS were also found to be resistant to autologous CFS. Of the 64 sensitive strains, 54 v/ere killed by autologous CFS, but 10 NHS-sensitive strains derived from 7 patients were resistant to CFS from those individuals. Figure 14 shows an agar plate with a strain of pseudomonas that has been tested for sensitivity to NHS and to autologous CFS. The strain

FIGURE 14

THE BACTERICIDAL EFFECT OF AUTOLOGOUS CYSTIC FIBROSIS SERUM AND NORMAL HUMAN SERUM ON ONE CF PSEUDOMONAS STRAIN

Nutriant agar plate after 18 hours incubation

AUTOLOGOUS CF SERUM

20 pi f romundiluted sample

20 pi from s amp 1e diluted lin 100

NORMAL HUMAN SERUM

i s s e n s i t i v e t o Nl-lS, w i t h f i v e c o l o n i e s g r o w i n g from th e

u n d i l u t e d s a m p l e a n d a few more from t h e d i l u t e d s a m p l e .

The o r g a n i s m i s n o t k i l l e d by a u t o l o g o u s CFS in t h i s

c a s e , w i t h a p p r o x i m a t e l y 2 0 0 c o l o n i e s g r o w i n g f r o m t h e

u n d i l u t e d s a m p l e a n d c o n f l u e n t g r o w t h f ro m t h e s a m p l e

d i l u t e d 1 i n 1 0 0 .

2. INTERFERENCE BY CYSTIC FIBROSIS SERA WITH NORMAL HUMAM SERUM KILLING OF P.AERUGINOSA

The seven patients' sera that were not bactericidal for autologous strains of pseudomonas were tested for blocking activity. Six of the seven sera neutralised the bactericidal effect of NHS on the eight strains of pseudomonas from those six patients. (One serum that failed to kill its own two strains had no effect on NHS). The blocking potency of these six sera was determined in serial doubling dilution tests, and the blocking titre was defined as the highest dilution of CFS that resulted in a 1,000-fold increase in viable count, compared with the control tube containing the test strain with NHS and Hanks solution to the same final volume. Titres of 32, 32, 16, 16, 8 and 4 were obtained for the six patients. Table XXVII shows the results obtained with one CF serum containing a blocking factor.

3. STUDIES OF SERA WITH BLOCKING ACTIVITY AND THE AUTOLOGOUS STRAINS OF PSEUDOMONAS

The s i x CF s e r a w i t h ' b l o c k i n g ' a c t i v i t y h a d n o r m a l

concentrations of complement, and normal or raised

TABLE XXVII

EFFECT OF CYSTIC FIBROSIS SERUM FROM PATIENT B ON THE BACTERICIDAL

ACTIVITY OF NIIS FOR PSEUDOMONAS STRAIN 130b*

Test organism incubated with: Viable count cfu/ml

Hanks' so lu tio n 11

1 X 107

1CF serum + Hanks' so lu tion |

11*5 X 105

1NHS + Hanks' so lu t io n |

NHS + CFS undiluted111

1 X 107

1 X i—■ o

2*5 X 1—•

NHS + CFS d ilu ted 1 in (1

1 X 1—>

16 | 1

7 X 105

32 |i 2*5 X 105

2*5 X 103i

( 128 | 2 X CMO

* Autologous s t r a in for p a t ien t B. t T it re = 32

HS = normal humanFS = c y s t ic f i b r o s i s serum from patient B.

a m o u n t s o f .T.gG, IgM and I g A . The s e r a w o re b a c t e r i c i d a l

f o r t h e f o u r s e r u m - s e n s i t i v e s t r a i n s o f t h e r e f e r e n c e

p a n e l and t o t h r e e o t h e r N H S - s e n s i t i v e CF s t r a i n s . The

e i g h t , s t r a i n s o f p s e u d o m o n a s c o n c e r n e d w e r e t y p i c a l , o f

i s o l a t e s r e c o v e r e d f ro m CF s p u t a ( C h a p t e r 6 ) ; f i v e grew

a s m u c o i d c o l o n i e s , and a l l showed m u l t i p l e

a g g u l t i n a t i o n r e a c t i o n s w i t h O - a n t i s e r a . T h e s e

o r g a n i s m s w e r e r e p e a t e d l y r e t e s t e d f o r s e n s i t i v i t y t o

NHS a n d r e m a i n e d s e n s i t i v e . They w e r e a l s o k i l l e d by

o t h e r CF s e r a , b o t h f r o m p a t i e n t s c o l o n i s e d by

p s e u d o m o n a s ( n = 1 2 ) and f r o m t h o s e f r e e o f t h e o r g a n i s m

( n = 6 ) .

4 . CHARACTERISATION OF BLOCKING ACTIVITY

a ) S t r a i n - s p e c i f i c i t y

The s e r a an d s t r a i n s o f t h e s i x p a t i e n t s w i t h a

b l o c k i n g t i t r e w e r e c r o s s - t e s t e d f o r b a c t e r i c i d a l an d

b l o c k i n g a c t i v i t y . By d e f i n i t i o n a se r u m c o n t a i n i n g a

f a c t o r t h a t i n t e r f e r e s w i t h t h e k i l l i n g o f a p a r t i c u l a r

s t r a i n by NKS, d o e s n o t k i l l t h a t s t r a i n . T a b l e X X V I I I

sh o w s t h a t b l o c k i n g a c t i v i t y w as s e l e c t i v e f o r t h e

p a t i e n t ' s own s t r a i n i n t h r e e c a s e s (A, B and C) b u t

c r o s s - r e a c t i o n s w e r e o b s e r v e d w i t h t h e s e r a and s t r a i n s

o f t h e o t h e r t h r e e p a t i e n t s (D, E and F ) .

b) A b s o r p t i o n o f c y s t i c f i b r o s i s se r u m

A l i q u o t s o f f o u r CF s e r a w i t h b l o c k i n g a c t i v i t y w ere

a b s o r b e d u s i n g t h e a u t o l o g o u s s t r a i n s o f p s e u d o m o n a s .

The s e r a r e t a i n e d b l o c k i n g a c t i v i t y when s u b s e q u e n t l y

t e s t e d w i t h a u t o l o g o u s o r h e t e r o l o g o u s s t r a i n s .

T A B L E X X V I I I

PATTERN OF 'BLOCKING' AND BACTERICIDAL ACTIVITY OF SERA FROM S I X CF PATIENTS FOR AUTOLOGOUS AND HETEROLOGOUS STRAINS OF

PSEUDOMONAS AEURINOSA.

Serum from CF patient

Strain of P.aeruginosa from stated patient157aA

A + + - - + - • • • -B + + + + + - • • ♦ -

C + - - - + - m • • +D - - - - - + • • • -E - - - - • • • - + -

F - - - - • • • - • • • ++ = p r e s e n c e o f b l o c k i n g f a c t o r ( d e f e c t i v e k i l l i n g ) - = a b s e n c e o f b l o c k i n g f a c t o r ( n o r m a l k i l l i n g ). . . = n o t d o n e

c ) S e r u m f r a c t i o n s

I g G - r i c h f r a c t i o n s o f two CF s e r a o b t a i n e d by

i o n - e x c h a n g e c h r o m a t o g r a p h y i n t e r e f e r e c l w i t h t h e

b a c t e r i c i d a l a c t i o n o f WHS on a u t o l o g o u s s t r a i n s o f

p s e u d o m o n a s , b u t f r a c t i o n s w i t h o u t IgG d i d n o t e x h i b i t

b l o c k i n g a c t i v i t y ( T a b l e X X I X ) . No i n t r i n s i c

b a c t e r i c i d a l a c t i v i t y w a s f o u n d i n t h o s e f r a c t i o n s , e v e n

i n t h e p r e s e n c e o f e x c e s s c o m p le m e n t ( G u i n e a - p i g

c o m p l e m e n t , W e l l c o m e ) , when t e s t e d a g a i n s t a u t o l o g o u s o r

r e f e r e n c e s t r a i n s .

5 . CLINICAL CONDITION OF PATIENTS WITH BLOCKING FACTORS IN

C l i n i c a l d a t a f o r t h e s i x p a t i e n t s w i t h a b l o c k i n g

f a c t o r i n s e r u m a r e g i v e n i n T a b l e XXX. One p a t i e n t w as

i n a v e r y p o o r c o n d i t i o n w i t h a Shwachman s c o r e o f 35

an d d i e d d u r i n g t h e s t u d y . T h r e e p a t i e n t s had.

m o d e r a t e l y s e v e r e d i s e a s e w i t h s c o r e s o f 50 t o 5 5 , and

two w e r e i n g o o d c o n d i t i o n w i t h a s c o r e o f 7 5 . T h e r e

w a s no c o r r e l a t i o n b e t w e e n b l o c k i n g f a c t o r t i t r e and

c l i n i c a l s c o r e , r e s p i r a t o r y f u n c t i o n , o r d u r a t i o n o f

c o l o n i s a t i o n by p s e u d o m o n a s .

TABLE XXIXINTERFERENCE WITH BACTERICIDAL ACTION OF NORMAL HUMAN SERUM ON AUTOLOGOUS STRAINS OF

PSEUDOMONAS BY TWO CYSTIC F I B R O S I S SERA AND FRACTIONS OF THOSE SERA OBTAINED BYION-EXCHANGE CHROMATOGRAPHY

P a t i e n t S t r a i n o f p s e u d o m o n a s f r o m s t a t e d p a t i e n t

I n c u b a t i o nm i x t u r e

P r e s e n c e o f IgG i n s e r u m

f r a c t i o n

V i a b l ec o u n t c f u / m l

A 1 5 7 a H anks s o l u t i o n 1 . 5 x 1 0 5CFS + Hanks 1 x 1 0 6NHS + Hanks 50

NHS + CFS 2 x 1 0 5

NHS + f r a c t i o n (4 Y e s 1 . 5 x 1 0 5(5 Y e s 5 x 1 0 4(6 No 350

D 1 4 6 c H anks s o l u t i o n 2 . 5 x 1 0 5CFS + Hanks 1.6 x 1 0 6NHS + Hanks 30

NHS + CFS 4 . 5 x 1 0 5

NHS + f r a c t i o n (4 Y e s 9 x 1 0 5(5 Y es 6 . 5 x 105(6 No 50

NHS = n o r m a l human s e r u m CFS = c y s t i c f i b r o s i s s e r u m

T A B L E X X X

CLINICAL DATA OF S I X CYSTIC F I B R O S I S PATIENTS WITH BLOCKING ACTIVITY IN SERUM

P a t i e n t S e x Age % p r e d i c t e d FEVi

% p r e d i c t e d FVC

C l i n i c a l s c o r e

( r e f )

B l o c k i n gf a c t o rt i t r e

D u r a t i o n o f c o l o n i s a t i o n

byp s e u d o m o n a s

( y r s )A M 16 45 66 75 16 3

B F 31 26 32 35 32 1 . 5

C M 24 18 35 50 8 9

D F 22 23 27 50 4 3+

E M 20 24 39 55 16 14

F M 24 46 61 75 32 11

+ = a t l e a s t t h r e e y e a r s b u t no d a t a on f i r s t i s o l a t i o n o f o r g a n i s m .

F. DISCUSSION

1 . S E R U M - S E N S I T I V I T Y O F P . A E R U G I N O S A

G r a m - n e g a t i v e b a c t e r i a f r o m l o c a l i s e d i n f e c t i o n s a r e

u s u a l l y s e n s i t i v e t o t h e b a c t e r i c i d a l a c t i o n o f n o r m a l

h u m a n s e r u m , b u t i n s o m e c h r o n i c i n f e c t i o n s t h e o r g a n i s m

m a y b e c o m e s e r u m - r e s i s t a n t . T h i s m a y b e d u e t o

f u n c t i o n a l a b s e n c e o f a n t i g e n , a n t i b o d y o r c o m p l e m e n t .

A n t i g e n m a y b e m a s k e d b y o t h e r c o m p o n e n t s o n t h e c e l l

s u r f a c e , o r l o s t b y m u t a t i o n ; a n t i b o d y m a y b e d e f i c i e n t

o r u n a b l e t o f i x c o m p l e m e n t , o r m a y f a c e c o m p e t i t i o n f o r

b i n d i n g s i t e s f r o m a b l o c k i n g a n t i b o d y . C o m p l e m e n t

f a c t o r s m a y b e d e f i c i e n t o r f a i l t o b i n d t o t h e

b a c t e r i a l c e l l s u r f a c e . I n t h e c a s e o f N e i s s e r i a

g o n o r r h o e a e , p a t h o g e n i c s e r u m - r e s i s t a n t s t r a i n s a r e

r e s p o n s i b l e f o r d i s s e m i n a t e d d i s e a s e , a n d t h e m e c h a n i s m

m a y b e a c h a n g e i n t h e a n t i g e n i c s t r u c t u r e o f t h e c e l l

w a l l l i p o p o l y s a c c h a r i d e w i t h l o s s o f t h e l o c u s f o r l y t i c

a n t i b o d y - * - 8 2 # i n s o m e p a t i e n t s w i t h c h r o n i c u r i n a r y

t r a c t i n f e c t i o n s w i t h E s c h e r i c h i a c o l i a b l o c k i n g

a n t i b o d y o f t h e I g G c l a s s w a s f o u n d i n s e r u m , w h i c h w a s

r e m o v e d b y a b s o r p t i o n o f s e r u m w i t h t h e a p p r o p r i a t e

o r g a n i s m - 1 - 8 8 # i n c o n t r a s t t o o t h e r G r a m - n e g a t i v e

b a c t e r i a , p s e u d o m o n a s f r o m t h e e n v i r o n m e n t a n d m o s t

c l i n i c a l s p e c i m e n s a r e s e r u m - r e s i s t a n t , a l t h o u g h a f e w

s t r a i n s f r o m c h r o n i c i n f e c t i o n s m a y b e s e r u m

s e n s i t i v e J - 7 7 # i n c p h o w e v e r , t h e m a j o r i t y o f s t r a i n s

a r e s e r u m - s e n s i t i v e ^ ^ , 1 7 9 ( C h a p t e r 6 ) . A r e l a t i v e

i n a b i l i t y o f C F s e r a t o k i l l a u t o l o g o u s s t r a i n s o f

P . a e r u g i n o s a w a s ( f i r s t n o t e d b y H0 . i b y S O i l i n g - ^ 9 i n

t h r e e o u t o f 1 7 CP' p a t i e n t s s t u d i e d ( 1 7 * 6 %) T h o m a s s c n 5

D e m k o l 7 9 [ f o u n d a s i m i l a r d e f e c t i n t h e . s e r a o f f i v e o u t

o f <12 p a t i e n t s ( 1 1 * 9 % ) a n d n o t e d t h a t t h e s e f i v e

p a t i e n t s w e r e i n a p o o r c l i n i c a l c o n d i t i o n . I n t w o

c a s e s t h e y d e m o n s t r a t e d t h a t C F s e r a c o u l d ' b l o c k ' t h e

b a c t e r i c i d a l a c t i v i t y o f n o r m a l h u m a n s e r u m a g a i n s t t h e

a u t o l o g o u s s t r a i n . T h e p r e s e n t s t u d y h a s s h o w n t h a t CF

s e r a c a n k i l l s t r a i n s o f p s e u d o m o n a s f r o m t h e

e n v i r o n m e n t a n d f r o m o t h e r C F p a t i e n t s i n a s i m i l a r

m a n n e r t o n o r m a l h u m a n s e r u m .

2 . B L O C K I N G F A C T O R S I N C Y S T I C F I B R O S I S S E R U M

N a t u r a l b a c t e r i c i d a l a n t i b o d y t o p s e u d o m o n a s i s

l a r g e l y t h e I g M c l a s s - * - ^ . A s m a l l n u m b e r o f p a t i e n t s ,

( s e v e n o u t o f 5 1 i n d i v i d u a l s , 1 3 * 7 % ) , h a d a s e l e c t i v e

i n a b i l i t y t o k i l l a u t o l o g o u s s t r a i n s o f p s e u d o m o n a s t h a t

w e r e s e n s i t i v e t o NHS a n d t o s e r u m f r o m o t h e r C F

p a t i e n t s . A ' b l o c k i n g f a c t o r ' t h a t c o u l d b e t i t r a t e d

w a s f o u n d i n s i x C F s e r a , a n d i n t e r e f e r e d w i t h t h e

b a c t e r i c i d a l a c t i o n o f N H S o n t h e p a t i e n t ' s o w n s t r a i n

o f p s e u d o m o n a s . T h i s f a c t o r w a s d e m o n s t r a t e d t o b e

a s s o c i a t e d w i t h t h e I g G f r a c t i o n o f s e r u m , b u t w a s n o t

r e m o v e d f r o m t h e s e r u m b y a b s o r p t i o n w i t h t h e

a p p r o p r i a t e p s e u d o m o n a s s t r a i n .

T h e p r e s e n c e o f b l o c k i n g f a c t o r s i n s e r u m d i d n o t

r e l a t e t o t h e c l i n i c a l c o n d i t i o n o f t h e p a t i e n t i n t h i s

s m a l l g r o u p , i n c o n t r a s t t o t h e s t u d y o f T h o m a s s e n &

D e m k o l ’7 9 > ^ s p S e u < j 01T10na s r e m a i n s l o c a l i s e d w i t h i n t h e

r e s p i r a t o r y t r a c t i n C F , t h e p r e s e n c e o f a b l o c k i n g

a n t i b o d y i n s e r u m i s u n l i k e l y t o b e i m p o r t a n t

c l i n i c a l l y . I t w o u l d b e i n t e r e s t i n g , h o w e v e r , t o l o o k

f o r a s i m i l a r b l o c k i n g a n t i b o d y i n b r o n c h i a l s e c r e t i o n s .

D e f e c t i v e k i l l i n g o f p s e u d o m o n a s w i t h i n t h e b r o n c h i a l

t r e e w o u l d d e c r e a s e t h e r a t e o f b a c t e r i a l c l e a r a n c e , a n d

m i g h t b e a f a c t o r i n t h e i n i t i a l c o l o n i s a t i o n o f t h e

l u n g b y p s e u d o m o n a s .

I n C F p a t i e n t s w i t h b l o c k i n g f a c t o r s i n s e r u m , t h e

p s e u d o m o n a s s t r a i n h a d b e c o m e ' r e s i s t a n t ' t o t h e s e r u m

o f i t s h o s t , a n d t h e p o s s i b l e m e c h a n i s m s h e r e a r e

l i m i t e d . A c h a n g e i n b a c t e r i a l s u r f a c e a n t i g e n s w o u l d

a f f e c t t h e s e n s i t i v i t y o f t h e o r g a n i s m t o o t h e r s e r a ,

b u t i n t h e p r e s e n t s t u d y t h e o r g a n i s m s r e m a i n e d

s e n s i t i v e t o o t h e r s e r a . T h e C F p a t i e n t s s e r a w i t h

b l o c k i n g f a c t o r s w e r e b a c t e r i c i d a l t o o t h e r s e n s i t i v e

s t r a i n s , s o a d e f i c i e n c y o f l y t i c a n t i b o d y o r c o m p l e m e n t

i s u n l i k e l y , a n d t o t a l i m m u n o g l o b u l i n a n d c o m p l e m e n t

l e v e l s w e r e n o r m a l . A p o s s i b l e e x p l a n a t i o n m a y b e t h e

p r e s e n c e o f a b l o c k i n g a n t i b o d y t h a t a r i s e s a s a

c o n s e q u e n c e o f l o n g - s t a n d i n g b a c t e r i a l c o l o n i s a t i o n .

B l o c k i n g f a c t o r s w e r e d e m o n s t r a t e d i n t h e s e r a o f

t w o p a t i e n t s w i t h c h r o n i c . p s e u d o m o n a s i n f e c t i o n ( n o t C F )

b y G u t t m a n a n d Waisbrenl89s T h e s e w e r e i d e n t i f i e d a s

I g G w h i c h w a s a b s o r b e d f r o m t h e s e r u m b y t h e a u t o l o g o u s

s t r a i n . T h e s e a u t h o r s s u g g e s t e d a m e c h a n i s m o f

c o m p e t i t i v e i n h i b i t i o n b y I g G o f n a t u r a l l y t i c I g M

b i n d i n g t o t h e b a c t e r i a l c e l l s u r f a c e , t h e b o u n d I g G

b e i n g u n a b l e t o f i x c o m p l e m e n t a s s u c c e s s f u l l y a s

m u l t i v a l e n t I g M . I n t h a t s t u d y o n e s e r u m was t e s t e d

a g a i n s t a o a n e l o f s t r a i n s r e p r e s e n t i n g t h e F i s c h e r

s e r o t y p e s a n d t h e p a t t e r n o f b a c t e r i c i d a l a c t i v i t y o f

t h e p a t i e n t ' s s e r u m w a s s i m i l a r t o t h a t o b s e r v e d w i t h

M B S . K i l l i n g o f o t h e r s e n s i t i v e s t r a i n s w a s n o t b l o c k e d

b y t h e s e r u m e v e n w h e n t h e s t r a i n s w e r e o f t h e s a m e

s e r o t y p e . T h e a u t h o r s c o n c l u d e d t h a t t h e a n t i g e n

b i n d i n g s i t e o f t h e b l o c k i n g f a c t o r w a s n o t t h a t

r e c o g n i s e d b y r a b b i t t y p i n g s e r a . T h i s m e c h a n i s m d o e s

n o t s e e m t o a p p l y t o b l o c k i n g f a c t o r s i n C F s e r u m a s

t h e y w e r e n o t a l w a y s s t r a i n - s p e c i f i c a n d w e r e u n a f f e c t e d

b y a b s o r p t i o n w i t h t h e p s e u d o m o n a s s t r a i n a n d t h e r e f o r e

n o t b o u n d t o a c e l l u l a r s i t e . T h o m a s s e n & D e m k o l 7 9 a l s o

s t a t e t h a t a b s o r p t i o n v j i t h t h e a u t o l o g o u s s t r a i n f a i l e d

t o r e m o v e b l o c k i n g a c t i v i t y . I g G f r a c t i o n s i n t h i s

s t u d y h a d n o i n t r i n s i c b a c t e r i c i d a l a c t i v i t y , e v e n i n

t h e p r e s e n c e o f e x c e s s c o m p l e m e n t . An a l t e r n a t i v e

e x p l a n a t i o n i s t h a t t h e I g G b l o c k i n g a n t i b o d y i s

d i r e c t e d a g a i n s t t h e n a t u r a l l y o c c u r r i n g b a c t e r i c i d a l

I g M . T h e s e l e c t i v e n a t u r e o f t h e d e f e c t i n

s e r u m - k i l l i n g o f p s e u d o m o n a s i n C F h a s n o t b e e n

d o c u m e n t e d e l s e w h e r e . I t i m p l i e s t h a t w i t h i n t h e

n a t u r a l b a c t e r i c i d a l I g M o f t h e C F p a t i e n t t h e r e i s a

s u b g r o u p s p e c i f i c t o t h e d o m i n a n t s t r a i n o f p s e u d o m o n a s

i n t h a t p a t i e n t , a n d t h a t b l o c k i n g I g G a n t i b o d y e f f e c t s

o n l y t h i s p a r t i c u l a r s u b - g r o u p o f I g M .

A r e c e n t r e p o r t a l s o d e s c r i b e s a n a n t i b o d y o f t h e

I g G c l a s s f r o m C F s e r u m w h i c h f u n c t i o n s t o t h e a d v a n t a g e

o f p s e u d o m o n a s ! 9 Q . S e r u m f r o m C F p a t i e n t s i s k n o w n t o

i n h i b i t p h a g o c y t o s i s a n d k i l l i n g of : p s e u d o m o n a s b y b o t h

r a b b i t a n d h u m a n a l v e o l a r m a c r o p h a g e s , a n d t h e

i n h i b i t i o n h a s b e e n a t t r i b u t e d t o a h e a t - s t a b l e s e r u m

f a c t o r 9 5 ̂ e f f i c i e n t p h a g o c y t o s i s o f p s e u d o m o n a s b y

p o l y m o r p h o n u c l e a r l e u c o c y t e s o r m a c r o p h a g e s r e q u i r e s

a n t i b o d y f o r o p s o n i s a t i o n . P i c k e t a l l - 9 0 i n v e s t i g a t e d

t h e p o s s i b i l i t y t h a t t h e i n h i b i t i o n o f p h a g o c y t i c

a c t i v i t y b y C F s e r u m w a s d u e t o t h e p r e s e n c e o f a

d e f e c t i v e a n t i b o d y . T h e s e a u t h o r s u s e d a f f i n i t y

c h r o m a t o g r a p h y w i t h l i p o p o l y s a c c h a r i d e a n t i g e n f r o m a

p s e u d o m o n a s o f F i s c h e r i m m u n o t y p e 4 ( I A T S t y p e 1 ) t o

i s o l a t e I g G f r o m 9 C F p a t i e n t s s e r u m . T h e s e C F I g G

a n t i b o d i e s d e c r e a s e d t h e r a t e o f p h a g o c y t o s i s o f t h e

s a m e p s e u d o m o n a s s t r a i n b y a l v e o l a r m a c r o p h a g e s f r o m

h e a l t h y v o l u n t e e r s . I n t r a c e l l u l a r k i l l i n g o f t h e

o r g a n i s m w a s r e d u c e d t o a l e s s e r e x t e n t . T h e C F I g G

m o l e c u l e s w e r e s h o w n t o c o a t t h e b a c t e r i a v e r y w e l l , b u t

d i d n o t a t t a c h t o t h e m a c r o p h a g e m e m b r a n e . A d e f e c t i n

t h e F c e n d s o f t h e h e a v y c h a i n s o f t h e s e I g G m o l e c u l e s

w a s p r o p o s e d t h a t r e d u c e d t h e i r a b i l i t y t o b i n d t o t h e

a l v e o l a r m a c r o p h a g e m e m b r a n e F c r e c e p t o r s .

T h e d e f e c t i v e I g G f r o m C F s e r u m d e s c r i b e d b y P i c k e t

a l l 9 0 r e d u c e d t h e a b i l i t y o f a l v e o l a r m a c r o p h a g e s f r o m

h e a l t h y i n d i v i d u a l s t o p h a g o c y t o s e p s e u d o m o n a s o f o n e

p a r t i c u l a r i m m u n o t y p e . T h e p r e s e n t s t u d i e s h a v e l o o k e d

o n l y a t s e r u m - k i l l i n g o f p s e u d o m o n a s , a n d n o t

i n v e s t i g a t e d t h e r o l e o f p h a g o c y t i c c e l l s . T h e a n t i b o d y

d e s c r i b e d b y F i c k e t a l w a s f o r t h e i r n m u n o t y p e F i s h e r 4

( I A T S t y p e 1 ) , w h i c h w a s r e p r e s e n t e d i n o n l y 2 « 9 ? > o f t h e

t y o a b l e s t r a i n s , a n d i n o n l y 2 * 1 ? > o f t h o r e a c t i o n s o f

p o l y a g g l u t i n a b l e s t r a i n s s t u d i e d i n C h a p t e r 6 , ( T a b l e

X X I I ) . T h i s a n t i b o d y a d h e r e d w e l l t o t h e b a c t e r i a l

c e l l , u n l i k e t h e b l o c k i n g f a c t o r s o f t h e p r e s e n t s t u d y .

A g r e a t d e a l o f f u r t h e r w o r k w i l l b e n e e d e d t o e l u d i c a t e

t h e r o l e o f a b n o r m a l I g G i n t h e h o s t d e f e n c e s o f C F

p a t i e n t s a g a i n s t p s e u d o m o n a s c o l o n i s a t i o n .

F . SUMMARYT h e b a c t e r i c i d a l a c t i o n o f s e r u m f r o m 6 1 a d u l t

p a t i e n t s w i t h c y s t i c f i b r o s i s a g a i n s t a u t o l o g o u s a n d

h e t e r o l o g o u s s t r a i n s o f P s e u d o m o n a s a e r u g i n o s a h a s b e e n

s t u d i e d . C F s e r u m h a d a s i m i l a r b a c t e r i c i d a l a c t i o n t o

n o r m a l h u m a n s e r u m a g a i n s t a r e f e r e n c e p a n e l o f s t r a i n s .

S i x C F s e r a h a d a s e l e c t i v e i n a b i l i t y t o k i l l a u t o l o g o u s

s t r a i n s o f p s e u d o m o n a s w h i c h w e r e s e n s i t i v e t o N H S a n d

t o s e r a f r o m o t h e r C F p a t i e n t s . T h e s i x s e r a h a d n o r m a l

l e v e l s o f c o m p l e m e n t a n d i m m u n o g l o b u l i n a n d w e r e

b a c t e r i c i d a l t o o t h e r s t r a i n s . A t i t r a t a b l e b l o c k i n g

f a c t o r w a s p r e s e n t i n t h e s e s e r a w h i c h i n t e r f e r e d w i t h

t h e b a c t e r i c i d a l a c t i o n o f NHS o n t h e a u t o l o g o u s s t r a i n .

T h i s f a c t o r w a s p r e s e n t i n t h e I g G c o n t a i n i n g f r a c t i o n s

o f s e r u m o b t a i n e d b y i o n - e x c h a n g e c h r o m a t o g r a p h y , b u t

w a s n o t r e m o v e d f r o m t h e s e r u m b y a b s o r p t i o n w i t h t h e

p s e u d o m o n a s s t r a i n .

C F s e r a m a y f a i l t o k i l l s e n s i t i v e s t r a i n s o f

p s e u d o m o n a s b e c a u s e o f t h e d e v e l o p m e n t o f a b l o c k i n g I g G

a n t i b o d y a g a i n s t n a t u r a l l y o c c u r r i n g b a c t e r i c i d a l I g M

a n t i b o d y .

Chapter 8

CONCLUSIONS AND GENERAL DISCUSSION

A . SUMMARY O F P O S I T I V E F I N D I N G S

1 . T e n d a y s t r e a t m e n t w i t h i n t r a v e n o u s g e n t a m i c i n a n d

t i c a r c i l l i n , o r g e n t a m i c i n a n d c a r b e n i c i 1 1 i n i n I S C F

p a t i e n t s s i g n i f i c a n t l y i n c r e a s e d m e a n P E F R , F E V ^ a n d F V C

c o m p a r e d w i t h b a s e l i n e v a l u e s .

2 . T e n d a y s t r e a t m e n t w i t h i n t r a v e n o u s g e n t a m i c i n a n d

a z l o c i l l i n , o r g e n t a m i c i n a n d c a r b e n i c i l l i n i n 2 0 C F

p a t i e n t s s i g n i f i c a n t l y i n c r e a s e d m e a n P E F R , F E V ^ , F V C

a n d p a t i e n t s o w n a s s e s s m e n t o f w e l l - b e i n g w h e n c o m p a r e d

w i t h b a s e l i n e v a l u e s .

3 . T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e i n t h e d e g r e e o f

i m p r o v e m e n t i n r e s p i r a t o r y f u n c t i o n b e t w e e n t h e g r o u p s

o f p a t i e n t s t r e a t e d w i t h e i t h e r a z l o c i l l i n o r

t i c a r c i l l i n c o m p a r e d w i t h c a r b e n i c i l l i n i n e a c h o f t h e

a b o v e t r i a l s .

4 . D e s p i t e a d e q u a t e s e r u m l e v e l s , s p u t u m p e n e t r a t i o n b y a l l

t h r e e p e n i c i l l i n s a n d b y g e n t a m i c i n w a s p o o r .

5 . A e r o s o l g e n t a m i c i n a n d c a r b e n i c i l l i n g i v e n t w i c e d a i l y

f o r s i x m o n t h s i n 1 7 C F p a t i e n t s s i g n i f i c a n t l y i n c r e a s e d

m e a n P E F R , FEV] _ a n d F V C c o m p a r e d w i t h p l a c e b o . T h e

f r e q u e n c y o f h o s p i t a l a d m i s s i o n s m a y b e r e d u c e d b y t h e

a c t i v e t r e a t m e n t .

6 . R e t r o s p e c t i v e a n a l y s i . s o f 4 1 p a t i e n t s o n a e r o s o l

c h e m o t h e r a p y s h o w e d a r e d u c t i o n i n t h e f r e q u e n c y o f

h o s p i t a l , a d m i s s i o n s c o m p a r e d w i t h t h e y e a r b e f o r e

t r e a t m e n t .

7 . 3 0 p a t i e n t s , s e l e c t e d a t r a n d o m , o n a e r o s o l a n t i b i o t i c s

h a d a h i g h e r i n c i d e n c e o f p e n i c i l l i n - r r e s i s t a n t

p s e u d o m o n a s o v e r a 6 m o n t h s p e r i o d t h a n 3 0 p a t i e n t s n o t

r e c e i v i n g t h i s t r e a t m e n t o v e r t h e s a m e p e r i o d . T h i s

r e s i s t a n c e t o p e n i c i l l i n s v z a s a t e m p o r a r y p h e n o m e n o n

t h a t d i d n o t p r e s e n t a c l i n i c a l p r o b l e m .

8 . An a i r c o m p r e s s o r w i t h h i g h f l o w r a t e i s r e q u i r e d t o

c r e a t e a f i n e a e r o s o l o f a e r o d y n a m i c m a s s m e d i u m

d i a m e t e r o f 3 m i c r o n s o r l e s s f r o m a n t i b i o t i c s o l u t i o n s .

9 . M o s t s t r a i n s o f P . a e r u g i n o s a i s o l a t e d f r o m CE1 p a t i e n t s

l o o s e t h e O - s e r o t y p e r e a c t i o n , e x p r e s s P A a n t i g e n a n d

a r e s e n s i t i v e t o n o r m a l h u m a n s e r u m . ( T h e ' c y s t i c '

f o r m )

1 0 . T h e s e t h r e e c h a r a c t e r i s t i c s o f l o s s o f O - s e r o t y p e

r e a c t i o n , p r e s e n c e o f p o l y a g g l u t i n a t i n g a n t i g e n a n d

s e n s i t i v i t y t o s e r u m a r e s i g n i f i c a n t l y a s s o c i a t e d w i t h

e a c h o t h e r .

1 1 . P a t i e n t s r e c e n t l y c o l o n i s e d b y p s e u d o m o n a s t e n d t o

h a v e o r g a n i s m s w i t h s e r o l o g i c a l c h a r a c t e r i s t i c s s i m i l a r

t o e n v i r o n m e n t a l o r g a n i s m s a n d t o t h o s e f r o m o t h e r

s o u r c e s . ( T h e 1 c o m m o n ' f o r m )

1 2 . P a t i e n t s w i t h o r g a n i s m s p o s s e s s i n g o n e o r t w o o f t h e

a b o v e p h e n o t y p e c h a n g e s , ( t h e ' i n t e r m e d i a t e ' f o r m ) a r e

g e n e r a l l y i n a p o o r c l i n i c a l c o n d i t i o n .

1 3 . C F s e r u m h a s s i m i l a r b a c t e r i c i d a l a c t i v i t y t o n o r m a l

h u m a n s e r u m a g a i n s t h e t e r o l o g o u s s t r a i n s o f

P . a e r u g i n o s a .

1 4 . S i x o u t o f 5 1 C F s e r a h a d a s e l e c t i v e i n a b i l i t y t o

k i l l a u t o l o g o u s s t r a i n s o f p s e u d o m o n a s .

1 5 . A t i t r a t a b l e b l o c k i n g f a c t o r w a s f o u n d i n t h e a b o v e

s i x s e r a w h i c h i n t e r e f e r e d w i t h t h e b a c t e r i c i d a l a c t i o n

o f n o r m a l h u m a n s e r u m o n t h e a u t o l o g o u s s t r a i n o f

p s e u d o m o n a s .

1 6 . T h i s b l o c k i n g f a c t o r w a s a s s o c i a t e d w i t h t h e I g G

f r a c t i o n o f s e r u m a n d v / a s n o t r e m o v e d b y a b s o r p t i o n o f

t h e s e r u m w i t h t h e a p p r o p r i a t e s t r a i n o f p s e u d o m o n a s .

B. DISCUSSION AND PROSPECTS FOR FUTURE WORKT h e p r e s e n t s t u d i e s h a v e p r o v i d e d c l e a r e v i d e n c e

t h a t t h e r e i s a t h e r a p e u t i c e f f e c t o f t r e a t m e n t o f a c u t e

e x a c e r b a t i o n s o f p u l m o n a r y d i s e a s e i n C F p a t i e n t s w i t h

P . a e r u g i n o s a i n f e c t i o n , u s i n g i n t r a v e n o u s

a n t i p s e u d o m o n a l a n t i b i o t i c s i n h o s p i t a l . T h e r e w a s no

s u g g e s t i o n t h a t t h e r e w a s a g r e a t e r c l i n i c a l r e s p o n s e t o

a n a n t i b i o t i c w h i c h h a d g r e a t e r a c t i v i t y t o w a r d s

P . a e r u g i n o s a i n v i t r o .

A c o n t r o l l e d t r i a l o f c h e m o t h e r a p y a n d h o s p i t a l c a r e

( i n c l u d i n g p h y s i o t h e r a p y ) v e r s u s h o s p i t a l c a r e a l o n e

s h o u l d n o w b e u n d e r t a k e n . T h i s i s e x t r e m e l y i m p o r t a n t ,

b o t h b e c a u s e o f t h e e n o r m o u s c o s t o f a n t i b i o t i c s a n d t h e

t r a u m a o f i n t r a v e n o u s i n f u s i o n s t o t h e p a t i e n t s , a l l o f

w h i c h m i g h t p r o v e t o b e u n n e c e s s a r y . S u c h a t r i a l

w o u l d h a v e t o i n c l u d e a s u f f i c i e n t l y l a r g e n u m b e r o f

p a t i e n t s t o p r o d u c e c o n c l u s i v e d a t a , s o t h a t w e r e t h e

r e s u l t p o s i t i v e i n f a v o u r o f c h e m o t h e r a p y , i t n e e d n e v e r

b e r e p e a t e d . T h i s b a s i c q u e s t i o n n e e d s t o b e a n s w e r e d

b e f o r e f u r t h e r s t u d i e s o n i n d i v i d u a l r e g i m e n s a r e

u n d e r t a k e n , a n d b e f o r e t h e q u e s t i o n o f r e g u l a r t r e a t m e n t ,

r e g a r d l e s s o f h e a l t h ( a s s u g g e s t e d b y H y f i b y e t a l l 9 1 )

v e r s u s t r e a t m e n t o n l y d u r i n g e x a c e r b a t i o n s c a n b e

c o n s i d e r e d .

F u r t h e r w o r k i s o b v i o u s l y n e c e s s a r y t o e s t a b l i s h t h e

m o s t e f f i c i e n t n e b u l i z e r s y s t e m f o r a e r o s o l a n t i b i o t i c s ,

a n d t h e p r a c t i c a l d e t a i l s o f r e g i m e n s t o b e u s e d . A

s i n g l e d r u g m a y b e a s e f f e c t i v e a s a c o m b i n a t i o n a n d

l e s s t i m e c o n s u m i n g t o n e b u l i z e . T h e d o s e t o b e u s e d ,

a n d t h e n u m b e r of : d o s e s p e r d a y i s n o t e s t a b l i s h e d .

T h e r e m a y a l r s o b e a r o l e t o r a e r o s o l a n t i b i o t i c s i n t h o

t r e a t m e n t o f a c u t e e x a c e r b a t i o n s o f C F p u l m o n a r y

disease.T h e h y p o t h e s i s t h a t a n t i g e n i c v a r i a t i o n o c c u r s i n

P . a e r u g i n o s a d u r i n g t h e c o u r s e o f c h r o n i c p u l m o n a r y

i n f e c t i o n i n C F , r e q u i r e s t h e t e s t o f a l o n g i t u d i n a l

s t u d y o f p a t i e n t s i n i t i a l l y f r e e o f t h e o r g a n i s m . T h e

c h e m i c a l n a t u r e o f t h e p s e u d o m o n a s s u r f a c e a n t i g e n s

i n v o l v e d i n t h e p h e n o m e n a o f p o l y a g g l u t i n a t i o n , l o s s o f

0 - s e r o t y p e a n d s e r u m s e n s i t i v i t y n e e d s t o b e e l u c i d a t e d .

T h e s e r o l o g i c a l a n d g e n e t i c b a s i s o f t h e a s s o c i a t i o n

b e t w e e n t h e s e p h e n o m e n a i s u n k n o w n .

T h e p r e s e n c e o f b l o c k i n g f a c t o r s w h i c h i n t e r f e r e d

w i t h n o r m a l h u m a n s e r u m k i l l i n g o f C F p s e u d o m o n a s i s

j u s t o n e o f m a n y r e p o r t s o f i s o l a t e d d e f e c t s i n t h e h o s t

d e f e n c e s a g a i n s t t h i s o r g a n i s m i n C F . T h e r e l e v a n c e ,

i f a n y , t o t h e p a t h o g e n e s i s o f t h e d i s e a s e i s o b s c u r e .

I f s u c h f a c t o r s w e r e f o u n d i n b r o n c h i a l s e c r e t i o n s t h e y

m i g h t b e w o r t h y o f f u r t h e r i n v e s t i g a t i o n .

APPENDIX 1

VALIDATION OF THE SERUM-SENSITIVITY ASSAY

a) Size of the innoculum.b) Length of the incubation period.c) Age of the broth culture.d) Dilution of normal human serum.e) Definition of serum-sensitivity.

V A L I D A T I O N O F T H E S E R U M - S E N S I T I V I T Y A S S A Y

A series of experiments were performed to establish the conditions for the serum-sensitivity test before commencing the main studies. NHS and strain 7/1, a serum-sensitive varient of laboratory strain 7 (the propagating strain for phage 7) were used throughout.

a ) S I Z E O F T H E I N N O C U L U M

MethodAn o v e r n i g h t b r o t h c u l t u r e o f s t r a i n 7/1

( a p p r o x i m a t e l y 1 0 ^ o r g a n i s m s / m l ) w a s d i l u t e d

l o g a r i t h m i c a l l y i n H a n k s s o l u t i o n t o 1 i n 1000.Aliquots of the neat culture and each dilution were incubated with equal volumes (0*5mls) of NHS, and with controls of Hanks solution at 37^0. At each time interval 20 ul of the incubation mixture was spotted onto NA plates, a further 20 ul was diluted logarithmically in Hanks solution to 1 in 10 million, and 20 ul of each dilution plated for viable count. Samples were taken immediately, and at 15 minutes,1 hrs, 2 hrs, 4 hrs and 24 hrs after starting the incubation. Plates were incubated at 37^C for 18 hours and the colonies counted.Results

The viable counts at each dilution of the overnight broth culture are shown in Table XXXI. The neat culture, and the dilution of 1 in 10 were affected very little by NHS, whereas at a dilution of 1 in 1000

TABLE XXXI

THE EFFECT OF THE SIZE OF THE INNOCULUM ON THE VIABLE COUNT

IN THE SERUM SENSITIVITY TEST

Viable count in cfu/ml for each dilution of the overnight broth culture of pseudomonas 7/1.

Lengthof

incubat ion period

1 hr2 hr 4 hr

1 x 108 1 x 107

2*5 x 107

1-5 x 107

1 x 108

1 in 102 x 107 2 x 106

4*5 x 106

1 x 103 7*5 x 103

1 in 100 2*5 x 104

4 x 103

1*5 x 102

1 in 1000 5*5 x 104

24 hr 1*9 x 1010 8 x 106 7*5 x 105 5 x 102

killing was almost complete by 15 minutes. At a dilution of 1 in 100 a viable count of 150 organisms/ml (3 colonies from a 20 ul aliquot) was obtained at 1 hour.Conclusion

Overnight broth cultures were diluted in 1 in 100 to give easily readable viable counts.

b) LENGTH OF INCUBATION PERIOD Method

Equal volumes (1 ml) of a 1 in 100 overnight broth culture of strain 7/1 and of NHS were incubated at 37^C. Aliquots were taken for viable counts as described above, immediately, and at ihr, 1 hr, 4 hrs, 7 hrs, and 24 hrs after the start of incubation. Control tubes contained Hanks solution instead of NHS.Results

The results are shown in Figure 15. By 1 hour the viable count had fallen by 1000 fold, and a further decrease of 10 fold was seen at 4 hours. By 7 hours the organism was beginning to recover and had recovered completely in 24 hours.Conclusion

Incubation period of 1 hour was considered optimal for the bactericidal effect of serum to bedemonstrated.

le cou

nt (cf

F I G U R E 1 5

EFFECT OF THE LENGTH OF THE INCUBATION PERIOD

ON VIABLE COUNT IN THE SERUM-SENSITIVITY TEST

Strain 7 /1Normal human serum

Length of incubation period (hours)

c ) AGE OF BROTH CULTURE

MethodAn overnight broth culture was diluted 1 in 50 in

broth and kept in a water bath at 37^C for 24 hours. At intervals, aliquots were removed and diluted 1 in 10 in Hanks solution and tested immediately for serum sensitivity, as described above, using an incubation period of 1 hour. Control tubes contained Hanks solution instead of serum.Results

Control tubes gave a viable count of 5x10^ to 8-5x107 over the three time intervals studied (4, 7 and 24 hours), whereas tubes containing NHS gave viable counts of 100 - 3000 at the same times.Conclusion

The age of the broth culture is not important. An overnight broth culture that has been kept a further 24 hours remains serum sensitive. In practice, overnight broth cultures were never kept for longer than a further 6 hours.

d ) D I L U T I O N O F NORMAL HUMAN S E R U M

M e t h o d

NHS was diluted serially in Hanks solution to a dilution of 1 in 64. Equal volumes of each dilution of serum and a 1 in 100 overnight broth culture of strain 7/1 were incubated at 37°C for 1 hour. Viable counts were determined as above, with control tubes of Hankssolution instead of serum.

R e .suit sThe final incubation mixture contained 50% serum. A

further dilution of 1 in 4 was bactericidal to strain 7/1, such that the viable count was reduced by a 1000 fold compared to the control. These results are shown in Table XXXII together with actual colony counts on the agar plate at two dilutions of the test mixture. Conclusion

Normal human serum was bactericidal to strain 7/1 up to a dilution of 1 in 8 of the final incubation mixture. All experimental work used NHS at a final dilution of 1 in 4 or less.

e) DEFINITION OF SERUM-SENSITIVITYSerum-sensitive cultures were defined as those where

the viable count was reduced by 1000 fold in NHS compared to the control in Hanks solution. For convenience in large numbers of experiments it was decided to count aliquots at two dilutions of the incubation mixture, neat and 1 in 100, since these gave satisfactory results on repeated tests with sensitive strains 7/1, 0985, 2361 and 003. Serum-sensitivity was also defined in terms of actual colony counts in agar plate in this system. Thus serum-sensitive cultures were those where 50 colonies or less grew from 20 ul of the undiluted serum mixture. Partially sensitive cultures gave counts of 51 to 200 colonies in the undiluted sample, and often recovered when diluted. Resistant cultures gave confluent growth in both samples.

TABLE XXXII

THE EFFECT OF DILUTION OF NORMAL HUMAN SERUM ON IT'S

BACTERICIDAL ACTION TOWARDS STRAIN 7/1

1Dilution of | NHS in | incubation | mixture |

Viable count from

a) Undiluted incubation mixture

on20 p 1 1 1 1 1

agar plate lofb) 1 in 100 dilution of incubation mixture

Viable count cfu/ml

1Neat | 1

1 in 2 | 1 1111

1 in 4 | | 811I 0 400

11 in 8 | 1 100

111 1 5000

11 in 16 | 1 Confluent

2 8 x 1031

1 in 31 | 1 Confluent111 14 7 x 104

11 in 64 |

11Confluent

35 5 x 105

Control | 1 1

Confluent

50 6*5 x 105

APPENDIX II

ASSAYS AND LABORATORY PROCEDURES NOT PERFORMED BY THE AUTHOR

a) Preparation of O-antisera for serotyping of P.aeruginosa.

b) Analysis of aerosol particle size.c) Antibiotic assays.d) Initial isolation of pseudomonas from sputum.e) Measurement of immunoglobulin levels in CF sera.

ASSAYS AMD LABORATORY PROCEDURES MOT PERFORMED BYTHE AUTHOR

a ) Preparat ion of f i -an t i ser a for serotyoing of

P.aeruginosa

The O-serotype strains used by the Central Public Health Laboratory are given in Table XXXIII. Details of the preparation of strains are as follows

The serotype strain is plated on nutrient agar, incubated at 37^C overnight, and five colonies are added to 5mls of nutrient broth. After incubation at 37^C lor 4 hours the culture is added to lOOmls of nutrient broth in a 500ml bottle for adequate aeration, and incubated for a further 18 hours at 37^C. The culture is boiled in a sterilizer for 1 hour, centrifuged at 3000 rpm for 45 minutes, the cell deposit washed once in saline and resuspended in 20mls of saline to give a final concentration of 2 to 4x10^ cells/ml. The suspension is stored at 4^C. Rabbits of 2.5 to 3 Itilograms in size are injected intravenously at 3-4 day intervals with the following doses:-

0'25mls, 0*5mls, lml, l*5mls, 2mls and 2mls.A test bleed of 5mls of blood is made after the

first 2ml injection, and if the titre is satisfactory the animal is bled out by heart puncture three days after the final injection. Serum is separated and passed through a 0*45 micron membrane filter, 0*1% (w/v) sodium azide is added as a preservative and serum is stored at 4^C.

TABLE XXXIII

O-SEROLOGICAL TYPING OF PSEUDOMONAS AERUGINOSA

O-TYPE STRAINS

C.P .H .L .1Type

Source I ATS2 ATCC No.

0:1 Habs 0:1 333480:2a Habs 0:2 333490:2b Veron _30:3 Habs 0:3 333500:4 Habs 0:4 333510: 5d Veron 0 :5 40:6 Habs 0:6 333540: 6a C.P.H.L. -

0:6c C.P.H.L. -

0: 6d Wahba (14) -0:7 Habs 0:7 333530:8 Habs 0:8 333550:9 Habs 0:9 333560:10 Habs 0:10 333570:11 Habs 0:11 333 5 80:12 Habs 0:12 333590:13 Sandvik ( I I ) 0:13 333600:14 Verder & Evans (V) 0:14 333610:15 Lanyi (170022) 0:15 333620:16 Lanyi (Ps 11) 0:16 333630:17 Meitert 0:17 33364

1 Central Public Health Laboratory (London).- Internat ional Antigenic Typing Scheme ̂ Sub-type not represented.

4 Type s t r a in of IATS = Habs 5 ca l led 5 c by Veron (ATCC 33352). We find that 5 c c ro s s - r e a c t s with other types so we used 5 d in preference.

5 Numbers in brackets = au thor ' s o r ig in a l type or s t r a in number.

E a c h s e r u m i s t i t r a t e d w i t h t h e h o m o l o g o u s s t r a i n

u s i n g t h e m i c r o t i t r e m e t h o d . T i t r e s a r e g e n e r a l l y o f

t h e o r d e r o f 3 2 0 t o 1 2 8 0 . C o m m o n c r o s s - r e a c t i o n s

a m o n g s t t y p e s t r a i n s a r e : -

0 : 1 / 0 : 5 / 0 : 1 6

0 : 7 / 0 : 8

0 : 4 / 0 : 1 1 .

S e r a a r e u s u a l l y r e n d e r e d s p e c i f i c b y a s i n g l e

a b s o r p t i o n .

S u b - t y p e s e r a 0 : 2 / 0 : 5 / 0 : 1 6

S p e c i f i c s e r a a r e o b t a i n e d b y t h e f o l l o w i n g

a b s o r p t i o n s : -

2 a s e r u m a b s o r p e d w i t h s t r a i n 1 6

2 b " " " " 5 c

5 d " " " " 5 c

1 6 " ............... 5 d

F o r g r e a t e r e f f i c i e n c y w i t h l a r g e n u m b e r s o f t e s t s ,

f o u r p o o l s o f p o l y v a l e n t s e r a a r e m a d e , a n d o r g a n i s m s

t e s t e d a g a i n s t t h e s e b e f o r e t e s t i n g w i t h t h e i n d i v i d u a l

s e r a f r o m e a c h p o s i t i v e p o o l .

P o o l I I I I I I I V

S e r u m 0 : 1 0 : 2 a 0 : 6 0 : 9

0 : 3 0 : 2 b 0 : 1 5 0 : 1 0

0 : 4 0 : 5 d 0 : 1 7 0 : 1 1

0 : 7 0 : 1 6 0 : 1 2

0 : 8 0 : 1 3

S e r u m 0 : 1 4 i s t e s t e d s e p a r a t e l y w i t h s t r a i n s n o t

a g g l u t i n a t e d b y a n y o f t h e t y p e s e r a .

b ) A N A L Y S I S O F A E R O S O L P A R T I C L E S I Z E

T h i s w a s p e r f o r m e d b y M i s s M C l a y a t t h e D e p a r t m e n t

o f T h o r a c i c M e d i c i n e , R o y a l F r e e H o s p i t a l . D e t a i l s o f

m e t h o d s a r e g i v e n i n C h a p t e r 5 .

C) A N T I B I O T I C A S S A Y S

T h e s e w e r e p e r f o r m e d b y M r H W i n g f i e l d a t t h e

D e p a r t m e n t o f M i c r o b i o l o g y a t t h e B r o m p t o n H o s p i t a l .

D e t a i l s o f m e t h o d s a r e g i v e n i n C h a p t e r 4 .

d ) I N I T I A L I S O L A T I O N O F P S E U D O M O N A S FROM C F S P U T U M

T h i s w a s p e r f o r m e d b y M r D E R o b e r t s , o f t h e H o s t

D e f e n c e U n i t , C a r d i o t h o r a c i c I n s t i t u t e .

e ) A S S A Y O F I M M U N O G L O B U L I N L E V E L S ON C F S E R A

T h i s w a s p e r f o r m e d b y t h e b i o c h e m i s t r y l a b o r a t o r y

o f t h e B r o m p t o n H o s p i t a l c o u r t e s y o f D r M W K e m p .

a c k n o w l e d g e m e n t s

I a m i n d e b t e d t o t h e f o l l o w i n g : -

D r M a r g a r e t E H o d s o n M S c MD F R C P DA, S e n i o r L e c t u r e r a n d

H o n o r a r y P h y s i c i a n , B r o m p t o n H o s p i t a l , w h o w a s m y o v e r a l

s u p e r v i s o r a n d m e n t o r , a n d w h o s u p e r v i s e d t h e c l i n i c a l

w o r k .

D r T L P i t t P h D , P r i n c i p a l M i c r o b i o l o g i s t , C e n t r a l P u b l i c

H e a l t h L a b o r a t o r y , f o r t e a c h i n g m e a b o u t p s e u d o m o n a s a n d

s u p e r v i s i n g t h e l a b o r a t o r y w o r k .

D r J C B a t t e n MD F R C P , P h y s i c i a n , B r o m p t o n H o s p i t a l , f o r

a l l o w i n g m e t o s t u d y t h e p a t i e n t s u n d e r h i s c a r e , a n d f o r

a w a k e n i n g m y i n t e r e s t i n c y s t i c f i b r o s i s i t s e l f .

T h e s t a f f o f t h e G r a m N e g a t i v e S e p s i s U n i t , D i v i s i o n o f

H o s p i t a l I n f e c t i o n , C e n t r a l P u b l i c H e a l t h L a b o r a t o r y ,

C o l i n d a l e f o r l a b o r a t o r y f a c i l i t i e s , a n d p a r t i c u l a r l y M r s B

H y d e f o r t e c h n i c a l a s s i s t a n c e .

M r D R o b e r t s F I M L S , H o s t D e f e n c e U n i t , C a r d i o t h o r a c i c

I n s t i t u t e , f o r i n i t i a l i s o l a t i o n o f p s e u d o m o n a s c u l t u r e s

f r o m s p u t u m .

D r J G r a n g e MD F R C P a t h , D e p a r t m e n t o f M i c r o b i o l o g y ,

C a r d i o t h o r a c i c I n s t i t u t e , f o r l a b o r a t o r y f a c i l i t i e s .

M i s s M R e h a h n , G r o u p S t a t i s t i c i a n , f o r s t a t i s t i c a l a d v i c e .

T h e M e d i c a l A r t a n d P h o t o g r a p h i c D e p a r t m e n t s o f t h e R o y a l

M a r s d e n H o s p i t a l f o r f i g u r e s a n d i l l u s t r a t i o n s .

M r H W i n g f i e l d , M i c r o b i o l o g y D e p a r t m e n t , B r o m p t o n H o s p i t a l ,

f o r a n t i b i o t i c a s s a y s .

M i s s M C l a y B S c , D e p a r t m e n t o f T h o r a c i c M e d i c i n e , R o y a l

F r e e H o s p i t a l , f o r a e r o s o l p a r t i c l e s i z e a n a l y s i s .

" 1 1 9

M i s s S H o c k l e y f o r p r e p a r i n g t h e m a n u s c r i p t . .

T h e F r a n c o s a n d A u g u s t u s M e w m a n F o u n d a t i o n f o r f i n a n c i a l

s u p p o r t w h i l e c o n d u c t i n g t h i s s t u d y .

BIBLIOGRAPHY

1 . W o o d R E , B o a t T F , D o e r s h u k C F .

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2 . d i S a n t ' A g n e s e P A , D a v i s P B .

C y s t i c f i b r o s i s i n a d u l t s . 7 5 c a s e s a n d a r e v i e w o f 2 3 2

c a s e s i n t h e l i t e r a t u r e .

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3 . K u l c z y c k i L L , M u r p h y T M , B e l l a n t i J A .

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0. B e a r n <\G .

G e n e t i c s o l : C y s t i c F i b r o s i s .

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9 . T h o m p s o n GW.

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C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n , 1 9 8 0 : 2 8 1 - 9 1 .

1 0 . D i S a n t ' A g n e s e P A , D a r l i n g R C , P e r e r a G A , S h e a E .

A b n o r m a l e l e c t r o l y t e c o m p o s i t i o n o f s w e a t i n c y s t i c f i b r o s i s

o f t h e p a n c r e a s .

P e d i a t r i c s , 1 9 5 3 ; 1 2 : 5 4 9 - 6 3 .

1 1 . R e i d L .

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F o u n d a t i o n , 1 9 8 0 : 1 9 8 - 2 1 4 .

1 2 . H o d s o n C J , F r a n c e N E .

P u l m o n a r y c h a n g e s i n c y s t i c f i b r o s i s o f t h e p a n c r e a s . A

r a d i o - p a t h o l o g i c a l s t u d y .

C l i n R a d i o l 1 9 6 2 ; 1 3 : 5 4 - 6 1 .

1 3 . D a y G , M e a r n s M B .

B r o n c h i a l l a b i l i t y i n c y s t i c f i b r o s i s .

A r c h D i s C h i l d 1 9 7 3 ; 4 8 : 3 5 5 - 9 .

1 4 . O r m e r o d L P , T h o m s o n R A , A n d e r s o n C M , S t a b l e f o r t h D E .

T h o r a x 1 9 3 1 ; 3 5 : 7 6 8 - 7 2 .

R e v e r s i b l e a i r w a y s o b s t r u c t i o n i n c y s t i c f i b r o s i s .

1 5 . M c a r n s M B , L o n g b o t t o m J L , B a t t e n J C .

P r e c i p i t a t i n g a n t i b o d i e s t o A s p e r g i l l u s f u m i g a t u s i n

c y s t i c f i b r o s i s .

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1 6 . N e l s o n L A , C a l . l e r a m e M L , S c h w a r t z R H .

A s p e r g i l l o s i s a n d a t o p y i n c y s t i c f i b r o s i s .

Am R e v R e s n i r D i s 1 9 7 9 ; 1 2 0 : 3 6 3 - 7 3 .

1 7 . H a n d w e r g e r 3 , R o t h J , G o r d e n P , d i S a n t ' A g n e s e P A ,

C a r p e n t e r D F , P e t e r G .

G l u c o s e i n t o l e r a n c e i n c y s t i c f i b r o s i s .

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1 3 . R o d m a n HM, M a t t h e w s LW.

H y p e r g l y c a e m i a i n c y s t i c f i b r o s i s : a r e v i e w o f t h e

l i t e r a t u r e a n d o u r o w n p a t i e n t e x p e r i e n c e .

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p a p e r . U n i v e r s i t y o f M i n n e s o t a , D e p a r t m e n t o f P e d i a t r i c s ,

1 9 8 1 , p 6 7 - 7 S .

1 9 . M i t c h e l l - H e g g s P , M e a r n s M, B a t t e n J C .

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O b s t r u c t i v e a z o o s p e r m i a a s a d i a g n o s t i c c r i t e r i o n f o r t h e

c y s t i c f i b r o s i s s y n d r o m e .

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C y s t i c f i b r o s i s a n d p r e g n a n c y .

L a n c e t 1 9 8 0 ; 2 : 8 4 2 - 4 .A n a t i o n a l s u r v e y .

22. P r i o r J , C r a w f o r d A D .

S y s t e m i c a m y l o i d o s i s c o m p l i c a t i n g c y s t i c f i b r o s i s .

P. r J D i s C h e s t 1 9 8 0 ; 7 4 : 8 4 - 6 .

2 3 . G i b s o n L E , C o o k e R E .

A t e s t f o r c o n c e n t r a t i o n o f e l e c t r o l y t e s i n s w e a t i n c y s t i c

f i b r o s i s o f t h e p a n c r e a s u t i l i s i n g p i l o c a r p i n e b y

i o n t o p h o r e s i s .

P e d i a t r i c s 1 9 5 9 ; 2 3 : 5 4 5 - 9 .

2 4 . H o d s o n M E , B e l d o n I , D u n c a n F , B a t t e n J C .

S w e a t t e s t s i n t h e d i a g n o s i s o f c y s t i c f i b r o s i s i n a d u l t s ,

M o n o g r P e d i a t r 1 9 8 1 ; 1 4 : 2 1 4 .

2 5 . H a d o r n 8 , Z o p p i G , S c h m e r l i n g D H , P r a d e r M, M c I n t y r e I ,

A n d e r s o n C M .

Q u a l i t a t i v e a s s e s s m e n t o f e x o c r i n e p a n c r e a t i c f u n c t i o n i n

i n f a n t s a n d c h i l d r e n .

J . P e d i a t r 1 9 6 8 ; 7 3 : 3 9 - 5 0 .

2 6 . D a n d o n a P , H o d s o n M E , B e l l J , R a m i d i a l L , B e l d o n I , B a t t e n

S e r u m i m m u n o r e a c t i v e t r y p s i n i n c y s t i c f i b r o s i s .

T h o r a x 1 9 8 1 ; 3 6 : 6 0 - 6 2 .

2 7 . J u n g l e e D , P e n k e t h A , K a t r a k A , H o d s o n M S , B a t t e n J C , D a n d o n a

S e r u m p a n c r e a t i c l i p a s e a c t i v i t y i n c y s t i c f i b r o s i s .

B r M e d J 1 9 8 3 , I n p r e s s .

2 8 . P r y o r J A , W e b b e r B A , H o d s o n ME , B a t t e n J C .

E v a l u a t i o n o f t h e f o r c e d e x p i r a t i o n t e c h n i q u e a s a n a d j u n c t t o

p o s t u r a l d r a i n a g e i n t h e t r e a t m e n t o f c y s t i c f i b r o s i s .

B r M e d J 1 9 7 9 ; 2 : 4 1 7 - 8 .

2 9 . W o o d R E , W a n n e r A , n i r s c h J , F a r r e l l PM.

T r a c h e a l m u c o c i l i a r y t r a n s p o r t i n p a t i e n t s w i t h c y s t i c

C i b r o s i s a n d i t s s t i m u l a t i o n b y t e r b u t i l i n e .

Am R e v R e s p i r D i s 1 9 7 5 ; 1 1 1 : 7 3 3 - 3 .

3 0 . P e n k e t h A R L , K n i g h t R K , H o d s o n M E , B a t t e n J C .

M a n a g e m e n t o f p n e u m o t h o r a x i n a d u l t s w i t h c y s t i c f i b r o s i s .

T h o r a x 1 9 8 2 ; 3 7 : 8 5 0 - 3 .

3 1 . S a k u l a A .

B r o n c h i a l a s t h m a d u e t o a l l e r g y t o p a n c r e a t i c e x t r a c t , a

h a z a r d i n t h e t r e a t m e n t o f c y s t i c f i b r o s i s .

B r J D i s C h e s t 1 9 7 7 ; 7 1 : 2 9 5 - 9 .

3 2 . Z e n t l e r - M u n r o P L , F i n e D R , B a t t e n J C , N o r t h f i e l d T C .

Ho w c i i n e t i d i n e i m p r o v e s l i p i d d i g e s t i o n a n d s o l u b i l i s a t i o n

i n c y s t i c f i b r o s i s .

M o n o g r P e d i a t r 1 9 8 1 ; 1 4 : 1 7 5 .

3 3 . A l l e n J D , M a s o n A , M o s s A D .

N u t r i t i o n a l s u p p l e m e n t a t i o n i n t r e a t m e n t o f c y s t i c f i b r o s i s

o f t h e p a n c r e a s .

Am J D i s C h i l d - 1 9 7 3 ; 1 2 6 : 2 2 - 6 .

3 4 . D o d g e J A , S a l t e r D G , Y a s s a , J G .

E s s e n t i a l f a t t y a c i d d e f i c i e n c y d u e t o a r t i f i c a l d i e t i n

B r M e d J 1 9 7 5 ; 2 : 1 9 2 - 3 .

3 5 . H o d s o n M E , H e a r n s M B , B a t t e n J C .

' M e c o n i u m i l e u s e q u i v a l e n t ' i n a d u l t s w i t h c y s t i c f i b r o s i s o f

t h e p a n c r e a s : a r e p o r t o f 6 c a s e s .

Br Med J 1 9 7 6 ; 2 : 7 9 0 - 1 .

36. St raker G , Kut liner M .

Psychological compensation in the individiuil with a

1. l f: e-threateninn i l l n e s s : a study of: adolescents with

S A f r M e d J 1 9 8 0 ; 5 1 : 6 1 - 2 .

3 7 . S h w a c h i n a n H , C a t z e l P , P a t t e r s o n P R , S t o p p l e m e n M.

• M u c o v i s c i d o s i s : An e v a l u a t i o n o f c o n t i n u o u s a n d p r o l o n g e d

a n t i b i o t i c t h e r a p y .

Am J D i s C h i l d 1 9 5 4 ; 8 8 : 3 8 0 - 3 .

3 3 . M e a r n s M B .

T r e a t m e n t a n d p r e v e n t i o n o f p u l m o n a r y c o m p l i c a t i o n s o f

c y s t i c f i b r o s i s i n i n f a n c y a n d e a r l y c h i l d h o o d .

A r c h D i s C h i l d 1 9 7 2 ; 4 7 : 5 - 1 1 .

3 9 . P o g u e R E , W a r w i c k W J .

C y s t i c F i b r o s i s . A n e w c h a l l e n g e t o i n t e r n a l m e d i c i n e .

M i n M e d 1 9 6 9 ; 5 2 : 1 5 5 1 - 5 .

4 0 . D i S a n t ' A g n e s e P A , A n d e r s o n D H .

C e l i a c s y n d r o m e

Am J D i s C h i l d 1 9 4 6 ; 7 2 : 1 7 - 6 1 .

4 1 . S h w a c h m a n H , C r o c k e r A C , F o l e y G E , P a t t e r s o n P R .

A u r e o m y c i n t h e r a p y i n t h e p u l m o n a r y i n v o l v e m e n t o f

p a n c r e a t i c f i b r o s i s ( m u c o v i s c i d o s i s ) .

N E n g l J M e d 1 9 4 9 ; 2 4 1 : 1 8 5 - 9 2 .

4 2 . M e a r n s M B , H u n t G H , R u s h w o r t h R .

B a c t e r i a l f l o r a o f r e s p i r a t o r y t r a c t i n p a t i e n t s w i t h

c y s t i c f i b r o s i s , 1 9 5 0 - 7 1 .

A r c h D i s C h i l d 1 9 7 2 ; 4 7 : 9 0 2 - 7 .

4 3 . M c a r n s MB .

N a t u r a l h i s t o r y o f p u l m o n a r y i n f e c t i o n i n c y s t i c f i b r o s i s .

I n : S t u r g e s s J M , c d . P e r s p e c t i v e s i n c y s t i c f i b r o s i s .

P r o c e e d i n g s o f 3 t h I n t e r n a t i o n a l C y s t i c F i b r o s i s C o n g r e s s ,

T o r o n t o , C a n a d a : C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n , 1 9 8 0

3 2 5 - 3 3 4 .

4 4 . D o g g e t t R G , H a r r i s o n G M, S t i l l w e l l R N , W a l l i s E S .

An a t y p i c a l P s e u d o m o n a s a e r u g i n o s a a s s o c i a t e d w i t h c y s t i c

f i b r o s i s o f t h e p a n c r e a s .

J P e d i a t r 1 9 5 6 ; 6 8 : 2 1 5 - 2 1 .

4 5 . M a y J R , H e r r i c k N C , T h o m p s o n D .

B a c t e r i a l i n f e c t i o n i n c y s t i c f i b r o s i s .

A r c h D i s C h i l d 1 9 7 2 ; 4 7 : 9 0 8 - 1 3 .

4 6 . D o g g e t t R G , H a r r i s o n G M , C a r t e r R E .

M u c o i d P s e u d o m o n a s a e r u g i n o s a i n p a t i e n t s w i t h c h r o n i c

i l l n e s s .

L a n c e t 1 9 7 1 ; 1 : 2 3 6 - 7 .

4 7 . B u r n s MW, M a y J R .

B a c t e r i a l p r e c i p i t i n s i n s e r u m o f p a t i e n t s w i t h c y s t i c

f i b r o s i s .

L a n c e t 1 9 6 8 ; 1 : 2 7 0 - 2 .

4 8 . R o b e r t s D E , C o l e P .

U s e o f r e l a t i v e m e d i a i n b a c t e r i o l o g i c a l i n v e s t i g a t i o n o f

p a t i e n t s w i t h c h r o n i c s u p p u r a t i v e r e s p i r a t o r y i n f e c t i o n .

L a n c e t 1 9 8 0 ; 1 : 7 9 6 - 7 .

\G e o r g e R H , G i l l e t t A P , R o y c r o f t C A .

H a e m o p h i l u s i n f e c t i o n s i n c y s t i c f i b r o s i s .

L a n c e t 1 9 8 0 ; 2 : 2 6 2 .

5 0 . H u a n g MH, H i l l e r H J , M a o r i C M , C a p i t a n o M, C u n d y H R .

C a r b e n i c i 1 1 i n i n p a t i e n t s w i t h c y s t i c f i b r o s i s . C l i n i c a l

p h a r m a c o l o g y a n d t h e r a p e u t i c e v a l u a t i o n .

J P e d i a t r 1 9 7 1 ? 7 8 : 3 3 8 - 4 5 .

5 1 . D u n c a n F R , H o d s o n M E , B a t t e n J C .

C y s t i c f i b r o s i s - s u r v i v a l i n t o a d u l t l i f e .

E u r J P e d i a t r 1 9 8 1 ? 1 3 7 : 1 2 5 .

5 2 . W r i g h t P F , K h a w K T , O x m a n MN, S h w a c h m a n H.

E v a l u a t i o n o f t h e s a f e t y o f a m a n t a d i n e - h y d r o c h l o r i d e , a n d

t h e r o l e o f r e s p i r a t o r y v i r a l i n f e c t i o n s i n c h i l d r e n

w i t h c y s t i c f i b r o s i s .

J I n f e c t D i s 1 9 7 6 ; 1 3 4 : 1 4 4 - 9 .

5 3 . P e t e r s e n N T , H o i b y N , M o r d h o r s t C H , L i n d K , F l e n s b o r g E W,

B r u u n B .

R e s p i r a t o r y i n f e c t i o n s i n c y s t i c f i b r o s i s c a u s e d b y v i r u s ,

c h y l a m y d i a a n d m y c o p l a s m a - p o s s i b l e s y n e r g i s m w i t h

P s e u d o m o n a s a e r u g i n o s a .

I n : S t u r g e s s J M , e d . P e r s p e c t i v e s i n c y s t i c f i b r o s i s .

T o r o n t o , C a n a d a : C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n , 1 9 8 0

3 4 6 - 5 1 .

5 4 . E f t h i m i o u J , T a y l o r P , T a y l o r AG, H o d s o n M E , B a t t e n J C .

T h e i m p o r t a n c e o f v i r u s e s , M y c o p l a s m a p n e u m o n i a e a n d

L e g i o n e l l a p n e u m o p h i l a i n a c u t e r e s p i r a t o r y e x a c e r b a t i o n s

i n p a t i e n t s w i t h c y s t i c f i b r o s i s .

B J D i s C h e s t 1 9 8 1 ; 7 5 : 3 1 8 .

5 5. f J o 1 s o 1 n w D S , K n t z SM.

Legionnaire ' s d i s e a s e complicating the pulmonary

les ion of c y s t i c f i b r o s i s .

I n : S t u r g e s s J M , c d . P e r s p e c t i v e s i n c y s t i c f i b r o s i s .

T o r o n t o , C a n a d a : C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n ,

1 9 8 0 : 1 9 a .

5 6 . B o x e r b a u m B .

I s o l a t i o n o f r a p i d l y - g r o w i n g m y c o b a c t e r i a i n p a t i e n t s

J P e d i a t r 1 9 8 0 ; 9 6 : 6 8 9 - 9 1 .

5 7 . E f t h i m i o u J , H o d s o n M E , B a t t e n J C .

M y c o b a c t e r i a l i n f e c t i o n s i n c y s t i c f i b r o s i s , a r e p o r t o f

t h r e e c a s e s .

( P e r s o n a l c o m m u n i c a t i o n ) .

5 8 . G e d d e s AM.

" O t h e r i n f e c t i o n s " .

I n : S a b a t h L D e d . ' P s e u d o m o n a s A e r u g i n o s a : t h e o r g a n i s m ,

d i s e a s e s i t c a u s e s a n d t h e i r t r e a t m e n t ' .

B e r n e ; H a n s H u b e r , 1 9 8 0 : 3 9 - 1 0 2 .

5 9 . W o l f f S M , B e n n e t t J V .

G r a m - n e g a t i v e r o d b a c t e r a e m i a .

N E n g l J M e d 1 9 7 4 ; 2 9 1 : 7 3 3 - 4 .

6 0 . R h a m e F S .

" T h e e c o l o g y a n d e p i d e m i o l o g y o f P s e u d o m o n a s a e r u g i n o s a " .

I n : S a b a t h LD e d .

' P s e u d o m o n a s a e r u g i n o s a : t h e o r g a n i s m , d i s e a s e s i t c a u s e s

a n d t h e i r t r e a t m e n t ' . B e r n e ; H a n s H u b e r , 1 9 8 0 : 3 1 - 5 1 .

5 1 . P h i l l i p s I .

I <;]e n i: i f L c a l : i o n o <; P s e u J o m o n a s A o r n g i n o a a i n t h g c l i n i c s 1

l a b o r a c o r y .

.T M e d M i a r ; o b i o l 1 9 6 9 ; 2: 9 - 1 6 .

6 2 . Xi nq F0, Xa r d MX, Raney D F .

T w o s i m p l e m e d i a t i e r d e m o n s t r a t i o n o f o v o c y a n i n a n d

f l u o r e s c i n .

.7 L a b C l i n M e d 1 9 5 4 ; 4 4 : 3 0 1 - 7 .

6 3 . X i n g A, P h i l l i p s I .

T h e i d e n t i f i c a t i o n o f p s e u d o m o n a d s a n d r e l a t e d b a c t e r i a i n a

c l i n i c a l l a b o r a t o r y .

J M e d M i c r o b i o l 1 9 7 8 ; 1 1 : 1 6 5 - 1 7 6 .

6 4 . G o v a n J R W .

P y o c i n t y p i n g o f P s e u d o m o n a s A e r u g i n o s a .

I n : B a r g a n T , N o r r i s J R e d s . ' M e t h o d s i n M i c r o b i o l o g y ' .

L o n d o n : A c a d e m i c P r e s s , 1 9 8 0 : V o l u m e 1 0 , 6 1 - 9 1 .

6 5 . P i t t T L .

A s t u d y o f t h e h e a t - l a b i l e a n t i g e n s o f P s e u d o m o n a s

A e r u g i n o s a

P h D T h e s i s , C N A A , 1 9 7 9 .

6 6 . P i t t T L .

S t a t e o f t h e A r t : T y p i n g P s e u d o m o n a s A e r u g i n o s a

J H o s p I n f e c t 1 9 8 0 ; 1 : 1 9 3 - 9 .

6 7 . L a n y i 3 , B e r g a n T .

S e r o l o g i c a l t y p i n g o f P s e u d o m o n a s A e r u g i n o s a

I n : R p i ' r r p r t , N o r r i s J R e d s . ' M e t h o d s i n M i c r o b i o l o g y '

L o n d o n : A c a d e m i c P r e s s , 1 9 8 0 : V o l u m e 1 0 , 9 3 - 1 6 8

6 0 . D o q a e t 1 RG , H a r r i s o n GM , Wa 1 1 i s E S .

Comparison of: some p ro p e r t ie s of Pseudomonas aeruginosa

i s o l a t ed from in f e c t i o n s in persons with and v;ithout c y s t i c

fLbrosis.J B a c t e r i d 1 9 6 4 ; 8 7 : 4 2 7 - 3 1 .

69. Martin DR.

M u c o i d v a r i a t i o n i n P s e u d o m o n a s a e r u g i n o s a i n d u c e d b y t h e

a c t i o n o f p h a g e .

J M e d M i c r o b i o l 1 9 7 3 ? 6 : 1 1 1 - 8 .

7 0 . E v a n s L R , L i n k e r A .

P r o d u c t i o n a n d c h a r a c t e r i s a t i o n o f t h e s l i m e p o l y s a c c h a r i d e

o f P s e u d o m o n a s a e r u g i n o s a .

J B a c t e r i d 1 9 7 3 ; 1 1 6 : 9 1 5 - 2 4 .

7 1 . L a m J , C h a n R , L a m K , C o s t e r t o n J W .

P r o d u c t i o n o f m u c o i d m i r o c o l o n i e s b y P s e u d o m o n a s a e r u g i n o s a

w i t h i n i n f e c t e d l u n g i n c y s t i c f i b r o s i s .

I n f e c t I m m u n 1 9 8 0 ; 2 8 : 5 4 6 - 5 6 .

7 2 . C a s h H A , W o o d s D E , M c C u l l o u g h B , J o h a n s e n WG, B a s s J A .

A r a t m o d e l o f c h r o n i c r e s p i r a t o r y i n f e c t i o n w i t h

Am R e v R e s p i r D i s 1 9 7 9 ; 1 1 9 : 4 5 3 - 9 .

7 3 . K e l l y NM, F a l k n e r F R , K e a n e C T , F i t z g e r a l d F X , T e m p a n y E .

M u c o i d G r a m - n e g a t i v e b a c i l l i i n c y s t i c f i b r o s i s .

L a n c e t 1 9 8 3 ; 1 : 7 0 5 .

7 4 . G o v a n J R W .

M u c o i d s t r a i n s o f P s e u d o m o n a s a e r u g i n o s a : t h e i n f l u e n c e o f

c u l t u r e m e d i u m o n t h e s t a b i l i t y o f m u c u s p r o d u c t i o n .

J Med M i c r o b i o l 1 9 7 5 ; 8 : 5 1 3 - 2 2 .

7 5 . G o v a n J R W , F v f e J A M .

M u c o i d P s e u d o m o n a s a e r u g i n o s a a n d c y s t i c f i b r o s i s :

r e s i s t a n c e o f m u c o i d f o r m t o c a r ’o e n i c i l . l i n , f l u c l o x a c i 1 1 i n

a n d t o b r a m c y i n a n d t h e i s o l a t i o n o f m u c o i d v a r i a n t s i n

v i t r o .

J A n t . i m i c r o b C h e m o t h e r 1 9 7 8 ; 4 : 2 3 3 - 2 4 0 .

7 6 . T h o m a s s e n M J , D e m k o C A , B o x e r b a u m B , S t e r n R C , K u c h e n b r o d p J .

M u l t i p l e i s o l a t e s o f P s e u d o m o n a s a e r u g i n o s a w i t h d i f f e r i n g

a n t i m i c r o b i a l s u s c e p t i b i l i t y p a t t e r n s f r o m p a t i e n t s w i t h

J I n f e c t D i s 1 9 7 9 ; 1 4 0 : ( 3 7 3 - 3 8 0 .

7 7 . S e a l e TW, T h i r k i l l H , T a r p a y M , F l u x M , R e n n e r t OM.

S e r o t y p e s a n d a n t i b i o t i c s u s c e p t i b i l i t i e s o f P s e u d o m o n a s

a e r u g i n o s a i s o l a t e s f r o m s i n g l e s p u t a o f c y s t i c f i b r o s i s

p a t i e n t s .

J C l i n M i c r o b i o l 1 9 7 9 ; 9 : 7 2 - 8 .

7 8 . S l a c k MD , M i c h o l s VJW.

T h e p e n e t r a t i o n o f a n t i b i o t i c s t h r o u g h s o d i u m a l g i n a t e a n d

t h r o u g h t h e e x o p o l y s a c c h a r i d e s o f a m u c o i d s t r a i n o f

L a n c e t 1 9 8 1 ; 2 : 5 0 2 - 3 .

7 9 . H 0 i b y N .

P s e u d o m o n a s a e r u g i n o s a i n f e c t i o n i n c y s t i c f i b r o s i s .

R e l a t i o n s h i p b e t w e e n m u c o i d s t r a i n s o f P s e u d o m o n a s

a e r u g i n o s a a n d t h e h u m o r a l i m m u n e r e s p o n s e .

A c t a P a t h o l M i c r o b i o l S c a n d 1 9 7 4 ; 8 2 : 5 5 1 - 8 .

8 0 . P o l l a c k M, A n d e r s o n S E .

T o x i c i t y o f P s e u d o m o n a s A e r u g i n o s a e x o t o x i n A f o r h u m a n

m a c r o p h a g e s .

I n f e c t I m m u n 1 9 7 8 ; 1 9 : 1 0 9 2 - 6 .

8 1 . R o e E A , J o n e s R J .

I n t r a c e l l u l a r k i l l i n g o f d i f f e r e n t s t r a i n s o f P s e u d o m o n a s

a e r u g i n o s a b y h u m a n l e u c o c y t e .

B r J E x p P a t h o l 1 9 7 4 ; 5 5 : 3 3 6 - 4 3 .

8 2 . M a r r i e T J , H a r d i n g G K , R o n a l d A R , D i k k e m a J , L a m J , H o b a n S ,

C o s t e r t o n J W .

I n f l u e n c e o f m u c o i d o n a n t i b o d y c o a t i n g o f P s e u d o m o n a s

A e r u g i n o s a .

J I n f e c t D i s 1 9 7 9 ; 1 3 9 : 3 5 7 - 6 1 .

8 3 . M c C a r t h y M , R o u r k MH, S p o c k A .

B a c t e r a e m i a i n p a t i e n t s w i t h c y s t i c f i b r o s i s .

C l i n P e d i a t r ( P h i l a ) 1 9 8 0 ; 1 9 : 7 4 6 - 8 .

8 4 . T a u s s i g L M , B e l m o n t e MM, B e a u d r y P H .

S t a p h y l o c o c c u s a u r e u s e m p y e m a i n c y s t i c f i b r o s i s .

J P e d i a t r 1 9 7 4 ; 8 4 : 7 2 4 - 7 .

8 5 . R e y n o l d s H Y .

P u l m o n a r y h o s t d e f e n c e s i n r a b b i t s a f t e r i m m u n i s a t i o n w i t h

P s e u d o m o n a s a n t i g e n s : t h e i n t e r a c t i o n o f b a c t e r i a ,

a n t i b o d i e s , m a c r o p h a g e s a n d l y m p h o c y t e s .

J I n f e c t D i s 1 9 7 4 ; 1 3 0 : s l 3 4 - 4 2 .

8 6 . W o o d s D E , B a s s J A , J o h a n s o n WG, S t r a u s D C .

R o l e o f a d h e r e n c e i n t h e p a t h o g e n e s i s o f P s e u d o m o n a s

a e r u g i n o s a l u n g i n f e c t i o n i n c y s t i c f i b r o s i s p a t i e n t s .

I n f e c t Immun 1 9 8 0 ; 3 0 : 6 9 4 - 9 .

8 7 . Y c a t e s D B , S t u r g e s s J M , K a h n S R , L e v i s o n H , A s p e n M.

M u c o c i l i a r y t r a n s p o r t i n t h e t r a c h e a o f p a t i e n t s w i t h c y s t i c

f i b r o s i s .

A r c h D i s C h i l d 1 9 7 6 ; 5 1 : 2 8 - 3 3 .

8 8 . R u t l a n d J , C o l e P J .

M a s a i m u c o c i l i a r y c l e a r a n c e a n d c i l i a r y b e a t f r e q u e n c y i n

c y s t i c f i b r o s i s c o m p a r e d w i t h s i n u s i t i s a n d b r o n c h i e c t a s i s .

T h o r a x 1 9 8 1 ; 3 6 : 6 5 4 - 3 .

8 9 . d i S a n t ' A g n e s e P A , D a v i s P B .

R e s e a r c h i n c y s t i c f i b r o s i s .

N E n g l J M e d 1 9 7 6 ; 2 9 5 : 4 8 1 - 5 .

9 0 . S p o c k A , H e i c k HM, C r e s s H , L o g a n W S .

A b n o r m a l s e r u m f a c t o r i n p a t i e n t s w i t h c y s t i c f i b r o s i s o f

t h e p a n c r e a s .

P e d i a t r R e s 1 9 6 7 ; 1 : 1 7 3 - 7 .

9 1 . B o w m a n B H , L o c k h a r t L H , M c C o m b s M L .

O y s t e r c i l i a r y i n h i b i t i o n b y c y s t i c f i b r o s i s f a c t o r .

S c i e n c e 1 9 6 9 ; 1 6 4 : 3 2 5 - 6 .

9 2 . S a n d e r s o n M J , S l e i g h MA .

S e r u m p r o t e i n s a g g l u t i n a t e c i l i a a n d m o d i f y c i l i a r y c o

o r d i n a t i o n .

P e d i a t r R e s 1 9 8 1 ; 1 5 : 2 1 9 - 2 8 .

9 3 . R u t l a n d J R , P e n k e t h A R L , G r i f f i n W, H o d s o n M E , B a t t e n J C ,

C o l e P .

C y s t i c f i b r o s i s s e r u m d o e s n o t i n h i b i t h u m a n c i l i a r y b e a t

f r e q u e n c y .

Am R e v R e s p i r D i s 1 9 8 3 ( i n p r e s s ) .

9 4 . B i q g a r WD, H o l m e s B , G o o d R A .

O p s o n i c d e f e c t i n p a t i e n t s w i t h c y s t i c f i b r o s i s o f t h e

p a n c r e a s .

P r o c N a t l A c a d S c i 1 9 7 1 ; 6 8 : 1 7 1 6 - 9 .

9 5 . T h o m a s s e n M J , D e m k o C A , W o o d R E , T a n d l e r B , D e a r b o r n D G ,

B o x e r b a u m B , K u c h e n b r o d P J .

U l t r a s t r u c t u r e a n d f u n c t i o n o f a l v e o l a r m a c r o p h a g e s f r o m

c y s t i c f i b r o s i s p a t i e n t s .

P e d i a t r R e s 1 9 8 0 ; 1 4 : 7 1 5 - 2 1 .

9 6 . C h u r c h J A , K e e n s T G , W a n g C l , O ' N e a l M, R i c h a r d s W.

N o r m a l n e u t r o p h i l a n d m o n o c y t e c h e r n o t a x i s i n p a t i e n t s v / i t h

J P e d i a t r 1 9 7 9 ; 9 5 : 2 7 2 - 4 .

9 7 . R a e b u r n J A , G r a n t M, G o r m l e y I P , S u t h e r l a n d R .

S t i m u l a t i n g p h a g o c y t i c c e l l f u n c t i o n i n c y s t i c f i b r o s i s .

E u r J P e d i a t r 1 9 8 1 ; 1 3 7 : 1 2 4 .

9 8 . G i b b o n s A , A l l e n J D , H o l z e l A , M c F a r l a n e H.

C e l l - m e d i a t e d i m m u n i t y i n p a t i e n t s w i t h c y s t i c f i b r o s i s .

B r M e d J 1 9 7 6 ; 1 : 1 2 0 - 2 .

9 9 . H ^ J i b y N , M a t h i e s e n L .

D i s t r i b u t i o n o f B a n d T l y m p h o c y t e s i n r e l a t i o n t o t h e

h u m o r a l i m m u n e r e s p o n s e .

A c t a P a t h o l M i c r o b i o l S c a n d ( B ) 1 9 7 4 ; 8 2 : 5 5 9 - 6 6 .

1 0 0 . L i e b e r m a n J , K a n e s h i r o W.

A b n o r m a l r e s p o n s e o f c u l t u r e d l y m p h o c y t e s t o

p h y t o h a e m a g g l u t i n i n a n d a u t o l o g o u s s e r u m i n c y s t i c f i b r o s i s .

Am R e v R e s p i r D i s 1 9 7 7 : 1 1 6 ; 1 0 4 7 - 5 5 .

. 1 0 1 . S o r e n s e n R U , S t e r n R C , P o l m a r S R .

T j y m p h o c y t i c r u s p o n s i v e n e s s t o P s e u d o m o n a s a e r u g i n o s a i n

c y s t i c f i b r o s i s : r e l a t i o n s h i p t o s t a t u s o f p u l m o n a r y

d i s e a s e i n s i b l i n g p a i r s .

J P e d i a t r 1 9 7 8 ; 9 3 : 2 0 1 - 5 .

1 0 2 . M a t t h e w s W J , W i l l i a m s M , O l i p h i n t B , G e h a R , C o l t e n H R .

H y p o g a m m a g l o b u . l i n a e m i a i n p a t i e n t s w i t h c y s t i c f i b r o s i s .

N E n g l J M e d 1 9 8 0 ; 3 0 2 : 2 4 5 - 9 .

1 0 3 . M a r t i n e z - T e l l o F J , B r a u n D G , B l a n c VIA.

I m m u n o g l o b u l i n p r o d u c t i o n i n b r o n c h i a l m u c o s a a n d b r o n c h i a l

l y m p h n o d e s p a r t i c u l a r l y i n c y s t i c f i b r o s i s o f t h e p a n c r e a s .

J I m m u n o l 1 0 6 8 ; 1 0 1 : 9 8 9 - 1 0 0 3 .

1 0 4 . W a l l w o r k J C , M c F a r l a n e H .

T h e S I g A s y s t e m a n d h y p e r s e n s i t i v i t y i n p a t i e n t s w i t h c y s t i c

f i b r o s i s .

C l i n A l l e r g y 1 9 7 6 ; 6 : 3 4 9 - 5 8 .

1 0 5 . K l i n g e r J D , S t r a u s D C , H i l t o n C B , B a s s J A .

A n t i b o d i e s t o p r o t e a s e s a n d e x o t o x i n A o f P s e u d o m o n a s

A e r u g i n o s a i n p a t i e n t s w i t h c y s t i c f i b r o s i s : d e m o n s t r a t i o n

b y r a d i o i m m u n o a s s a y .

J I n f e c t D i s 1 9 7 8 ; 1 3 8 : 4 9 - 5 8 .

1 0 6 . Y o u n g L S .

R o l e o f a n t i b o d y i n i n f e c t i o n s d u e t o P s e u d o m o n a s A e r u g i n o s a

J I n f e c t D i s 1 9 7 4 ; 1 3 0 : S l l l - 1 1 8 .

1 0 7 . H p y l b y N .

A c t a P a t h o l M i c r o b i o l S c a n d (C) 1 9 7 7 S u p p l . 2 6 2 .

52. 3 b

1 0 8 . F e r n a n d e s P B , K i m C , C u n d y K R f H u a n g H d .

A n t i b o d i e s t o c e i l e n v e l o p e p r o t e i n s o f P s e u d o m o n a s

a e r u g i n o s a i n c y s t i c f i b r o s i s p a t i e n t s .

I n f e c t I m m u n 1 9 8 1 ; 3 3 : 5 2 7 - 3 2 .

1 0 9 . P e t e r s o n P K , K i m Y , S c h m e l i n y D , L i n d e m a n n M, V e r h o e f J ,

Q u i e P G .

C o m p l e m e n t - m e d i a t e d p h a g o c y t o s i s o f P s e u d o m o n a s A e r u g i n o s a .

J L a b C l i n M e d 1 9 7 3 ; 9 2 : 883-894.

1 1 0 . H o d s o n M E .

I m m u n o l o g i c a l a b n o r m a l i t i e s i n c y s t i c f i b r o s i s :

c h i d d e n o r e g g ?

T h o r a x 1 9 8 0 ; 3 5 : 8 0 1 - 8 0 6 .

1 1 1 . C o t e s J E

L u n g F u n c t i o n ( 4 t h E d i t i o n )

O x f o r d : B l a c k w e l l , 1 9 7 9 .

1 1 2 . S h w a c h m a n H , K u l c z y c k i L L .

L o n g - t e r m s t u d y o f 1 0 5 p a t i e n t s w i t h c y s t i c f i b r o s i s :

s t u d i e s m a d e o v e r a 1 4 y e a r p e r i o d .

Am J D i s C h i l d 1 9 5 3 ; 9 6 : 6 - 1 5 .

1 1 3 . H a b s I .

U n t e r s u c h u n g e n u b e r d i e O - a n t i g e n e v o n P s e u d o m o n a s

a e r u g i n o s a .

Z H y g 1 9 5 7 ; 1 4 4 : 2 1 8 - 2 2 8 .

1 1 4 . P i t t T L , E r d m a n Y J .

T h e s p e c i f i c i t y o f a g g l u t i n a t i o n r e a c t i o n s o f P s e u d o m o n a s

a e r u g i n o s a w i t h 0 a n t i s e r a .

J Med M i c r o b i o l 1 9 7 3 ; 1 1 : 1 5 - 2 3 .

1 1 5 . G A P c o n f e r e n c e r e p o r t : C h r o n i c b r o n c h i t i s a n d c y s t i c

f i b r o s . i s .

A t l a n t a : C y s t i c F i b r o s i s F o u n d a t i o n , 1 9 7 7 .

1 1 6 . P i t c h e r - W i l m o t t RW, L e v i n k s y R J , G o r d o n I , T u r n e r MW, M a t t h e w

DJ.P s e u d o m o n a s i n f e c t i o n , a l l e r g y a n d c y s t i c f i b r o s i s .

A r c h D i s C h i l d 1 9 8 2 ; 5 7 : 5 3 2 - 6 .

1 1 7 . M a r k s M I .

T h e p a t h o g e n e s i s a n d t r e a t m e n t o f p u l m o n a r y i n f e c t i o n s i n

p a t i e n t s w i t h c y s t i c f i b r o s i s .

J P e d i a t r 1 9 8 1 ? 9 3 : 1 7 3 - 9 .

1 1 3 . B o x e r b a u m B , D o e r s h u k G F , P i t t m a n S , M a t t h e w s LW.

E f f i c a c y a n d t o l e r a n c e o f c a r b e n i c i l l i n i n p a t i e n t s

A n t i m i c r o b A g e n t s C h e m o t h e r 1 9 6 8 ; 3 : 2 9 3 - 5 .

1 1 9 . P h a i r J P , T a n J S , W a t a n a k u n a k o r n C , S c h w a b L , S a n d e r s L W .

C a r b e n i c i l l i n t r e a t m e n t o f p s e u d o m o n a s p u l m o n a r y

i n f e c t i o n . U s e i n c h i l d r e n w i t h c y s t i c f i b r o s i s .

Am J D i s C h i l d 1 9 7 0 : 1 2 0 ; 2 2 - 2 5 .

1 2 0 . M a r k s M I , P r e n t i c e R , S w a r s o n R , C o t t o n E K , E i c k h o f f T C .

C a r b e n i c i 1 1 i n a n d g e n t a m i c i n : p h a r m a c o l o g i c s t u d i e s i n

p a t i e n t s w i t h c y s t i c f i b r o s i s a n d p s e u d o m o n a s p u l m o n a r y

i n f e c t i o n s .

J P e d i a t r 1 9 7 1 : 7 9 : 8 2 2 - 8 .

1 2 1 . M c C r a e WM.

T r e a t m e n t o f p s e u d o m o n a s i n f e c t i o n i n c y s t i c f i b r o s i s u s i n g

t o b r a m y c i n .

S c o t t Med J 1 9 7 6 ; 2 1 : 6 3 - 9 .

1 2 2 . P a r r y M F , d o u FiC, M o r l i n o M, G n s r l a n P F , O r e s CW,

D e n n i n g C P .

T r e a t m e n t o E p u l m o n a r y i n f e c t i o n s i n p a t i e n t s w i t h

c y s t i c f i b r o s i s : a c o m p a r a t i v e s t u d y o f t i c a . r c i l l i n

a n d g e n t a m i c i n .

J P e d i a t r 1 9 7 7 ; 9 0 : 1 4 4 - 8 .

1 2 3 . B e a u d r y P H , M a r k s M I , M c D o u g a l l D , D e s m o n d K , R a n g e l , R .

I s a n t i - p s e u d o m o n a s t h e r a p y w a r r a n t e d i n a c u t e r e s p i r a t o r y

e x a c e r b a t i o n s i n c h i l d r e n w i t h c y s t i c f i b r o s i s ?

J P e d i a t r 1 9 8 0 ; 9 7 : 1 4 4 - 7 .

1 2 4 . R a b i n H R , H a r l e y F L , B r y a n L E , E l f r i n g G L .

E v a l u a t i o n o f a h i g h - d o s e t o b r a m y c i n a n d t i c a r c i l l i n

t r e a t m e n t p r o t o c o l i n c y s t i c f i b r o s i s b a s e d o n i m p r o v e d

s u s c e p t i b i l i t y c r i t e r i a a n d a n t i b i o t i c p h a r m a c o k i n e t i c s .

T o r o n t o , C a n a d a : C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n ,

1 9 8 0 : 3 7 0 - 5 .

1 2 5 . M a r t i n A J , S m a l l e y C A , G e o r g e R H , H e a l i n g D E , A n d e r s o n CM.

G e n t a m i c i n a n d t o b r a m y c i n c o m p a r e d i n t h e t r e a t m e n t o f

m u c o i d p s e u d o m o n a s l u n g i n f e c t i o n s i n c y s t i c f i b r o s i s .

A r c h D i s C h i l d 1 9 8 0 ; 5 5 : 6 0 4 - 7 .

1 2 6 . M i l l e r N E , R i e w e r t s E r i k s e n K , F e d d e r s e n C e t a l .

C h e m o t h e r a p y a g a i n s t P s e u d o m o n a s a e r u g i n o s a i n c y s t i c

f i b r o s i s . A s t u d y o f c a r b e n i c i l l i n , a z l o c i l l i n o r

p i p e r a c i l l i n i n c o m b i n a t i o n w i t h t o b r a m y c i n .

Eur J R e s p i r D i s 1 9 8 2 ; 6 3 : 1 3 0 - 9 .

1 2 7 . H o d s o n M E , W i n g f i e l d H J , B a t t e n J C .

T o b r a m y c i n a n d c a r b o n i c i l l i n c o m p a r e d w i t h g e n t a m i c i n a n d

c a r b e n i c i 1 1 i n i n t h e t r e a t m e n t o f i n f e c t i o n w i t h P s e u d o m o n a s

a e r u g i n o s a i n a d u l t p a t i e n t s w i t h c y s t i c f i b r o s i s .

B r J D i s C h e s t 1 9 8 3 ; 7 7 : 7 1 - 7 .

1 2 8 . R e d d i n g G J , R e s t u c c i a R , C o t t o n EI< , B r o o k s J G .

S e r i a l c h a n g e s i n p u l m o n a r y f u n c t i o n s i n c h i l d r e n h o s p i t a l i s e d

Am R e v R e s p i r D i s 1 9 8 2 ; 1 2 6 : 3 1 - 3 6 .

1 2 9 . S t o k e s E J , W a t e r w o r t h P lM .

A n t i b i o t i c s e n s i t i v i t y t e s t s b y d i f f u s i o n m e t h o d s .

A s s o c i a t i o n o f C l i n i c a l P a t h o l o g i s t s B r o a d s h e e t 1 9 7 2 ; 5 5 .

1 3 0 . P e r k i n s A .

S t a t i s t i c s o f P l a t e A s s a y s .

R e e v e s D S , P h i l l i p s I , W i l l i a m s J D , W i s e R , e d s .

I n : L a b o r a t o r y M e t h o d s i n A n t i m i c r o b i a l C h e m o t h e r a p y .

E d i n b u r g h , L o n d o n , N e w Y o r k : C h u r c h i l l L i v i n g s t o n e , 1 9 7 8 :

1 5 5 - 1 6 7 .

1 3 1 . S u t h e r l a n d R , B u r n e t t J , R o l i n s o n G N .

A l p h a - c a r b o x v - 3 t h i e n y l m e t h y l p e n i c i l l i n , ( B R L 2 2 8 8 ) a n e w

s e m i - s y n t h e t i c p e n i c i l l i n : i n v i t r o e v a l u a t i o n .

A n t i m i c r o b A g e n t s C h e m o t h e r 1 9 7 0 ; 3 9 0 - 5 .

1 3 2 . N e u H C , W i n s h e l l E B .

I n v i t r o s t u d i e s o f a s e m i - s y n t h e t i c p e n i c i l l i n , 6 - D

( - ) a l p h a c a r b o x y 3 t h i e n y l a c e t a m i d o p e n i c i l l a m i c a c i d

( B R L 2 2 8 8 ) , a c t i v e a g a i n s t p s e u d o m o n a s .

A n t i m i c r o b A g e n t s C h e m o t h e r 1 9 7 0 ; 3 8 5 - 8 9 .

1 3 3 . W a l d F,R, S t a n d i f o r d H C , T a t e m BA r C a l i a FM, H o m i c k R D .

B T 3 - P 1 6 5 4 , t i c a r c i l . l i n a n d c a r b e n i c i l . l i n : i n v i t r o

c o m p a r i s o n a l o n e a n d i n c o m b i n a t i o n w i t h g e n t a m i c i n

a g a i n s t P s e u d o m o n a s a e r u g i n o s a .

A n t i m i c r o b A g e n t s C h e m o t h e r 1 9 7 5 ; 7 : 3 3 6 - 3 4 0 .

1 3 4 . W i s e R , G i l l e t t A P , A n d r e w s J M , B e d f o r d K A .

A c t i v i t y o f a z l o c i l l i n a n d m e x l o c i l l i n a g a i n s t G r a m

n e g a t i v e o r g a n i s m s : c o m p a r i s o n w i t h o t h e r

p e n i c i l l i n s .

A n t i m i c r o b A g e n t s C h e m o t h e r 1 9 7 3 ; 1 3 : 5 5 9 - 5 6 5 .

1 3 5 . K o n i g H B , M e t z g e r K G , M u r m a n R P , O f f e H A , S c h a c h t P ,

S c h r o c k W.

A z l o c i l l i n - a n e w p e n i c i l l i n a g a i n s t P s e u d o m o n a s

a e r u g i n o s a a n d o t h e r G r a m - n e g a t i v e b a c t e r i a .

I n f e c t i o n 1 9 7 7 ; 5 : 1 7 0 - 1 8 2 .

1 3 6 . H y a t t A C , C h i p p s B E , K u m o r KM, M e l l i t s E D , L i e t m a n P S ,

R o s e n s t e i n B J .

A d o u b l e - b l i n d c o n t r o l l e d t r i a l o f a n t i - p s e u d o m o n a s

c h e m o t h e r a p y o f a c u t e r e s p i r a t o r y e x a c e r b a t i o n s i n

p a t i e n t s w i t h c y s t i c f i b r o s i s .

J P e d i a t r 1 9 8 1 ; 9 9 : 3 0 7 - 1 1 .

1 3 7 . W i e n t z e n R , P r e s t i d g e C B , K r a m e r R I , M c C r a c k e n G H , N e l s o n J D .

A c u t e p u l m o n a r y e x a c e r b a t i o n s i n c y s t i c f i b r o s i s . A d o u b l e

b l i n d t r i a l o f t o b r a m y c i n a n d p l a c e b o t h e r a p y .

Am J D i s C h i l d 1 9 8 0 ; 1 3 4 : 1 1 3 4 - 8 .

1 3 3 . B e r g a n T , M i c h a l s e n H .

P h a r m a c o k i n e t i c s o f A z l o c i l l i n i n c h i l d r e n w i t h c y s t i c

f i b r o s i s .

A r z n e i m i t t l e f o r s c h 1 9 7 9 ; 2 9 : 1 9 5 5 - 7 .

1 3 9 . L o d e H , H i e s t r a t n U , K o e p n e P , L a n g m a a c k H .

A z l o c i l l . i n a n d m e z l o c i l l i n : t w o n e w s e m i s y n t h e t i c

p e n i c i l l i n s .

I n f e c t i o n 1 9 7 7 ; 5 : 1 6 3 - 1 6 9 .

1 4 0 . D a i k o s G K , G i a m a r e l l o u H, H a d j i i p o l y d o r o u K , K a n e l l a k o p o u l o u K

P s e u d o m o n a s i n f e c t i o n s o f t h e l u n g t r e a t e d w i t h

a z l o c i l l i n .

A r z n e i m i t t l e f o r s c h 1 9 7 9 ; 2 9 : 1 9 7 6 - 8 .

1 4 1 . S m i t h A L .

A n t i b i o t i c t h e r a p y : e f f i c a c y a n d a s s e s s m e n t .

T o r o n t o , C a n a d a : C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n , 1 9 8 0 :

3 8 2 - 8 .

1 4 2 . S a g g e r s B A , L a w s o n D .

I n v i v o p e n e t r a t i o n o f a n t i b i o t i c s i n t o s p u t u m i n

A r c h D i s C h i l d 1 9 6 8 ; 4 3 : 4 0 4 - 9 .

1 4 3 . B e r g o g n e - B e r e z i n E .

P e n e t r a t i o n o f a n t i b i o t i c s i n t o t h e r e s p i r a t o r y t r e e .

J A n t i m i c r o b C h e m o t h e r 1 9 8 1 ; 8 : 1 7 1 - 4 .

1 4 4 . B r y a n t R E , H a m m o n d D .

I n t e r a c t i o n o f p u r u l e n t m a t e r i a l w i t h a n t i b i o t i c s u s e d

t o t r e a t p s e u d o m o n a s i n f e c t i o n s .

A n t i m i c r o b A g e n t s C h e m o t h e r 1 9 7 4 ; 6 : 7 0 2 - 7 .

1 4 5 . B a r n e s P , W a t e r w o r t h P M .

N e w c a u s e o f p e n i c i l l i n t r e a t m e n t f a i l u r e .

Br Med J 1 9 7 7 ; 1 : 9 9 1 - 3 .

•2.4 2

1 4 6 . ' i a l m b j p r g A S , A l f r e d s o n H, K u s o f f s k y E , S t r a n d v i k B .

A z l o c i l l i n a n d g e n t a m i c i n i n r e s p i r a t o r y t r a c t

i n f e c t i o n s w i t h P s e u d o m o n a s a e r u g i n o s a i n p a t i e n t s w i t h

S c a n d J I n f e c t D i s ( S u p p l ) 1 9 8 1 ; 2 9 : 6 4 - 6 9 .

1 4 7 . L e v y J , B a r a n D , K l a s t e r s k y J .

A n t i - p s e u d o m o n a s a c t i v i t y o f a z l o c i l l i n d u r i n g p u l m o n a r y

i n f e c t i o n i n p a t i e n t s w i t h c y s t i c f i b r o s i s .

J A n t i m i c r o b C h e m o t h e r 1 9 8 2 ; 1 0 : 2 3 5 - 8 .

1 4 8 . B a r a c h A L , M o l o m u t M, S o r o k a M.

I n h a l a t i o n o f p r o m i n i n e x p e r i m e n t a l t u b e r c u l o s i s .

Am R e v T u b e r c u l o s i s 1 9 4 2 ; 4 6 : 2 6 8 - 7 6 .

1 4 9 . L e p p e r MH, H o f m a n S , B l a t t N e t a l .

E f f e c t o f e i g h t a n t i b i o t i c s u s e d s i n g l y a n d i n c o m b i n a t i o n o n

t r a c h e a l f l o r a f o l l o w i n g t r a c h e o s t o m y i n p o l i o m y e l i t i s .

A n t i b i o t C h e m o t h e r 1 9 5 4 ; 4 : 8 2 9 - 8 4 3 .

1 5 0 . K l a t e r s k y J , H u y s m a n s E , W e e r t s D , H e n s g e n s C , D a n e a u D .

E n d o t r a c h e a l l y a d m i n i s t e r e d g e n t a m i c i n f o r t h e p r e v e n t i o n o f

i n f e c t i o n s o f t h e r e s p i r a t o r y t r a c t i n p a t i e n t s w i t h

t r a c h e o s t o m y . A d o u b l e - b l i n d s t u d y .

C h e s t 1 9 7 5 ; 6 5 : 6 5 0 - 4 .

1 5 1 . K l a t e r s k y J , H e n s g e n s C , N o t e r m a n J , M o u a w a d E ,

M e u n i e r - C a r p e n t i e r F .

E n d o t r a c h e a l a n t i b i o t i c s f o r t h e p r e v e n t i o n o f i n f e c t i o n s i n

t r a c h e o s t o m i s e d u n c o n s c i o u s p a t i e n t s . A c o m p a r a t i v e s t u d y

o f g e n t a m i c i n a n d a m i n o s i d i n - p o l y m i c i n B c o m b i n a t i o n .

C h e s t 1 9 7 5 ; 6 8 : 3 0 2 - 6 .

“1 4 3

1 5 2 . P i n e s A , R a a f n t H , P l u c i n s k i K .

G e n t a m i c i n a n d c o . l i . s t i n i n c h r o n i c p u r u l e n t b r o n c h i a l

infection.Bi : M e d J 1 9 6 7 ; 2 : 5 4 3 - 5 .

1 5 3 . R o s e H D f P e n d h a r k e r 'MB, S n i d e r G L , K o r y R C .

E v a l u a t i o n o f s o d i u m c o l i s t i m e t h a t e a e r o s o l i n G r a m - n e g a t i v e

i n f e c t i o n s o f t h e r e s p i r a t o r y t r a c t .

J C l i n P h a r m a c o l 1 9 7 0 ; 1 0 : 2 7 4 - 8 1 .

1 5 4 . D a l l y M B , K u r r l e S , B r e s l i n A B X .

V e n t i l a t o r y e f f e c t s o f a e r o s o l g e n t a m i c i n .

T h o r a x 1 9 7 8 ; 3 3 : 5 4 - 6 .

1 5 5 . N o l a n G , M c l v o r P , L e v i s o n H , F l e m i n g P C , C o r e y M , G o l d R.

A n t i b i o t i c p r o p h y l a x i s i n c y s t i c f i b r o s i s : I n h a l e d

c e p h a l o r i d i n e a s a n a d j u n c t t o o r a l c l o x a c i l l i n .

J P e d i a t r 1 9 8 2 ; 1 0 1 : 6 2 6 - 3 0 .

1 5 6 . P r i n c e A S , N e u H C .

A c t i v i t i e s o f n e w b e t a - l a c t a m a n t i b i o t i c s a g a i n s t i s o l a t e s o f

P s e u d o m o n a s a e r u g i n o s a f r o m p a t i e n t s w i t h c y s t i c f i b r o s i s .

A n t i m i c r o b A g e n t s C h e m o t h e r 1 9 8 1 ; 2 0 : 5 4 5 - 5 .

1 5 7 . R i c h m o n d MH.

" R e s i s t a n c e o f P s e u d o m o n a s a e r u g i n o s a t o a n t i b i o t i c s " .

I n : S a b a t h , L D e d . " P s e u d o m o n a s a e r u g i n o s a , t h e o r g a n i s m ,

d i s e a s e s i t c a u s e s a n d t h e i r t r e a t m e n t . B e r n e , S t u t t g a r t ,

V i e n n a . H a n s H u b e r ; 1 9 8 0 : 1 7 6 - 9 2 .

1 5 8 . B r y a n L E .

R e s i s t a n c e t o a n t i m i c r o b i a l a g e n t s . I n : D o g e t t RG e d .

’ P s e u d o m o n a s a e r u g i n o s a : c l i n i c a l m a n i f e s t a t i o n s o f i n f e c t i o n

a n d c u r r e n t t h e r a p y ' . L o n d o n : A c a d e m i c P r e s s , 1 9 7 9 : 2 1 9 - 2 6 5 .

l L A A

1 5 9 . L o r i a n V , A t k i n s o n B A S , A m a r a l L .

E f f e c t o f s u b - i n h i b i t o r y c o n c e n t r a t i o n s o f a n t i b i o t i c s o n

P s e u d o m o n a s a e r u g i n o s a : t h e M I C / M A C r a t i o . I n : S a b a t h L P o d .

P . a e r u g i n o s a , t h e o r g a n i s m , d i s e a s e s i t c a u s e s a n d t h e i r

t r e a t m e n t " . B e r n e , S t u t t g a r t , V i e n n a . H a n s H u b e r ; 1 9 8 0 :

1 9 3 - 2 0 5 .

1 6 0 . L a k e K B , V a n D y c k J J , R u m s f e l d J A .

C o m b i n e d t o p i c a l p u l m o n a r y a n d s y s t e m i c g e n t a m i c i n : t h e

q u e s t i o n o f s a f e t y .

C h e s t 1 9 7 5 ; 6 8 : 6 2 - 4 .

1 6 1 . M o r r o w P E .

A e r o s o l c h a r a c t e r i s a t i o n a n d d e p o s i t i o n .

Am R e v R e s p i r D i s 1 9 7 4 ; 1 0 9 : s 8 8 - 9 9 .

( S u p p l e m e n t , C o n f e r e n c e o n t h e S c i e n t i f i c B a s i s o f R e s p i r a t o r y

T h e r a p y ) .

1 6 2 . N e w h o u s e M T , R u f f i n R E .

D e p o s i t i o n a n d f a t e o f a e r o s o l i s e d d r u g s .

C h e s t 1 9 7 8 ; 7 3 : 9 3 6 - 4 2 .

1 6 3 . B r a i n J D , V a l b e r g P A .

S t a t e o f t h e A r t : D e p o s i t i o n o f a e r o s o l i n t h e r e s p i r a t o r y

t r a c t .

Am R e v R e s p i r D i s 1 9 7 9 ; 1 2 0 : 1 3 2 5 - 7 3 .

1 6 4 . P a v i a D , T h o m a s s o n M L , C l a r k e SW, S h a n n o n H S .

E f f e c t o f l u n g f u n c t i o n a n d m o d e o f i n h a l a t i o n o n p e n e t r a t i o n

o f a e r o s o l i n t o t h e h u m a n l u n g .

T h o r a x 1 9 7 7 ; 3 2 : 1 9 4 - 7 .

1 G 5 . A l d e r s o n P O , S c c k e r - ' d a l k e r R H , S t r o m i n g e r D 3 , M a r k h a m J ,

M i l l R L .

P u l m o n a r y d e p o s i t i o n o f a e r o s o l s i n c h i l d r e n w i t h c y s t i c

f i b r o s i s .

J P e d i a t r 1 9 7 4 ; 3 4 : 4 7 9 - 3 4 .

1 6 6 . L o u r e n j : o R V , L o d d e n k e m p e r R , C a r t o n RW.

P a t t e r n s o f d i s t r i b u t i o n a n d c l e a r a n c e o f a e r o s o l i n

p a t i e n t s w i t h b r o n c h i e c t a s i s .

Am R e v R e s p i r D i s 1 9 7 2 ; 1 0 6 : 3 5 7 - 6 6 .

1 6 7 . C l a y M , P a v i a D , N e w m a n S P . C l a r k e S VI.

E f f i c a c y o f j e t n e b u l i z e r s i n t h e p r o d u c t i o n o f

t h e r a p e u t i c a e r o s o l s .

T h o r a x 1 9 8 2 ; 3 7 : 7 8 8 .

1 6 8 . N e w m a n S P , P a v i a D , f r l o r e n F , S h e a h a n N F , C l a r k e S W.

D e p o s i t i o n o f p r e s s u r i s e d a e r o s o l s i n t h e h u m a n

r e s p i r a t o r y t r a c t .

T h o r a x 1 9 8 1 ; 3 6 : 5 2 - 5 .

1 6 9 . H p i b y N , O i l i n g S .

A c t a P a t h o l M i c r o b i o l S c a n d ( C ) 1 9 7 7 ; 8 5 : 1 0 7 - 1 1 4 .

1 7 0 . Z i e r d t C H , S c h m i d t P J .

D i s s o c i a t i o n i n P s e u d o m o n a s a e r u g i n o s a .

J B a c t e r i o l 1 9 7 5 ; 8 7 : 1 0 0 3 - 1 0 1 0 .

1 7 1 . D o g g e t t R G .

I n c i d e n c e o f m u c o i d P s e u d o m o n a s a e r u g i n o s a f r o m c l i n i c a l

s o u r c e s .

A p p l M i c r o b i o l 1 9 6 9 ; 1 8 : 9 3 6 - 7 .

112 . B e r g an T , WfiL b y N .

e p i d e m i o l o g i c a l m a r k e r s f o r P s e u d o m o n a s a e r u g i n o s a :

R e l a t i o n s h i p b e t w e e n c o n c o m m i t e n t m u c o i d a n d n o n - m u c o i d

s t r a i n s f r o m t h e r e s p i r a t o r y t r a c t i n c y s t i c f i b r o s i s .

A c t a P a t h o l M i c r o b i o l S c a n d ( 3 ) 1 9 7 5 : 8 3 : 5 5 3 - 6 0 .

1 7 3 . Z i e r d t C H , W i l l i a m s R L .

S e r o t y p i n g o f P s e u d o m o n a s a e r u g i n o s a i s o l a t e s f r o m p a t i e n t s

w i t h c y s t i c f i b r o s i s o f t h e p a n c r e a s .

J C l i n M i c r o b i o l 1 9 7 5 ; 1 : 5 2 1 - 5 2 6 .

1 7 4 . W i l l i a m s R J , G o v a n J R W .

P y o c i n e t y p i n g o f m u c o i d s t r a i n s o f P s e u d o m o n a s a e r u g i n o s a

i s o l a t e d f r o m c h i l d r e n w i t h c y s t i c f i b r o s i s .

J M e d M i c r o b i o l 1 9 7 3 ; 6 : 4 0 9 - 1 2 .

1 7 5 . D i a z F , M o s o v i c h L L , N e t e r E .

S e r o g r o u p s o f P s e u d o m o n a s a e r u g i n o s a a n d t h e i m m u n e r e s p o n s e

o f p a t i e n t s w i t h c y s t i c f i b r o s i s .

J I n f e c t D i s 1 9 7 0 ; 1 2 1 : 2 6 9 - 7 4 .

1 7 6 . H ^ i b y N , R o s e n d a l K .

E p i d e m i o l o g y o f P s e u d o m o n a s a e u r i n g o s a i n f e c t i o n i n p a t i e n t s

t r e a t e d a t a c y s t i c f i b r o s i s c e n t r e .

A c t a P a t h o l M i c r o b i o l S c a n d ( B ) , 1 9 8 0 ; 8 8 : 1 2 5 - 1 3 1 .

1 7 7 . Y o u n g L S , A r m s t r o n g D .

H u m a n i m m u n i t y t o P s e u d o m o n a s a e r u g i n o s a . 1 . I n v i t r o

i n t e r a c t i o n o f b a c t e r i a , p o l y m o p h o n u c l e a r l e u k o c y t e s a n d s e r u m

f a c t o r s .

J I n f e c t D i s 1 9 7 2 ; 1 2 6 : 2 5 7 - 7 6 .

1 7 8 . Dn M a t t c o C S , H a m m e r MC , B a l t c h A L , S m i t h R P , S u t p h e n N T ,

• l i e h e I s o n P B .

S u s c e p t i b i l i t y o f P s e u d o m o n a s a e r u g i n o s a t o s e r u m

b a c t e r i c i d a l a c t i v i t y . A c o m p a r i s o n of : 3 m e t h o d s w i t h

c l i n i c e i l c o r r e l a t i o n s .

J L a b C l i n M e d 1 9 8 1 ; 9 3 : 5 1 1 - 8 .

1 7 9 . T h o m a s s e n M J , D e r n k o C A .

S e r u m b a c t e r i c i d a l e f f e c t o n P s e u d o m o n a s a e r u g i n o s a

i s o l a t e s f r o m c y s t i c f i b r o s i s p a t i e n t s .

I n f e c t I m m u n 1 9 8 1 ; 3 3 : 5 1 2 - 5 1 8 .

1 8 0 . K e l l y NiM, T e m p a n y E , F a l k i n e r F R , F i t z g e r a l d M X ,

O ' B o y l e C , K e a n e C T .

D o e s p s e u d o m o n a s c r o s s - i n f e c t i o n o c c u r b e t w e e n c y s t i c

f i b r o s i s p a t i e n t s ?

L a n c e t 1 9 8 2 ; 2 : 6 8 8 - 9 .

1 8 1 . T u r n e r M J .

A n t i g e n i c v a r i a t i o n .

I n : S m i t h H , S k e h e l J J , T u r n e r M J e d s . T h e m o l e c u l a r

b a s i s o f m i c r o b i a l p a t h o g e n i c i t y .

L i f e S c i e n c e s R e s e a r c h R e p o r t 1 6 , 1 9 8 0 : 1 3 3 - 1 5 8 .

1 8 2 . S c h n e i d e r H , M c L e o d G r i f f i t h s J , W i l l i a m s G D , P i e r G B .

I m m u n o l o g i c a l b a s i s o f s e r u m r e s i s t a n c e o f N e i s s e r i a

g o n o r r h o e a e .

J G e n M i c r o b i o l 1 9 8 2 ; 1 2 8 : 1 3 - 2 2 .

1 8 3 . T e j e d o r C , F o u I d s J , Z a s l o f f M.

B a c t e r o p h a g e s i n s p u t u m o f p a t i e n t s w i t h b r o n c h o p u l m o n a r y

p s e u d o m o n a s i n f e c t i o n s .

I n f e c t Immun 1 9 8 2 ; 3 6 : 4 4 0 - 1 .

CL 4 8

1 8 4 . S a d o f f J C .

C e l l w a l l s t r u c t u r e s o f P s e u d o m o n a s a e r u n i n o s a w i t h

i m m u n o l o g i c s i g n i f i c a n c e : A b r i e f r e v i e w .

J I n f e c t D i s 1 9 7 4 ; 1 3 0 : ( S u p p i ) s 6 1 - 6 4 .

1 8 5 . Y o u n g L S .

H u m a n i m m u n i t y t o P s e u d o m o n a s a e r u g i n o s a I I R e l a t i o n s h i p

b e t w e e n h e a t - s t a b l e o p s o n i n s a n d t y p e - s p e c i f i c l i p o p o l y

s a c c h a r i d e s .

J I n f e c t D i s 1 9 7 6 ; 1 2 6 : 2 7 7 - 8 7 .

1 8 6 . M a y e r MM.

C o m p l e m e n t a n d c o m p l e m e n t f i x a t i o n .

I n : R a b a t E A , M a y e r MM, e d s . E x p e r i m e n t a l I m m u n o c h e m i s t r y

S p r i n g f i e l d , I l l i o n o i s : C h a r l e s C T h o m a s , 1 9 6 1 : 1 3 3 - 2 4 0 .

1 8 7 . O u c h t e r l o n y O .

D i f f u s i o n - i n - g e l m e t h o d s f o r i m m u n o l o g i c a l a n a l y s i s . I n :

K e l l o s P , e d . P r o g r e s s i n A l l e r g y , B a s e l : K a r g e r ; 1 9 5 8 :

V o l u m e 5 1 - 7 8 .

1 8 8 . T a y l o r PW.

An a n t i b a c t e r i a l f a c t o r i n t h e s e r u m o f t w o p a t i e n t s w i t h

i n f e c t i o n o f t h e u p p e r u r i n a r y t r a c t .

C l i n S c i 1 9 7 2 ; 4 3 : 2 3 - 3 0 .

1 8 9 . G u t t m a n R M, W a i s b r e n B A .

B a c t e r i a l b l o c k i n g a c t i v i t y o f s p e c i f i c I g G i n c h r o n i c

P s e u d o m o n a s a e r u g i n o s a i n f e c t i o n .

C l i n E x p I m m u n o l 1 9 7 5 ; 1 9 : 1 2 1 - 1 3 0 .

1 9 0 . F i c k R B , N a e g e l G P , M a t t h a y R A , R e y n o l d s H Y .

C y s t i c f i b r o s i s p s e u d o m o n a s o p s o n i n s .

I n h i b i t o r y n a t u r e i n a n i n v i t r o p h a g o c y t i c a s s a y .

J C l i n I n v e s t 1 9 8 1 ; 5 8 : 8 9 9 - 9 1 4 .

191. hoiby M, F r l i ci B, Jensen K, Koch C, Miller EE, St/jvrinq E,

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