Review o f Literature

REVIEW OF LITERATURE

HISTORICAL BACKGROUND OF PNEUMONIA

Pneumon~a an anclent d~sease known to Hlppocrates was understood

clearly only after the isolation of Pneumococcus in the year 1880 and

establlshrng Pneumococcr as ~ t s causative agent in humans by

Welchselhaum In 1886 In the year 1889 Townsend and Coolrdge reported

1000 cases of pneumonla treated In the Massachusetts General Hosprtal

wlth a mortalrty rate of 25% Sulfapyr~dlne was prescrrbed for the treatment

of pneumonra In the year 1939 followed by report of the use of serum

therapy Type-specrf~c rabb~t antrserum was used for serum therapy by

Lohman In the year 1939 Trllett et al In 1944 reported the use of 40 000-

100 000 un~ts of pen~crllrn a day for 4 days to cure both bacteremic and

nonbacterem~c pneumococcal pneumonla Th~s amazing discovery had a

remarkable effect In the treatment of cases of pneumonla (Austr~an 1999)

The d~scovery of typ~ng of pneumococcl by uslng raprd techniques

followed - slnce the knowledge of typlng was not necessary In treating cases

of pneumonla, ~t was totally drscarded for few years With the result in the

year 1950 Re~man Mote "Unfortunately slnce the determrnatlon of types of

pneumococcl w t ~ c h cause pneumonla IS no longer of practical necessity for

therapy the procedure has been almost entlrely abandoned It was felt that

among the cases of pneumonla in the age group of 15-60 without any co-

ex~st~ng infectron bacter~olog~c studres were unnecessary This ult~mately

led to the concept of adm~ss~on of very few cases of pneumonia in any

hosp~tal (Austr~an 1999)

Finally, the physicians concluded that prophylaxis IS the only

alternative to those at risk of a fatal outcome from pneumococcal infection.

Thls led to the advent of production of pneumococcal vaccines.

EPIDEMIOLOGY

Pneumonia in industrialised countries with special reference to

Pneumococcal pneumonia:

Acute respiratory infections, particularly pneumonia, are the leading

cause of morbidity and mortality worldwide. The leading cause of bacterial

pneumonia is Spneumoniae in most hospltalised patlents (Ort et al. 1983).

Desplte the avallabllity of effective antibiotics, morbldlty IS still high During

the past 10 years, the overall incidence of bacterernlc pneumococwl

pneumonia has Increased (Plouffe et al, 1996; Breiman et al, 1990. Hedlund

et al, 1995; Foster et al, 1994). Reports from a study conducted at Ohio,

state an Increased Incidence of 1.2 fold between the year 1991 and 1993

(Plouffe et at, 1996) among persons above 65 years of age. In Sweden, the

lncldence of pneumonia among persons 65 years of age and older was 11.7

per 1000 in the year 1995 (Ortqv~st, 1999). Globally, pneurnococcal

pneumonia is responsible for nearly 1.2 mllllon deaths per year and nearly

40% of all pneumonia deaths in children less than 5 years of age

(Mulholland, 1999; WHO, 1994). In the United States, 500,000 cases of

pneumonia are reported annually (Watson, 2000).

Annual mortality rate of 1-5% has been reported worldwide for lower

respiratory tract infections in infants (Tyeryar et al, 1978). Studies conducted

at Scotland reports a mortality rate of 24% (McKenzie et al, 2000). Case

fatality rates with bacteremic pneumococcal pneumonia in adults are 2040%

in the United States. It was reported to be 1 in 10 among all age groups

above 20 years of age (Mufson et al 1999) denoting an Increasing rate w~th

advancing age Case fatality rate was 4 5% In ch~ldren aged 4 years or

younger Reports of case fatality rates from Stockholm and Sweden were 7%

and 11% respectively (Ortqv~st 1993 Ortqvist 1990) Mortality rate of

7 36% due to pneumococcal pneumonia has been reported by Marrie

(1999) In a study conducted by Davis et al (1995) In 3 paediatrlc centres

from Sydney a mortality rate of 6 6% was reported

Pneumonia in developing countries:

Every year, 10 million deaths occur in children less than 5 years of

age in low income countries. Out of this, approximately 3 million deaths are

caused by pneumonia (WHO, 1998). Data on mortality are poorly recorded

~n some developing countries and hence the rates may be even higher. In

countries like Gambia, most of the cases admitted to the paediatric hospitals

are reported to be due to pneumonia (Leowski, 1986; Denny, 1986; de

Francisco et al, 1993). The Incidence of pneumonia in the developing

country is up to 10 times hlgher than that in developed countries like United

States (Mccracken, 2003). The mortality rate goes unnoticed despite being

high ~n developing countries since there IS paucity of infoination describ~ng

the pattern of disease. Annual incidence of pneumonia in children less than

5 years is 3-4 per 100 in industrialised countries but it IS estimated to be 10-

20 per I00 in low income countries (Shann, 1996). Studies from a rural area

of West Africa, reveal a case fatality rate of 1% in children w~th pneumonia.

An annual attack rate of pneumonia In chlldren less than 5 years was 4090

out of 100,000 per year in rural area of West Africa (O'dempsey et al, 1996)

According to a study conducted by Board on Science and Technology for

International Development (BOSTID) in Argentrna, Pakistan, Phillpplnes,

Tharland and Uruguay, intidence ranged from between 12.7 to 16.8 new

ep~sodes of acute respiratory lnfectlon per 100 child-weeks at rlsk. 3ates of

lower respiratory infectlons varied from 0.2 to 3.4 new episodes per 100

child-weeks at risk (Selwyn, 1990).

Mortality rate reported among Israeli adults with pneurnococcal

bacteremla was 27.8% (Raz et al, 1997). In a study undertaken in Argentina,

Braz~l, Chile, Colombia, Mexico and Uruguay in under-fives, out of 3,393

children with systemic pneurnococcal infectlons, 1578 had pneumonia.

Around 63.8% of them were under 2 years of age (Hortal et al, 2000)

Indian scenario:

In India, like the other developing nations acute respiratory tract

Infections continue to be the cause of morbidity and mortality. It is the

second most common cause of mortality In children after acute diarrhoea.

According to a compiled data ~nvolving maliy centres in Indla, a rate of 15-

20% mortality rates in infancy (Registrar General of India, 1987) with acute

lower respiratory tract infections accounting for 20-24% deaths were

recorded (Pocket Book of Health Statist~cs in India, 1980). Datta-Banik et al

(1969) and Gupta et al (1982) reported 5-8 episodes of acute respiratory

tract infections per year in urban children and 3-5 episodes in the rural areas.

Stuciies conducted by the lnvasive Bacterial Infection Surveillance

(IBIS) group which Included 6 hospitals in lndia for a period of 4 years in

3,686 pat~ents with suspected pneumonia, a case fatality rate of 19% was

reported Mortality from invasive pneurnococcal disease in lndia has

exceeded 20% in patients excluding those with underlying illnesses (IBIS

Group, 1999). Roy et al (1991) have reported a case fatality rate of 17.6%

during infancy in children with acute respiratory tract infection who were

admitted to a hospital in Calcutta. Morbidity due to ARI was found to be

12 5% in children under 5 years of age from rural area of Delhi by Chhabra et

al (1993). Sehgal et al (1997) have reported a case fatality rate of 10.45%

Acute respiratory infection is responsible for one million deaths and an attack

rate of 3-7/childlyear has been reported. Of these 10-15% are due to acute

lower respiratory tract infections (Reddiah and Kapoor, 1988).

CONTRIBUTORY FACTORS TO PNEUMOCOCCAL PNEUMONIA

The factors responsible for the high incidence of pneumococcal

disease among young children in developing countries are not fully known.

Factors like age, sex, colonisation, nutritional status and underlying

~nfectrons have been found to have causal relationsh~p wlth the spectrum of

pneumococcal infection.

i .Age:

Information regarding high prevalence of Pneumococcal pneumonia in

certa~n ages suggests that disease IS common at the extremes of life. The

inc~dence of pneumococcal bacteremia is relatively high among infants upto

2 years of age and low among teenagers and young adults; rates increase

steadrly beginning at around 55 years (Musher, 1998). Durlng the flrst year

of Ilfe, pneumonia and bronch~olltrs are most common (Glezen et al, 1973)

In children younger than one year of age the annual incidence of invasive

pneumococcal disease was found to be 5541100,000 and ~t was 2401100,000

in chrldren younger than 5 years according to a report from West Africa

(O'dempsey et al, 1996).

In children less than 2 years old, S.pneumoniae is the leading cause

of death (WHO, 1997). It is responsible for at least 1.2 million deaths

annually (Shann and Steinhoff, 1999). Studies conducted at Soweto. South

Afrlca report that an annual incidence ~n children younger than 5 years of age

increased from 6111 00,000 in 198611 987 to 13011 00,000 in 199611 997

(Karstaedt et al, 2000).

According to a study by Scott et al (1996) different serotypes vaned

with regard to age. Serotype 1 was associated with a progressive decline In

relative risk through adulthood whereas, serotype 3 infection increased in the

7Ih decade of life. Serotype 8 had a relative preference for adults. During the

flrst decade of life, serotype 23 and 18 took over. However, no stat~stically

significant Interactions were noted according to thls study.

2.Sex:

Pneumococcal d~sease has a consistent preference for males and the

reason is largely unexplained One of !?e reasons could be the early

reporting and hospitalization of male chlld In contrast to a female child, In

certain low soclo-economlc communities in developing countries. Some

studies report that male female ratlo varies with serotype. Based on the

study by Scott et al (1906), the proportion of all ~solates that were recovered

from male patients was 0 64 (male.female ratio = 1.8:l) The variation of

males between different serogroups was not marked and the overall

association between sex and serogroup was not statistically significant. A

sllght preference for females were seen In 2 serogroups 14 and 23 (Scott et

al, 1996). The male:female ratio reported from a study in a rural area of

West Afrlca was 1.4:l (O'Dempsey et al, 1996).

3.Nasopharyngeal colonization:

S.pneurnon;ae, the most common causative agent of pneumonia,

colonizes the nasopharynx and can be isolated from 5-10% of healthy adults

and from 20-40% of healthy children. Children are more likely to be carriers

of pneumococci than the adults (Musher, 1998). This colon~zation again has

also shorn to be dependent on factors like age, sex, geographical

predeliction, overcrowding and socioeconom~c status (O'dempsey et al,

1996, Howard et al, 1988). Pneumococcal lnfectlon is usually followed by

colon~zation of the nasopharynx, whrch is an important risk factor for the

development of the disease (Gleblnk, 1989). S.pneumoniae can be ~solated

in 25-60% of nasopharyngeal cultures obtained from healthy carriers

(Ingvarsson et al, 1982 and Anianson et al, 1992). In a study conducted at

Pondicherry, South India, in healthy school children between 5-10 years, a

prevalence rate of 24.3% for S.pneu,non~ae colonization was noted

(Kanungo et al, 2000). In another study of nasopharyngeal colonization

among South Indian infants, prevalence rates of 54%, 64.1% and 70.2% in

Infants by age 2 months, 4 and 6 months respectively were reported thus

(Coles et al, 2004) explalnlng the potential r~sk for pneunonia in these

Infants. Studies from Gambia reported a colon~zat~on rate of 76 1% In healthy

ch~ldren (Lloyd-Evans et al, 1996).

4.Malnutrition:

Malnutrition has been incriminated as one of the factors responsible

for the development of pneumonia in children in low income countries (Wolf

and Fleer, 2000). In malnourished children the biological integrity of the

respiratory tract mucosa may be compromised leading to alteration in the

colonisation rate which is one of the predisposing factors for pneumowccal

disease. Studies have proved that mucosal immunity plays an important role

In inhibiting pneumococcal colon~zat~on (Stenfors and Raisanen, 1993).

Decreased serum retinol concentrations can result in impaired mucosal

immunity decreasing the secretory antibody (IgA) which is a nonspecific

barrier defense (Chandra, 1988; Biesalski and Stom, 1992; Semba, 1998;

Sir~sinha et al, 1980). This clearly suggests that malnourished pat~ents are at

a high risk and this association behveen malnutrition and infection together

with insufficient health care services in the community has been responsible

for the high mortality among chlldren in low income countries (Wolf and

Fleer, 2000). Studies also suggest that reversal of Vitamin A deficiency may

reduce the rate of colonization and ultimately decrease the morbidity rates

associated with the infection (Coles et al, 2001).

5.Underlying infections:

Pneumococcal infections are associated with certain predisposing

illnesses and usually ~t occurs at the extremes of age (Burman et al, 1985).

The usual risk factors associated in an adult include alcoholism, Human

Immunodeficiency Virus (HIV) infection, splenectomy, multiple myeloma,

connective tissue disease, steroid therapy, dlabetes mellltus and intravenous

drug use (Burman et al, 1985; Janoff et al, 1992; Musher, 1992) In children,

Increased incidence has been assoc~ated with sickle cell anaemia (Musher,

1992 Barrett-Conner, 1971; Wong et al, 1992) Increased incidence of

pneumococcal infection can also be due to defects in host defense

mechan~sm like congenital or acquired defects in antibody production,

neutropenia, dysfunction of white blood cells (WBC), complement

deficienc~es or splenic dysfunction (Musher, 1995).

In patients infected with HIV, respiratory tract infections are very

common (Nathoo et al, 1993; Bobat et al, 1998). Studies from rural Zambia

and urban Zimbabwe suggests that HIV infection was a strong predictor of

severe morbidity and mortality from acute lower respiratory tract infection

(Smyth et al, 1997; Nathoo et al, 1993) in part~cular due to S.pneumoniae. A

study conducted to determine the impact of HIV on the epidemiology of

invasive pneumococcal infection in South Africa revealed that the burden of

severe invasive pneumococcal disease was 41.7 fold more In HIV infected

compared with uninfected children (Madhl et al, 2000) Another study

conducted In South Africa reported a high incidence of pneumococcal

bacteremla in children which doubled due to the impact of HIV epidemic

(Karstaedt et al, 2000)

The other underlying conditions predisposing pneumococcal d~sease

Include hospitalization, c~rrhos~s of the liver, renal insuffic~ency, viral

resp~ratory Infections, allergies, cigarette smoking and COPD (Musher,

1998)

ETIOLOGICAL AGENTS OF PNEUMONIA

Identifying the etiologic agent responsible for pneumonia IS

challenging mainly because of diff~culty in acquiring adequate material for

diagnosis of the infection and also due to lack of reliable diagnostic methods

(Isaacs, 1989; Lode et al, 1993; Shann, 1986). Difficulty in identifying the

causative agent in pneumonia is a major drawback In instituting speciflc

treatment and preventive measures.

In a study by Socan et al (1999) on the mlcroblal etlology of

community acqulred pneumonia In hospltalised adult patlents (>I5 years)

S pneumonrae was the bacterium ~solated most frequently (5 7%) 9 5% had

Chlamyd~a pneumonlae 5 7% had Mycoplasma pneumon~ae and 24 1 % had

vlral lnfectlon Bacterial pneumonia was dlagnosed In 39 8% patlents of

bhich 23 had concurrent vlral infection Pneumonia caused only by vlral

agents were dlagnosed In 28 patlents out of the total 211 patlents whlch

formed the study population

Etiology of pneumonia in children:

Pneumonia is a major cause of morbidity and mortality in children in

low income countries. In children, nonbacterial pneumonias are the

frequently seen pulmonary Infections (Correa, 1996) Respiratory syncytial

virus is the commonest agent particularly in infants, followed by

parainfluenza virus, adenovirus and influenza virus. Another leading cause

of pneumonia is Mycoplasma pneumoniae in chlldren (Boyer et al, 1992)

over 5 years of hge. Chlamydia pneumoniae has been reported recently as

an Important cause of community acquired pneumonia among children,

between 5-14 years cf age (Grayson 1994). With respect to the bacterial

pneumonlas, S.pneumoniae appears to be the predominant etiological agent

(Correa, 1996). Incidence of pneumonia due to Haemophilus influenzae has

decreased significantly owing to the success of H.rnfluenzae type b (Hib)

vaccine in the developed nations. However, it is still an important cause of

pneumonias in some developing countries where this vaccine is not yet

~vailable (Klein, 1992). Concurrent viral infection along with bacterial

pneumonia has been reported in 25-75% of children from a children's

hospltal of Northern California, Oakland (Turner et al, 1987). Several studies

have reported S.pneumoniae and H.influenzae as the leading bacterial cause

and RSV, the leading viral cause of pneumonia (Silverman et al, 1977;

Shann, 1986; Avila et al, 1990; Forgie et al, 1991; Greenwood, 1992; Forgie

et al, 1992; Adegbola et al, 1994; Sutmoller et al, 1995; Falade et al, 1997).

Studies from Afr~ca, Asia and Latrn America have reported S.aureus as

another common organism, in children with prior vlral respiratory infection

(Gonzaga et al, 1990). Rarely, Gram negative bac~lli have been incriminated

as a cause ot pneumonia in normal Infants and children in developed

countries (Bang et al, 1993). Moraxella catarrhalis is currently recognised as

the third most common pathogen of respiratory tract after S.pneumoniae and

H.influenzae (Wolf and Fleer, 2000).

Studles from develop~ng countr~es lncludlng lndla have shown that

S pneumoniae and H influenzae are the 2 most predominant bacter~a Isolated

from acute lower respiratory tract ~nfect~ons (Berman 1991) The estimated

case fatallty rate of bacter~al pneumon,a due to Spneumon~ae and

H influenzae In develop~ng countries is 50% more than that due to resplratory

syncyt~al virus and para~nfluenza vlrus (PIV) (Berman 1991) From a report

from Pondlcherry lnd~a 2096 of the ~solates of H ~nfluenzae were non-type b

stralns Prevalence rate of acute lower resplratory tract ~nfectlons was 6 4%

due to Chlamydra pneumoniae (Chaudhry et al 1998) Serological evidence

of pneumonia due to Legionella pneumophila has been reported In 4 out of

45 pat~ents In Bombay Overall incidence of 16 6% of pneumonia associated

w~th Mycoplasma pneumoniae was reported In 1972 by Ayyagarl et al

Detect~on of M pneumoniae has been posltlve by culture In 10 5% of cases

24 samples out of 43 by PCR and 20 out of 43 samples by culture

(Ramamoorth~ et al 1996 N~sar et a1 1998)

Pandey et a1 (1998) reported an incidence of 30% by using a panel of

tests like culture, antigen and antibody detection assays to demonstrate

M.pneumoniae infection in children below 5 years of age. John et a1 (1991)

reported 38% of upper respiratory ~nfect~on (URI) cases and 49% of lower

respiratory infection (LRI) due to vlral agents by conver~tional methods.

Agents associated were RSV (32%) followed by PIV (10.8%), influenza

vlruses (1.5%) and adenovirus (3.6%). Study from Lucknow, India reported

RSV infect~ons in 5%, PIV in 7%, influenza viruses in 4% and adenovirus in

5% (Jain et al, 1991) V~ral etiology was reported in 43.5% of lower

respiratory tract infection where RSV had the maximum association (60%),

followed by influenza v~rus (14.5%), PIV (11.5%) and adenovirus (1.5%)

(Ma~treyi et al, 2000).

In HIV Infected ch~ldren who are at a greater r~sk of develop~ng

pneumonla S pneumoniae IS agaln one bf the most frequently Isolated

agents (Madh~ et al 2000) Reports from Southern Afr~ca suggests that

pneumonla due to H influenzae S aureus Klebaella sps, E COB and

Salmonella sps are also seen In HIV Infected ch~ldren (Nathoo et al 1993)

Clinical features:

The signs and symptoms of bacterial pneumonia vary with bacterial

pathogen, the age of the patient and the severity of the disease (Klein, 1998).

Infants: In infants, it is characterized by a mild upper respiratory tract

~nfect~on wth stuffy nose, fretfulness and diminished appetite. This mild

~llness of several days duration ends with abrupt onset of fever of 3g°C or

higher, restlessness and respiratory distress. The patient appears ill with

moderate-to-severe air hunger often with cyanosis. The respiratory distress

is man~fested by grunting, flaring of the alaenasi, retractions of the

supraclavicular, intercostal and subcostal areas, tachypnea and lachycard~a

(Behrman et al, 1992). This is reflected by the proliferation of bacteria and

the inflammatory response In the alveol~.

Children: The signs and symptoms are similar to those of adults.

Symptoms and signs of pneumonla in children may be classified for

convenience into five categories: nonspecific manifestations of Infection and

toxic~ty; general signs of lower respiratory tract disease; signs of pneumonia;

signs of pleural fluid and signs of extrapulmonary disease (Klein, 1998).

Respiratory rates are correlated inversely w th age dur~ng the I* 3

years of l~ fe and vary between a medlan of 47 breathslm~n ~n the l d month of

llfe to 381m1n at the end of 1' year to 28Im1n by 3 years of age In case of

older ch~ldren the respiratory rate varies between 15 and 25im1n Based on

these flndlngs, to dlagnose ALRl tachypnea IS deflned as 50 breathslm~n In

Infants I-; 1 months of age 401m1n In ch~ldren 1-4 years of age and 30lm1n In

children 5 years of age or older (Korpp~ 1995)

Radiographic findings:

Failure to recognize various rad~ological presentations by the

physicians is a problem often compl~cat~ng the diagnosis of pneumococcal

pneumonia. Pneumococcal pneumonla can appear with a variety of

radiological patterns leading to difficulty in making a specific bacteriologic

diagnosis ~n patients with bacterial bronchopneumonia (Kantor, 1981)

However, in pat~ents wth a classic lobar presentation, the clinical symptoms

and roentgenographic findings are often characteristic, and an etiological

diagnosis can be made, based on these findings (Davies et al, 1996).

The radiographic diagnosis of pneumonia is made on the basis of

pulmonary perihilar linear opacities or infiltrates (airways disease) andlor

consolidatlon (airspace disease) (Friis et al, 1990; Swischuk et at, 1986;

Khamapirad et al, 1987).

In most cases of pneumococcal pneumonia, the chest X-ray reveals

an area of infiltration involving less than a full segment. Whereas, In young

adults, true consolidation with an alr bronchogram is seen. A moth-eaten,

less homogenous appearance IS seen In pneumonia superimposed on

severe chronic lung disease. In half of the cases, segmental or lobar

consolidatlon is apparent (Musher, 1992)

General laboratory findings:

In majority of the patlents wth pneun~ococcal pneumonia, white blood

cell count (WBC) is >12,000 cellslpl. However, the count can be as low as

~ 6 0 0 0 cellslpl in 5-10% of persons hospitalized for pneumococcal

pneumonia. This decreased count is often not because of bone marrow

suppression and tbe outcome of thls findlng may be fatal (Musher, 1998) A

study by Korppi et al (1993)reports elevation of C-reactive protein

concentrations (CRP), erythrocyte sedimentation rate (ESR) levels and WBC

counts by 25%, 40% and 36% respectively in patients with pneumonia. This

f~ndlng of elevated WBC counts are independent of age. C-reactive protein is

more often elevated in patients wth bacterial infection alone than in those

patlents with viral or mixed viral-bacterial infections. However, correlation of

pneumococcal etiology with nonspecific indicators of a bacterial infection like

elevated WBC, CRP amd ESR is poor (Korppi et al, 1993).

Microbiological diagnosis:

Optimum treatment of any infection requires definition of the etiologic

agent. For the diagnosis, efforts should be made to obtain adequate and

appropriate clinical materials. Viral and bacterial pathogens responsible for

lower respiratory tract infections produces a variety of cllnical spectrum

ranging from mild illness to life threatening pneumonias. Using various

clinical specimens, isolation of that particular pathogen is possible.

Alternative technique for conventional tube cultures to culture virus, which

take several days to weeks for isolation and identification, is the

centrifugation enhanced culture combined with indirect immunofluorescence

stalning on direct smears as described by Gleaves et al (1984) for isolation

of RSV.

Apart from culture, other rapid methods include antigen detection

assays These assays are simple and rap~d techniques where the antigens

of the pathogen is detected from the clinical specimens like serum, urine,

sputum, pleural fluid etc. Antigen can be detected by various techniques llke

Co-agglutination, Counter-immunoelectrophores~s, Latex agglutination,

lmmunofluorescenca tests and Enzyme immunoassay (Coonrod and Rytel

1973; Chattopadhya et al, 1992. Whitby et al 1985; Chaudhry et a l 1998)

lrnmunofluorescent stainlng with monoclonal antibodies is also a preferred

method (Broor et al, 1999).

Detection of antibodies to various antigens has been demonstrated by

enzyme immunoassays, complement fixation test and micro-

immunofluorescence test. The other methods employed for the diagnosis

include antigen capture enzyme immunoassay and monoclonal immunoblot

assay (Kok et al, 1988; Madsen et al, 1988, Hirschberg and Holme, 1991).

Molecular techniques available include detect~on by nucleic acid

probes or PCR, DNA probe hybridization uslng specific primers and probes

which help in the rapid detect~on of respiratory viruses (Sullender and Wertz,

1991; Paton et al, 1992; Class et al, 1992; Gilbert et at, 1996; Maitreyi et al,

2000) Multiplex quantitative reverse transcription PCR enzyme hybridization

is another molecular technique which has been descr~bed for rapid and

s~multaneous detection of multiple viruses.

PNEUMOCOCCUS

In the year 1880, George Miller Sternberg isolated pneumowccus for

the f~rst time. It was also isolated by Louis Pasteur in the same year. In the

year 1882, Friedlander identified a bacterium from the lungs section which

was s~milar to pneumococci by Grem's technique. Initially, this bacterium

was named as Diplococcus pneumoniae (1926) based on its appearance in

Gram-stained sputum. In the year 1974, the organism was renamed

Streptococcus jneumoniae since it grew in chains in a liquid medium

(Austrian, 1999).

Typing of pneumococcl based on the nature of the capsular

polysaccharide was discovered helping in the serum therapy of

pneumococcal pneumonia Followng this, pneumococcal vaccine was

introduced in 1927 based on the d~scovery of the immunogenicity of

pneumococcal capsular polysaccharides by Schieman and Casper (Austrian,

1999).

Some of the major contributions of molecular biology and medic~ne

resulted from studies of pneumococcus. Studies with S.pneumoniae has !ed

to the identification of the first polysaccharide antlgen, genetic recombination

mechanisms, identification of DNA as the genetic matertal, m~crob~al

hormone inducing and controlling the physiological state of competence and

identification of the first bacterial autolysin (Tomasz, 1999). Apart from all

these findings, S.pneumoniae has played an important role in the history of

antimicrobtal therapy.

Finally, the history of pneumococci can be concluded with a quotation

from Benjamin White's Monograph (1937), "opportunities for enquiries into

the ways of pneumococcus are by no means exhausted. The prosecution of

future studies on the life, activities of pneumococcus cannot fail to bring new

gold to the abundant store of bullion already accumulated" (Austrian, 1999).

General properties:

S.pneumoniae is a Gram positive coccus that replicates in chains in

liquid medium. They usually occur in pairs or In short chains. The coccus is

nearly 1 pm in diameter. The diplococcus is ovoid or lanceolate shaped, with

their distal ends narrowed. They are nonmotile and nonsporing (Duguid and

Ross,1 989).

Pneumococcus is an aerobtc and facultative anaerobe. It requlres alr

or hydrogen with 5-10% COz with an optimum temperature of 37% for its

growth. When pneumococci grows on media containing glucose vhich

stimulates growth (energy source) there is the formation of acid which leads

to cessation of growth. This could be prevented by addition of blood and

serum which was shown to protect them from hydrogen peroxide formed

durlng ~ t s growth (Colrnan, 1990).

Pneumococc~ produce alpha haemolysin which breaks down Hb

producing a green colour and as a result pneumococcal colonles are

surrounded by a green zone durlng growth on blood agar plates Colonies

on blood agar are small transparent and glittering that tend to d ~ p down in

the centre and resemble a doughnut or draughtsrnan form Colonles of type

3 are usually larger and have a mucold appearance After anaerobic

lncubatlon on blood agar and a subsequent period at 6°C zones of a-

haernolysls appear around the colonles especially for the colonies close to

the dlscs contaln~ng antlblotlcs which act on the cell wall of the bacterium

Pneumococci tends to die in cultures within 24-48 hours. Once the

bacterium dies, it gets autolysed. The blle or other detergents activate the N-

acetylmuramyl-L-alan~ne amidase wtlch IS responsible for the lysis of

cultures of pneumococci.

Important tests used routinely in a laboratory to identify pneumococci

Include (1) Alpha haemolysls on blood agar, (2) Catalase negativity, (3)

Solubility in bile salts, (4) Susceptibility to ethyl hydrocupreine (Optoch~n)

hydrochloride, (5) lnulin fermentation.

More than 98% of pneumococcal isolates are susceptible to optoch~n

Reports of optochln resistant pneumococcl have been described (Kontlalnen

and Slvonen, 1987, Munoz et al, 1990) Alternative methods have been

evaluated to ldentlfy pneumococcl such as the pneumosllde test (BD

Diagnostics, Cockeysville, MD) and Accuprobe S.pneumoniae culture

identiflation test which is a DNA-based test (Geneprobe, Sandiego).

Animal pathogenicity:

Pneumococci isolated from infective conditions are mostly virulent for

the mouse except serotype 14 which is avirulent (Duguid and Ross, 1989).

Peritonitis, septicemia and death of the mouse results on intraperitoneal

inoculation of pneumococci. Because of this fact, mouse serves as a

selective animal model facilitating the isolation of scanty pneumococci from

clinical specimens. Currently, much of the molecular work such as studies on

mutations In the various genes of pneumococci are carr~ed out using mouse

as the animal model (Berry et at, 1999). Studies on virulence of d~fferent

mutants of S.pneumoniae are also carried out in mice after intranasal or

intraper~toneal challenge (Berry et at, 1989) Rabbits are also hlghly

susceptible to pneumococci and are predomnantly used to ralse ant~bodies

against various antigens of pneumococci like pneumolys~n and capsular

polysaccharides (Cima Cabal, 1999; Lund, 1978).

Structure:

Cell wall:

The outermost structure on the surface of the non-encapsulated

pneumococcus, is composed of an electron-dense outer and inner band, and

enclosing a less electron dense band, represents the pneumococcal cell wall.

Whereas in an encapsulated pneumococci, this trilaminated structure is

covered fram outside with a less structured layer of polysacchar~de capsule

(Tomasz, 2000).

Pneurnococcal replication:

Pneumococc~ divide in a single plane in the central equatorial region

of the cell and the site of incipient septum is marked by a morphological

alteration at the cell wall, which appears as a "hump" of the cell wall located

at the middle of the cell surface. The equatorial ring is made of cell wall

material and the next event involves the division of the eqyatorial ring into

two which may be due to the action of autolysin. This splitting event

coincides with the formation of septum. The cell wall enveloping the dividing

bacterium between the equatorial ring and the septa1 tip is one generation

younger in biosynthetic age than the cell wall between the equatorial rings

and the left or right poles of the bacterium (Tomasz, 2000). It is known that

pneumococcus may engage in a new cycle of cell division before the physical

separation from the daughter cell is accomplished. This leads to the

appearance of a cham of cells in cultures

Pneumococcal cell organisation:

Plasma membrane:

Plasma membrane represents the five osn~iophylic bands ~nslde the

cell wall which stain with different intens~ty It IS a unit membrane, with band 1

representing the inner leaflet, band number 3 the outer leaflet, band number

5, the inner surface of the cell wall and band number 4, represents the

structural or functional analogue of periplasmic space found In Gram

negative bacteria. A number of bridges or invaginations connect the plasma

membrane and the cell wall representing a close anatomical andlor functional

interd~gitation of the plasma membrane and the cell wall in pneumococcl

Cytoplasm:

Pneumococci has a cytoplasm containing an evenly distributed, large

number of electron dense particles wth the dlameter of approximately 150A.

representing the ribosomes and polysomes (Tomasz, 2000).

Membranous organelles:

These organelles are usually referred to as "mesosomes" or

"chondrioids". However, the function of these organelles are not known. It is

simply referred as intracellular membranes. It is reported that due to

peculiarity of these intracellular membranes In their assocratron \nth septa

and alignment of pairs of these intracellular membranes with the dividing

chromosome, it may be involved with the equatorial biosynthesis of cell wall

or rn the separation of chromosomes during cell div~sion (Tomasz, 2000).

Nuclear region:

It is represented by the centrally located low electron densrty region

which is filled with packed fibrils of uniform 25-30 A width. These fibrils

represent the appearance of bacterial chromosome.'

Morphology of S.pneumonrae was made possible with the help ot high

resolution electron microscopic study of a laboratory strain R6 which is a

derrvative of R36A. This strain represents the prototype of the

pneumococcus and has been extensively used for the characterization of the

complete genome of S.pneumoniae. The clinical isolate, serotype 2 was the

parent strain of R36A (Tomasz, 2000)

Chromosome structure:

By fragmentation of genomic DNA of S.pneumoniae with low-

frequency-cleadage restriction endonucleases and separation of the

fragments by field-inversion gel electrophoresis (FIGE) it is possible to get a

DNA-finger-print of a strain. This process helps in constructing a physical

and genetic map of the R6 laboratory strain The ability to separate large

fragments of DNA by pulsed-field gel electrophoresis has provided the

technology to map bacter~al chromosomes. Genetrc markers are located on

the map of chromosome of S.pneumon1ae Th~s method IS being used to

compare the profiles of several strains and also for typing S.pneumoniae

strains (Lefevre et al, 1993). Genes in S pneumoniae were identified by DNA

sequence sampling and gene disruption system was developed to identrfy

several potential lethal targets for antibiotic intervention (Baltz et al, 2000).

VIRULENCE FACTORS

The subcapsular surface components ,i.e, the cell wali and

Iipopolysaccharide) of bacteria are the rmportant determinants of

inflammat~on apart from the other components like pili and polysaccharide

capsule. Evidences are available, indicating the role of pneumococcal

proteins in the pathogenesis of disease, either as mediators of inflammation

or by directly attacking host tissues (Paton et al, 1993). The three major

proteins contributing to the virulence of the organism include the toxin

pneumolysin, the major pneumococcal autolysin and pneumococcal surface

protein A) Other enzymes contributing to the virulence of S pneumoniae are

neuraminidase, hyaluronidase and lgAl protease

Surface components of pneumococci:

Capsule:

The presence of capsule was first reported as type-specific soluble

substance in filtrates of pneumococcal cultures and in the sera and urine of

infected person. Later on, this specific-soluble substance was k n o w as type

specific capsular polysaccharide (CPS). Discovery of the antigenicity of CPS

led to the development of pneumococcal vaccines. This was the flrst non-

protein substance discovered to be antigenic in humans (Kamerling, 2000).

Based on the nature of its capsular polysacchar~de. Spneumonrae

has been divided into several serotypes Two different systems of

nomenclature exlst for the pneumococcal serotypes viz, the Danish system

and the American System. Danish system is based on the cross-reactions

between d~fferent types and the cross-reactive types are included under a

common serogroup, with individual serotypes within each group distinguished

by the trailing letter Whereas, according to the American system, numbering

of serotypes are based on the order at which they are discovered and it does

n ~ t recognize antigenic cross-reactivity among types (Kamerling, 2000)

There are 90 serotypes to date, which are distinguished by chemical

differences in their capsular polysacchar~des and also by the ability of the

immune system of rabbits to recognize these structural differences and to

respond with specific antibodies against the antigens of each different type

(Shapiro, 1991) Typing methods include Capsular reaction test, Latex

agglutination, Co-agglutination and Capillary precipitation. Newer methods

include the use of DNA probes and DNA sequence-based subtyping

(Henrichsen, 1999).

PATHWAY OF BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES

TC;: phog lucomut ase

UTP ~~ i~o~I~Pho~pha le Uridylyl transferare

UDP-Glc + Ppi ""4 UD-Glc dehydrogenase

NaDH

UDP-GI Type 3 capsular polysacchande synthase

Biosynthetic pathway for type 3 CPS

Fig.1. Proposed biosynthetic pathway for type 3 CPS (Dillard et al, 1995)

Using the traditonal methods of structural analysis, the structure of

CPS was analysed in the year 1964 and was re-examined by Larm and

Lindberg in the year 1976 (Kamerling, 2000).

As a virulence factor:

Capsular polysaccharide is one of the component of S.pneumoniae responsible for the virulence of the bacterium. The function of capsular

polysaccharide (CPS) is to protect the pneumococci from phagocytosis by

polymorphonuclear leukocytes.

As early as 1928, Griffith reported that unencapsulated pneumococcal

variants were avirulent (Garcia et al, 2000). Loss of the capsule is

accompanied by a 100,000-fold reduction of the virulence of S.pneumoniae

and it was found that nonencapsulated pneumococci were readily

phagocytized M e n added to a suspension of leukocytes In normal serum,

whereas mucoid, capsulated organisms were resistant to phagocytosis and

multipl~ed rapidly. Although the chemical composition of the capsule plays

an ~mportant role in the virulence, a quantitative relationship between the

amount of type-specific polysaccharide and v~rulence has been reported

(Garcia et al, 2000).

Host defense mechanism agalnst S.pneumoniae IS based on humoral

immun~ty, that IS, antibodies directed to the CPS can protect humans to

infections with viable pneumococci (Kamerling, 2000). Function of CPS in

preventing ingestion and Killing by phagocytic cells is due to hidlng the FC of

IgG that reacts with cell wall and the C3b that is fixed during that reaction or

by the alternative complement pathway (Musher et al, 2000).

Virulence of pneumococci is determined by the chemical composition

of the capsule and to a lesser extent, its size (Knecht et al, 1970).

CPS, which is purified, does not elicit any inflammation when it is

~nstilled directly into the lung in contrast to unencapsulated pneumowcci,

vhich have inflammatory properties (Tuomanen et at, 1987). However, CPS

is not required for inflammation but favour the progression of the infection

due to its property of inhibiting phagocytosis. Owing to its antigenic nature, it

is able to elicit the production of serotype-specific, protective antibody.

Currently, a total of 90 serotypes of S.pneumoniae has been recognized

which includes 21 serogroups.

Pneumococcal capsular types vary significantly in their virulence.

Type 3 is highly virulent because of formation of a larger capsule ~ 4 t h the

requirement of only one colony forming unit to produce a lethal infection in a

mouse. Also, the case fatality rate 1s 50% for a penicillin treated type 3

bacteremlc pneumococcal pneumonia (Austrian, 2000) whereas type 37

which also has the same capsular slze as that of type 3 is a rare cause of

any lnfectlon and a larger dose 1s required to produce an infection in mouse.

Serotypes 3, 4 14 and 19 predominate in ~ t s isolation from the blood

stream or they are more vlrulent In case of infants and chlldren lnfectlon

can occur no matter what the type colonized - serotypes 6, 14, 19F and 23F

are seen to be common (Gray et al, 1980) Whereas in adults thls does not

hold true Stud~es have been conducted to determ~ne the virulence of each

serotype and ~t has been found that the serotypes 1,2 3,5,8 and 12F appear

to be most vlrulent (Heffron 1979 Austra~n 1981)

Genetic analysis:

Capsular polysaccharide of S.pneumon~ae has an Important role in the

development of molecular genetla. Genetic exchange of DNA among

S.pneumoniae strains seems to have an important role in the generation of

new strains and in the evolution of capsular serotypes (Henrichsen, 1995;

dan Dam et al, 1990).

Genetic evidence indicated that the genes responsible for capsular

polysaccharide biosynthesis were closely linked in the pneumococcal

chromosome and could be transferred as a unit during transformation (Garcia

et al, 2000). Inter-type transformation takes place when the donor DNA

displaces the resident capsular genome and it was assumed that this

interchange was mediated by homologous sequences flanking the type-

specific gene cluster.

Role of capsular polysaccharide in diagnosis:

Soluble pneumococcal capsular polysaccharide was f~rst shown in the

circulation of patients wth pneumococcal pneumonia more than 50 years ago

(Coonrod and Drennan, 1976). Due to the insensitivity of the tests available

for detecting circulating polysaccharide, efforts to evaluate the balance

between antigen and antibody in the serum in pneurnococcal pneumonia

were hindered.

Capsular polysaccharide antigen has been detected from various

clln~cal samples like urine, transtracheal secretion, serum, CSF and sputum

by techniques like Co-agglut~nation, Latex agglutination and Counter

~mrnunoelectrophoresis (Coonrod and Rytel, 1973; Kronvall, 1973; Burman et

al, 1991; Singhal et al, 1996).

Studies on measurement of anticapsular antibodies have been

reported by various workers by enzyme-linked immunosorbent assay (ELISA)

(Barett et al, 1980; Shyarnala et al, 1988, Korppi et al, 1992; Korppi et al.

1993 and Musher et al, 2000). ELISA has been used to measure IgM, IgG

and IgA antibody concentrations.

Immune complexes containing type specific CPS has been studied in

the sera of children with lower respiratory tract infection by Korppi et al

(1998). Holloway et al (1993) has also developed ELlSA for the

demonstration of circulating pneumococcal IgG immune complexes in

patients with community acquired pneumonia. From the dissociated

circulating Immune complexes pneumococcal capsular antigen was

demonstrated by latex agglutinat~on.

Role of capsular polysaccharide in prevention:

Pneumococcal polysaccharide vaccines

Although llcensed vaccines against invasive pneumococcal disease

are available in the United States, Europe and other countries, their use IS

l~m~ted (Poland, 1999). Currently available pneumococcal vaccines,

manufactured by Merck and Company (Pneumovax 23; Nest Po~nt

Pennsylvania), Lederle Laboratories (Pnu-Immune 23; Wayne, New Jersey)

,and Pasteur Merieux (Pneumo 23, Lyon, France) contain 25 ~g of each of 23

purlfled capsular polysaccharide antigens of S pneumoniae (Serotypes 1. 2,

3, 4, 5, 6B. 7F, 8, 9N, 9V, IOA, I l A , 12F, 14, 158, 17F, 18C, 19A, 19F, 20,

22F, 23F and 33F). According to the data from surveillance conducted by

the Centres for Disease Control and Prevention (CDC), these 23 capsular

types represent atleast 85-90% of the pneumococcal serotypes causing

invasive infections among children and adults in the United States.

Pneurnococcal capsular polysaccharide antigens induce serotype-

specific antibodies that enhance opsonlzation, complementdependent

phagocytosis and killing of pneumococci by leukocytes and other phagocytic

cells. Concentration of antibodies to pneumowccal polysaccharides begin to

increase within 1 week afler vaccination and remain elevated for over 5 years

In healthy adults (Mufson et al, 1983; Mufson et al, 1987; Musher et al,

1993). The overall efficacy against lnvasive pneumococcal disease among

immunocompetent persons greater than 65 years of age is 75% (Butler et al,

1993).

Recently, prevention of pneumococcal infection by vacc~nation has

become Important In light of the emergence and spread of drug-res~stant

strains of pneumococci (Hoffman et al, 1995, Butler et al, 1996). Innovative

strategies to improve the protectlon prov~ded by the pneumococcal

polysaccharide vacclne are under evaluation.

Limitations of Pneumococcal polysaccharide vaccines:

Though the pneumococcal polysaccharide vaccine is effective against

pneumococcal invasive Infections in adult patients, immunization has not

been protective ~n infants and young children as these vaccines are not

sufficiently immunogenic in these age groups (Fedson et al, 1999; Makela et

al, 1980; Karma et al, 1985; Riley et al, 1986). Therefore, this vaccine is not

recommended for children <2 years of age (Eskola, 2000). It is known to

c ~ n f e r only limited protectlon to patients with certain underlying illnesses

Conjugate Pne~mococcal vaccines:

Development of pneumococcal conjugate vacclnes offers the potentla1

beneflts of prevent~on of pneumococcal d~sease In populations that are

unable to generate an adequate Immune response to polysacchar~de

vacclnes (Bruyn and Van Furth, 1991) By conjugating a capsular

polysaccharlde wlth an lmmunogenlc proteln the Immune response changes

from a T-cell Independent response, wh~ch IS poorly developed In ch~ldren <2

years of age, to a T-cell dependent response enhanc~ng protective antlbody

formation and ~mmunologic memory in infants and young children (Watson,

2000). Common protein carriers include diphtheria toxoid, tetanus toxoid, the

CRM 197 nontoxic cross-reactive variant of diphtheria toxin (Pnc CRM) and

men~ngococcal outer membrane protein complex (Pnc-OMPC).

Measurement of serum antibody concentrations help in the evaluation of the

immunogenicity of pneumococcal conjugate vaccines.

Pneumococcal conjugate vaccines have proven to b e safe in clinical

trials. Two separate efficacy field trials of pneumococcal conjugate vaccines

were initiated in 1995 at Northern Kalser Permanente and in Finland.

Efficacy of 2, 7-valent conjugate vaccines against otitis media were evaluated

in Flnland (Eskola et al, 1998). A surveillance trial was conducted in

Northern California, vhich evaluated the efficacy of pneumococcal CRM

conjugate vaccine against the primary endpoint of invasive pneumococcal

infections caused by serotypes included in the vaccine in -38,000 ~nfarts

from a multiethnic population. The efficacy of Pnc CRM vaccine aga~nst

pneumonia and otitis media evaluation produced a significant (p < 0.01)

reduction in ali episode of otitis media among fully vaccinated children

(Shinefield and Black, 2000). Based on a study on the evaluation of

irnmunogenicity and reactogenicity of a pneumococcal conjugate vaccine

adm~n~stered comb~ned with a H.inf/uenzae type b conjugate found well-

tolerated, safe and immunogenic when adm~nistered as a separate or as a

comblned 7V PncIHbOC lnjection (Choo et al, 2000)

LlPOTElCHOlC AND TElCHOlC ACID

Lipoteichoic acids (LTAs) and teichoic acids (TAs) are the polymers of

+he cell wall membrane complex in a Gram positive bacteria. LTAs and TAS

of pneumococci are unique because they possess identical repeat and chain

structures unlike other Gram positive bacteria vhich are structurally and

biosynthetically distinct entities (Fischer, 2000).

Previously, pneumococcal TA was described as C-polysaccharide by

Tillet et al in the year 1930. After 13 years, pneumococcal LTA was isolated

by Goebel et al and named as lipocarbohydrate or pneumococcal F-antigen

because of its fatty acid content and immunological properties (Fischer,

2000). The two polymers differ immunologically, as Forssman antigenicity is

associated with LTA (Briles et al, 1973; Behr et al, 1992). By

~mmunoelectron microscopy, it was proved that C-polysaccharide is uniformly

distributed on both inside and outside of the cell walls and LTA on the

surface of the cytoplasmic membrane (Sorensen et al, 1988). N-acetyl

galactosamine and choline are the components of C-polysaccharide

(Tomasz, 1967; Kilpper-Balz et al, 1985; Garcia et al, 1986). However, it is

known that choline is an essential growth factor of pneumococci.

As a virulence factor:

Cell wall has the highest ability to cause inflammation than the

capsule or cytoplasm. Signs and symptoms of infection induced by cell wall

mlmlc that of livlng bacteria in animal models of invasive pneumococca!

infections (Tuomanen et al, 1985; Tuomanen et al, 1987, Ripley-Petzoldt et

a1 1988)

The TAs and LTAs are strongly associated with acute inflammation by

activating the alternative pathway of the complement cascade and also binds

the acute phase reactant C-reactive protein. It also activates the

procoagulant activity on the surface of endothelial cells, promotes cytokine

release initiating the influx of leukocytes (Winkelstein et al, 1978; Riesenfeld-

Orn et al, 1989; Geelen et al, 1993). Recently, it has been reported that IL-2,

an important component inducing cell mediated immunity, is induced by

pneumococcal cell wall (Cleveland et al, 1996).

Tha peptidoglycan portion of the pneumococcal cell wall 1s a potent

stimulus of blood brain barrier permeability. It is also cytotoxic to cil~ated

cells of the choroid plexus, neurons and can induce sleep (Spellerberg et al,

1995)

AUTOLYSIN

Autolysins are enzymes that degrade different bonds in the

peptidoglycan and eventually cause the lysis and death of the cell. Substrate

and bond specificities are exhibited by these enzymes. Most of the

organisms contain lytic enzymes.

S.pneumoniae contalns a powerful autolytic enzyme, an amidase,

which is the best characterized autolysin described so far (Lopez et al, 2000).

The major pneumococcal autolysin is a 36-KDa N-acetyl muramic and L-

alanine amidase located in the cell envelope (Paton et al, 1993) The

enzyme is bound to choline residues of lipoteichoic acid (Forssman antigen)

which in turn is anchored to the cell membrane. Cells growing in

ethanolarnine-containing medium, contain an inactive form of the amidase

which can be activated by incubating at low temperature with choline-

containing cell walls (Tomasz, 1981).

Attempts to isolate autolysin-defective mutants were tried which

helped in cloning and sequencing a gene (lyt A) coding for a bacterial

autolysin (Garcia et al, 1985). Cloning of lyt A also helped in the isolation of

genes encoding the cell wall lytic enzymes from pneumococcal

bacteriophages (Garcia et at, 1986; Garcia et al, 1987; Garcia et al, 1988).

Possible role as a virulence factor:

Activity of the autolytic enzyme or its regulation can give rise to

remarkable changes affecting the phenotypic properties of a clinical isolate of

pneumococcus It can also influence the pathogenic role of such strains

resulting in hlgher morbidity and mortality from this type of Infection

(Tuomanen et al, 1988). Such atyp~cal stralns of pneumococci are clinically

important as they are often associated wth more invasive types of infections

like meningitis (Salyers and Whitt, 1994).

Autolysin-induced lysis of a proportion of the Invading pneumococcl

can injure the host by releasing increased concentrations of potent toxins

and hydrolytic enzymes locally (Paton et al, 1993).

lmmun~zation of mice with purified autolysin resulted in the production

of antibodies capable of inhibiting autolysis of both rough and encapsulated

pneumococcal cultures (Berry et al, 1989). Autolysin can indirectly act as a

virulence factor by releasing the cytoplasmic protein, pneumolysin into the

external medium which happens when pneumococci undergo autolysis. But

when autolysis is inhibited by growth of pneumococci in the presence of anti-

autolysin antibody, pneumolysin activity stays cell-associated.

NEURAMINIDASES

Most of the fresh clinical isolates of pneumococci have the ability to

synthesize one or more neuraminidases. Camara et al (1994) have cloned

and sequenced a neuraminidase encoding gene (nan-A) from S.pneumoniae.

Molecular weight of nanA was determined to be 107 KDa. It is assumed that

neuraminidase can be released from the cells either by proteolytic cleavage

or after cellular autolysis. It was found later that S.pneumoniae produced

more than one neuraminidase enzyme. The gene responsible for this was

identified and designated as nan-8, which is located on the pneumococcal

chromosome. The molecular weight of nan-B was found to be 74.5 KDa.

Role as a virulence factor:

Neuraminidase cleaves terminal salicyltc acid residues present in

glycolipids, glycoproteins and oligosaccharide on cell surfaces or in body

fluids. This activity results in great damage to the host (Paton et al, 1993). It

can also unmask potential cell surface receptors for putative pneumococcal

adhesins (Krlvan et al, 1988). It is also noted that both coma and bacterem~a

can occur significantly more often in patients with pneumococcal mentngitis

when the concentration of N-acetylneuraminlc acid in the cerebrosptnal fluid

is elevated (O'Toole et al, 1971).

HYALURONIDASE

Hyaluronidase enzyme is produced by almost all strains of

S.pneumoniae. Substrate of thls enzyme is hyaluronic acid which is found

associated with connective tissue and extracellular matrix. This enzyme is

secreted by 99% of clinical isolates of S.pneumoniae during log-phase

growth in vitro. Characterization of hyaluronidase gene has been done from

a type 23 pneumococcus (Berry et al, 1994). Hyaluronidase enzyme has

been produced from recombinant E.coli carrying this gene. Molecular weight

of the purified hyaluronidase enzyme was 89 KDa. Western blot analysis

using antiserum raised against the purified 89 KDa hyaluronidase indicated

that the E.coli clone also expressed the 107-KDa form of the enzyme and this

antiserum labeled a 107 KDa protein in partially-purified hyaluronidase

preparations from S.pneumoniae (Paton et al, 2000). The enzyme act~vity is

cell-associated, which is consistent with the presence of the Gram-positive

cell surface anchorage domain (LPXTGE) near its C-terminus

As a virulence factor:

Role of hyaluronidase enzyme in the pathogenesis of pneumococcal

Infection remains unclear. Role of hyaluronidase in a nasal colonization

model is currently being assessed. Hyaluronic acid being the substrate of

thls enzyme might have a role in pathogenesis facilitating the spread of

infection, providing a greater microbial access to host tissue for colonization.

It can also help in the migration of the organism like translocation from the

lung to the vascular system, between tissues. However, it has not been able

to demonstrate any protection in a mouse immunization I challenge model

(Paton et al, 2000).

IgA I PROTEASE

S.pneumoniae colonizing the mucosal surfaces produces a protease

enzyme which has the ability to cleave human IgA I at a specific point w~thin

the hinge region, providing intact Fab and Fc fragments (Paton et al, 1993).

S.pneumon~ae gene encoding IgA I protease has been cloned. But

due to instability its further characterization has not been possible. Further

analysis of a more stable clone is needed M i c h was isolated (Camara,

19921. .

As a virulence factor:

Till now, no definite evidence is available to prove the involvement of

any of these proteases in pathogenesis of the disease. This is mainly

because the enzymes are highly specific and do not cleave IgA from any

animal species commonly used as models for disease (Paton et al, 1993).

PNEUMOCOCCAL SURFACE PROTEIN A (PspA)

Pneumococcal surface protein A is a pneumococcal virulence protein

(Yother et al, 1991). It is antigenic in nature and can elicit the production of

antibodies which is being explored currently in the production of vaccines It

serves as a good protein candidate for the preparation of pneumococcal

vaccines. It is also known that PspA is serologically variable (Mc Daniel et

al, 1987; Crain et al, 1990). Because of this property, a PspA based vaccine

may need to contain PspAs of more than one pneumococcus to be able to

protect against all pneumococci.

As a virulence factor:

According to a study it was observed that an encapsulated mutant

strain lacking PspA expression fixed more complement than the isogenic

parent strain expressing PspA even though they were found to have identical

levels of capsular polysaccharide (de Velasco et al, 1995). It was also

observed that infections of nonimmune mice with PspA' capsular type 3

pneurnococci caused greater early activation of serum complement than did

infections with a ~ s p A ' isogenic parent. These findings suggest that PspA is

able to decrease the consumption of complement by pneumococci, ultimately

reducing complement mediated clearance and phagocytosis of pneumococcl

(Briles et al, 2000).

PNEUMOCOCCAL SURFACE ADHESIN A (PsaA)

PsaA has a molecular weight of 37KDa and was detected for the 1'

time by Russell et a1 (1990) using monoclonal antibodies. Experiments have

proved that immunization with purified PsaA protected mice from challenge

with virulent S.pneumoniae (Talkington et al, 1996). Sequence analysis of

the clonal PsaA gene has been determined and it was found that there exists

a homology with putative lipoprotein adhesins of S.sanguis and

S.parasanguis (Sampson et al, 1994). Location of PsaA on S.pneumoniae is

not clear. Since it is able to elicit a protective antibody in humans, it is being

tried as a candidate for non-serotype-dependent vaccine antigen.

PNEUMOLYSIN

Production of haemolysin by pneumococci was first reported 9

decades ago (Paton et al, 1993). It is a pore-forming, thiol-activated toxin

produced by S.pneumoniae. Pneumolysin belongs to the family of sulphydryl

(SH)-activated t~aemolysins (Kanclerski and Mollby, 1987). These thiol-

activated toxins are produced by 4 genera of Gram positive bacteria,

Streptococcus, Listeria, Clostridium and Bacillus.

General properties:

Although an intracellular prote~n, rt IS always detected extracellularly in

broth cultivation and released only when the bacterium undergoes autolys~s

due to autolysin or lyt~c agent. Pneumolysin is oxygen-labrle whlch is only

apparent in crude preparations (Andrew et al, 2000). When pure, the toxlns

are not oxygen-labile and no longer activated by thiol-reduc~ng agents since

they are fully active. It is antigenic and irreversibly inactivated by treatment

with cholesterol (Paton et al, 1993).

Mode of action:

All the thiol-activated cytolysins are known to have a common mode of

action which involves an interaction with cholesterol in the target cell-

membrane leading to insertion of the toxin into the lipid bilayer and lateral

diffus~on and oligomerization of 20-80 toxin molecules ending up in the

format~on of arc and ring structures which are assumed to be transmembrane

pores (Bhakdi et al, 1986). Presence of transmembrane pores results in cell

lysis or modulation in cell activity, thereby leading to leakage of solutes from

erythrocytes, nucleated cells and liposomes (Andrew et al, 2000)

The binding of pneumolysin is independent of temperature, whereas

oltgomer~zation is dependent on it. The mechanism of binding of

pneumolysin to mammalian cells is not clear. Cholesterol may be the cell

receptor (Alouf and Geoffroy, 1991) based on the evidence that cells which

do not contain cholesterol In their cell membranes are insensitive to lys~s. A

recent report by Jacobs et a1 (1998) suggests that the role of cholesterol is

more involved mth pore formation than mth binding.

Regarding the mode of action, it is unclear whether insertion of

pneumolysin into the target membrane precedes oligomer~zation or

oligomerization occurs on the membrane surface before insertion (Andrew et

al 2000)

Structure:

Pneumolysin is 53 KDa polypeptide of 471 ammoacids and is

produced by almost all strains of pneumococci (Paton et al, 1993; Wheeler et

al, 1999). Complete nucleotide sequence of the pneumolysin gene has been

carr~ed out. The N-terminal aminoacid sequence of purified pneumolys~n was

observed to be NH2-A-N-K-A-V-N-D-F-I-L-A-M-N-Y-D-K as demonstrated by

Walker et al (1987). Cloning of pneumolysin gene has been useful in

carrylng out a number of ~nvest~gations of the structure-function relationships

of the thiol-act~vated toxins. These toxins are called hydrophilic channel

form~ng proteins.

Predicted aminoacid sequence of pneumolysin reveals that the protein

contalns a single cysteine residue at position 428. Substitution of cysteine

with serlne or glyc~ne resulted in reduced cytolytic activity. It has been

demonstrated that 4th doma~n mediates b~nd~ng and self-association of toxin.

So, cysterne region is Important in the orrentation of pneumolysrn, for the

functioning of the channels and cell bind~ng. Pneumolysin contains 8

histidine residues wh~ch are Important In ant~celluiar activity and pore

formatron (Andrew et al, 2000).

Biological properties:

The role of haemolysln In the pathogenes~s of pneumococcal infection

wab reported for the first time by Shumway who observed an Increased

erythrocytic osmotic fragility and hemoglobinemia in rabbits on intravenous

injections of the purified hemolysin (Shumway and Klebanoff, 1971).

Pneumolysin has a variety of toxic effects on different cell types.

It is known that pneumolysin causes lysis of all eukaryotic cells that

have cholesterol in their membrane. Pneumolysin can also injure bronchial

epithelial cells, alveolar epithelial cells and pulmonary arterial endothelial

cells. It is known that these cells are involved in lung-capillary barrier,

destruction by the toxin may lead to the histopathological conditions of early

pneumococcal pneumonia like alveolar flooding and hemorrhage (Mitchell

and Andrew, 2000).

Pneumolysin, at low doses is known to inhibit the respiratory burst of

human polymorphonuclear leukocytes (PMNL) (Paton and Ferrante, 1983).

This is also assoc~ated with reduced ability to take up and kill opsonized

pneumococci. Other properties of pneumolysin included the inhibition of

chernotaxis, random migration of PMNL, respiratory burst, degranulation,

bactericidal activity and phospholipid methylation of human monocytes. All

these effects of pneurnolysin could be blocked when pneumolysin

preparation was pretreated with cholesterol. Inflammatory cytokines TNF-a

and ILI-P was produced when human monocytes were stimulated by very low

doses of pneumolysin Pneumolysin is a potent activator of phospholipase A

In pulmonary artery endothel~al cells. Same phenomenon within the host

during an infection can contribute to direct lung injury and to inflammatory

response. Activation of PMNL and release of toxic molecules can injure

pulmonary tissue (Mitchell and Andrew, 2000). Pneurnolysin has detrimental

effects on ciliated epithelium. The toxln has been found to inhibit the

beatings of cilia on human respiratory epithelial cells and disrupt the

monolayers of cultured epithelial cells from the upper respiratory tract and

alveoli. Breaching of the endothelial barr~er facilitates the development of

pneumococcal bacteremia which is associated with high fatality rate.

Pneurnolysin Increases the alveolar permeability in isolated perfused rat

lungs. Alveolar epithelium acts as a limiting membrane providing a barrier to

tissue invasion by bacteria. Damage to alveolar epithelium can also be one

of the factor contributing for pathogenesis of pneumococcal ~nfection

(Mitchell and Andrew, 2000). Ventricular surface of the brain and cerebral

aqueducts are lined by ciliated ependymal cells which acts as a barrier

between the CSF which is infected during meningitis and neuronal tissue.

These cilia may protect the neuronal tissue from damage during infection by

allowing continual movement of the CSF and preventing margination of

bacteria durlng meningitis. Perturbation of cilia1 function can play a role in

the pathophysiology of pneumococcal meningitis and pneumolysin has

effects on host tissue that could compromise nonspecific host defence

mechanisms (Mitchell and Andrew, 2000).

It is indicated that pneurnolysin plays an important role in

sensorineural hearlng loss, a complication of pneumococcal meningitis This

is studied in guineapig model. Electrophysiological and histological damage

occurred when pneumolysin was perfused through the Scala tympani (Comis

et al, 1993).

Pneumolysin when added to human serum activates the classical

complement pathway in the absence of specific antibody resulting in

decreased serum opsonic activity. This activatton is due to the nonspecific

binding of IgG Fc by the toxin (Mitchell et al, 1991). Function of pneumolysin

to dctivate the complement pathway cannot be blocked by incubation with

cholesterol (Paton et al, 1984). All these studies demonstrate that

pneumolysin can interfere with opsonization, phagocytos~s and killing of

pneumococci and also block the establishment of humoral immune response

to the infection (Paton et al, 1993).

It is also known that significant antipneumolysin antibody titres or

circulating pneumolysin immune complexes are found in sera from the

majority of patients with pneumococcal pneumonia. Antibody levels against

pneumolysin evaluated by neutralrsation test revealed a titer of more than 4-

fold In patients with invasive pneumocoml infections than controls (Bhaskar

et al, 1999). Antipneumolysin levels were lowest in children under 1 year of

age and adults over 70 years of age in whom the risk of pneumocoml

pneumonia is high. Patients with pneumonia had low levels of

antipneumolysin in acute phase sera than in age-matched healthy controls,

indicating that individuals with lower pneumolysin antibody levels may be at

greater risk of contracting pneumococcal pneumonia (Paton et al, 1993).

Genetic analys~s of pneumolysin virulence factor:

Studies with cloned pneumolysin gene has enabled the construction of

a defined pneumolysin-negative derivative of S.pneumoniae type 2 by

insertion-dupl~cation mutagenesis. Virulence of pneumolysin-negative

derivative was compared with its isogenic parental type in a mouse intranasal

and intraperitoneal challenge model. Intravenous challenge with the w~ld

type straln resulted in an ovelwhelrn~ng bacteremia (IO~-IO~ organ~srnslml of

blood) and the animal died within 24 hours. Whereas the mutant

pneumococci had a bacteremia (105-10~rganismslml) that persisted for a

week In several cases wthout any detrimental effect (Paton et al, 1993).

lntratracheal challenge wth pneumolysin negative mutant resulted in

lobar pneumonia in mice rather than the bronchopneumonia seen with

intranasal inoculation. Pneumolysin-negative mutant had decreased ability

to grow and multiply within the lungs and invade the blood stream. Infection

with the wild type pneumococci led to the leakage of serum albumin into the

alveolar air space increasing the permeability of the alveolar capillary barrier

and induced separation of the tight junctions of the epithelial cells and

aaherence of pneumococci. This mechanism may be important In the

invasion process. But infection with pneurnolysin-negative mutant did not

induce the same separation of tight junctions or adherence of pneumococci

(Mitchell and Andrew, 2000).

Association of pneumolysln to meningitis-associated sensorineurai

deafness has been studied using pneumolysin-negative mutant. When

purifled pneumolysin was perfused through the guinea pig cochlea,

substantial electrophysiological and ultrastructural damage occurred (Comis

et al, 1993). Also there was pneumococcal invasion of the scala tympani and

ultrastructural damage to the organ of corti when meningitis was established

experimentally in a guinea pig model by subarachnoid inoculation (Winter et

al, 1996). Pneumolysin negative mutant in this model induced an

inflammatory response in the CSF which was similar to the wild type.

Infection with the wild type also caused a progressive hearing loss at all

frequencies tested. Studies proved that pneumolysin mediated the local

cochlear damage.

In another study by Berry et al (1999), a comparattve virulence of the

type 2 S.pneumoniae strain D39 and derivatives containing insertion-

duplication, deletion or point mutations in the ply gene was examined. Based

on the survival time after i.p. challenge and the numbers of pneumococci in

blood, lungs, brain, llver or spleen at various times after challenge, virulence

of the strain was assessed. A derivative of the type 2 S.pneumoniae strain

D39 in which the portion of the pneumolysin gene encodlng aminoacids 55 to

437 was deleted in-frame was constructed. The virulence of this strain (A

ply) was compared with those of wild type D39, a pneumolysin insertion-

duplicat~on mutant (PLN-A), and a derivative (PdT) carrying a toxin gene with

three point mutations known to abolish both cytolytic activity and complement

activation. PdT was intermediate in virulence between D39 and either PLN-A

or A ply in a niouse, intraperitoneal challenge model. This study provided

unequivocal evidence that pneumolysin has an additional property that is not

abolished by point mutations which reduce cytotoxicity and complement

activation to virtually undetectable levels. This property contributes

significantly to the pathogenesis of disease.

Though pneumolysin clearly plays a role In the pathogenes~s of

pulmonary, ocular and systemic infections by the pneumococcus, its role in

other infections is not very clear. It is known that although pneumolysin does

not have a role in the generation of inflammation in meningitis, it does have

role In the hearing loss associated with the ~nfection (Winter et al, 1996).

All these studies confirm the involvement of pneumolysin in the

pathogenesis of pneumococcal infections. However, the finding that, the

nactivation of the pneumolysin gene s~gnificantly reduced, but did not totally

inhibit, the ability of pneumococci to kill their host indicates that other

pneumococcal products are also involved (Paton et al, 1993).

Role of pneurnolysin i n diagnosis of pneurnococcal infections:

Pneumolysin, has an advantage over CPS as an antigen. It is

common in most or all strains and capable of eliciting an antibody response

and hence an ideal target for diagnosing pneumococcal disease. It occurs in

99% of clinical isolates of S.pneumoniae. Less than 5% cross reaction

between streptolysin and pneumolysin is seen (Kalin et al, 1987).

Systematic study of pneumolysin began with the work on examining

extracts obtained from frozen and ground pneumococci as well as from the

cells dissolved in sodium cholate. Antigenicity of pneumolysin was

demonstrated It was found that haernolyt~c extracts could be ~nact~vated by

ox~dat~on and react~vated by reduction Haemolys~n has been synthestzed

from pneumococcl (Stratn Dl39lR) (M~tchell and Andrew 2000)

Pneumolysln was extracted and pur~fred by Shumway and Klebanoff (1971)

whch Included sonlcat~on, ac~d preclpltatton, ammonrum sulphate

prec~p~tat~on, adsorb~ng on to d~ethyl amno ethyl cellulose concentratlon by

ultraf~ltrat~orl fract~onat~on wth Sephadex G-100 and concentratlon

Kanclersk~ and Mollby (1987) have produced and purtf~ed pneumolys~n

They demonstrated that pneumolys~n was produced by 112 of 113 cl~n~cal

~solates of S pneumonlae In the study The culture of S pneumon~ae was

concentrated by f~ltrat~on 10 t~rnes before centr~fugat~on followed by

pur~f~cat~on of the cellular content by on-exchange chromatography covalent

th~opropyl gel chromatography and gel flitratton Y~eld of 66% and a speclflc

act~vrty of 1 400 000 haemolyt~c unlts per mg of prote~n was reported

Pur~f~cat~on of pneumolys~n helped In further studtes on th~s haemolys~n

A pneumolysin-based agglutination test has been developed by Cima-

Cabal et a1 (1999) to differentiate pneumococcl from other related human and

a, .~mal pathogenic bacter~al strams.

Pneumolys~n has been detected from sputum samples by Western blot

which was reported to be almost as sens~tive as PCR for the non-cultural

detection of pneumococci in S.pneumoniae culture positive sputa from

patients with chest infections (Wheeler et al, 1999). According to a study

(Kearns et al, 2000), pneumolysin detection has led to the identification of

atypical isolates of S.pneumoniae. The atypical ~solates w r e examined by

real-time PCR method for the pneumolysin gene. Using the first defined 559-

bp and 649-bp regions of the pneumolysin gene as nested primers, the ability

of PCR to detect S.pneumoniae in blood was tested by Rudolph et a1 (1993)

which seemed to be a promising alternative for definitive diagnosis.

Polymerase chain reaction assay based on the amplification of

pneumolysin gene fragments in patient sera was developed for the diagnosis

of acute pneumococcal pneumonia by Salo et al (1995) reporting a high

specificity and sensitivity.

Frasen and Tunewall (1969) have detected antibodies against crude

preparations of pneumolysin. The sensitivity and reproducibility of this assay

were not sat~sfactory.

Detection of antibod~es to pneumolysin by a neutralization test using

crude pneumolysin has been done by Bhaskar et al (1999) in invasive

childhood infections. Enzyme linked ~mmunosorbent assay (ELISA) has

been used for measurement of antibodies against highly purifled

pneumolysin by various workers (Kalin et al, 1987; Kanclerski et al, 1988;

Korppi et al, 1992). Detect~on of IgG response to pneumolysln by ELISA

hdve proved to be an aid in the laboratory diagnosis of pneumococcal

pneumonia.

For quantlficatlon of c~rculating Immune complexes (IC) containing

specific antipneumococcal immunoglobulins, ELISA has been developed by

Korppl et al (1998) for the diagnosis of pneumococcal infections. These ICs

containing antibodies to pneumolysin, a protein antigen detected by ELISA

has proved to be more sensitive than other serological methods for the

diagnosis of pneumococcal lower respiratory infection (Korppi et al, 1998).

Based on a study by Toikka et al (1999), it was concluded that a combination

of several methods are needed for the detection of invasive pneumococcal

infection.

Role of pneumolysin in prevention of pneumococcal infection

Future pneumococcal vaccine formulation using pneumolysin:

Studies on the immunogenicity of genetically obtained pneumolysin

toxoids and their conjugation to S.pneumoniae type 19F polysaccharide

conducted by Paton et al (1991) revealed that significant anti-pneumolysoid

titers could be obtained and the immunogenicity of the 19F polysaccharide

moiety was markedly enhanced compared to that of unconjugated

polysaccharide. Conjugation resulted in conversion of 19F polysaccharide

Into an antigen capable of inducing a booster effect. Studies have proved

that pneumolysin toxoids can be a su~table candidate for development of

pneumococcal vaccine as it was able to confer significant degree of

protection in mice (Alexander et at, 1994).

HOST DEFENSE MECHANISMS

Specific or nonspecific ~mmunolog~cal mechanism can be exhibited in

a host. Usually pneumococcal infection does not occur without prior

nasopharyngeal colonization. This colonization is influenzed by various

factors like age, exposure, overcrowding, the season, etc. To colonize,

pneumococcal cells have to get attached to the nasopharyngeal epithelial

cells with the help of adhesin M i c h mediate adherence. Chlldren are more

likely to be carriers of pneumococci than the adults (Gray et al, 1980). A

patient may carry upto four different serotypes of pneumococci. Once

colonized, pneumowccus has the potential to become invasive M e n it

migrates out of the nasopharynx (Musher et al, 2000), to the middle ear of the

paranasal sinuses, bronchial tree, or the blood stream too. The nonspecific

mechanisms which inhibit the pneumococci to invade include, laminar airflow

across mucous layers that filter the inspired air, the glotteal reflex, laryngeal

closure, the cough reflex and ciliary mechanisms. If the normal clearance

mechanism fail to remove pneumococci that have been migrated beyond the

nasopharynx, next comes the secondary defense line consisting of

professional phagocytes which will ingest the bacteria. Again the

pneumococci is w l l protected from this act because of the presence of a

polysaccharide capsule which inhibits phagocytosis. Anticapsular antibody

produced by the host provides serotype-specific protection against

pneumococcal infection. Antibodies appear either afler colonization or

infection following immunization. In persons who have diminished lower

airway clearance, there is a risk of developing pneumonia before antibody is

produced. The incidence of invasive pneumococcal infection is greatly

increased in patients with decreased antibody synthesis or decreased activity

of polymorphonuclear cells and macrophages. In an adult, it is related to

immunocompromised conditions like HIV infection. This increased incidence

is seen in developing countries. Other conditions underlying serious

pneumococcal infections include malnutr~t~on, alcoholism, diabetes and those

wi:h hepatic or renal insufficiency, myeloma and lymphoma. Absence of

spleen further predisposes the infection to a fulminant state resulting in

pneumococcal sepsis.

Antibiotic resistance in S.pneurnoniae:

S.pneurnoniae is a major cause of pneumonia, meningitis, acute otitis

media and acute sinusitis (Austrian, 1994; Musher, 1995). Before the

introduction of penicillin, mortality and morbidity rates of pneumococcal

infect~ons were very high. The only antimicrobial agent used to treat

pneumococcal infections then was sulphonamides. Resistance to

sulphonamides were reported as early as 1943 There was a dramatic

reduction in the death rates once penicillin was introduced. Benzyl penicill~n

soon became the drug of choice for treating pneumococcal infections (Barry,

1 999).

Slowly, reports stating the development of resistance to tetracycline,

chloramphenicol and erythromycin came up. During 1980s and 1990s,

resistance to the alternative drugs became more prevalent As these drugs

had limited use in the treatment of pneumococcal infections, there was little

selective pressure on the normal susceptible population of pneumococci

Penicillin resistance was first reported from the natives of New Gu~nea in the

year 1967 (Jacobs et al, 1978). In 1980s penicillin-resistant pneumococci

emerged in many countries. Currently, the prevalence of resistant

pneumococci to penicillin and other antibiotics IS increasing globally

(Pallares et al, 2000). Antimicrobial consumption is a major factor

contributing to the development of resistance to penicillin and other

antibiotics (Arason et al, 1996; Baquero, 1996; Kanungo et al, 2001).

Mechanism of resistance

Penicillin:

Minimum inhibitory concentrations (MIC) of penicillin of penicillin-

susceptible S.pneumoniae are 5 0.06 pglml. Strains with MlCs from 0.1-1.0

pglml are termed as intermediately susceptible and isolates with MIC > 2.0

pglml are considered as truly resistant to penicillin. Penicillin-intermediate

p~eumococci are not truly resistant to penicillin in all clinical settings; at the

same time, they are not susceptible when used to treat meningitis or other

infections for which low concentrations may be achieved at the site of

infection. The truly reslstant isolates of S.pneumoniae has to be treated wth

alternat~ve drugs (Barry, 1999).

Development of peniclllln reslstance ~n clinical lsolates of

Spneumoniae IS due to the alterat~on of penlclllin blndlng protelns (PBPs)

PBPs from penlclllin-resistant Spneumonfae have reduced affinities for the

antlblotlc molecule (Z~ghelbolm and Tomasz 1980) PEPS catalyze

Important steps in the assembly of peptldoglycan wth the transpept~dat~on

between 2 pept~de s~de chams of the mucln subunlts It Interacts wth p- lactams enzymatically by formlng a covalent complex vla the active site

serlne Alterations In PBPs of reslstant stralns result In decreased afflnlty to

the antrblotlc so that hlgher penlclllln concentrations are requlred for blndlng

and rnhib~tlon of the enzyme (Hakenbeck et al 2000) It was also found that

hlgh-level reslstance to peniclllln lnvolved gradual remodeling of several (3-

4) of the h~gh molecular weight PBFs In parallel wlth the stepwse increase In

the resistance level This was demonstrated by genei~c transformat~on of

reslstance using the cllnical isolates as DNA donors (Zlghelbo~m and

Tornasz 1980, Clonlng and sequencing of these reslstant PBP genes

id~n t~ f ied mosarc sequences In each one of these lndlcatlng a heterologous

recombination (Dowson et al 1989) Development of reslstance to penlclllln

among pneumococci have occurred ~ndependently at several times glvlng

rlse to unique penlcillin-res~stant clones or genetlc lineages (Munoz et al

1992) The beglnnrng of a reslstance clone IS marked by the lncorporatlon of

one of such altered low-affln~ty PBP genes Through cell dlvislon this llneage

wlth low level reslstance expands which agaln gets lnvolved In a znd recornblnat~onal event resulting In the modlflcat~on of another of the high

molecular welght PBP genes In the recipient pneumococcus Th~s process

further continues resulting ~n a stra~n with full blown reslstance to penlclllln

and an increased MIC. But the genetic background of the bacterium remalns

unaltered.

Five PBPs have been described: IA , 18, 2A, 28 and 2X (Musher et al,

2000) Changes in PBP 1A and 2A may explain low level (intermediate)

penlclllln decreased susceptibility; higher levels of reslstance to penicillin

requlres alterations of PBPs I A , 2X and 28 In contrast, high level

reslstance to th~rd generation cephalosporins may result from altered PBPs

1A and 2X Alternat~ve mechanisms leading to res~stance not ~nvolving

PBPs, IS the mutat~ons In genes llke CiaH and CiaR (Musher et al, 2000)

Macrolldes and related antlblotlcs.

Development of reslstance to erythromycln IS by 2 mechanisms

Pneumococc~ that are resistant by vlrtue of an efflux system show low-level

res~stance to erythromycln and are suscept~ble to the llncosamlde

cllndamycln Hlgh level reslstance occurs due to altered target sltes and

those stralns are resistant to cl~ndamycln as well as erythromycln (Leclercq

and Courvalln 1993 Sutcliffe et al 1996) When the lnvitro actlvity of

d~ffxent antlb~ot~cs were compared against 3 phenotypes of pneumococcl

the macrol~des such as clarithromycln dlrlthromyc~n and roxithromyc~n

showed complete cross-res~stance Whereas the streptogramln

qulnuprlstin-dalfoprlstln and the ketolide telithromycln did not show such

cross resistance However both can be used in areas where erythromycln-

resistant stralns are prevalent (Barry 1999)

Quinolones:

Quinolone-resistance in pneumococci has been reported among

patient isolates (Janolr et al, 1996; Tankovic et al, 1996) To develop a hlgh

level resistance atleast 2 mutations are requ~red. The f~rst, Par C, mutation

results in low level resistance to ciprofloxacin (MIC 4 or 8 pglml). These

mutants undergo the 2& mutation, Mhlich involves DNA gyrase (gyr A)

resulting in high level resistance to ciprofloxacin (MIC 16-64 pglml) (Barry,

1999) Resistance to qu~nolones can arise through point mutations in any

subunit of DNA gyrase. The isolate that has undergone the 2" mutatlon

usually are relatively resistant to all other fluoroquinolones except some of

the newer agents llke trovafloxacln and moxafloxacin may have MlCs below

achtevable levels. H~gh level reslstance to ciprofloxacin and related

quinolones are extremely rare

Rifampicin:

Rifampicin reslstance has risen in several different species of bacterla

due to alterations in 1 or more regions in the target of the antib~ot~c, the p- suburiit of RNA polymerase encoded by rpoB. Resistance to rifampicln has

been shorn to evolve by polnt mutatlon In a number of nontransformable

specles (Enr~ght et al, 2C00) R~fampic~n-resistant pneumococci is very rare

Tetracycline:

Tetracycline belng a broad spectrum antib~ot~c, is used in the

treatment of both Gram posltlve and Gram negative infections (Chopra et 31,

1981). Its wdespread use has resulted tn development of resistance in

pneumococci and other bacteria (Widdowson and Klugman, 2000).

Tetracycline reststance gene In pneumococci tet(M), was 1' described

~n Spneumon~ae by Burdett, lnamine and Rajagopalan (1982) Later on,

tet(0) was also described from South Africa The tet(0) and tet(M) genes are

closely related, sharing -76% nucleotide sequence homology and 77%

aminoacid homology (Widdowson and Klugman, 2000).

Trimethoprim:

Trimethoorim, usually in combination with sulfamethoxazole is one of

the most commonly used antibiotics globally

Tr~methoprim inhibits dlhydrofolate reductase (DHFR), thus blocking

the reduction of dihydrofolate to tetrahydrofolate, an important precursor for

the synthesis of purines, thymidylate and certain aminoac~ds and v~tam~ns

Cloning and sequencing of the trimethoprim resistance determinant from

these pneumococcal stralns lndlcated that an altered chromosome-encoded

DHFR was responsible for the observed resistance. Comparison of DHFR

sequences from pneumococcal strains wth various suscept~bilities to

trimethoprim, together with slte-directed mutagenesis, showed that

substitut~on of so leu cine-100 with a leucine residue resulted in trimethoprim

resistance (Pikis et al, 1998).

Sulphonamide resistance in S.pneumoniae can arise by alterations to

the chromosomally encoded dihydropterate synthase (sulA) (Enright et al.

2000).

Clones of antibiotic resistant pneumococci:

Pneumococcal genetic lineages that have achieved massive

gecgraphic spread across continental boundaries have been described. The

most w~dely spread of these is the SpanishlUSA serotype 23F clone.

Spanish serotype 23F strains are resistant to penicillin, tetracycline,

chloramphenicol and variably resistant to erythromycin and trlmethoprim-

sulfamethoxazole (Tomasz, 1999) Th~s clone has spread to numerous

countr~es In Europe the Un~ted K~ngdom Un~ted States South Afrlca South

Amer~ca and some reglons In the Far East (Mc Gee et a1 2000)

Pneumococc~ belong~ng to th~s clone are not only wdespread but also

represents a very large proportion of pen~c~ll~n-res~stant pneumococcl In a

glven epldemlolog~c sett~ng Isolates belong~ng lo th~s clone have been

recovered from all normally sterlle s~tes In lnvaslve paedlatrlc as well as adult

dlsease from different countr~es (Tomasz, 1999)

The second pneumococcal clone is the FrenchlSpanish clone,

resistant to penicillin and trimethopr~m-sulfamethoxazole and expresses

either serotype 14 or 9 also has widely spread all over the world.

A third clone of S.pneumon~ae expressing capsular serotype 6B

carries multidrug res~stant genes. It was identifled in Spain, the UK and

particularly in Iceland (Soares et al, 1993).

Reports of antimicrobial resistance among S.pneumoniae:

Clinical resistance to penic~ll~n in S pneumoniae was l* reported In

Boston In the year 1965. Resistance was later reported from Australla in

1967 and especially In South Afr~ca In 1977 where stralns with both a hlgh

level of penicillin resistance and multiple antibiotic resistance were reported

(Jacobs et al, 1978).

Table 1.Penicillin Resistance of S.pneumoniae from Europe

Country Resistance Year Reference

Poland Germany Switzerland Northern Ireland Belglum Iceland England Spain Romanla France Hungary Finland

Jeljaszewicz et al Milatovlc et al Wust et al Lafong et al Verhaegen et a1 Kristinsson et al Ridgway et al Fenoll et al Millar et al Geslin et al Marton Nissinen and Leinonen

In Portugal, penicillin resistance increased fron~ 4.6% in 1989 to

17.9% In 1993 (Pato et al, 1995) in France it increased from 3.2% in 1987 to

20% in 1992 (Bedos et al, 1996) and In the Unlted States it Increased from

around 5% in the 1980s to 33 5% In 1996 (Thornsberry et al, 1997). But the

incidence of panic~llin-resistant pneumococci remained stable at very low

levels in other areas of Europe Denmark (<2%) Germany (1 8%), Belgium

(2-4%), Sweden (1 7%), Finland (1.7%), Great Britain (1 53.9%) and Italy

(5 5%) (Hedlund et al, 1995; Johnson et al, 1996; Marchese et al, 1995.

Nielsen and Henrichsen, 1996; Nissinen et a l 1995; Re~nert et al, 1995;

Verhaegen et al, 1995). Mult~res~stance to antib~ot~cs has been prominent ln

Brita~n from 1985 onwards (Colman et al, 1998).

Studies from Edinburgh C~ty Hospital, UK, In the year 1995, reported a

very low prevalence rates of penicillin (2:3%) and erythromycin (2.5%)

resistance than those reported elsewhere in the UK (Kalima et al, 1999)

Studles from North America reports a prevalence rate of 68% for

sensitive, 15% for intermediate and 17% for resistance to pencillin among

S.pneumoniae isolates (Barry et al. 1999) during the period of 1996-1997.

Indian scenario:

Epidemiologic character~stics of lnvaslve pneumococcal infections In 6

hospitals In lndla were studled for a perlod of 4 years by the lnvasive

Bacterial lnfectlon Surveillance Group (IBIS) The antlmlcroblal suscept~bil~ty

pattern of 307 lsolates of Spneumon~ae were tested 56 3% were

nonsuscept~ble to co-trlmoxazole 16 6% to chloramphen~col 4 2% to

erythromyc~n 1 3% to oxacllhn and none of the lsolates were resistant to

cefotaxlme (IBIS Study 1999)

The antlbiotlc susceptibilities of 94 isolates of S.pneumoniae obtalned

fr0.n outpatients with respiratory tract lnfectlons were determined by disk

dlffuslon and E-test In the Union Territory of Pondicherry in South India. 6%

of the pneumococcal isolates showed reduced suscept~bility to penicillin (MIC

2 0.125 pglml) and 3% showed reduced susceptibility to cefotaxime (MIC z 0 25 pglml). Resistance to tetracycline (MIC z 4 pglml) and chloramphenicol

(MIC 2 16 pglml) was found in 37% and 4% of the isolates respectively.

Resistance to erythromycin was not found in any of the isolates. 15% of the

pneumococcal isolates showed reduced susceptibility to TMP-SMZ (MIC = 32

[1.6 i 30.4) pglml) and 52% were resistant (MIC 2 64 [3.2 i 60.81 vglml)

(Larsson et al, 1999; Kanungo et al, 2001).

SEROTYPES AND ANTIBIOTIC RESISTANCE

Rates of antibiotic resistance are increasing continuously in the

following years. In the present decade, reslstance to penicillrn has spread

throughout the world This causes concern and knowledge of its evolution

could be of interest for those countries Were lncreaslng resistance is being

observed

A study from Spain, revealed that penicillin resistance was present in

strains of all serogroupslserotypes, the h~ghest rate being found in serogroup

19 and 23 strains Erythromycin resistant rate (48.4%) appeared to be

associated wlth serotype 19 (17.7%) Two rnultiresistant isolates, one

serotype 19 and one nontypeable were resistant to rifarnplcin (,Lopez et al,

1999).

Studles on the impact of human ~mmunodef~c~ency vlrus type 1 on the

disease spectrum of S pneumoniae in South African chlldren revealed that

there was no d~fference in pen~clllln reslstance between HIV-infected and

HIV unlnfected chlldren among the paedlatrlc sero groups (6, 9 14 19 or 23)

64 of 115 (55 6%) vs 99 of 44 (43 2%) respectively, or among other of

serogroups 2 of 27 (7 4%) vs 3 of 33 (9 1 %) (Madhl et al 2000)

Out of 768 (14%) isolates which were rnsusceptlble to 1 or more

antlblotic, 591 of these belonged to serotypes 6, 9, 14, 9 or 23 from a study

by Colman et a1 (1998) in Brltaln Representatives of type 14 resistant to

erythromyc~n were prominent from 1986 onwards Also there was an

Increase In the number of multl-res~stant pneurnococci from 1985 Type 23

was found non-suscept~ble to penlclllln, chlorarnphen~col and tetracycline

Table 2. Predominant Penicillin Resistant Serotypes from Different Countries

Country Predominant Penicillin Reference, year

Ta~wan Europe Thailand Spain Israel Asia Gaza Bangladesh

resistant serotypes 23F,l9F,6B 6,9,14,19,23 23F,9V 19,23 23,19,9,4,6 23,19,6 6,14,19,23 14

Hsueh et al (1999) Priviteria (1994) Dejsirilert et al (1999) Lopez et al(1999) Raz et al(1997) Song et a1 (1999) El-Astal et al (1997) Saha et a1 (1 999)

LABORATORY DIAGNOSIS OF PNEUMOCOCCAL PNEUMONIA

The causes of pneumonia, are st111 ~ncompletely def~ned, pr~mar~ly

because of the d~fficulty In obtaln~ng adequate and appropr~ate samples for

testlng and the lack of rellable diagnostic methods Th~s serves as the maln

drawback because knowledge of causat~ve agents In d~fferent patlent

populations and settlngs IS cruclal for the emplr~cal treatment of the lnfect~on

Recent development of newer d~agnost~c methods, such as serology and

PCR testing have complemented the use of blood and pleural fluid cultures to

assess the etiology of pneumonia.

Sputum examination and culture:

An etiologic role for the pneumococcus in pneumonia is strongly

suggested by the demonstration of large numbers of polymorphonuclear

leukocytes and slightly elongated Gram positive cocci in pairs and chains in

tbr? sputum. Diagnosis can be confirmed by the identification of

S.pneumoniae in sputum culture. But sputum is rarely used as a diagnostic

specimen in children as they do not produce it. In older children and

adolescent, the diagnosis is complicated by lndlgenous flora of the mouth

and pharynx. Correlation between the results of sputum culture, smear and

blood culture in pneumococcal pneumonia IS poor (Parkinson et al, 1992,

Weissenbacher et al, 1990). Some physlclans completely reject sputum

culture and rely on a characteristic sputum Gram's staln or more invasive

procedures. A study by Ort et a1 (1983), suggested a correlation between

Gram's stain and culture data with radiographic findings. However, diagnosis

by sputum culture relies on the quality of the specimen provided, the care

with which the relevant purulent component is sampled (Musher, 1998).

Throat swabs and nasopharyngeal swabs have limited use as

diagnostic procedure, as both sample the normal flora of the upper

respiratory tract.

Blood culture

Fewer than 5% of children wth pneumonia have cu!ture positive

bacteremia (Anonymous 1988) Blood culture is speciflc but the sensittvity is

very low It IS posltlve only In 20-25% of cases (Austrian 1981 Mac Farlane

et al 1982 Kalin and Llndberg 1983) In ambulatory patlents wth

pneumonia the range IS between 3-10% (Parkinson et al 1992 Mason and

Jacobs 1994) Blood cultures are often negatlve from pattents who have

received prior anttmicrobral therapy It has been shorn that drugs can be

transferred along wth blood into the culture broth and suppress the growth of

bacterla (Pazln et al 1982) In patrents wth pleural effusron proport~ons of

posltive blood culture IS found to be hlgh as reported by Hortal et al (1990)

Modern automated systems often y~eld pos~trve blood cultures wthln 12

hours after the sample IS obtatned (Musher 1998)

lnvasive diagnostic techniques:

For dlagnoslng bacterlal pneumonia, isolation of bacter~a from

consolidated lung parenchyma IS a speclflc method (Adegbola et al 1994

Falade et al, 1997) These techniques are usually performed In patlents wth

clln~cal deterloratron or In whom non-rnvaslve methods fall to provlde a

dlagnosls (Parkinson et al, 1992)

In developing countries 50-70% of lung asplrates from those who have

not recelved prior antlb~otlcs are posltlve for bacterla (Funkhouser et al

1991) A range of 46-50% IS reported by other workers (Kalra et al, 1981)

Thls technique, ~f not undertaken wth due care IS often associated wlth

compl~cat~ons of pneumothorax Although, the results of lung puncture

obtalned provlde a deflnltlve dlagnosls of bacterlal pneumonla, ~t IS not

performed routinely as ~t IS lnvaslve and IS regarded as unethical In most

cases (Wolf and Fleer 2000)

Other methods to determine the bacterlal cause of pneumonla In

chrldren Include specimens taken by deep tracheal suctlon and broncho-

alve~lar lavage. Both these methods cannot be employed as routine

methods but can be considered in children requiring endotracheal lntubatlon

(Wolf and Fleer, 2000). Broncheoalveolar lavage and flexlble bronchoscopy

are relatively safe, effective and its diagnostic yield ranges from 27-75% in

children with various underlying diseases (Winthrop et al, 1990).

Antigen detection assays:

A number of rapid diagnostic methods have been developed based

mainly on antigen detection from various clirtical specimens. The various

antigens of S.pneumoniae which has been detected from varlous clinical

specimens include pneumolysln and capsular polysaccharide

Antigen detection assays have several advantages over bacterial

culture Samples for ant~gen detection usually do not need any special

transport media and the tests are simple and can be performed directly aftar

sampling These assays are less influenced by concomitant use of

antimicrobial drugs (Lankinen et al, 1997)

Pneumolysin, a protein antlgen, found in all stra~ns of S pneumoniae is

an Ideal target for antigen detection Studies have proved ~ t s presence in

large amounts in pneumococcus-induced inflammatory processes, in which it

may be an important virulence factor (Walker et al, 1987).

The routlne presumptlve ldentificatlon of pneumococcal colonies relies

primarily on thelr sensitivity to optochin and secondarily on their bile

solub~lity But there are reports where pneumococcl has shown res~stance to

optochin Such misinterpretations can be avoided by performing the

pneumolys~n-based agglutination test It was found to be easy, rapld, cost-

effective and accurate (100% speciflc and 95% sensitive) in identify~ng

pneumococci from other related human and anlmal pathogenic bacteria

(Clma-Cabal et a1 1999) Kearns et al (2000) reported that pneumolysin-

mediated agglut~nation w l l be a rapld and reliable new method for the

identification of S pneumon~ae including atypical stralns

Demonstration of pneunococcal capsular polysaccharide antlgens in

blood, urine or sputum has decreased the time for diagnosis and increased

the diagnostic sensitivity Counter immunoelectrophores~s, Co-agglutination,

Latex agglutination have been employed to detect capsular polysaccharide

antigen and positive results with these methods have indicated a

pneumococcal etiology inspite of negative cultures.

Capsular polysaccharide is a type-specific antigen of pneumowccl

detected by utilizing 'Omniserum' (Statens Serum Institut, Copenhagen,

Denmark), a mixture of rabblt polyclonal antibodies to all known capsular

serotypes The drawback is the cross reaction between Omniserum and the

antlgens of viridans Streptococci (Holmberg et al, 1985). Pneumococcal

polysacchar~des have been detected from urine by Counter

~mrnunoelectropnoresis by Coonrod and Rytel (1973) The test was found to

be less sensitive for the detection of polysaccharide types 7 and 14 than for

other types. Rates of antigen detection varies in different studies. 46% of

sputa revealed the presence of antigen by latex agglutination (Holloway et al,

1991) Singhal et al (1996) has reported a sens~tivity of 100% and a

specificity of 97.1% for detection of pneumococcal antlgens dlrectly In

untreated CSF by modifled latex agglutination test. Pneumococcal capsular

antigen was detected by latex agglutination tests by Boersma et al (1993).

reprtlng a sensitivity of 89% A sensitivity of 78% 100% and 71% was

reported from Baltimore, Sao Paulo and Calro sltes for the diagnosis of

lnvaslve pneumococcal Infection by monovalent latex agglutination tests

using alkalinized, unconcentrated urine samples (Harrison et al, 1996)

Coagglutination for the detection of capsular antigen using sputum and

concentrated urine had a sensitivity of 79% and 12% respectively (Burman et

al, 1991). Whitby et al (1985) reported 74% sensitivity and 98% specificity

by CoA, 71% and 99% by LA and 67% and 98% by Counter

~mmunoelectrophoresis for pneumococcal antigen detection.

Molecular methods:

Molecular based techniques provides rap~d detection of Infectious

dlseases (Cartwight, 1992) lnvitro DNA amplification by the polymerase

chain reaction (PCR) has gained considerable importance in the field of

diagnostic microbiology for its exquisite sensitivity and speclfic~ty (Saiki et al,

1988) According to a study by Toikka et al (1999) blood-based pneumolystn

PCR was compared to blood culture and detection of pneumolysin Immune

complexes, as well as to detection of antibodies to pneumolysin and to C-

polysaccharide, for the diagnosis of pneumococcal infection In febr~le

children. In vitro sens~tivity of the PCR was found to be 10 CFU and 1 CFU

after southern hybridization of the PCR product. It was concluded that

pneumolysin PCR was the most sensitive assay, but its clinical value is

reduced by the fact that 3 blood fractions were needed. Rudolph et al (1993)

studied 16 adults with culture-proven pneumococcal pneumonia using nested

PCR, with primers designed from pneumolyin and autolysin genes. He

reported a invitro sensitivity of 10 fg or 200 CFU for whole bacteria and 20

CFU for buffy coat samples. The sensit~vity of the assay was 37.5% for the

vhole blood and 75% for the buffy coat. 25 adults from Gambia wth

suspcted pneumonia were examined by PCR with primers detecting the

autolysin gene sequence, lnvitro sensitlvity was 50 fg or 3 CFU (Hassan-

King et al, 1994). Salo et al (1995) studied 20 serum samples from adult

patlents with blood culture positive acute pneumococcal pneumonia by using

nested PCR method. In vitro sensitivity was found to be 24 fg. All 20

samples were posltive by PCR with a specificity of 94%. Using whole-blood

PCR, 36 paediatric patients were studied wth suspected bacterem~a, using

the prlmers and probes derlved from penicillin-binding proteln 28 gene The

lnvitro sensitivity was 100 fg or 1 CFU (Zhang et al, 1995). Based on a

prospective study to evaluate the accuracy of pneumolysin PCR of serum, for

the detect~on of pneumococcal infections In ch~ldren, the lnvltro senslt~v~ty of

the pneumolys~n PCR assay was found to be 10 CFU whereas the cllnlcal

sensrtlvlty of blood and CSF culture pos~t~ve samples from 13 pat~ents was

100% It was noted that although pneumolysln PCR was sensltlve ~t was not

very useful for the detect~on of deep-seated pneumococcal lnfect~ons

because a h~gh rate of pos~tlv~ty was seen In the controls (Dagan et a1 1998)

Also, it was proved that pneumolysin PCR IS currently not a feasible

method for routlne d~agnos~s of lnvasive pneumococcal infection in children

Garc~a et al (1999) reported a sensitivity of 911.3% and specificity of 96.5%

for PCR with samples obta~ned by thorac~c needle asp~ratlon (TNA) for

d~agnosis of pneumococcal pneumonia. It was noted that PCR assay was

more sensitive than TNA culture. part~cularly In patlents who had recelved

prior ant~biotic therapy Though PCR was considered as a sensitive and

speclflc techn~que, ~t has not proved to be cost-effect~ve. Western blot

detection of pneumolysin was shown to be almost as sensitive as PCR In 27

sputum samples whlch were culture positive for S.pneurnoniae obtained from

pat~ents having chest infections Western blot and PCR was found to be

mois sensitlve than Counter current ~mmunoelectrophores~s (CIE) for

pneumococcal capsular polysacchar~de ant~gens on the same specimens

The sensitivities noted were loo%, 85% and 67% for PCR, pneumolysln-

immunoblotting and capsular polysacchar~de-CIE respectively. Pneumolysin

detection seems to be a promising technique for the noncultural diagnosis of

pneurnococcal chest infection (Wheeler et al, 1999).

Serological diagnosis of pneumococcal pneumonia:

W ~ t h the development of vaccines to pneumococcl, estimation of

antibodies in the vaccines to determine the level of protection has become

essential. Antibodies against various type of antigens of S.pneumon~ae have

been detected in children and adults Serologic assays for diagnosing

pneumococcal pneumonia have been used less than other methods.

Antlbodles against type-specif~c capsular polysacchar~de (IgG), C-

polysaccharide (IgG, IgM), pneumolysln (IgG) and phosphoryl chollne (IgG,

IgM) have been detected by Burman et al (1991). Significant increase in

antibodies against all pneumococcal antigens were noted.

Serum IgM and IgG antlbodles agalnst pneumococcal polysaccharlde

antlgens have been detected by enzyme-l~nked ~mmunosorbent assay

(ELISA) (Barrett et al 1980) Shyamala et al (1988) has descr~bed an

enzyme-~mmunoassay (EIA) for ant~bodles to pneumococcal

polysacchar~des Assay uses the 14-valent vacclne (Pneumovax) as the

source of antlgen EIA was developed to allow the quant~tat~on of the human

response to ~mmun~zatlon wth pneumococcal polysacchar~de All

nonlmmunlzed subjects tested had IgG ant~bod~es In serum and lmmun~zed

subjects hsd IgG In the~r serum wlth a rlse In IgM and IgA spec~flc ant~body

~o~icentrat~ons at a mean of 6 weeks post-~mmun~zatlon

Modification of a direct ELISA for the detection of IgG and M

antibodies to pneumococcal capsular polysaccharide was reported by

Messina et al (1985) Antlbody responses to serotypes 1, 3, 9N and 23F

were measured Direct ELISA was also performed for the determination of

ant~body responses In ind~viduals immun~zed with 14-valent pneumococcai

vacclne. It was noted that direct ELISA was a sens~t~ve and specific assay.

A sensitivity of 83% was noted in patients with blood culture posltive

pneumococcal pneumonia by ELlSA in paired samples for the detection of

antibodies to pur~fied pneumolysin. Significant rise in ELlSA titre was found

in 17% of the patients (Kanclerski et al, 1988). Kalin et al (1987) reported a

sensitivity of 95% by ELlSA to detect antibodies to pneurnolysin. A two-fold

rise in antibody titres were noted in bacteremic pneumococcal pneumonia

cases.

Ant~body response to CPS, pneumolysin and C-polysacchar~de

antigens of S.pneumoniae were measured by Korppi et al (1992). Findings

of this study suggested that each serologlcal test for pneumococcal etiology

was insensitive and to get an optimal result, a large panel of pneumococcal

antigen and antibody must be used Another study reported a 3-fold rise of

antibody titre to CPS, 3-fold rise of antibodies to C-polysacchar~de and a 2-

fold rise in IgG antibody titre for pneumolysin antigen (Korppi et al, 1993).

Circulatlng pneurnococcal IgG immune complexes in patients wth

comrnun~ty acquired pneumonia were demonstrated by Holloway et al (1993).

E L J A was developed for quantitat~on of circulat~ng immune complexes (ICs)

contaming specific antipneumococcal immunoglobulin G. According to thls

study, no correlation was found between ICs and serum IgG antibody levels.

In the year 1998, Korppi et al evaluated the usefulness of ICs in the

diagnosis of pneumococcal lower respiratory infection in children.

Pneumococcal immune complexes containing antibodies to species-specific

C-polysaccharide, type-specific capsular polysaccharide and pneumolysin

were studied. Measurement of ICs were more sensitive than other

serologlcal methods except in infants to diagnose pneumococcal IOWW

respiratory infections.