PCR-where have we gone? Manuel Cuenca-Estrella Spanish National Centre for Microbiology Diagnostic Issues for Clinicians 4th TIMM

PCR-where have we gone?

Manuel Cuenca-EstrellaSpanish National Centre for Microbiology

Diagnostic Issues for Clinicians4th TIMM

Clinical Infectious Diseases 2008; 46:1813-21


Early diagnosis of the infection may be improved if several different diagnostic techniques are combined together. With this approach, the quantification of another fungal component, the beta-glucan, has been included as a diagnostic criterion for probable invasive fungal infection


Early diagnosis of the infection may be improved if several different diagnostic techniques are combined together. With this approach, the quantification of another fungal component, the beta-glucan, has been included as a diagnostic criterion for probable invasive fungal infection

And, what about the diagnostic

PCR? Not until a PCR system is developed Not until a PCR system is developed that has been externally validated for that has been externally validated for blood, tissue, or BAL fluidblood, tissue, or BAL fluid

None PCR system has been None PCR system has been externally validated so far. externally validated so far.

Please keep workingPlease keep working

• Ligth Cycler SeptiFast– Walet et al. CMI 2009.

• 72 Sepsis. Three cases of candidemia, SF detects 1/3– Von Lilienfeld-Toal M. JCM 2009

• 119 FN, – 2 Candida, one by BC and one by SF – 2 A. fumigatus, by SF only

The PCR commercial The PCR commercial systems systems

• Cepheid, Affigene Aspergillus Tracer: Real-time PCR amplification for the qualitative determination of Aspergillus spp. DNA in human whole blood and plasma samples.

• Myconostica, MycAssayTM Aspergillus: It is a CE- marked, real-time PCR assay for the detection of Aspergillus DNA in lower respiratory tract samples

Mengoli et al 2009 Lancet Infect Dis 9:89-96Mengoli et al 2009 Lancet Infect Dis 9:89-96

16 publications, 1,620 patients (EORTC and prospective)

Two o more POSITIVE PCR: 75% (95%CI 54-88) sensitivity87% (78-93) specificityDOR: 21.33 (6.86-466.30); LR+: 6.04; LR-: 0.28

One POSITIVE PCR88% (75-95%) sensitivity75% (63-84) specificityDOR: 22.11 (7.77-62.92); LR+: 3.53; LR-: 0.15

Differences in patient cohortsDifferences in methods

• Samples and volume (blood vs. tissues)

• Extraction• Targets to amplify• Internal control• Quantitative real time, Nested PCR,

Tandem PCR? • Serial determinations• Aspergillus fumigatus so far

The PCR problems The PCR problems

The MIQE guidelines: Minimum information for publication of

quantitative real-time PCR experiments

Bustin SA, Clinical Chemistry 2009

• 60 different items:– Experimental design– Samples– PCR validation – Interpretation

• Some of them should be essential and others desirable

Working Group “Towards a standard for Aspergillus PCR”

http://www.isham.org/Groups.htmlhttp://www.isham.org/Groups.htmlhttp://www.isham.org/Groups.htmlhttp://www.isham.org/Groups.html

Now in progress. Dozens of labs Now in progress. Dozens of labs Now in progress. Dozens of labs Now in progress. Dozens of labs

http://www.isham.org/doc/ASP_PCR_NEWSLETTER_NOV_2007.doc










Samples and volume Samples and volume

LSV: 1 mL, 17/17 aspergillosis and only 3 FP

Samples and volume Samples and volume

Use of PCR on the combination of serum and whole blood specimens for the earlier diagnosis of invasive aspergillosis in haematology patients

16 aspergillosis of 102 patients

9/16 are detected by PCR (56%)Combination detects 12 days earlier

48th ICAAC, Abstract M-1721, Morrisey et al

Combination of PCR and GMBarnes et al Journal of Clinical

Pathology 2009125 AI high risk patients studied prospectively

EORTC/MSG criteria1 year follow up (multiple determination)

8% of proven of probable IFI, 12% if PCR was includedDiagnostic driven strategy: Decrease in antifungal use and cost saving

Combination of PCR and GMBarnes et al Journal of Clinical

Pathology 2009125 AI high risk patients studied prospectively

EORTC/MSG criteria1 year follow up (multiple determination)

8% of proven of possible IFI, 12% if PCR was includedDiagnostic driven: Decrease in antifungal use and cost saving

PCR PCR + GMSensitivity Single sp. Multiple sp.

87.5%75%

100%87.5%

Specificity

98% 100%

Serial determinations of Aspergillus fumigatus DNA by PCR + GM for early detection

•Neutropenic patients in high risk for aspergillosis between 2004 and 2005•Clinical, radiological and microbiological data (GM and cultures)•Whole blood and serum twice a week (four samples weekly) in 83 patients • A total of 2,244 clinical samples were tested

Hospital 12 de Octubre and Centro Nacional de Microbiología

Cuenca-Estrella et al. JCM 2009

Evaluation of serial determinations of Aspergillus

fumigatus DNA by PCR

•Neutropenic patients in high risk for aspergillosis between 2004 and 2005•Clinical, radiological and microbiological data (GM and cultures)•Whole blood and serum twice a week (four samples weekly) in 83 patients • A total of 2,244 clinical samples were tested



•Samples were analyzed blindly

•Criteria of positive PCR were establish

•Cases were revised by external reviewers

•Clinical and PCR results were faced up


12 cases of aspergillosis according to

EORTC/MSG 2008 (14,4%):•1 proven

•9 probable•2 possible


Evaluation of serial determinations of Aspergillus fumigatus DNA by

PCR

1 sample + by PCR

2 samples + by PCR

> 3 samples +

by PCR

Positive 11/12 11/12 9/12FalseNegative 1 1 3

Negative 57/71 67/71 69/71Falsepositive

14 4 2


PCR

1 sample + by PCR

2 samples + by PCR

> 3 samples +

by PCR

Sensitivity 91,6% 91,6% 75,0%

Specificity 80,3% 94,4% 97,2%

PPV 43,9% 73,3% 81,8%

NPV 98,3% 98,5% 95,8%


PCR

1 - Specificity1,00,80,60,40,20,0

Se

ns

itiv

ity

1,0

0,8

0,6

0,4

0,2

0,0

REFERENCE LI NEPOSI TI VE GM

>or= THREE POSI TI VE PCR

>or= TWO POSI TI VE PCR

ONE POSI TI VE PCR

1 PCR + 0,860

2 PCR + 0,930

3 PCR + 0,861

GM 0,81


PCR

PCR__2_POSITIVE_OR_MORE$ = (Yes)

TerminalNode 1

Class = YesClass Cases %NO 4 26.7Yes 11 73.3

W = 15.00N = 15

PCR__2_POSITIVE_OR_MORE$ = (NO)

TerminalNode 2

Class = NOClass Cases %NO 67 98.5Yes 1 1.5

W = 68.00N = 68

Node 1Class = Yes

PCR__2_POSITIVE_OR_MORE$ = (Yes)Class Cases %NO 71 85.5Yes 12 14.5

W = 83.00N = 83

Relative cost: 0.140ROC: 0.93Relative risk: 5.04Prediction success: 93.98


PCR

2 + samples/a week

PCR2R$ = (SI)

TerminalNode 1

Class = SIClass Cases %NO 4 26.7SI 11 73.3

W = 15.00N = 15

PCR2R$ = (NO)

TerminalNode 2

Class = NOClass Cases %NO 66 97.1SI 2 2.9

W = 68.00N = 68

Node 1Class = SI

PCR2R$ = (SI)Class Cases %NO 70 84.3SI 13 15.7

W = 83.00N = 83

Value = 5,04 veces


PCR2 + samples/a

week

Relative cost: 0.140ROC: 0.97Relative risk: 6.92Prediction success: 95.18

PCR__2_POSITIVE_OR_MORE$ = (Positive)

TerminalNode 1

Class = AspergillosisClass Cases %

Aspergillosis 11 73.3No_Aspergillosis 4 26.7

W = 15.00N = 15

GM$ = (Positive)

TerminalNode 2

Class = AspergillosisClass Cases %


W = 1.00N = 1

GM$ = (Negative)

TerminalNode 3

Class = No_AspergillosisClass Cases %


W = 67.00N = 67

PCR__2_POSITIVE_OR_MORE$ = (Negative)

Node 2Class = No_Aspergillosis

GM$ = (Positive)Class Cases %


W = 68.00N = 68

Node 1Class = Aspergillosis

PCR__2_POSITIVE_OR_MORE$ = (Positive)Class Cases %


W = 83.00N = 83


PCR



PCR

15 patients had two consecutive PCR positive

11/15 were finally diagnosed of aspergillosis

DNAemia preceded GM and CT in 7 patients under ITZ prophylaxis:21 days before CT68 days before GM

Silent and prolonged DNAemia of Aspergillus detected by Real-Time PCR in neutropenic patients receiving antifungal prophylaxis

48th ICAAC, Abstract M-692


PCR48th ICAAC, Abstract M-692

0

2

4

6

8

10

12

Week1

Week3

Week5

Week7

Week9

Fever

PCR

CT

GM

ITZPositiv

e

ITZ Prophylaxis

>38 ºC


PCRMennink-Kersten JCM 2006

Little is known of the kinetics of fungal components. GM and other fungal antigens are released when Aspergillus is found in exponential growth phase, while fungal DNA is released when the hyphae break up, a phenomenon which occurs naturally by autolysis when the amount of nutrients is limited or when antifungal agents are present


PCR

Risk of IFI

ProphylaxisGM and CT

when symptoms

Serial PCR and treatment

PCR-base preemptive therapy. N=198Empirical antifungal therapy L-AMB. N=211

One PCR+ or 120 h FN vs. 120 h FN

Other Fungal Infections Candida and Aspergillus

Hebart et al. BMT 2009

PCR-base preemptive therapy. N=198Empirical antifungal therapy L-AMB

One PCR+ or 120 h FN vs. 120 h FN112 pt (57%) vs. 76 pt. (36.7%) AF therapy

IFI proven/probable

AF therapy

IFI in treated

Mort. 30 days

PCR-based

12/4 (8%)

112 (57%)

16/112 (14.3%)

1.5%

Empirical

16/1 (8%)

76 (36.7%)

12/76 (15.8%)

6.3%



PCR-base preemptive therapy. N=198Empirical antifungal therapy L-AMB

One PCR+ or 120 h FN vs. 120 h FN112 pt (57%) vs. 76 pt. (36.7%) AF therapy

IFI proven/probable

AF therapy

IFI in treated

Mort. 30

days

PCR-based

12/4 (8%)

112 (57%)

16/112 (14.3%)

1.5%

Empirical

16/1 (8%)

76 (36.7%)

12/76 (15.8%)

6.3%

15 candidiasis, 8 aspergillosis, and 5 mixed infections



23 patients with proven infection

23 patients with proven infection

Real time PCR to detect Rhizopus, Mucor, Rhizomucor and Cunninghamella

Rabbit model of pulmonary zygomycosis (BAL and biopsies)

Sensitivity 100% PCR vs 67% culture

1-10 sporangiospores/mL, detection limit

Useful for clinical diagnose

Clinical strains and validation in a murine model. Real time PCR to detect S. apiospermum or S.

prolificansS. apiospermum S. prolificans

Strains on cultures 100% 100%

Lung samples 97.2% 95.5%Serum 81.8% 85%Blood 54.5% 83.3%

Clinical strains and validation in a murine model. Real time PCR to detect Fusarium solani

F. solani

Strains on cultures 100%

Lung samples 95.6%

Serum 88.8%

Blood 55.5%

Real Time PCR to detect Fusarium solani. In press

=+ + Not classifiedNON

EORTC criteria

What is the significance of positive PCR results in blood or serum

specimens?Screening of infection?

PCR in tissues. Proven IFI

Nuclear rDNA 18S 5.8S Nuclear rDNA 26SITS I ITS II

Its-1 Its-4PCR

Sequencing

Data Base ITS, ID… and GeneBank

Its-1

Its-4

Yeast or filamentous fungi

Panfungal PCRCultures or tissues

A total of 105 positive by ME and negative by culture deep site biopsies were analyzed. 2006-

200984/105, 80% were positive by panfungal PCR

Species N/%

A. fumigatus 33 (40%)Other Hyalohyphomycetes 22 (26.2%)

Mucorales 7 (8.3%)Candida spp. 5 (6%)Scedosporium spp. 3 (3.6%)Black Fungi 3 (3.6%)Mixed infections 5 (6%)

Preliminary results of panfungal PCR. Spanish National Reference Lab

Conclusions

Aspergillus PCR in blood and serum:–No useful commercial methods are available–A standard is needed–If screening, high S and NPV–If diagnosis, high E and PPV–Combination with other techniques (GM and B-G)–Early diagnosis (infection vs. disease)–Keep working (prophylaxis and treatment)

Conclusions

PCR for other species in blood and serum

–It could be useful for candidiasis

PCR in tissues:–Proven infections–Panfungal or specific–Useful for emerging species

Documents

PCR-where have we gone? Manuel Cuenca-Estrella Spanish National Centre for Microbiology Diagnostic Issues for Clinicians 4th TIMM