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PCR-where have we gone?
Manuel Cuenca-EstrellaSpanish National Centre for Microbiology
Diagnostic Issues for Clinicians4th TIMM
Clinical Infectious Diseases 2008; 46:1813-21
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
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
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
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
Hospital 12 de Octubre and Centro Nacional de Microbiología
Cuenca-Estrella et al. JCM 2009
•Samples were analyzed blindly
•Criteria of positive PCR were establish
•Cases were revised by external reviewers
•Clinical and PCR results were faced up
Hospital 12 de Octubre and Centro Nacional de Microbiología
12 cases of aspergillosis according to
EORTC/MSG 2008 (14,4%):•1 proven
•9 probable•2 possible
Cuenca-Estrella et al. JCM 2009
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
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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%
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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 %
Aspergillosis 1 100.0No_Aspergillosis 0 0.0
W = 1.00N = 1
GM$ = (Negative)
TerminalNode 3
Class = No_AspergillosisClass Cases %
Aspergillosis 0 0.0No_Aspergillosis 67 100.0
W = 67.00N = 67
PCR__2_POSITIVE_OR_MORE$ = (Negative)
Node 2Class = No_Aspergillosis
GM$ = (Positive)Class Cases %
Aspergillosis 1 1.5No_Aspergillosis 67 98.5
W = 68.00N = 68
Node 1Class = Aspergillosis
PCR__2_POSITIVE_OR_MORE$ = (Positive)Class Cases %
Aspergillosis 12 14.5No_Aspergillosis 71 85.5
W = 83.00N = 83
Evaluation of serial determinations of Aspergillus fumigatus DNA by
PCR
Hospital 12 de Octubre and Centro Nacional de Microbiología
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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
Evaluation of serial determinations of Aspergillus fumigatus DNA by
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%
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%
15 candidiasis, 8 aspergillosis, and 5 mixed infections
Other Fungal Infections Candida and Aspergillus
Hebart et al. BMT 2009
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