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

And, what about the diagnostic PCR? 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 Not until a PCR system is developed that has been externally validated for blood, tissue, or BAL fluid Clinical Infectious Diseases 2008; 46:1813-21

None PCR system has been externally validated so far. Please keep working

The PCR commercial systems 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 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

16 publications, 1,620 patients (EORTC and prospective) Mengoli 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) sensitivity 87% (78-93) specificity DOR: 21.33 (6.86-466.30); LR+: 6.04; LR-: 0.28 One POSITIVE PCR 88% (75-95%) sensitivity 75% (63-84) specificity DOR: 22.11 (7.77-62.92); LR+: 3.53; LR-: 0.15 Differences in patient cohorts Differences in methods

The PCR problems 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

Bustin SA, Clinical Chemistry 2009 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.html Working Group “Towards a standard for Aspergillus PCR” Now in progress. Dozens of labs

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

48th ICAAC, Abstract M-1721, Morrisey et al Samples and volume 48th ICAAC, Abstract M-1721, Morrisey et al 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

Combination of PCR and GM Barnes et al Journal of Clinical Pathology 2009 125 AI high risk patients studied prospectively EORTC/MSG criteria 1 year follow up (multiple determination) 8% of proven of probable IFI, 12% if PCR was included Diagnostic driven strategy: Decrease in antifungal use and cost saving

Combination of PCR and GM Barnes et al Journal of Clinical Pathology 2009 125 AI high risk patients studied prospectively EORTC/MSG criteria 1 year follow up (multiple determination) 8% of proven of possible IFI, 12% if PCR was included Diagnostic driven: Decrease in antifungal use and cost saving PCR PCR + GM Sensitivity Single sp. Multiple sp. 87.5% 75% 100% Specificity 98%

Serial determinations of Aspergillus fumigatus DNA by PCR + GM for early detection Cuenca-Estrella et al. JCM 2009 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

Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR Cuenca-Estrella et al. JCM 2009 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 Hospital 12 de Octubre and Centro Nacional de Microbiología

1 proven 9 probable 2 possible Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR Cuenca-Estrella et al. JCM 2009 12 cases of aspergillosis according to EORTC/MSG 2008 (14,4%): 1 proven 9 probable 2 possible Hospital 12 de Octubre and Centro Nacional de Microbiología

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 9/12 False Negative 1 3 57/71 67/71 69/71 positive 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% 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 0,860 2 PCR + 0,930 3 PCR + 0,861 GM 0,81

Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR 2 + samples/a week Relative cost: 0.140 ROC: 0.93 Relative risk: 5.04 Prediction success: 93.98

2 + samples/a week Value = 5,04 veces Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR 2 + samples/a week Value = 5,04 veces

Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR Relative cost: 0.140 ROC: 0.97 Relative risk: 6.92 Prediction success: 95.18

Hospital 12 de Octubre and Centro Nacional de Microbiología Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR 48th ICAAC, Abstract M-692 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 CT 68 days before GM Silent and prolonged DNAemia of Aspergillus detected by Real-Time PCR in neutropenic patients receiving antifungal prophylaxis Hospital 12 de Octubre and Centro Nacional de Microbiología

Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR 48th ICAAC, Abstract M-692 ITZ Prophylaxis >38 ºC Positive

Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR Mennink-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

Other Fungal Infections Candida and Aspergillus Hebart et al. BMT 2009 PCR-base preemptive therapy. N=198 Empirical 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 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 76 (36.7%) 12/76 (15.8%) 6.3% PCR-base preemptive therapy. N=198 Empirical antifungal therapy L-AMB One PCR+ or 120 h FN vs. 120 h FN 112 pt (57%) vs. 76 pt. (36.7%) AF therapy

Other Fungal Infections Candida and Aspergillus 15 candidiasis, 8 aspergillosis, and 5 mixed infections Hebart et al. BMT 2009 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 76 (36.7%) 12/76 (15.8%) 6.3% PCR-base preemptive therapy. N=198 Empirical antifungal therapy L-AMB One PCR+ or 120 h FN vs. 120 h FN 112 pt (57%) vs. 76 pt. (36.7%) AF therapy

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 Clinical strains and validation in a murine model. Real time PCR to detect S. apiospermum or S. prolificans S. apiospermum S. prolificans Strains on cultures 100% Lung samples 97.2% 95.5% Serum 81.8% 85% Blood 54.5% 83.3%

Real Time PCR to detect Fusarium solani. In press 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%

Screening of infection? EORTC criteria = + Not classified NON What is the significance of positive PCR results in blood or serum specimens? Screening of infection?

PCR in tissues. Proven IFI

Panfungal PCR Cultures or tissues Yeast or filamentous fungi ITS I ITS II Nuclear rDNA 18S 5.8S Nuclear rDNA 26S Its-1 Its-4 PCR Sequencing Its-1 Its-4 Data Base ITS, ID… and GeneBank

Preliminary results of panfungal PCR. Spanish National Reference Lab A total of 105 positive by ME and negative by culture deep site biopsies were analyzed. 2006-2009 84/105, 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 Mixed infections

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 PCR in tissues: It could be useful for candidiasis PCR in tissues: Proven infections Panfungal or specific Useful for emerging species