PCR-where have we gone?

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PCR-where have we gone?
Manuel Cuenca-Estrella Spanish National Centre
for Microbiology Diagnostic Issues for
Clinicians 4th TIMM
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Clinical Infectious Diseases 2008 461813-21
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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 461813-21
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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 461813-21
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None PCR system has been externally validated so
far. Please keep working
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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

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Mengoli et al 2009 Lancet Infect Dis 989-96
16 publications, 1,620 patients (EORTC and prospective)
Two o more POSITIVE PCR 75 (95CI 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
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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

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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

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http//www.isham.org/Groups.html
Working Group Towards a standard for Aspergillus PCR
Now in progress. Dozens of labs
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Samples and volume
LSV 1 mL, 17/17 aspergillosis and only 3 FP
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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
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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
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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 87.5
Specificity 98 100
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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
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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
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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
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Evaluation of serial determinations of
Aspergillus fumigatus DNA by PCR
1 sample by PCR 2 samples by PCR gt 3 samples by PCR
Positive 11/12 11/12 9/12
False Negative 1 1 3
Negative 57/71 67/71 69/71
False positive 14 4 2
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Evaluation of serial determinations of
Aspergillus fumigatus DNA by PCR
1 sample by PCR 2 samples by PCR gt 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
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Evaluation of serial determinations of
Aspergillus fumigatus DNA by PCR
1 PCR 0,860
2 PCR 0,930
3 PCR 0,861
GM 0,81
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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
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Evaluation of serial determinations of
Aspergillus fumigatus DNA by PCR
2 samples/a week
Value 5,04 veces
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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
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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
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Evaluation of serial determinations of
Aspergillus fumigatus DNA by PCR
48th ICAAC, Abstract M-692
ITZ Prophylaxis
gt38 ºC
Positive
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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
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Evaluation of serial determinations of
Aspergillus fumigatus DNA by PCR
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Other Fungal Infections Candida and Aspergillus
Hebart et al. BMT 2009
PCR-base preemptive therapy. N198 Empirical antifungal therapy L-AMB. N211
One PCR or 120 h FN vs. 120 h FN
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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 (8) 76 (36.7) 12/76 (15.8) 6.3
PCR-base preemptive therapy. N198 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
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Other Fungal Infections Candida and Aspergillus
Hebart et al. BMT 2009
15 candidiasis, 8 aspergillosis, and 5 mixed
infections
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. N198 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
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23 patients with proven infection
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23 patients with proven infection
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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
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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 100 100
Lung samples 97.2 95.5
Serum 81.8 85
Blood 54.5 83.3
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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
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EORTC criteria
What is the significance of positive PCR results
in blood or serum specimens?
Screening of infection?
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PCR in tissues. Proven IFI
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Panfungal PCR Cultures or tissues
Yeast or filamentous fungi
ITS I
ITS II
Nuclear rDNA 18S
5.8S
Nuclear rDNA 26S
PCR
Sequencing
Data Base ITS, ID and GeneBank
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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 3 (3.6)
Mixed infections 5 (6)
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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)
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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