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

2. Clinical Infectious Diseases 2008; 46:1813-21

3. 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:

4. 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:

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

6. 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

7. 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: ( ); LR+: 6.04; LR-: 0.28
One POSITIVE PCR
88% (75-95%) sensitivity
75% (63-84) specificity
DOR: ( ); LR+: 3.53; LR-: 0.15
Differences in patient cohorts
Differences in methods

8. 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

9. 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

10. Working Group “Towards a standard for Aspergillus PCR”
Working Group “Towards a standard for Aspergillus PCR”
Now in progress. Dozens of labs

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

12. 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

13. 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

14. 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%

15. 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 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

16. Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR
Cuenca-Estrella et al. JCM 2009
Neutropenic patients in high risk for aspergillosis between 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

17. 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

18. 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

19. 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%

20. Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR
0,860
2 PCR +
0,930
3 PCR +
0,861 GM
0,81

21. 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

22. 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

23. 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

24. 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

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

26. 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

27. Evaluation of serial determinations of Aspergillus fumigatus DNA by PCR

28. 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

29. 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

30. 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

31. 23 patients with proven infection

32. 23 patients with proven infection

33. 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

34. 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%

35. 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%

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

37. PCR in tissues. Proven IFI

38. 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

39. 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
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

40. 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)

41. 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