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Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia.

Church DL, Ambasta A, Wilmer A, Williscroft H, Ritchie G, Pillai DR, Champagne S, Gregson DG - Can J Infect Dis Med Microbiol (2015 Sep-Oct)

Bottom Line: Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.PJ real-time PCR improved detection of PJ in immunocompromised patients.Abstract available from the publisher.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, Calgary Laboratory Services, Departments of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia ; Division of Medical Microbiology and the University of British Columbia, Vancouver, British Columbia ; Medicine, Alberta Health Services and the University of Calgary, Calgary, Alberta, and Department of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia.

ABSTRACT

Background: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.

Methods: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ-]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.

Results: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.

Conclusion: PJ real-time PCR improved detection of PJ in immunocompromised patients.

No MeSH data available.


Related in: MedlinePlus

Comparison of the Pneumocystis jirovecii (PJ) real-time polymerase chain reaction (PCR) assay to that of immunofluorescence assay (IFA) to confirm the diagnostic cut-off of the assay. Ct Cycle threshold; IQR Interquartile range; NN Negative according to both IFA and PJ real-time PCR; NP Negative according to IFA and positive according to PJ real-time PCR; PP Positive according to both IFA and PJ real-time PCR
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f1-cjidmm-26-263: Comparison of the Pneumocystis jirovecii (PJ) real-time polymerase chain reaction (PCR) assay to that of immunofluorescence assay (IFA) to confirm the diagnostic cut-off of the assay. Ct Cycle threshold; IQR Interquartile range; NN Negative according to both IFA and PJ real-time PCR; NP Negative according to IFA and positive according to PJ real-time PCR; PP Positive according to both IFA and PJ real-time PCR

Mentions: Figure 1 compares range and CIs for the PJ real-time assay Ct compared with the detection of PJ cysts using IFA. The 95% upper CI for PJ+ BAL fluid samples that were detected by both the IFA and molecular assays was 26 cycles. The lower and upper 95% CIs for BAL fluid samples that were PJ− according to IFA, but PJ+ according to molecular assay, was 30 and 37 cycles, respectively. Therefore, the reportable range for the PJ real-time assay was established as follows: Ct ≤30 cycles = true positive PJ result; Ct ≥30 but <37 = low positive result that may be indicative of early infection or colonization in which clinical correlation is required to establish the presence of disease; and >37 = negative result.


Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia.

Church DL, Ambasta A, Wilmer A, Williscroft H, Ritchie G, Pillai DR, Champagne S, Gregson DG - Can J Infect Dis Med Microbiol (2015 Sep-Oct)

Comparison of the Pneumocystis jirovecii (PJ) real-time polymerase chain reaction (PCR) assay to that of immunofluorescence assay (IFA) to confirm the diagnostic cut-off of the assay. Ct Cycle threshold; IQR Interquartile range; NN Negative according to both IFA and PJ real-time PCR; NP Negative according to IFA and positive according to PJ real-time PCR; PP Positive according to both IFA and PJ real-time PCR
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644010&req=5

f1-cjidmm-26-263: Comparison of the Pneumocystis jirovecii (PJ) real-time polymerase chain reaction (PCR) assay to that of immunofluorescence assay (IFA) to confirm the diagnostic cut-off of the assay. Ct Cycle threshold; IQR Interquartile range; NN Negative according to both IFA and PJ real-time PCR; NP Negative according to IFA and positive according to PJ real-time PCR; PP Positive according to both IFA and PJ real-time PCR
Mentions: Figure 1 compares range and CIs for the PJ real-time assay Ct compared with the detection of PJ cysts using IFA. The 95% upper CI for PJ+ BAL fluid samples that were detected by both the IFA and molecular assays was 26 cycles. The lower and upper 95% CIs for BAL fluid samples that were PJ− according to IFA, but PJ+ according to molecular assay, was 30 and 37 cycles, respectively. Therefore, the reportable range for the PJ real-time assay was established as follows: Ct ≤30 cycles = true positive PJ result; Ct ≥30 but <37 = low positive result that may be indicative of early infection or colonization in which clinical correlation is required to establish the presence of disease; and >37 = negative result.

Bottom Line: Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.PJ real-time PCR improved detection of PJ in immunocompromised patients.Abstract available from the publisher.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, Calgary Laboratory Services, Departments of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia ; Division of Medical Microbiology and the University of British Columbia, Vancouver, British Columbia ; Medicine, Alberta Health Services and the University of Calgary, Calgary, Alberta, and Department of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia.

ABSTRACT

Background: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.

Methods: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ-]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.

Results: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.

Conclusion: PJ real-time PCR improved detection of PJ in immunocompromised patients.

No MeSH data available.


Related in: MedlinePlus