Validation of the MycAssay Pneumocystis Kit for Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Specimens by Comparison to a Laboratory Standard of Direct Immunofluorescence Microscopy, Real-Time PCR, or Conventional PCR

Abstract

dPneumocystis jirovecii pneumonia is a significant cause of morbidity and mortality in AIDS patients as well as those with non-HIV immunosuppressive diseases. To aid diagnosis, the commercial MycAssay Pneumocystis real-time PCR assay (Myconostica, Ltd., Manchester, United Kingdom) targeting the mitochondrial ribosomal large subunit (mtLSU) has been developed to detect P. jirovecii in bronchoalveolar lavage (BAL) specimens. Here, we validated this assay against a laboratory standard of direct immunofluorescence microscopy, a cdc2 real-time PCR assay, or conventional PCR and sequencing of mtLSU. While more sensitive than any of these three assays analyzed individually, the MycAssay Pneumocystis assay demonstrated 100% sensitivity, 100% specificity, a 100% negative predictive value, and a 100% positive predictive value for detecting the presence of P. jirovecii in BAL specimens compared to the laboratory standard. Of note, two samples with positive cycle threshold (C T ) values according to the MycAssay Pneumocystis assay lacked exponential amplification curves and thus were deemed negative. Also negative according to the laboratory standard, these samples highlight the importance of examining the amplification curves, in addition to noting the C T values, when interpreting positive results. Comparison of the MycAssay Pneumocystis assay to a laboratory standard establishes this assay to be a highly sensitive and specific method for the detection of P. jirovecii in bronchoalveolar lavage specimens. The approach may also be useful for the clinical laboratory validation of other sensitive real-time PCR assays.

South African guideline for the management of community-acquired pneumonia in adults

“…Immunohistochemical stains (IFA and silver stains) are most commonly requested on expectorated and induced sputum samples, but the sensitivity of these tests is poor (≤60%) and they are inadequate to rule out the diagnosis of PCP (92). Sensitive PCR assays (93)(94)(95)(96)(97), including a commercial assay (98,99), have been developed and evaluated on a variety of respiratory specimens. Unfortunately, HIV-associated PCP has not been well represented in these evaluation studies.…”

Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia

“…The first assays used for molecular diagnosis of PCP used "in-house" PCR. In recent years, commercial kits were made available by several companies, but few studies evaluated their performances (5,(7)(8)(9)(10) and none compared them. The main objective of this study was to evaluate the concordance between four real-time PCR assays, three commercial kits and the in-house PCR (11), for the detection of P. jirovecii DNA in pulmonary samples.…”

Rapid diagnostic test for respiratory infections

“…A negative PCR result would exclude infection and colonisation by Pneumocystis. 57 The drawback of the conventional PCR and of the nested PCR is the lack of quantification, the determination of which would be a reflection of the fungal load present and could thus help to differentiate between colonisation and acute infection. This problem can be overcome with real-time quantitative PCR; nonetheless, clear cut-off points for distinguishing between colonisation and illness have yet to be established.…”

“…In general, a threshold cycle, or an elevated initiation cycle for the exponential phase of the amplification, represents a low fungal load and is possibly more suggestive of a colonisation than an infection. 57 In these situations of a positive PCR, serological tests can be added (such as the detection in serum of 1-3 beta-d-glucan), which, even without being specific to Pneumocystis, increases in fungal infections and can supplement a positive PCR result in a non-invasive respiratory sample (sputum or even saliva). 58 The negative predictive value of the beta-d-glucan assay is extremely high, and it can be employed in patients on whom bronchoscopy cannot be performed or for whom there is a slight suspicion of infection by Pneumocystis.…”