A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

Abstract

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r 2 of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 l reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log 10 copies/20 l reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

“…The greatest advantages of PCR can be seen in its sensitivity, specificity and speed with test results being available within hours [9,10]. Real-Time PCR and microarray assays have been investigated for the detection and identification of pathogenic fungi [11,12,13]. However, these methods are the most of the time, unavailable in the routine of mycology laboratories of developing countries.…”

ConcurrentPneumocystis jiroveciiand pulmonary histoplasmosis in an undiagnosed HIV patient

“…Though there is no easy road to definite diagnosis-making in complicated patients, the confirmation of diagnosis is important and could be life-saving. Recently, there is a proposed role of multiplex real-time PCR on biological secretions as it can detect concomitant OIs in a highly efficient manner7; however, the experience with such testing is limited.…”

“…Though there is evidence regarding the usefulness of molecular testing like multiplex real-time PCR in opportunistic coinfections, the clinical experience with such testing is still limited. Moreover, unavailability of these tests in usual clinical settings is another potentially limiting factor 7. Therefore, a multiplex real-time PCR could not be performed in our patient.…”

Diagnostic Methods for Histoplasmosis: Focus on Endemic Countries with Variable Infrastructure Levels

“…For nearly two decades, ITS targets have been used to detect and identify fungal yeasts in culture and specimens [8•, 9, 27], and these loci were recently recommended as the universal DNA barcode marker for fungi [38••]. Molecular ITS pan-fungal assays have been utilized to detect H. capsulatum DNA in formalin-fixed paraffin-embedded (FFPE) tissues [31] in various assays, including a TaqMan® quantitative PCR (qPCR) (Applied Biosystems, Carlsbad, CA) [39, 40] and a multiplex qPCR that concurrently detects closely related pathogenic yeasts [36]. Sensitivities of these qPCR assays in detecting H. capsulatum DNA in human specimens were 70 % [40], 86 % [36] and 95 % [39], and specificities ranged between 96 % [39] and 100 % [40].…”