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

• Published in: Journal of clinical microbiology Vol. 52; no. 4; pp. 1168 - 1176
• Main Authors: Gago, Sara, Esteban, Cristina, Valero, Clara, Zaragoza, Oscar, Puig de la Bellacasa, Jorge, Buitrago, María José
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.04.2014
• Subjects: AIDS-Related Opportunistic Infections - diagnosis; Bronchoalveolar Lavage Fluid - microbiology; Cryptococcus - isolation & purification; Cryptococcus neoformans; DNA, Fungal - chemistry; DNA, Fungal - genetics; DNA, Ribosomal - genetics; DNA, Ribosomal Spacer - genetics; Histoplasma - isolation & purification; Histoplasma capsulatum; Human immunodeficiency virus; Humans; Lung Diseases, Fungal - diagnosis; Molecular Diagnostic Techniques - methods; Multiplex Polymerase Chain Reaction - methods; Mycology; Pneumocystis; Pneumocystis carinii - isolation & purification; Pneumonia - microbiology; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Sensitivity and Specificity; Time Factors
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.02895-13

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 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl 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.