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 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.

FIG 1

Quantification standard curve obtained by using serial 1:10 dilutions of different concentrations of DNA from Pneumocystis jirovecii (A), Histoplasma capsulatum (B), and Cryptococcus neoformans (C). Error bars represent the percent coefficient of variation. Standard curves were constructed after a color compensation experiment.

FIG 2

Dependence of fluorescence signal on the number of cycles in the multiplex real-time PCR assay for the patient with mixed infection. (A) Fluorescence detection in the 6-carboxyfluorescein (FAM) fluorescence channel, based on positive signal for the Histoplasma capsulatum molecular beacon probe. (B) Fluorescence detection in the Cyan 500 fluorescence channel. The positive signal is for the Cryptococcus neoformans/C. gattii molecular beacon probe. (C) Fluorescence detection in the 6-carboxy-hexachlorofluorescein (HEX) fluorescence channel. The positive signal is for the Pneumocystis jirovecii molecular beacon probe. (D) Fluorescence detection of Cy5 dye as end label to the specific pICJP molecular beacon probe.