期刊
JOURNAL OF CLINICAL MICROBIOLOGY
卷 52, 期 4, 页码 1168-1176出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JCM.02895-13
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资金
- Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III [PI11/00412]
- Fondo de Investigaciones Biomedicas of the Spanish Ministry of Science and Innovation [FI10/00464, FI12/00095]
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 mu l reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log(10) copies/20 mu 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.
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