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.
J Clin Microbiol
; 52(4): 1168-76, 2014 Apr.
Article
em En
| MEDLINE
| ID: mdl-24478409
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Infecções Oportunistas Relacionadas com a AIDS
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Cryptococcus
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Pneumocystis carinii
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Reação em Cadeia da Polimerase Multiplex
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Reação em Cadeia da Polimerase em Tempo Real
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Histoplasma
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Pneumopatias Fúngicas
Tipo de estudo:
Diagnostic_studies
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Evaluation_studies
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Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article