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Their overlapping clinical manifestations delay diagnosis in developed and resource-limited settings, with greater impact where access to rapid diagnostic tools is limited. This study evaluated the performance of a multiplex quantitative PCR (qPCR) assay for simultaneous detection of Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jirovecii, and Mycobacterium tuberculosis complex in respiratory samples from PLWHA. A cross-sectional study was conducted at the Instituto Nacional de Infectologia Evandro Chagas (Rio de Janeiro, Brazil) between December 2023 and March 2025. Adults with HIV infection (CD4\u00a0&amp;lt;\u00a0200 cells\/mm\u00b3) and clinical or radiological evidence of pulmonary infection were enrolled. Standard diagnostic methods included culture, microscopy, antigen detection (CrAg-LFA, urinary Histoplasma antigen, TB-LAM), GeneXpert\u00ae MTB\/RIF, and commercial qPCR for P. jirovecii. The multiplex assay targeted the ITS region (H. capsulatum and C. neoformans), mtLSU (P. jirovecii), and IS6110 (M. tuberculosis). Among 198 screened participants, 88 met inclusion criteria (mean CD4 count: 56 cells\/\u00b5l). Diagnosis included TB (56.8%), pneumocystosis (34.1%), histoplasmosis (10.2%), and cryptococcosis (11.4%); 25% presented coinfections. The multiplex qPCR showed 100% sensitivity for histoplasmosis, 80% for pneumocystosis, and 40% for both cryptococcosis and TB, with specificities \u2265 82% and 100% for TB. Overall accuracy ranged from 66% to 86%. Mortality was 12.5%, highest among cryptococcosis cases (20%). The assay provided accurate detection of H. capsulatum and P. jirovecii, although performance was limited for TB and cryptococcosis, supporting its use as a complementary molecular tool for differential diagnosis in PLWHA.<\/jats:p>","DOI":"10.1093\/mmy\/myag108","type":"journal-article","created":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T11:37:14Z","timestamp":1791027434000},"source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of a multiplex qPCR assay for rapid diagnosis of fungal pneumonia and tuberculosis in people with advanced HIV disease"],"prefix":"10.1093","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-2448-3198","authenticated-orcid":false,"given":"Thiago Brasil","family":"de Moura da Costa","sequence":"first","affiliation":[{"name":"Funda\u00e7\u00e3o Oswaldo Cruz, Instituto Nacional de Infectologia Evandro Chagas, laborat\u00f3rio de Micologia , Rio de Janeiro, RJ 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