Direct detection and rapid speciation and subspeciation of Mycobacterium avium complex using five-target multiplex PCR and clinical correlations in HIV-positive patients in Tehran, Iran

Disseminated Mycobacterium avium complex (DMAC) infection is a major AIDS-defining condition with diagnostic challenges due to nonspecific symptoms. This study, for the first time in Iran, aimed to discriminate MAC organisms in 100 HIV-positive patients directly from clinical specimens and assess their clinical significance, epidemiological characteristics, and associated risk factors through a detailed review of medical and demographic records. Clinical specimens (blood, sputum, and stool) were collected, and routine clinical evaluations were performed. Acid-fast bacilli were detected using Ziehl-Neelsen (ZN) staining, and multiplex conventional PCR assay was used for typing. Analytical sensitivity and specificity of the multiplex PCR were assessed. Data were analyzed using SPSS statistical software version 19 and analysis of variance (ANOVA test). A total of 28 MAC strains, consisting of 9 isolates of M. avium subsp. paratuberculosis, 6 isolates of M. avium subsp. avium, 11 isolates of M. intracellulare, and 2 isolates of M. genavense were detected in 100 HIV-positive patients. All 28 cases were identified as disseminated infections, including 14 with pulmonary involvement, 9 with gastrointestinal manifestations, and 3 presenting with skin abscesses. M. avium subsp. avium and M. intracellulare were identified in patients with disseminated infections who experienced treatment failure. The multiplex PCR demonstrated high analytical sensitivity and specificity. A significant relationship was found between alkaline phosphatase (ALP) levels, age, CD4 count, education level, treatment adherence, and opportunistic infection incidence (P<0.01). In conclusion, multiplex PCR demonstrated a rapid and reliable assay for direct detection of MAC species and subspecies, outperforming traditional diagnostic approaches.