Targeting novel genes for simultaneous detection of five fungal and bacterial agents from BAL samples using multiplex PCR assay

The main purpose of our study was to evaluate multiplex PCR assay targeting novel genes for detection of five fungal and bacterial agents in BAL samples; because many fungi and bacteria that cause respiratory infections have similar clinical symptoms, diagnosing and differentiating them are therefore essential to controlling and treating them. A total of 100 BAL specimens from a mycobacterium and mycology laboratory were collected from patients suspected of having TB or other respiratory diseases. Novel DNA targets for Aspergillus, Nocardia, Cryptococcus, and Streptomyces were found using modified comparative genomic analysis. Afterward, the primers were designed based on novel targets, and the sensitivity and specificity of the newly designed primers were evaluated. These primers, along with specific primers for M. tuberculosis (SDR), were used in a multiplex PCR assay. The results showed the culture test to be more sensitive than the PCR assay in detecting M. tuberculosis. However, in the detection of Aspergillus, the PCR assay was more sensitive than the culture test. We also found one positive culture and two positive PCR assays for Nocardiosis. Cryptococcal infections and Streptomyces associated with lung diseases were not identified by the culture test nor by the PCR assay. The multiplex PCR is one of the cheapest molecular diagnostic tests readily available for BAL samples in clinical laboratories. This assay can be used for early reports of the causative agents and for treating patients with appropriate drugs at an early stage.