A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.

Diagnostic Challenges and Prospects Associated With Zoonotic Tuberculosis of Central Nervous System

Introduction: The diagnosis of Tuberculous Meningitis (TBM) has remained a challenge due to its insidious onset and the failure of conventional diagnostic tests. The present study aimed to identify the mycobacterial pathogen in the CSF of patients with TBM and a poor prognosis. Methods: We retrospectively recruited 224 TBM and 34 non-TBM patients admitted to the Central India Institute of Medical Sciences, Nagpur, India, in 2014. The CSF samples of these patients were subjected to a duplex PCR assay for the species-specific identification of the causative pathogen. Results: M. bovis and infection with M.tuberculosis were detected in 7% (18) and 32.9% (85) of the patients, respectively. Moreover, 14% (36) of the study samples were culture positive; however, the mycobacterial pathogens could not be differentiated to the species level. Conclusion: The present study findings emphasized the potentially vital importance of M. bovis identification for appropriate patient management. The obtained data also demonstrated the persistent significance of M. bovis, as a zoonotic pathogen.