MPT51 and MPT64-based antigen detection assay for the diagnosis of extrapulmonary tuberculosis from urine samples.

In view of WHO's "End-TB" strategy, we developed a non-invasive, urine-based ELISA, targeting 2 Mycobacterium tuberculosis antigens namely MPT51 and MPT64 for extrapulmonary TB (EPTB) diagnosis. Suspected EPTB patients (n = 137) [Pleural TB, Abdominal TB and Tuberculous meningitis] were categorized in "Definite" EPTB (n = 10) [Xpert-MTB/RIF and/or culture-positive], "Probable" EPTB (n = 77) and "Non-EPTB" (n = 50) groups using defined composite reference standards. ROC-curves were generated using ELISA results of "Definite" EPTB and "Non-EPTB" groups for both antigens independently and cut-off values were selected to provide 86.3% (95%CI:73.3-94.2) specificity for MPT51 and 92% (95%CI:80.8-97.8) for MPT64. The sensitivity of MPT51-ELISA and MPT64-ELISA was 70% (95%CI:34.7-93.3) and 90% (95%CI:55.5-99.7) for "Definite" EPTB group and 32.5% (95%CI:22.2-44.1) and 30.8% (95%CI:20.8-42.2) for "Probable" EPTB group, respectively. Combining the results of both ELISAs showed a 100% (95%CI:69.1-100) sensitivity in "Definite" EPTB group and 41.6% (95%CI:30.4-53.4) in "Probable" EPTB group, with an 80% (95%CI:66.3-89.9) specificity. The results demonstrated the potential of urine-based ELISAs as screening tests for EPTB diagnosis.

Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study.

Tuberculous Meningitis (TBM) diagnosis remains a grave challenge. We evaluated the utility of extracellular vesicles (EVs) as a source of cell-free transrenal-mycobacterial DNA (cf-Tr-MTB DNA) for TBM diagnosis from urine samples. We developed a qPCR-assay targeting a highly repetitive 36-bp sequence specific to Mycobacterium tuberculosis complex. EVs were isolated from urine samples of suspected TBM groups (n = 44) [categorized using composite reference standard as 'Definite' TBM (n = 8), 'Probable' TBM (n = 15), 'Possible' TBM (n = 21)] and 'Non-TBM' group (n = 26). cf-Tr-MTB DNA-based qPCR assay was applied to DNA isolated from EVs (EV-DNA) and EV-free-fraction (EV-free DNA). ROC-curves were generated using qPCR results of 'Definite' TBM and 'Non-TBM' category in both EV-DNA and EV-free DNA samples and cut-off values were selected to provide 100% (95%CI:69.1-100) specificity. The cf-Tr-MTB DNA assay gave a sensitivity of 54.5% (95%CI:38.8-69.6) for EV-DNA and 77.3% (95%CI:62.1-88.5) for EV-free DNA in the TBM group (n = 44). The combination of EV-DNA and EV-free DNA results (corresponding to performance cf-Tr MTB DNA assay in urine), gave an overall sensitivity of 81.8% (95%CI:67.2-91.8) in the TBM group. Our results confirmed EVs as one of the sources of cf-Tr-MTB DNA and we believe the cf-Tr-MTB DNA-based qPCR assay has a potential application for TBM diagnosis.