INTRODUCTION: Primary mitochondrial diseases (PMDs) comprise a large and heterogeneous group of genetic diseases that result from pathogenic variants in either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Widespread adoption of next-generation sequencing (NGS) has improved the efficiency and accuracy of mtDNA diagnoses; however, several challenges remain. AREAS COVERED: In this review, we briefly summarize the current state of the art in molecular diagnostics for mtDNA and consider the implications of improved whole genome sequencing (WGS), bioinformatic techniques, and the adoption of long-read sequencing, for PMD diagnostics. EXPERT OPINION: We anticipate that the application of PCR-free WGS from blood DNA will increase in diagnostic laboratories, while for adults with myopathic presentations, WGS from muscle DNA may become more widespread. Improved bioinformatic strategies will enhance WGS data interrogation, with more accurate delineation of mtDNA and NUMTs (nuclear mitochondrial DNA segments) in WGS data, superior coverage uniformity, indirect measurement of mtDNA copy number, and more accurate interpretation of heteroplasmic large-scale rearrangements (LSRs). Separately, the adoption of diagnostic long-read sequencing could offer greater resolution of complex LSRs and the opportunity to phase heteroplasmic variants.

INTRODUCTION: Diagnosis of extrapulmonary tuberculosis (EPTB) is an arduous task owing to different anatomical locations, unusual clinical presentations, and sparse bacillary load in clinical specimens. Although GeneXpert® MTB/RIF is a windfall in TB diagnostics including EPTB, it yields low sensitivities but high specificities in many EPTB specimens. To further improve the sensitivity of GeneXpert®, GeneXpert® Ultra, a fully nested real-time PCR targeting IS6110, IS1081 and rpoB (Rv0664) has been endorsed by the WHO (2017), wherein melt curve analysis is utilized to detect rifampicin-resistance (RIF-R). AREA COVERED: We described the assay chemistry/work design of Xpert Ultra and evaluated its performance in several EPTB types, that is, TB lymphadenitis, TB pleuritis, TB meningitis, and so on, against the microbiological reference standard or composite reference standard. Notably, Xpert Ultra exhibited better sensitivities than Xpert, but mostly at the compensation of specificity values. Moreover, Xpert Ultra exhibited low false-negative and false-positive RIF-R results, compared with Xpert. We also detailed other molecular tests, that is, Truenat MTBTM/TruPlus, commercial real-time PCR, line probe assay, and so on, for EPTB diagnosis. EXPERT OPINION: A combination of clinical features, imaging, histopathological findings, and Xpert Ultra are adequate for definite EPTB diagnosis so as to initiate an early anti-tubercular therapy.