Bandari, Aravind K.

Abstract Background Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. Methods We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Results Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%–28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%–94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. Conclusions We demonstrate the feasibility of magnetic bead–based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.

Abstract Primary immunodeficiencies (PIDs) are a rare and heterogeneous group of inherited genetic disorders that are characterized by an absent or impaired immune system. In this report, we describe the use of next-generation sequencing to investigate a male infant with clinical and immunological manifestations suggestive of a PID. Whole-exome sequencing of the infant along with his parents revealed a novel nucleotide variant (cytosine to adenine substitution at nucleotide position 252) in the coding region of the interleukin 2 receptor subunit gamma ( IL2RG ) gene. The mother was found to be a carrier. These findings are consistent with a diagnosis of X-linked severe combined immunodeficiency and represent the first such reported mutation in an Indian family. This mutation leads to an asparagine to lysine substitution ( p.Asn84Lys ) located in the extracellular domain of IL2RG, which is predicted to be pathogenic. Our study demonstrates the power of next-generation sequencing in identifying potential causative mutations to enable accurate clinical diagnosis, prenatal screening, and carrier female detection in PID patients. We believe that this approach, which is not a current routine in clinical practice, will become a mainstream component of individualized medicine in the near future.