Implementation of national whole-genome sequencing of Mycobacterium tuberculosis, National Public Health Laboratory, Singapore, 2019-2022.

The National Tuberculosis Programme (NTBP) monitors the occurrence and spread of tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in Singapore. Since 2020, whole-genome sequencing (WGS) of Mycobacterium tuberculosis isolates has been performed at the National Public Health Laboratory (NPHL) for genomic surveillance, replacing spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeats analysis (MIRU-VNTR). Four thousand three hundred and seven samples were sequenced from 2014 to January 2023, initially as research projects and later developed into a comprehensive public health surveillance programme. Currently, all newly diagnosed culture-positive cases of TB in Singapore are prospectively sent for WGS, which is used to perform lineage classification, predict drug resistance profiles and infer genetic relationships between TB isolates. This paper describes NPHL's operational and technical experiences with implementing the WGS service in an urban TB-endemic setting, focusing on cluster detection and genomic drug susceptibility testing (DST). Cluster detection: WGS has been used to guide contact tracing by detecting clusters and discovering unknown transmission networks. Examples have been clusters in a daycare centre, housing apartment blocks and a horse-racing betting centre. Genomic DST: genomic DST prediction (gDST) identifies mutations in core genes known to be associated with TB drug resistance catalogued in the TBProfiler drug resistance mutation database. Mutations are reported with confidence scores according to a standardized approach referencing NPHL's internal gDST confidence database, which is adapted from the World Health Organization (WHO) TB drug mutation catalogue. Phenotypic-genomic concordance was observed for the first-line drugs ranging from 2959/2998 (98.7 %) (ethambutol) to 2983/2996 (99.6 %) (rifampicin). Aspects of internal database management, reporting standards and caveats in results interpretation are discussed.

Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer.

The role of programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) in triple negative breast cancer (TNBC) remains to be fully understood. In this study, we investigated the role of PD-1 as a prognostic marker for TNBC in an Asian cohort (n = 269). Samples from patients with TNBC were labeled with antibodies against PD-L1 and PD-1, and subjected to NanoString assays to measure the expression of immune-related genes. Associations between disease-free survival (DFS), overall survival (OS) and biomarker expression were investigated. Multivariate analysis showed that tumors with high PD-1+ immune infiltrates harbored significantly increased DFS, and this increase was significant even after controlling for clinicopathological parameters (HR 0.95; P = 0.030). In addition, the density of cells expressing both CD8 and PD-1, but not the density of CD8-PD-1+ immune infiltrates, was associated with improved DFS. Notably, this prognostic significance was independent of clinicopathological parameters and the densities of total CD8+ cell (HR 0.43, P = 0.011). At the transcriptional level, high expression of PDCD1 within the tumor was significantly associated with improved DFS (HR 0.38; P = 0.027). In line with these findings, high expression of IFNG (HR 0.38; P = 0.001) and IFN signaling genes (HR 0.46; p = 0.027) was also associated with favorable DFS. Inclusion of PD-1 immune infiltrates and PDCD1 gene expression added significant prognostic value for DFS (ΔLRχ2 = 6.35; P = 0.041) and OS (ΔLRχ2 = 9.53; P = 0.008), beyond that provided by classical clinicopathological variables. Thus, PD-1 mRNA and protein expression status represent a promising, independent indicator of prognosis in TNBC.