An integrated method for targeted Oxford Nanopore sequencing and automated bioinformatics for the simultaneous detection of bacteria, fungi, and ARG.

AIMS: The use of metagenomics for pathogen identification in clinical practice has been limited. Here we describe a workflow to encourage the clinical utility and potential of NGS for the screening of bacteria, fungi, and antimicrobial resistance genes (ARGs). METHODS AND RESULTS: The method includes target enrichment, long-read sequencing, and automated bioinformatics. Evaluation of several tools and databases was undertaken across standard organisms (n = 12), clinical isolates (n = 114), and blood samples from patients with suspected bloodstream infections (n = 33). The strategy used could offset the presence of host background DNA, error rates of long-read sequencing, and provide accurate and reproducible detection of pathogens. Eleven targets could be successfully tested in a single assay. Organisms could be confidently identified considering ≥60% of best hits of a BLAST-based threshold of e-value 0.001 and a percent identity of >80%. For ARGs, reads with percent identity of >90% and >60% overlap of the complete gene could be confidently annotated. A kappa of 0.83 was observed compared to standard diagnostic methods. Thus, a workflow for the direct-from-sample, on-site sequencing combined with automated genomics was demonstrated to be reproducible. CONCLUSION: NGS-based technologies overcome several limitations of current day diagnostics. Highly sensitive and comprehensive methods of pathogen screening are the need of the hour. We developed a framework for reliable, on-site, screening of pathogens.

Population structure and antimicrobial resistance patterns of Salmonella Typhi and Paratyphi A amid a phased municipal vaccination campaign in Navi Mumbai, India.

We performed whole-genome sequencing of 174 Salmonella Typhi and 54 Salmonella Paratyphi A isolates collected through prospective surveillance in the context of a phased typhoid conjugate vaccine introduction in Navi Mumbai, India. We investigate the temporal and geographical patterns of emergence and spread of antimicrobial resistance. We evaluated the relationship between the spatial distance between households and genetic clustering of isolates. Most isolates were non-susceptible to fluoroquinolones, with nearly 20% containing ≥3 quinolone resistance-determining region mutations. Two H58 isolates carried an IncX3 plasmid containing blaSHV-12, associated with ceftriaxone resistance, suggesting that the ceftriaxone-resistant isolates from India independently evolved on multiple occasions. Among S. Typhi, we identified two main clades circulating (2.2 and 4.3.1 [H58]); 2.2 isolates were closely related following a single introduction around 2007, whereas H58 isolates had been introduced multiple times to the city. Increasing geographic distance between isolates was strongly associated with genetic clustering (odds ratio [OR] = 0.72 per km; 95% credible interval [CrI]: 0.66-0.79). This effect was seen for distances up to 5 km (OR = 0.65 per km; 95% CrI: 0.59-0.73) but not seen for distances beyond 5 km (OR = 1.02 per km; 95% CrI: 0.83-1.26). There was a non-significant reduction in odds of clustering for pairs of isolates in vaccination communities compared with non-vaccination communities or mixed pairs compared with non-vaccination communities. Our findings indicate that S. Typhi was repeatedly introduced into Navi Mumbai and then spread locally, with strong evidence of spatial genetic clustering. In addition to vaccination, local interventions to improve water and sanitation will be critical to interrupt transmission. IMPORTANCE Enteric fever remains a major public health concern in many low- and middle-income countries, as antimicrobial resistance (AMR) continues to emerge. Geographical patterns of typhoidal Salmonella spread, critical to monitoring AMR and planning interventions, are poorly understood. We performed whole-genome sequencing of S. Typhi and S. Paratyphi A isolates collected in Navi Mumbai, India before and after a typhoid conjugate vaccine introduction. From timed phylogenies, we found two dominant circulating lineages of S. Typhi in Navi Mumbai-lineage 2.2, which expanded following a single introduction a decade prior, and 4.3.1 (H58), which had been introduced repeatedly from other parts of India, frequently containing "triple mutations" conferring high-level ciprofloxacin resistance. Using Bayesian hierarchical statistical models, we found that spatial distance between cases was strongly associated with genetic clustering at a fine scale (<5 km). Together, these findings suggest that antimicrobial-resistant S. Typhi frequently flows between cities and then spreads highly locally, which may inform surveillance and prevention strategies.

Whole-genome sequencing of presumptive MDR-TB isolates from a tertiary healthcare setting in Mumbai.

OBJECTIVES: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB), proven to be a better alternative when compared with the combined sensitivity and specificity of all other modalities for diagnosis of tuberculosis (TB), aids epidemiological surveillance investigations by combining the current research with diagnostics. This study was conducted to identify and resolve operational challenges in performing WGS-based drug resistance testing (DRT) for MTB in a TB culture and drug susceptibility testing (DST) laboratory. Three critical, non-redundant steps for WGS-based DRT were tested: viz. DNA extraction, high-throughput paired-end next-generation sequencing (NGS), and genomic analysis pipeline for automated reporting of WGS-based DRT. METHODS: DNA was extracted from 100 liquid culture isolates on a mycobacterial growth indicator tube (MGIT) using DNEASY Ultraclean Microbial Kit (Qiagen, USA) as per the manufacturer's instructions. Illumina paired-end sequencing was performed. All analysis steps were automated using custom python scripts, requiring no intervention. Variant calling was performed as per the World Health Organization (WHO) technical guide. RESULTS: The number of cultures resistant to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin was 89, 88, 35, 67, and 73, respectively. Resistance to amikacin, kanamycin, and capreomycin was found in 15, 17, and 15 cultures, respectively. Seventy cultures were resistant to fluoroquinolones, four were resistant to ethionamide, and 12 were resistant to linezolid. Six cultures were resistant to only one of the 18 drugs tested. Seventy-five cultures were resistant to more than three anti-TB drugs. One culture was resistant to 13 of the 18 anti-TB drugs tested for this study. The maximum number of variants were observed in the rpoB gene (n = 93, 93%), wherein the Ser450Leu was the predominant mutation (n = 68, 73%). Ser315Thr was the most common variant (n = 86, 97%) that encoded resistance to isoniazid. The Lys43Arg variant encodes resistance to streptomycin and was the third most predominant variant (n = 65, 89%). In addition to the high levels of resistance observed in the dataset, we also observed a high proportion of Beijing strains (n = 63, 63%). CONCLUSION: Compared with results from routine diagnostics based on the 'Guidelines on Programmatic Management of Drug-Resistant TB (PMDT) in India', none of the samples had DST available for all 18 drugs. This represents a gap in PMDT guidelines. The WGS-DRT must be considered as the primary DST method after a sample is flagged rifampicin-resistant by cartridge-based nucleic acid amplification testing (CBNAAT). With several research studies currently underway globally to identify novel variants associated with drug resistance and classifiy their minimum inhibitory coefficients, WGS-DRT presents a scalable technology that updates analytical pipelines, relegating the need for changing microbiological protocols.

Detection of a novel mutation in the rpoB gene in a multidrug resistant Mycobacterium tuberculosis isolate using whole genome next generation sequencing.

BACKGROUND: Mycobacterium tuberculosis (Mtb) drug resistance is a global concern. Moreover, multiple drug resistant (MDR), extensively drug resistant (XDR), and totally drug resistant (TDR) Mtb cases are on the rise in developing countries like India. Most of these cases are identified only 3-6 months after initiation of treatment owing to incomplete/failed clinical response and incomplete information from phenotypic drug resistance assays and/or targeted Mtb mutation analysis. Here, we report the development of an in-house whole genome sequencing (WGS) assay and bioinformatics pipeline that helped resolve the phenotype-genotype discrepancy in a clinical isolate. METHODOLOGY AND RESULTS: A sample from a suspected drug resistant Mtb case tested by line probe assay (LPA) showed the absence of both the mutant and wild type alleles for an rpoB gene mutation site. An in-house next generation sequencing (NGS) assay was used for WGS of this isolate. Bioinformatics analysis revealed that the isolate harboured a novel insertional mutation in the 81-bp hotspot region of the rpoB gene and a S315T mutation in the katG gene, which could explain resistance to rifampicin and isoniazid, respectively. These results correlated with the clinical diagnosis, LPA, solid culture drug susceptibility testing, and pyrosequencing carried out on the sample. The WGS data also provided information regarding the isolate's lineage and indicated an absence of known mutations conferring resistance to other antitubercular drugs. CONCLUSION: WGS is a highly sensitive, specific, and unbiased approach for identification of all possible drug resistance-conferring mutations, which can help clinicians make more informed treatment-related decisions.

Rapid laboratory diagnosis of pulmonary tuberculosis.

BACKGROUND: Tuberculosis (TB) ranks as the second leading cause of death from an infectious disease worldwide. Early diagnosis of Mycobacterium tuberculosis in clinical samples becomes important in the control of TB both for the treatment of patients and for curbing of disease transmission to the others in the community. The study objective was to perform Ziehl-Neelsen (ZN) staining, fluorochrome staining, line probe assay (LPA), and loop-mediated isothermal amplification (LAMP) assay for rapid detection of pulmonary TB (PTB) and to compare the results of LPA and LAMP in terms of sensitivity, specificity, and turnaround time. METHODS: A total of 891 sputum samples from clinically diagnosed/suspected cases of TB were subjected to ZN and fluorochrome staining. Smear positive samples were subjected to LPA, and smear negative were cultured on Lowenstein-Jensen media. A total of 177 samples were subjected to liquid culture and LAMP. Conventional culture was considered as "gold standard" for calculation of parameters. RESULTS: Light-emitting diode fluorescence microscopy had the same sensitivity as ZN with similar high specificity. LPA was performed on 548 sputum samples which includes 520 smear positive and 28 smear negative culture positive samples and multidrug-resistant TB was detected in 32.64%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TB-LAMP on direct sputum samples was found to be 98.96%, 95%, 96%, and 98.70%, respectively, when compared with ZN smear microscopy. By considering culture as "gold standard," LAMP showed a sensitivity, specificity, PPV, and NPV of 98.94%, 96.34%, 96.90%, and 98.75%, respectively. The sensitivity and PPV of TB-LAMP were 98.97% and 96%, respectively, when compared with LPA. CONCLUSIONS: A successful rapid laboratory diagnosis of PTB is possible when one combines the available methodology of microscopy, culture as well as molecular techniques. The LAMP assay was found to be simple, self-contained, and efficacious for early diagnosis of suspected cases of PTB with advantages of having a high throughput, no requirements of sophisticated equipment, and complex biosafety facilities.