Rapid, culture-free detection of carbapenem-resistant Klebsiella pneumoniae in a case of bloodstream infection using genomics.

Timely diagnosis and treatment of sepsis is a major challenge faced by critical care specialists around the world. The traditional blood culture methods have a significant turnaround time which delays targeted therapy leading to poor prognosis. In the current study, we highlight the clinical utility of a genomics solution for diagnosis and management of bloodstream infections by combining the real-time DNA sequencing of Oxford Nanopore Technology with an automated genomic data analysis software. We identify a carbapenem-resistant Klebsiella pneumoniae directly from a blood sample in <24 hours and thereby prove the effectiveness of the test in early diagnosis of sepsis.

Whole-genome sequencing of clinical isolates from tuberculosis patients in India: real-world data indicates a high proportion of pre-XDR cases.

Treatment decisions for tuberculosis (TB) in the absence of full drug-susceptibility data can result in amplifying resistance and may compromise treatment outcomes. Genomics of Mycobacterium tuberculosis (M.tb) from clinical samples enables detection of drug resistance to multiple drugs. We performed whole-genome sequencing (WGS) for 600 clinical samples from patients with tuberculosis to identify the drug-resistance profile and mutation spectrum. We documented the reasons reported by clinicians for referral. WGS identified a high proportion (51%) of pre-extensively drug-resistant (pre-XDR) cases followed by multidrug-resistant tuberculosis (MDR-TB) (15.5%). This correlates with the primary reason for referral, as non-response to the first-line treatment (67%) and treatment failure or rifampicin resistance (14%). Multivariate analysis indicated that all young age groups (P < 0.05), male gender (P < 0.05), and Beijing strain (P < 0.01) were significant independent predictors of MDR-TB or MDR-TB+ [pre-extensively drug-resistant tuberculosis (XDR-TB) and XDR-TB]. Ser315Thr (72.5%) in the inhA gene and Ser450Leu in the rpoB gene (65.5%) were the most prevalent mutations, as were resistance-conferring mutations to pyrazinamide (41%) and streptomycin (61.33%). Mutations outside the rifampicin resistance-determining region (RRDR), Ile491Phe and Val170Phe, were seen in 1.3% of cases; disputed mutations in rpoB (Asp435Tyr, His445Asn, His445Leu, and Leu430Pro) were seen in 6% of cases, and mutations to newer drugs such as bedaquiline and linezolid in 1.0% and 7.5% of cases, respectively. This study on clinical samples highlights that there is a high proportion of pre-XDR cases and emerging resistance to newer drugs; ongoing transmission of these strains can cause serious threat to public health; and whole-genome sequencing can effectively identify and support precision medicine for TB. IMPORTANCE: The current study is based on real-world data on the TB drug-resistance profile by whole-genome sequencing of 600 clinical samples from patients with TB in India. This study indicates the clinicians' reasons for sending samples for WGS, which is for difficult-to-treat cases and/or relapse and treatment failure. The study reports a significant proportion of cases with pre-XDR-TB strains that warrant policy makers' attention. It reflects the current iterative nature of the diagnostic tests under programmatic conditions that leads to delays in appropriate diagnosis and empirical treatment. India had an estimated burden of 2.95 million TB cases in 2020 and 135,000 multidrug-resistant cases. However, WGS profiles of M.tb from India remains disproportionately poorly represented. This study adds a significant body of data on the mutation profiles seen in M.tb isolated from patients with TB in India, mutations outside the RRDR, disputed mutations, and resistance-conferring mutations to newer drugs such as bedaquiline and linezolid.

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.

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.

Effect of public-private interface agency in Patna and Mumbai, India: Does it alter durations and delays in care seeking for drug-sensitive pulmonary tuberculosis?

Background: Public-private interface agency (PPIA) intervention models in Patna (E. India) and Mumbai (W. India) for pulmonary drug-sensitive (DS) tuberculosis (TB) patients were evaluated over 2 years after maturity to examine effect on reduction of patient pathways and retention.  The models engaged private providers, diagnostic facilities and pharmacies into an effective network providing free diagnostic tests and treatment. Methods: A population-based retrospective study was undertaken to assess effectiveness of the PPIA model in care pathways of 64 (Patna) and 86 (Mumbai) patients through in-depth interviews conducted within 6 months of initiation treatments to identify types and facilities accessed, duration to diagnosis and treatment. Median durations based on facilities accessed were statistically analysed.  Comparisons were made with baseline values and endline pathways of patients accessing PPIA engaged/non-engaged facilities in private and public sectors. Results: Compared to non-engaged facilities, persons accessing engaged facilities at first point-of-care had shorter pathways (Mumbai: 32 vs 43 days), (Patna: 15 vs 40 days).  Duration for first care-seeking was considerably shorter for patients accessing PPIA in Patna and for both engaged and non-engaged private facilities in Mumbai (4 days).  Whilst PPIA engaged facilities diagnosed more cases than others, the RNTCP in Mumbai provided diagnosis early.  There was good retention of patients by PPIA-engaged (1 st) facilities (90% post-diagnosis in Patna) but this was affected by the hub-spoke referral system in Mumbai (13%). Second diagnosis is a common feature in Mumbai.  The spoke-hub model in Mumbai contributed considerably to treatment delay; PPIA-engaged providers were better at retaining patients post treatment initiation 11/25 (44%). Conclusion: PPIA-engaged facilities, accessed at onset, result in marked reduction in pathway durations.  Such initiatives should engage a critical mass of competent providers, proximal investigation facilities with enhanced disease awareness and literacy efforts amongst communities.  Patient movement should be minimized for early treatment and retention.

Discovery of small molecule binders of human FSHR(TMD) with novel structural scaffolds by integrating structural bioinformatics and machine learning algorithms.

BACKGROUND: The activation of follicle stimulating hormone receptor (FSHR) by FSH and the consequent downstream signaling activities are crucial for reproductive health. The role of FSHR in tumor progression as well as osteoporosis advancement has also been well established. Currently, steroid preparations of estrogen and progesterone are being used for managing fertility, in spite of the harmful side effects, as there has not been much success in identification of effective FSHR modulators. Structure-based drug design initiatives for identification of potent and specific FSHR modulators have been impeded due to the non-availability of the complete crystal structure of hFSHR complexed with FSH. METHODS: In this study, we have modeled the 3D structure of transmembrane domain (TMD) of hFSHR and identified molecules that demonstrate good binding affinity by virtual screening of drug-like library of compounds. The 3D structural and pharmacophoric features of the binders and non-binders obtained from virtual screening were further used to develop Support Vector Machine based classifier for TMD binding. Based on the observations from docking and SVM classification, a small molecule was identified for extensive MD simulations and in vitro assays for FSHR modulatory activity. RESULTS: The molecule selected based on docking score and SVM prediction was found to inhibit FSH-induced cAMP activity by 80% at 300 µM concentration. CONCLUSION: The study proposes 1,3-diphenyl-1H-pyrazole-5-carboxylate as a promising scaffold for the design of new and potent FSHR allosteric inhibitors.

Such a long journey: What health seeking pathways of patients with drug resistant tuberculosis in Mumbai tell us.

INTRODUCTION: The Indian Tuberculosis (TB) Programme currently faces the dual challenges of tackling increasing numbers of drug resistant (DR) TB cases and regulating practices of a pluralistic private sector catering to TB patients. A study of health seeking behaviour of DR-TB patients in such a situation, offers an opportunity to understand the problems patients face while interacting with health systems. METHODOLOGY: Forty-six DR-TB patients drawn from 15 high TB burden wards in Mumbai were interviewed using an open ended interview tool. Interviews were audio recorded and transcribed. Pathway schematics developed from analysis of patient records, were linked to transcripts. Open coding was used to analyse these units and themes were derived after collating the codes. RESULTS AND DISCUSSION: The paper presents themes interwoven with narratives in the discussions. These include awareness-action gap among patients, role of neighbourhood providers, responsiveness of health systems, the not-such a 'merry go round' that patients go/are made to go on while seeking care, costs of diagnostics and treatment, and how DR-TB is viewed as the 'big TB'. CONCLUSION: The recommendations are based on a preventative ethos which is sustainable, compared to interventions with top-down approaches, which get piloted, but fail to sustain impact when scaled up.