Culture-Free Whole Genome Sequencing of Mycobacterium tuberculosis Using Ligand-Mediated Bead Enrichment Method.

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.

Identification of DprE1 inhibitors for tuberculosis through integrated in-silico approaches.

Decaprenylphosphoryl-ß-D-ribose-2'-epimerase (DprE1), a crucial enzyme in the process of arabinogalactan and lipoarabinomannan biosynthesis, has become the target of choice for anti-TB drug discovery in the recent past. The current study aims to find the potential DprE1 inhibitors through in-silico approaches. Here, we built the pharmacophore and 3D-QSAR model using the reported 40 azaindole derivatives of DprE1 inhibitors. The best pharmacophore hypothesis (ADRRR_1) was employed for the virtual screening of the chEMBL database. To identify prospective hits, molecules with good phase scores (> 2.000) were further evaluated by molecular docking studies for their ability to bind to the DprE1 enzyme (PDB: 4KW5). Based on their binding affinities (< - 9.0 kcal/mole), the best hits were subjected to the calculation of free-binding energies (Prime/MM-GBSA), pharmacokinetic, and druglikeness evaluations. The top 10 hits retrieved from these results were selected to predict their inhibitory activities via the developed 3D-QSAR model with a regression coefficient (R2) value of 0.9608 and predictive coefficient (Q2) value of 0.7313. The induced fit docking (IFD) studies and in-silico prediction of anti-TB sensitivity for these top 10 hits were also implemented. Molecular dynamics simulations (MDS) were performed for the top 5 hit molecules for 200 ns to check the stability of the hits with DprE1. Based on their conformational stability throughout the 200 ns simulation, hit 2 (chEMBL_SDF:357100) was identified as the best hit against DprE1 with an accepted safety profile. The MD results were also in accordance with the docking score, MM-GBSA value, and 3D-QSAR predicted activity. The hit 2 molecule, (N-(3-((2-(((1r,4r)-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)acrylamide) could serve as a lead for the discovery of a novel DprE1 inhibiting anti-TB drug.

A ten-year experience of musculoskeletal tuberculosis at a tertiary care hospital in South India.

Background: The delayed identification and management of musculoskeletal tuberculosis (MSTB) poses substantial health challenges and leads to significant morbidity. This study aimed to collate ten years of hospital data and provide valuable insights into the clinical, diagnostics, and outcomes of the patients diagnosed with MSTB. Methods: A retrospective study was undertaken to review clinic records from 2013 to 2022 for all individuals diagnosed with MSTB in a tertiary care hospital in South India. Results: Over a decade, 400 cases of MSTB were diagnosed, revealing 57 % males and 43 % females with a mean age of 43.2 ± 18.9 years. Spinal TB constituted 72 % of cases, with the most common involvement of thoracic vertebrae (50.9 %). Extra-spinal MSTB accounted for 28 %, prevalent more in the pediatric age group (p < 0.05). Surgical intervention was required for 80 % of spinal TB cases and 58 % of extra-spinal MSTB cases. The average follow-up duration was two years, with 73 % completing treatment. Unfortunately, seven patients died, and three experienced relapse. Conclusion: Spinal TB is the most common type of MSTB and is predominant in young and middle-aged adults, while extra-spinal MSTB is more frequently observed in children. Where use of MRI facilitates early detection of spinal TB; histopathological and microbiological examination confirm the diagnosis. Combining anti-tubercular drugs with modern surgical approaches is essential for obtaining favorable outcomes and improving the quality of life of such patients. It is crucial to have advanced and affordable diagnostic facilities, along with increased public awareness, to reinforce tuberculosis control strategies.

Comparative evaluation of traditional and molecular diagnostic methods for malaria: An analysis of performance.

Purpose: As we edge closer to the eradication of malaria, several methods for detecting Plasmodium species have been developed, including peripheral blood smear examination (PBS), rapid diagnostic tests (RDTs), serological evaluations, fluorescent microscopy, polymerase chain reactions (PCRs), fluorescent in situ hybridization, and flow cytometry. The suitability of these tools for routine diagnosis requires evaluation, considering both their diagnostic accuracy and cost-effectiveness. Materials and Methods: Our study compared four diagnostic techniques for malaria: PBS, quantitative buffy coat (QBC), RDT, and PCR. We used PCR as the benchmark standard and statistically assessed the performance of PBS, QBC, and RDT against PCR in detecting malaria. Adopting a prospective observational approach, we collected blood samples from 117 patients exhibiting the symptoms suggestive of malaria. Results: The findings from our study showed that PBS had a positivity rate of 93.4%, with a 95% confidence interval (CI) of 0.881-0.987, indicating reliable results for a similar population. The QBC assay demonstrated an elevated positivity rate of 96.7% with a solid 95% CI of 0.930-1.000. Although the RDT had a slightly lower rate of 92.4%, it still delivered dependable results, presenting a significant 95% CI of 0.868-0.980, ensuring a robust diagnostic performance compared to PCR. Conclusion: PCR is a reliable test when the identification of the specific species is inconclusive. Conversely, the commonly used PBS occasionally overlooks positive malaria cases due to the specialized skills needed for accurate reading. The cost-effective RDT is feasible for field operations without the need for expert knowledge. However, it fails to differentiate between old and new infections. Meanwhile, the QBC test, known for its sensitivity and speed, can be consistently employed for malaria diagnosis in a tertiary care settings.

Lung abscess by Streptococcus intermedius: An unusual first case report from India.

A unique case report, probably first case from India, of lung abscess caused by Streptococcus intermedius in a previously untreated patient with Type 2 diabetes mellitus is reported here. The patient presented with non-productive cough and right-sided chest pain. Microbiological evaluation confirmed the presence of Streptococcus intermedius and the patient responded positively to antibiotic therapy. This case highlights the fact that S.intermedius may act as pathogen in immunocompromised individuals. So, a caution is needed by the medical fraternity before disregarding it as a commensal.

Impact of Whole-Genome Sequencing of Mycobacterium tuberculosis on Treatment Outcomes for MDR-TB/XDR-TB: A Systematic Review.

The emergence and persistence of drug-resistant tuberculosis is a major threat to global public health. Our objective was to assess the applicability of whole-genome sequencing (WGS) to detect genomic markers of drug resistance and explore their association with treatment outcomes for multidrug-resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB). METHODS: Five electronic databases were searched for studies published in English from the year 2000 onward. Two reviewers independently conducted the article screening, relevant data extraction, and quality assessment. The data of the included studies were synthesized with a narrative method and are presented in a tabular format. RESULTS: The database search identified 949 published articles and 8 studies were included. An unfavorable treatment outcome was reported for 26.6% (488/1834) of TB cases, which ranged from 9.7 to 51.3%. Death was reported in 10.5% (194/1834) of total cases. High-level fluoroquinolone resistance (due to gyrA 94AAC and 94GGC mutations) was correlated as the cause of unfavorable treatment outcomes and reported in three studies. Other drug resistance mutations, like kanamycin high-level resistance mutations (rrs 1401G), rpoB Ile491Phe, and ethA mutations, conferring prothionamide resistance were also reported. The secondary findings from this systematic review involved laboratory aspects of WGS, including correlations with phenotypic DST, cost, and turnaround time, or the impact of WGS results on public health actions, such as determining transmission events within outbreaks. CONCLUSIONS: WGS has a significant capacity to provide accurate and comprehensive drug resistance data for MDR/XDR-TB, which can inform personalized drug therapy to optimize treatment outcomes.

COVID-19 and tuberculosis coinfection: A case-control study from a tertiary care center in South India.

Context: Coronavirus disease 2019 (COVID-19) and tuberculosis (TB), are presently the major infectious diseases imposing a consequential public health threat and their coinfection has a significant impact on the outcome. Aims: To evaluate the clinical features and outcomes of COVID-19-TB coinfected cases compared to solely COVID-19-infected cases. Settings and Design: A retrospective observational study was conducted between August 1, 2020, to February 28, 2022, at a tertiary care hospital. Materials and Methods: In this case-control study, an equal number of gender-age-matched COVID-19 and TB coinfected patients and COVID-19 cases without TB were included using simple random sampling. Statistical Analysis Used: The data was analyzed using SPSS v 26. Categorical variables were compared using the Chi-square test, and an independent t-test or Mann-Whitney U test was applied for the quantitative variables in the univariate analysis. A P-value of less than 0.05 was considered significant. Results: A total of 27 patients were included in each group. Upper lobe involvement (44%) and pleural effusion (22%) were significantly more common in TB-COVID-19 cases when compared to the control group (7% and 4%, respectively; P < 0.05). Moreover, median levels of C-reactive protein and ferritin were significantly higher in TB-COVID-19 coinfection. Conclusions: Chest radiology and a higher level of certain biomarkers like C-reactive protein and ferritin can help to suspect TB in COVID-19 patients and vice-versa.

Etiology and Clinical Profile of Patients with a Tree-in-bud Appearance on High-resolution Computed Tomogram of the Thorax.

We read with great interest the article "the deadly duo of hypertension and diabetes in India: further affirmation from a new epidemiological study" by Metri et al.1 They rightly pointed out that the prevalence of hypertension in Indian patients with type 2 diabetes patients is high and therefore early screening and management of hypertension should be included in the treatment of patients with type 2 diabetes. We wish to share our study findings on the prevalence of hypertension in newly onset diabetes mellitus (DM). We find that the prevalence of hypertension in all males and females with DM was 44.59, 44.34, and 45.16%, respectively.2.