Genomic Sequencing from Sputum for Tuberculosis Disease Diagnosis, Lineage Determination, and Drug Susceptibility Prediction.

Universal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited, and targeted molecular assays are vulnerable to emerging resistance mutations. Improved protocols for direct-from-sputum Mycobacterium tuberculosis sequencing would accelerate access to comprehensive drug susceptibility testing and molecular typing. We assessed a thermo-protection buffer-based direct-from-sample M. tuberculosis whole-genome sequencing protocol. We prospectively analyzed 60 acid-fast bacilli smear-positive clinical sputum samples in India and Madagascar. A diversity of semiquantitative smear positivity-level samples were included. Sequencing was performed using Illumina and MinION (monoplex and multiplex) technologies. We measured the impact of bacterial inoculum and sequencing platforms on genomic read depth, drug susceptibility prediction performance, and typing accuracy. M. tuberculosis was identified by direct sputum sequencing in 45/51 samples using Illumina, 34/38 were identified using MinION-monoplex sequencing, and 20/24 were identified using MinION-multiplex sequencing. The fraction of M. tuberculosis reads from MinION sequencing was lower than from Illumina, but monoplexing grade 3+ samples on MinION produced higher read depth than Illumina (P < 0.05) and MinION multiplexing (P < 0.01). No significant differences in sensitivity and specificity of drug susceptibility predictions were seen across sequencing modalities or within each technology when stratified by smear grade. Illumina sequencing from sputum accurately identified 1/8 (rifampin) and 6/12 (isoniazid) resistant samples, compared to 2/3 (rifampin) and 3/6 (isoniazid) accurately identified with Nanopore monoplex. Lineage agreement levels between direct and culture-based sequencing were 85% (MinION-monoplex), 88% (Illumina), and 100% (MinION-multiplex). M. tuberculosis direct-from-sample whole-genome sequencing remains challenging. Improved and affordable sample treatment protocols are needed prior to clinical deployment.

Impact of COVID-19 vaccination on transmission risk of breakthrough infections: Lessons from adapted N95 mask sampling for emerging variants and interventions.

This study used an adapted N95 mask sampling to understand the effect of COVID-19 vaccination in the context of circulating variants on infected individuals to emit the virus into the air, a key risk factor of transmission. Mask, swab, and blood samples were collected from 92 COVID-19 patients vaccinated (Covishield/COVAXIN-partial/fully) or unvaccinated between July and September 2021 during the Delta-dominated period in Mumbai. Mask/swab samples were analyzed by reverse transcription polymerase chain reaction for viral RNA. Blood was evaluated for SARS-CoV-2 anti-spike and nucleocapsid antibody responses. At <48 h of diagnosis, 93% of the patients emitted detectable viral RNA, with 40% emitting >1000 copies in 30 min (high emitters). About 8% continued to be high emitters even after 8 days of symptom onset. No significant difference was observed in emission patterns between partial, full, and unvaccinated patients. However, when vaccinated patients were stratified based on spike protein neutralization and nucleocapsid immunoglobulin G (IgG), the group with moderate/high neutralization showed a significantly lower proportion of high emitters and viral RNA copies than the group with no/low neutralization, which further reduced in the group having antinucleocapsid IgG. In conclusion, mask sampling showed that Delta infections were associated with greater virus emission in patients, which was significantly reduced only in vaccinated patients with moderate/high SARS-CoV-2 neutralization, especially with evidence of past infection. The study demonstrated that mask sampling could be useful for understanding the transmission risk of emerging variants, screening vaccine/booster candidates, and guiding control interventions.

Genomics of postvaccination SARS-CoV-2 infections during the Delta dominated second wave of COVID-19 pandemic, from Mumbai Metropolitan Region (MMR), India.

The present study was initiated to understand the proportion of predominant variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in postvaccination infections during the Delta dominated second wave of coronavirus disease 2019 (COVID-19) in the Mumbai Metropolitan Region (MMR) in India and to understand any mutations selected in the postvaccination infections or showing association with any patient demographics. Samples were collected (n = 166) from severe/moderate/mild COVID-19 patients who were either vaccinated (COVISHIELD/COVAXIN-partial/fully vaccinated) or unvaccinated, from a city hospital and from home isolation patients in MMR. A total of 150 viral genomes were sequenced by Oxford Nanopore sequencing and the data of 136 viral genomes were analyzed for clade/lineage and for identifying mutations. The sequences belonged to three clades (21A, 21I, and 21J) and their lineage was identified as either Delta (B.1.617.2) or Delta+ (B.1.617.2 + K417N) or sub-lineages of Delta variant (AY.120/AY.38/AY.99). A total of 620 mutations were identified of which 10 mutations showed an increase in trend with time (May-October 2021). Associations of six mutations (two in spike, three in orf1a, and one in nucleocapsid) were shown with milder forms of the disease and one mutation (in orf1a) with partial vaccination status. The results indicate a trend toward reduction in disease severity as the wave progressed.

Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing.

BACKGROUND: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. METHODS: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. RESULTS: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. CONCLUSIONS: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

Building capacities of Auxiliary Nurse Midwives (ANMs) through a complementary mix of directed and self-directed skill-based learning-A case study in Pune District, Western India.

Auxiliary nurse midwives (ANMs) play a pivotal role in provision of maternal and newborn health at primary level in India. Effective in-service training is crucial for upgrading their knowledge and skills for providing appropriate healthcare services. This paper aims at assessing the effectiveness of a complementary mix of directed and self-directed learning approaches for building essential maternal and newborn health-related skills of ANMs in rural Pune District, India. METHODS: During directed learning, the master trainers trained ANMs through interactive lectures and skill demonstrations. Improvement and retention of knowledge and skills and feedback were assessed quantitatively using descriptive statistics. Significant differences at the 0.05 level using the Kruskal-Wallis test were analysed to compare improvement across age, years of experience, and previous training received. The self-directed learning approach fulfilled their learning needs through skills mall, exposure visits, newsletter, and participation in conference. Qualitative data were analysed thematically for perspectives and experiences of stakeholders. The Kirkpatrick model was used for evaluating the results. RESULTS: Directed and self-directed learning was availed by 348 and 125 rural ANMs, respectively. Through the directed learning, ANMs improved their clinical skills like maternal and newborn resuscitation and eclampsia management. Less work experience showed relatively higher improvement in skills, but not in knowledge. 56.6% ANMs either improved or retained their immediate post-training scores after 3 months. Self-directed learning helped them for experience sharing, problem-solving, active engagement through skill demonstrations, and formal presentations. The conducive learning environment helped in reinforcement of knowledge and skills and in building confidence. This intervention could evaluate application of skills into practice to a limited extent. CONCLUSIONS: In India, there are some ongoing initiatives for building skills of the ANMs like skilled birth attendance and training in skills lab. However, such a complementary mix of skill-based 'directed' and 'self-directed' learning approaches could be a plausible model for building capacities of health workforce. In view of the transforming healthcare delivery system in India and the significant responsibility that rests on the shoulder of ANMs, a transponder mechanism to implement skill building exercises at regular intervals through such innovative approaches should be a priority.

A time motion study of community mental health workers in rural India.

BACKGROUND: Community Health Workers (CHWs) are critical to providing healthcare services in countries such as India which face a severe shortage of skilled healthcare personnel especially in rural areas. The aim of this study is to understand the work flow of CHWs in a rural Community Mental Health Project (CMHP) in India and identify inefficiencies which impede their service delivery. This will aid in formulating a targeted policy approach, improving efficiency and supporting appropriate work allocation as the roles and responsibilities of the CHWs evolve. METHODS: A continuous observation Time Motion study was conducted on Community Health Workers selected through purposive sampling. The CHWs were observed for the duration of an entire working day (9 am- 3 pm) for 5 days each, staggered during a period of 1 month. The 14 different activities performed by the CHWs were identified and the time duration was recorded. Activities were then classified as value added, non-value added but necessary and non-value-added to determine their time allocation. RESULTS: Home visits occupied the CHWs for the maximum number of hours followed by Documentation, and Traveling. Documentation, Administrative work and Review of work process are the non-value-added but necessary activities which consumed a significant proportion of their time. The CHWs spent approximately 40% of their time on value added, 58.5% of their time on non-value added but necessary and 1.5% of their time on non-value added activities. The CHWs worked for 0.7 h beyond the stipulated time daily. CONCLUSION: The CHW's are "dedicated" mental health workers as opposed to being "generalists" and their activities involve a significant investment of their time due to the specialized nature of the services offered such as counselling, screening and home visits. The CHWs are stretched beyond their standard work hours. Non-value added but necessary activities consumed a significant proportion of their time at the expense of value-added activities. Work flow redesign and implementation of Health Management Information Systems (HMIS) can mitigate inefficiencies.

Deregulation of Genes Associated with Alternate Drug Resistance Mechanisms in Mycobacterium tuberculosis.

Alternate mechanisms of drug resistance involving intrinsic defense pathways play an important role in development of drug resistance. Deregulation of drug efflux, cellular metabolism, and DNA repair have been indicated to have effect on drug tolerance and persistence. Here we chose eight genes from these pathways to investigate their association with development of multidrug resistance (MDR). We generated mono drug resistant and MDR strains of rifampicin and isoniazid and examined the differential expression of genes belonging to efflux, DNA repair and cell wall lipid synthesis pathways. Rv1687c, recB, ppsD and embC genes showed significant (P <0.05) upregulation in mono-resistant (both rifampicin and isoniazid) as well as MDR strains. mmr showed significant upregulation with rifampicin resistance while Rv1457c showed significant upregulation only with mono-resistant strains. Highest expression change was observed with Rv1687c and ppsD. The study identified potential key genes that are significantly associated with development of drug resistance in vitro. These genes may help identify clinical strains predisposed to acquiring drug resistance in patients during the course of treatment or help in management of MDR forms of tuberculosis.

Family planning training needs of auxiliary nurse midwives in Jharkhand, India: Lessons from an assessment.

Background: Auxiliary nurse midwives (ANMs), who play a pivotal role in the provision of maternal health services including family planning services, must be adequately trained and skilled. Systematic assessment of their training needs helps in determining this adequacy. We did a systematic assessment of the training needs of ANMs in Jharkhand. Methods: We designed a multi-stakeholder study including (i) meeting with government officials and international development partners (n=1 5); (ii) structured observation of health facilities (n=1 7); (iii) review of health management information system data; (iv) interviews with ANMs (n=19); and (v) interviews with contraceptive users (n=31). The data were thematically analysed based on gaps in technical knowledge, skills and practices; communication and counselling; infrastructural gaps; uptake of contraceptives and emerging training needs and approaches. Results: The ANMs lacked knowledge, e.g. in natural contraceptive methods and skills, e.g. counselling and recordkeeping. Gaps in infrastructure further hampered the provision of uninterrupted service. The belief system of the community about contraception was perceived as a barrier. The lacunae in their knowledge and skills could be attributed to systemic issues such as quality of pre-service education, absence or poor implementation of in-service trainings or individual issues such as incompetence or apathy towards the provision of service. Government training to ANMs in family planning was inadequate. Conclusion: Our study found inadequacy in the training of ANMs in family planning. We suggest the need for systemic efforts with the life cycle approach to family planning, a mix of theory and practical training, appropriate duration of training, careful selection of trainers, a confidence-building approach and supportive environment to raise competencies of ANMs to help them manage their health facilities.

Appraisal of maternity management and family planning guidelines using the agree II instrument in India.

INTRODUCTION: Guideline development gathered pace in India after the inception of National Rural Health Mission (NRHM) in 2005. However, there is a lack of adequate information about guideline development process, review, and update. This paper reports on the systematic appraisal of Indian guidelines related to maternity management (MM) and family planning (FP) using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) instrument, which was one of the components of a pilot research in 2012-13. MATERIALS AND METHODS: Forty-four selected guidelines about MM and FP, identified through a consensus building workshop, were independently appraised by two appraisers with AGREE instrument having six different domains. Mean item scores, domain scores, and standardized scores were calculated by averaging the scores across the two appraisers. RESULTS: Most guidelines scored high in scope and purpose and clarity of presentation. However, they had little documentation about the development group member details, incorporation of patient views, evidence search method, method chosen for formulating recommendations, tools for application, potential barriers, cost implications, and information about the funding body. Nonclinical guidelines scored higher than clinical guidelines (P = 0.01) for MM in the domain applicability. Clinical FP guidelines scored higher than nonclinical guidelines in the domain of rigor of development (0.01). CONCLUSION: Despite being clinically sound, Indian guidelines score poorly due to weak documentation about their development process. It is recommended that the guideline development process be improved with systematic documentation for achieving standardization.

Kinetics of recA and recX induction in drug-susceptible and MDR clinical strains of Mycobacterium tuberculosis.

OBJECTIVES: To investigate and compare the expression of recA and recX, components of the SOS pathway, following rifampicin treatment in drug-susceptible and MDR clinical strains of Mycobacterium tuberculosis. METHODS: Strains (M. tuberculosis and Mycobacterium smegmatis) were subjected to rifampicin- and mitomycin-induced stress for 36 h followed by RNA extraction. recA and recX in the RNA extract were estimated using qRT-PCR. RESULTS: The MDR clinical strain induced faster (24 h) and higher (7-fold) levels of recA as compared with the drug-susceptible strain (36 h) in response to rifampicin. recX levels were found to rise with an increase in levels of recA; however, the levels were relatively higher than recA. CONCLUSIONS: Drug-susceptible and MDR strains have different kinetics of induction of DNA repair.

Long-term follow-up of contacts of drug-resistant tuberculosis cases in high-burden areas of Mumbai, India.

BACKGROUND: Drug-resistant tuberculosis (DRTB) is a significant public health threat particularly in high burden areas like Mumbai, India. Contacts of DRTB cases are highly vulnerable to infection and development of active disease. In this study we assess long-term outcomes of contacts of DRTB cases, focusing on active TB development and the potential role of IGRA, vitamin D status and supplementation. METHODS: A cohort of 262 DRTB contacts identified from a prior case-control study conducted in Mumbai were enlisted for the study. Interviews were conducted, and data were analysed using descriptive statistics and logistic regression. RESULTS: Of the 262 contacts, 34.73% had LTBI. Three contacts (1.36%) developed active TB, with a crude incidence rate of 4.64 per 1000 people. Vitamin D deficiency was prevalent in 75.3% of contacts, and all three TB cases were vitamin D deficient. Vitamin D supplementation showed a non-significant trend in reducing TB risk (OR = 0.56, p = 0.492). IGRA status did not significantly predict TB development. CONCLUSION: This study provides valuable insights into the long-term outcomes of contacts of DRTB cases. While baseline IGRA did not prove to predict development of active TB, association between vitamin D deficiency and TB development highlights the need for larger studies and development of more effective screening tools. The study contributes valuable information to TB control strategies in high-burden areas.

BCG immunotherapy as an adjunct to chemotherapy in BL-lL patients--its effect on clinical regression, reaction severity, nerve function, lepromin conversion, bacterial/antigen clearance and 'persister' M. leprae.

BACKGROUND AND OBJECTIVE: Multidrug therapy in leprosy has failed to eliminate the problem of persister bacilli. Clearance of bacterial antigens is extremely slow which could predispose to continued nerve damage even after release from treatment. In the present study the immunomodulatory efficacy of BCG vaccine administered post-MDT in BL-LL leprosy patients was investigated in depth with a view to determining if augmenting chemotherapy with immunotherapy would help in faster clearance of M. leprae/antigens, bring down the level of persisters and minimise the occurrence/severity of reaction and nerve damage. METHODS: This is a placebo-controlled study in treated BL-LL patients. The patients are matched with respect to age, sex, bacteriological index and history of reaction, stratified and allocated to the two groups. One group (Gr A) received two doses of BCG-MOSCOW (3-33 x 10(5) cells) and the other (Gr B) normal saline (0.85%), injected intra-dermally at 3 month intervals. The Primary outcomes assessed at the end of 6 months were bacterial/antigen clearance, lepromin conversion, granuloma clearance and the occurrence of persisters. The secondary outcomes were clinical regression, occurrence and severity of reaction and changes in nerve functions. MATERIAL: A total of 107 BL-LL patients comprised of 49 in Gr A and 58 in Gr B; of which 36 and 42 respectively completed the study as per protocol, and are included in the final analysis. FINDINGS: The study findings show that both the primary and the secondary out comes were comparable in the two groups. Two doses of BCG administered post-MDT (Gr A) did not significantly alter the level of persisters or help in hastening the bacterial/antigen clearance, clinical regression of lesions and granuloma clearance. Lepromin conversion rates were also comparable. While the frequency of lepra reaction/neuritis following the intervention was comparable, the severity of reactions was significantly higher in Gr A. On the positive side neural functions assessed by nerve conduction studies showed that deterioration of motor nerve conduction was significantly lower in the BCG arm. Since all patients developing moderate to severe reactions, immediately received a course of corticosteroids, it is possible that timely use of it might have helped.

Availability of treatment for eclampsia in public health institutions in Maharashtra, India.

Severe pre-eclampsia and eclampsia are common causes of maternal deaths worldwide and more so in developing countries. Magnesium sulphate (MgSO4) is now the most-recommended drug of choice to treat these conditions. Despite favourable policies for the use of MgSO4 treatment in India, eclampsia continues to take a high toll. This study examined the availability and use of MgSO4 treatment in the public health system and poor women's recent experiences with eclampsia treatment in Maharashtra state. A mix of qualitative and quantative methods was used. A facility-based survey of all secondary and tertiary healthcare facilities (n = 44) in 3 selected districts and interviews with public and contracted-in private sector obstetricians, health officials, and programme managers were conducted. A list of recently-delivering women from marginalized communities, with up to two livebirths, was drawn through a community-level survey in 272 villages covered by 60 subcentres selected at random. Mothers were selected for interviews, using maximum variation sampling, and interviews were conducted with 17% of the mothers who reported having experienced eclampsia; 61% of facilities had no stock of MgSO4, the stock-out position continuing from a period ranging from 3 months to 3 years while another 20% had some stock, although less than the expected minimum quantity. No treatment for eclampsia was provided in the recent 3 months at 73% facilities. Our survey of recently-delivering mothers recorded a history of eclampsia in 3.2% pregnancies/ deliveries. Interviews with 10 such mothers revealed that treatment for eclampsia has been sought from public as well as private hospitals and from traditional healers. However, facilities where women have received medical treatment are exclusively in the private sector. Almost all public and private care providers were aware of MgSO4 as the gold standard to treat eclampsia; however, it is unclear if they knew of its use to treat severe pre-eclampsia. The private care providers routinely used MgSO4 for eclampsia treatment while the public care providers seemed hesitant to use it fearing risks of complications. We stress the need for improved inventory control practices to ensure sustained availability of supplies and building confidence of care providers in using MgSO4 treatment for severe pre-eclampsia and eclampsia in public facilities, in addition to teaching expectant mothers how to recognize symptoms of these conditions.

Vitamin D and Zinc Supplementation to Improve Treatment Outcomes among COVID-19 Patients in India: Results from a Double-Blind Randomized Placebo-Controlled Trial.

Background: There remains a need to identify low-cost interventions to improve coronavirus disease 2019 (COVID-19) outcomes. Vitamin D and zinc play a role in respiratory infections and could hold value as part of therapeutic regimens. Objectives: To determine the effect of vitamin D or zinc supplementation on recovery from COVID-19. Methods: We conducted a double-blind, randomly assigned 2 x 2 factorial placebo-controlled trial with 1:1:1:1 allocation ratio, enrolling nonpregnant adults with COVID-19 from hospitals in Mumbai and Pune, India (NCT04641195). Participants (N = 181) were randomly assigned to vitamin D3 (180,000 IU bolus, then 2000 IU daily), zinc (40 mg daily), vitamin D3 and zinc, or placebo, for 8 wk. Participants were followed until 8 wk. The primary outcome was time to resolution of fever, cough, and shortness of breath. Secondary outcomes were duration of individual symptoms; need for assisted ventilation; duration of hospital stay; all-cause mortality; and blood biomarkers, including nutritional, inflammatory, and immunological markers. Results: We observed no effect of vitamin D or zinc supplementation on time to resolution of all 3 symptoms [vitamin D hazard ratio (HR): 0.92; 95% confidence interval (95% CI): 0.66, 1.30; P = 0.650; zinc HR: 0.94; 95% CI: 0.67, 1.33; P = 0.745)]. Neither vitamin D nor zinc supplementation was associated with secondary outcomes, except for increased endline serum vitamin D with vitamin D supplementation [median (interquartile range) difference between endline and baseline for vitamin D: 5.3 ng/mL (-2.3 to 13.7); for no vitamin D: -1.4 ng/mL (-5.6 to 3.9); P = 0.003]. We observed nonsignificant increases in serum zinc at endline following zinc supplementation. There was no evidence of interaction between vitamin D and zinc supplementation, no effect of either on hypercalcemia, and no adverse events. Conclusions: Results suggest that neither vitamin D nor zinc supplementation improves COVID-19 treatment outcomes in this population. However, much larger-scale evidence, particularly from populations with vitamin D or zinc deficiency and severe infection, is required to corroborate our findings. This trial was registered at ClinicalTrials.gov and the Clinical Trials Registry of India as NCT04641195 and CTRI/2021/04/032593 respectively.

Distribution of Mycobacterium leprae strains among cases in a rural and urban population of Maharashtra, India.

The elimination of leprosy continues to be a challenge, with the disease remaining endemic in several countries. India accounts for the highest number of cases, and the identification of child cases indicates recent transmission. Genetic markers, like variable-number tandem repeats (VNTRs) and single-nucleotide polymorphisms (SNPs), have been identified to track transmission of the pathogen Mycobacterium leprae. They were used to describe M. leprae strains detected in 48 skin biopsy specimens from leprosy patients in the state of Maharashtra in western India in rural and urban areas near Mumbai. Ninety-three percent of strains across both settings belonged to the SNP type 1D, with three of SNP type 1B being identified in patients living within 3 km of each other. The VNTR profiles of the Maharashtra strains clustered with those from Southern India reported previously and a few other Asian strains, indicating that the Indian strains are genotypically conserved at the level of many VNTR loci. Taken together, SNP and VNTR markers are sufficiently reliable and suitable for both localized and broad geographical genotype associations. VNTR profiles of additional cases may aid in distinguishing the SNP type 1B and 1D strains.

Drug resistance mutations and heteroresistance detected using the GenoType MTBDRplus assay and their implication for treatment outcomes in patients from Mumbai, India.

BACKGROUND: Only 5% of the estimated global multidrug resistant TB (MDRTB) load is currently detected. Endemic Mumbai with increasing MDR would benefit from the introduction of molecular methods to detect resistance. METHODS: The GenoType MTBDRplus assay was used to determine mutations associated with isoniazid and rifampicin resistance and their correlation with treatment outcomes. It was performed on a convenience sample comprising 88 onset and 67 fifth month isolates for which phenotypic drug susceptibility testing (DST) was determined by the Buddemeyer technique for an earlier study. Simultaneous presence of wild type and mutant bands was referred to as "mixed patterns" (heteroresistance). RESULTS: Phenotypically 41 isolates were sensitive; 11 isoniazid, 2 rifampicin, 2 pyrazinamide and 5 ethambutol monoresistant; 16 polyresistant and 78 MDR. The agreement between both methods was excellent (kappa = 0.72-0.92). Of 22 rifampicin resistant onset isolates, the predominant rpoB mutations were the singular lack of WT8 (n = 8) and mixed D516V patterns (n = 9). Of the 64 rifampicin resistant fifth month isolates, the most frequent mutations were in WT8 (n = 31) with a further 9 showing the S531L mutation. Mixed patterns were seen in 22 (34%) isolates, most frequently for the D516V mutation (n = 21). Of the 22 onset and 35 fifth month katG mutants, 13 and 12 respectively showed the S315T1 mutation with loss of the WT. Mixed patterns involving both S315T1 and S315T2 were seen in 9 and 23 isolates respectively. Seventeen of 23 and 23/35 inhA mutant onset and fifth month isolates showed mixed A16G profiles. Additionally, 10 fifth month isolates lacked WT2. Five onset and 6 fifth month isolates had both katG and inhA mutations. An association was noted between only katG but not only inhA resistance and poor outcome (p = 0.037); and additional resistance to ethambutol (p = 0.0033). More fifth month than onset isolates had mixed profiles for at least 1 gene (p = 0.000001). CONCLUSIONS: The use of the assay to rapidly diagnose MDR could guide simultaneous first- and second-line DST, and reduce the delay in administering appropriate regimens. Furthermore, detection of heteroresistance could prevent inaccurate "cured" treatment outcomes documented through smear microscopy and permit more sensitive detection of neonascent resistance.

Contracting in specialists for emergency obstetric care- does it work in rural India?

BACKGROUND: Contracting in private sector is promoted in developing countries facing human resources shortages as a challenge to reduce maternal mortality. This study explored provision, practice, performance, barriers to execution and views about contracting in specialists for emergency obstetric care (EmOC) in rural India. METHODS: Facility survey was conducted in all secondary and tertiary public health facilities (44) in three heterogeneous districts in Maharashtra state of India. Interviews (42) were conducted with programme managers and district and block level officials and with public and private EmOC specialists. Locations of private obstetricians in the study districts were identified and mapped. RESULTS: Two schemes, namely Janani Suraksha Yojana and Indian Public Health standards (IPHS) provided for contracting in EmOC specialists. The IPHS provision was chosen for use mainly due to greater sum for contracting in (US $ 30/service episode vs.300 US$/month). The positions of EmOC specialists were vacant in 83% of all facilities that hence had a potential for contracting in EmOC specialists. Private specialists were contracted in at 20% such facilities. The contracting in of specialists did not greatly increase EmOC service outputs at facilities, except in facilities with determined leadership. Contracting in specialists was useful for non emergency conditions, but not for obstetric emergencies. The contracts were more of a relational nature with poor monitoring structures. Inadequate infrastructure, longer distance to private specialists, insufficient financial provision for contracting in, and poor management capacities were barriers to effective implementation of contracting in. Dependency on the private sector was a concern among public partners while the private partners viewed contracting in as an opportunity to gain experience and credibility. CONCLUSIONS: Density and geographic distribution of private specialists are important influencing factors in determining feasibility and use of contracting in for EmOC. Local circumstances dictate balance between introduction or expansion of contracts with private sector and strengthening public provisions and that neither of these disregard the need to strengthen public systems. Sustainability of contracting in arrangements, their effect on increasing coverage of EmOC services in rural areas and overlapping provisions for contracting in EmOC specialists are issues for future consideration.

High fluoroquinolone resistance proportions among multidrug-resistant tuberculosis driven by dominant L2 Mycobacterium tuberculosis clones in the Mumbai Metropolitan Region.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined. METHODS: In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively. RESULTS: In total, 1016 MTBC strains were MDR, out of which 703 (69.2%) were pre-XDR and 45 (4.4%) were XDR. Cluster rates were high among MDR (57.8%) and pre-XDR/XDR (79%) strains with three dominant L2 (Beijing) strain clusters (Cl 1-3) representing half of the pre-XDR and 40% of the XDR-TB cases. L2 strains were associated with pre-XDR/XDR-TB (P < 0.001) and, particularly Cl 1-3 strains, had high first-line and FQ resistance rates (81.6-90.6%). Epidemic success analysis using THD showed that L2 strains outperformed L1, L3, and L4 strains in short- and long-term time scales. More importantly, L2 MDR and MDR + strains had higher THD success indices than their not-MDR counterparts. Overall, compensatory mutation rates were highest in L2 strains and positive selection was detected in genes of L2 strains associated with drug tolerance (prpB and ppsA) and virulence (Rv2828c). Compensatory mutations in L2 strains were associated with a threefold increase of THD indices, suggesting improved transmissibility. CONCLUSIONS: Our data indicate a drastic increase of FQ resistance, as well as emerging bedaquiline resistance which endangers the success of newly endorsed MDR-TB treatment regimens. Rapid changes in treatment and control strategies are required to contain transmission of highly successful pre-XDR L2 strains in the Mumbai Metropolitan region but presumably also India-wide.

Randomised trial to determine the effect of vitamin D and zinc supplementation for improving treatment outcomes among patients with COVID-19 in India: trial protocol.

INTRODUCTION: Presently, there are few population-level strategies to address SARS-CoV-2 infection except preventive measures such as vaccination. Micronutrient deficiency, particularly vitamin D and zinc deficiency, has been associated with dysregulated host responses, and may play an important role in COVID-19. METHODS AND ANALYSIS: We have designed a 2×2 factorial, randomised, double-blind, multi-centre placebo-controlled trial to evaluate the effect of vitamin D and zinc on COVID-19 outcomes in Maharashtra, India. COVID-19 positive individuals are recruited from hospitals in Mumbai and Pune. Participants are provided (1) vitamin D3 bolus (180 000 IU) maintained by daily dose of 2000 IU and/or (2) zinc gluconate (40 mg daily), versus placebo for 8 weeks. Participants undergo a detailed assessment at baseline and at 8 weeks, and are monitored daily in hospital or every 3 days after leaving the hospital to assess symptoms and other clinical measures. A final follow-up telephone call occurs 12 weeks post-enrolment to assess long-term outcomes. The primary outcome of the study is to time to recovery, defined as time to resolution of all of fever, cough and shortness of breath. Secondary outcomes include: duration of hospital stay, all-cause mortality, necessity of assisted ventilation, change in blood biomarker levels and individual symptoms duration. Participant recruitment commenced on April 2021. ETHICS AND DISSEMINATION: Ethical approval was obtained from institutional ethical committees of all participating institutions. The study findings will be presented in peer-reviewed medical journals. TRIAL REGISTRATION NUMBERS: NCT04641195, CTRI/2021/04/032593, HMSC (GOI)-2021-0060.