Maxine Caws’s research while affiliated with Liverpool School of Tropical Medicine and other places

Mycobacterium tuberculosis (Mtb) has co-evolved with humans for thousands of years causing variation in virulence, transmissibility, and disease phenotypes. To identify bacterial contributors to phenotypic diversity, we developed new RNA-seq and phylogenomic tools to capture hundreds of Mtb isolate transcriptomes, link transcriptional and genetic variation, and find associations between variants and epidemiologic traits. Across 274 Mtb clinical isolates, we uncovered unexpected diversity in expression of virulence genes which we linked to known and previously unrecognized regulators. Surprisingly, we found that many isolates harbor variants associated with decreased expression of EsxA (Esat6) and EsxB (Cfp10), which are virulence effectors, dominant T cell antigens, and immunodiagnostic targets. Across >55,000 isolates, these variants associate with increased transmissibility, especially in drug resistant Mtb strains. Our data suggest expression of key Mtb virulence genes is evolving across isolates in part to optimize fitness under drug pressure, with sobering implications for immunodiagnostics and next-generation vaccines.

Background Menstrual Health (MH) knowledge, attitude and practice (KAP) are highly affected by access to information regarding menstruation. Despite being included in the school curriculum, Sexual and Reproductive Health (SRH) education is often not delivered in practice. School-based educational interventions have been shown to be effective in promoting MH. Methods A school-based study was conducted in Indrawati rural municipality of Sindhupalchowk district in Nepal. 427 participants (175 boys and 252 girls), aged 11–13, completed a questionnaire evaluating MH KAP before receiving a structured training module on MH provided by experienced trainers from Putali Nepal using the Menstrupedia tool. The questionnaire was repeated one month after the training. Pre and post intervention scores were compared to determine the effect of the intervention. Focus group discussions were also conducted to understand the perceptions of participants toward SRH teaching. Association of independent socio-demographic with dependent variables knowledge and attitude towards menstrual health were analyzed using MANOVA test. The Wilcoxon signed-rank test was used to compare the median outcome of the pre and post-test attitude and knowledge. The maximum possible score was 6 for MH knowledge. The total attitude score ranged 14 to 70. Results The median knowledge score increased by 1 point (p = <0.001) and the median attitude score by 5 points (p = <0.001), one month after delivery of the intervention. Higher knowledge scores were significantly associated with Hindu religion, female gender, higher father’s literacy, and mothers in an informal occupation on multivariate analysis. Higher attitude scores were significantly associated with Hindu religion while lower attitude scores were associated with a mother in an informal occupation. Conclusion The Menstrupedia comic educational intervention improved knowledge and attitude towards menstruation among Nepali adolescents. A scale-up of the Menstrupedia based intervention would significantly change knowledge and attitude towards menstruation in Nepali adolescents.

Background In rural Nepal, poor road and transport networks and few testing laboratories impede tuberculosis diagnosis. A drone transport system was established to transport sputum samples to laboratories with advanced molecular diagnostic machines – GeneXpert MTB/RIF. This study explored the perceptions of using drones for tuberculosis diagnosis among community stakeholders, female community health volunteers, and healthcare providers from communities with drones implemented and without drone programs. Methods In December 2019, we conducted focus group discussions in two drone-implemented and three without drone programs. We purposively selected 40 participants: Female community health volunteers (n=16), community stakeholders (n=18), and healthcare providers (n=6). Focus group discussions employed semi-structured questions, which were audio-recorded, transcribed, and translated into English. Codebook thematic analysis was performed and charted using three levels of the socioecological model: individual, community, and health system. Results We identified four themes (i) Trust in drones underpins successful use for tuberculosis diagnosis; (ii) Drone-based sample transport optimised connectivity and accessibility for people with tuberculosis and healthcare providers; (iii) Drones create opportunities to improve community and health system, and (iv) External factors impede the use of drones to facilitate tuberculosis diagnosis. The study reported, at an individual level, people’s trust in drones mainly through community-based events. For local healthcare providers, drones reduce transport time, opportunity costs, and immediate cash costs of transport. At the community level, drone use creates opportunities to increase the skills of local people as drone pilots. At the health system level, drone transport increases efficient sputum sample delivery and provides opportunities to transport medicines and other biomedical samples. Perceived challenges of using drones were adverse weather, limitations in skilled human resources, and financial resources to operate drones sustainably. Conclusions Healthcare providers, female community health volunteers, and community stakeholders reported high levels of trust in drones and perceived their use for tuberculosis diagnosis and care to substantially benefit people with tuberculosis and providers in rural Nepal. There was a high level of demand for application to other healthcare services and wider geographical coverage, demonstrating drones as a potential tool for enhancing access to healthcare in geographically remote communities.

Antibiotic tolerance in Mycobacterium tuberculosis reduces bacterial killing, worsens treatment outcomes, and contributes to resistance. We studied rifampicin tolerance in isolates with or without isoniazid resistance (IR). Using a minimum duration of killing assay, we measured rifampicin survival in isoniazid-susceptible (IS, n=119) and resistant (IR, n=84) isolates, correlating tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs), and isoniazid-resistant mutations. Longitudinal IR isolates were analyzed for changes in rifampicin tolerance and genetic variant emergence. The median time for rifampicin to reduce the bacterial population by 90% (MDK90) increased from 1.23 days (IS) and 1.31 days (IR) to 2.55 days (IS) and 1.98 days (IR) over 15–60 days of incubation, indicating fast and slow-growing tolerant sub-populations. A 6 log10-fold survival fraction classified tolerance as low, medium, or high, showing that IR is linked to increased tolerance and faster growth (OR = 2.68 for low vs. medium, OR = 4.42 for low vs. high, p-trend = 0.0003). High tolerance in IR isolates was associated with rifampicin treatment in patients and genetic microvariants. These findings suggest that IR tuberculosis should be assessed for high rifampicin tolerance to optimize treatment and prevent the development of multi-drug-resistant tuberculosis.

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. We determined the in-vitro rifampicin survival fraction by minimum duration of killing assay in isoniazid susceptible (IS, n=119) and resistant (IR, n=84) M. tuberculosis isolates. Then we correlated the rifampicin tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal IR isolates collected from patients were analyzed for changes in rifampicin tolerance and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation. This indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log 10 -fold survival fraction enabled classification of tolerance as low, medium or high and revealed IR association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high, P -trend=0.0003). The high tolerance in IR isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Furthermore, the high rifampicin tolerant IR isolates have survival potential similar to multi-drug resistant isolates. These findings suggest that IR tuberculosis needs to be evaluated for high rifampicin tolerance to improve treatment regimen and prevent the risk of MDR-TB emergence.

Background Bacterial diversity could contribute to the diversity of tuberculosis infection and treatment outcomes observed clinically, but the biological basis of this association is poorly understood. The aim of this study was to identify associations between phenogenomic variation in Mycobacterium tuberculosis and tuberculosis clinical features. Methods We developed a high-throughput platform to define phenotype–genotype relationships in M tuberculosis clinical isolates, which we tested on a set of 158 drug-sensitive M tuberculosis strains sampled from a large tuberculosis clinical study in Ho Chi Minh City, Viet Nam. We tagged the strains with unique genetic barcodes in multiplicate, allowing us to pool the strains for in-vitro competitive fitness assays across 16 host-relevant antibiotic and metabolic conditions. Relative fitness was quantified by deep sequencing, enumerating output barcode read counts relative to input normalised values. We performed a genome-wide association study to identify phylogenetically linked and monogenic mutations associated with the in-vitro fitness phenotypes. These genetic determinants were further associated with relevant clinical outcomes (cavitary disease and treatment failure) by calculating odds ratios (ORs) with binomial logistic regressions. We also assessed the population-level transmission of strains associated with cavitary disease and treatment failure using terminal branch length analysis of the phylogenetic data. Findings M tuberculosis clinical strains had diverse growth characteristics in host-like metabolic and drug conditions. These fitness phenotypes were highly heritable, and we identified monogenic and phylogenetically linked variants associated with the fitness phenotypes. These data enabled us to define two genetic features that were associated with clinical outcomes. First, mutations in Rv1339, a phosphodiesterase, which were associated with slow growth in glycerol, were further associated with treatment failure (OR 5·34, 95% CI 1·21–23·58, p=0·027). Second, we identified a phenotypically distinct slow-growing subclade of lineage 1 strains (L1.1.1.1) that was associated with cavitary disease (OR 2·49, 1·11–5·59, p=0·027) and treatment failure (OR 4·76, 1·53–14·78, p=0·0069), and which had shorter terminal branch lengths on the phylogenetic tree, suggesting increased transmission. Interpretation Slow growth under various antibiotic and metabolic conditions served as in-vitro intermediate phenotypes underlying the association between M tuberculosis monogenic and phylogenetically linked mutations and outcomes such as cavitary disease, treatment failure, and transmission potential. These data suggest that M tuberculosis growth regulation is an adaptive advantage for bacterial success in human populations, at least in some circumstances. These data further suggest markers for the underlying bacterial processes that contribute to these clinical outcomes. Funding National Health and Medical Research Council/A*STAR, National Institutes of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the Wellcome Trust Fellowship in Public Health and Tropical Medicine.

Background Vietnam’s primary mechanism of achieving sustainable funding for universal health coverage (UHC) and financial protection has been through its social health insurance (SHI) scheme. Steady progress towards access has been made and by 2020, over 90% of the population were enrolled in SHI. In 2022, as part of a larger transition towards the increased domestic financing of healthcare, tuberculosis (TB) services were integrated into SHI. This change required people with TB to use SHI for treatment at district-level facilities or to pay out of pocket for services. This study was conducted in preparation for this transition. It aimed to understand more about uninsured people with TB, assess the feasibility of enrolling them into SHI, and identify the barriers they faced in this process. Methods A mixed-method case study was conducted using a convergent parallel design between November 2018 and January 2022 in ten districts of Hanoi and Ho Chi Minh City, Vietnam. Quantitative data were collected through a pilot intervention that aimed to facilitate SHI enrollment for uninsured individuals with TB. Descriptive statistics were calculated. Qualitative interviews were conducted with 34 participants, who were purposively sampled for maximum variation. Qualitative data were analyzed through an inductive approach and themes were identified through framework analysis. Quantitative and qualitative data sources were triangulated. Results We attempted to enroll 115 uninsured people with TB into SHI; 76.5% were able to enroll. On average, it took 34.5 days to obtain a SHI card and it cost USD 66 per household. The themes indicated that a lack of knowledge, high costs for annual premiums, and the household-based registration requirement were barriers to SHI enrollment. Participants indicated that alternative enrolment mechanisms and greater procedural flexibility, particularly for undocumented people, is required to achieve full population coverage with SHI in urban centers. Conclusions Significant addressable barriers to SHI enrolment for people affected by TB were identified. A quarter of individuals remained unable to enroll after receiving enhanced support due to lack of required documentation. The experience gained during this health financing transition is relevant for other middle-income countries as they address the provision of financial protection for the treatment of infectious diseases.

Background The psychosocial consequences of tuberculosis (TB) are key barriers to ending TB globally. We evaluated and compared stigma, depression, and quality of life (QoL) among people with TB diagnosed through active (ACF) and passive (PCF) case-finding in Nepal. Methods We prospectively recruited adults with TB diagnosed through ACF and PCF in four districts of Nepal between August 2018 and April 2019. Participants were interviewed at 8–12 weeks (baseline) and 22–26 weeks (follow-up) following treatment initiation. TB stigma was measured using an adapted Van Rie Stigma Scale (0 = no stigma to 30 = highest stigma). Depression was measured using a locally-validated Patient Health Questionnaire (PHQ-9). Mild and major depression were indicated by PHQ-9 scores 5–9 and ≥ 10, respectively. QoL was measured using the EuroQoL 5-Dimension 5-level (EQ-5D-5L) from 0 to 1 (optimal QoL); and self-rated health from 0 to 100 (optimal self-rated health). Results We recruited 221 participants (111 ACF; 110 PCF) with a mean age of 48 years (standard deviation [SD] = ± 16), of whom 147/221 (67%) were men. The mean TB stigma score was 12 (SD = 7.3) at baseline and 12 (SD = 6.7) at follow-up. The most commonly perceived elements of TB stigma at baseline were that people with TB experienced guilt (110/221, 50%) and feared disclosure outside their household (114/221, 52%). Self-rated health and EQ-5D-5L scores increased from baseline to follow-up (69.3 to 80.3, p < 0.001; 0.92 to 0.9, p = 0.009). Nearly one-third of participants (68/221, 31%) had mild or major depression at baseline. The proportion of participants with major depression decreased from baseline to follow-up (11.5% vs. 5%, p = 0.012). There was a moderate, significant positive correlation between depression and stigma scores ( r = 0.41, p < 0.001). There were no differences found in TB stigma, self-rated health, QoL, or prevalence of mild/major depression between ACF and PCF participants. Conclusions We found a substantial, persistent, and clustered psychosocial impact among adults with TB diagnosed through both ACF and PCF strategies in Nepal. These findings suggest an urgent need to develop effective, evidence-based psychosocial support interventions with the potential to be integrated with existing ACF strategies and routine TB service activities.

Background Tuberculosis (TB) continues to be a leading cause of morbidity and mortality worldwide. Past genome-wide association studies (GWAS) have explored TB susceptibility across various ethnic groups, yet a significant portion of TB heritability remains unexplained. Methods We conducted GWAS in the Singapore Chinese and Vietnamese, followed by a comprehensive meta-analysis incorporating 4 independent East Asian datasets, resulting in a total of 11,841 cases and 197,373 population controls. Findings We identified a novel susceptibility locus for pulmonary TB (PTB) at 22q12.2 in East Asians [rs6006426, OR (95%Cl) =1.097(1.066, 1.130), P meta =3.31×10 ⁻¹⁰ ]. The association was further validated in Europeans [OR (95%Cl) =1.101(1.002, 1.211), P =0.046] and was strengthened in the combined meta-anlaysis including 12,736 PTB cases and 673,864 controls [OR (95%Cl) =1.098(1.068, 1.129), P meta =4.33×10 ⁻¹¹ ]. rs6006426 affected SF3A1 expression in various immune cells ( P from 0.003 to 6.17×10 ⁻¹⁸ ) and OSM expression in monocytes post lipopolysaccharide stimulation ( P =5.57×10 ⁻⁴ ). CRISPR-Cas9 edited zebrafish embryos with osm depletion resulted in decreased burden of Mycobacterium marinum ( M.marinum ) in infected embryos ( P =0.047). Interpretation Our findings offer novel insights into the genetic factors underlying TB and reveals new avenues for understanding its etiology.

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. There is limited understanding of antibiotic tolerance in clinical isolates of M. tuberculosis . Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. In-vitro rifampicin survival fractions determined by minimum duration of killing assay in isoniazid susceptible (n=119) and resistant (n=84) M. tuberculosis isolates. Rifampicin tolerance was correlated with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal isoniazid-resistant isolates were analyzed for rifampicin tolerance based on collection time from patients and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation respectively. Increase in MDK90 time indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log 10 -fold survival fraction enabled classification of tolerance as low, medium or high and revealed isoniazid-resistance association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high, P -trend=0.0003). The high tolerance in longitudinal isoniazid-resistant isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Our study identifies a range of rifampicin tolerance and reveals that isoniazid resistance is associated with higher tolerance with growth fitness. Furthermore, rifampicin treatment may select isoniazid-resistant isolate microvariants with higher rifampicin tolerance, with survival potential similar to multi-drug resistant isolates. These findings suggest that isoniazid-resistant tuberculosis needs to be evaluated for rifampicin tolerance or needs further improvement in treatment regimen.