Multi-platform whole genome sequencing for tuberculosis clinical and surveillance applications.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis offers valuable insights for tuberculosis (TB) control. High throughput platforms like Illumina and Oxford Nanopore Technology (ONT) are increasingly used globally, although ONT is known for higher error rates and is less established for genomic studies. Here we present a study comparing the sequencing outputs of both Illumina and ONT platforms, analysing DNA from 59 clinical isolates in highly endemic TB regions of Thailand. The resulting sequence data were used to profile the M. tuberculosis pairs for their lineage, drug resistance and presence in transmission chains, and were compared to publicly available WGS data from Thailand (n = 1456). Our results revealed isolates that are predominantly from lineages 1 and 2, with consistent drug resistance profiles, including six multidrug-resistant strains; however, analysis of ONT data showed longer phylogenetic branches, emphasising the technologies higher error rate. An analysis incorporating the larger dataset identified fifteen of our samples within six potential transmission clusters, including a significant clade of 41 multi-drug resistant isolates. ONT's extended sequences also revealed strain-specific structural variants in pe/ppe genes (e.g. ppe50), which are candidate loci for vaccine development. Despite some limitations, our results show that ONT sequencing is a promising approach for TB genomic research, supporting precision medicine and decision-making in areas with less developed infrastructure, which is crucial for tackling the disease's significant regional burden.

Genotypic Distribution and the Epidemiology of Multidrug Resistant Tuberculosis in Upper Northern Thailand.

The epidemiology and genotypes of multidrug-resistant tuberculosis (MDR-TB), a global public health threat, remain limited. The genotypic distribution and factors associated with MDR-TB in upper northern Thailand between 2015 and 2019 were investigated. The DNA sequencing of rpoB, katG, and inhA promoter of 51 multidrug-resistant Mycobacterium tuberculosis isolates revealed nine patterns of the rpoB gene mutation distributed in seven provinces. The S531L mutation was the most common mutation in all provinces. The rpoB mutation in Chiang Rai, Chiang Mai, and Lampang was highly diverse compared to other areas. Here, the mutation profiles that have yet to be reported in northern Thailand (H526P, Q513P, and H526C) were detected in Chiang Rai province. The S315T katG mutation was the most common genotype associated with INH resistance, especially in Chiang Mai and Lampang. Further analysis of data from 110 TB patients (42 MDR-TB and 68 drug-susceptible TB) revealed that <60 years of age was a significant factor associated with MDR-TB (OR = 0.316, 95% CI 0.128−0.784, p = 0.011) and ≥60 years of age was a significant factor associated with the S315T katG-mutation (OR = 8.867, 95% CI 0.981−80.177, p = 0.047). This study highlighted the necessity for continuous surveillance and risk factor monitoring for effective control of MDR-TB.

Risk factors associated with large clusters of tuberculosis patients determined by whole-genome sequencing in a high-tuberculosis-burden country.

Whole-genome sequencing (WGS) analysis has great discriminative power for detecting similar molecular fingerprints of suspected tuberculosis (TB) clusters. The proportion of TB cases within clusters and the associated risk factors are important epidemiological parameters guiding appropriate outbreak control strategies in endemic settings. We conducted a hospital-based TB case-cohort study between 2003 and 2011 in the northernmost province of Thailand. We identified TB clusters by Mycobacterium tuberculosis WGS and analysed the risks of TB clustering and the characteristics of large clusters compared with small clusters. Among 1146 TB isolates, we identified 77 clusters with 251 isolates defined by a 5-single-nucleotide variant (SNV) cutoff and 112 clusters with 431 isolates defined by a 12-SNV cutoff. Twelve large clusters with 6 isolates or more in each cluster were identified by a 12-SNV cutoff. Sublineage 2.2.1 (both Ancestral and Modern) strains and imprisonment were independently associated with large clusters. Furthermore, although large clusters of Lineage 2.2.1/Ancestral strains included a high number of prisoners, Lineage 2.2.1/Modern strain clusters were only associated with treatment failures and drug resistance. Heterogeneity among lineage strains was observed with respect to large-cluster characteristics. Patients with an increased TB-transmission tendency should be priority targets for contact investigations and outbreak interventions to prevent ongoing transmission.

Ensuring tuberculosis treatment adherence with a mobile-based CARE-call system in Thailand: a pilot study.

Background: Medication non-adherence in tuberculosis (TB) patients is an obstacle to TB treatment. Directly observed treatment to monitor and ensure adherence still has some limitations in high TB-burden countries. Most digital adherence technologies emphasize medication-taking reminder functions; however, a bi-directional communication platform to provide patient-health workers with an interface that focuses on enhancing medication adherence is likely to improve medical adherence. A budgeted mobile-based system called CARE-call providing both functions was developed and evaluated concerning whether it could enhance medication adherence.Design: Mixed methods combined quantitative and qualitative approaches. One hundred TB patients were randomized into intervention or control groups. Medication adherence rates between the two groups were compared. A focus group discussion was conducted to obtain in-depth perspectives from the patients.Results: At a 90% adherence level, the number of non-adherence patients in the intervention group was significantly lower than that of the control group (7.5% vs. 27.5%, p= .037). The participants were satisfied with the functions provided by the system, especially the confidentiality of the monitoring process, which did not involve monitoring by imaging or video recording. The bi-directional communication enabled them to contact health staff when concerns arose during the treatment course. Poor mobile phone signals and fast battery drain were reported as major technical problems of the system.Conclusions: The CARE-call system was able to prevent non-adherence in this rural setting in Thailand. However, further investigation with a larger sample size should be conducted on whether the system can also improve successful TB treatment outcome.

A novel Ancestral Beijing sublineage of Mycobacterium tuberculosis suggests the transition site to Modern Beijing sublineages.

Global Mycobacterium tuberculosis population comprises 7 major lineages. The Beijing strains, particularly the ones classified as Modern groups, have been found worldwide, frequently associated with drug resistance, younger ages, outbreaks and appear to be expanding. Here, we report analysis of whole genome sequences of 1170 M. tuberculosis isolates together with their patient profiles. Our samples belonged to Lineage 1-4 (L1-L4) with those of L1 and L2 being equally dominant. Phylogenetic analysis revealed several new or rare sublineages. Differential associations between sublineages of M. tuberculosis and patient profiles, including ages, ethnicity, HIV (human immunodeficiency virus) infection and drug resistance were demonstrated. The Ancestral Beijing strains and some sublineages of L4 were associated with ethnic minorities while L1 was more common in Thais. L2.2.1.Ancestral 4 surprisingly had a mutation that is typical of the Modern Beijing sublineages and was common in Akha and Lahu tribes who have migrated from Southern China in the last century. This may indicate that the evolutionary transition from the Ancestral to Modern Beijing sublineages might be gradual and occur in Southern China, where the presence of multiple ethnic groups might have allowed for the circulations of various co-evolving sublineages which ultimately lead to the emergence of the Modern Beijing strains.

Evidence for Host-Bacterial Co-evolution via Genome Sequence Analysis of 480 Thai Mycobacterium tuberculosis Lineage 1 Isolates.

Tuberculosis presents a global health challenge. Mycobacterium tuberculosis is divided into several lineages, each with a different geographical distribution. M. tuberculosis lineage 1 (L1) is common in the high-burden areas in East Africa and Southeast Asia. Although the founder effect contributes significantly to the phylogeographic profile, co-evolution between the host and M. tuberculosis may also play a role. Here, we reported the genomic analysis of 480 L1 isolates from patients in northern Thailand. The studied bacterial population was genetically diverse, allowing the identification of a total of 18 sublineages distributed into three major clades. The majority of isolates belonged to L1.1 followed by L1.2.1 and L1.2.2. Comparison of the single nucleotide variant (SNV) phylogenetic tree and the clades defined by spoligotyping revealed some monophyletic clades representing EAI2_MNL, EAI2_NTM and EAI6_BGD1 spoligotypes. Our work demonstrates that ambiguity in spoligotype assignment could be partially resolved if the entire DR region is investigated. Using the information to map L1 diversity across Southeast Asia highlighted differences in the dominant strain-types in each individual country, despite extensive interactions between populations over time. This finding supported the hypothesis that there is co-evolution between the bacteria and the host, and have implications for tuberculosis disease control.

Mutations in rrs, rpsL and gidB in streptomycin-resistant Mycobacterium tuberculosis isolates from Thailand.

The objectives of this study were to characterise mutations in rrs, rpsL and gidB genes in Mycobacterium tuberculosis isolates from Thailand and to examine possible associations between mutations and strain genotypes. In total, 110 streptomycin (STR)-resistant M. tuberculosis isolates and 51 STR-susceptible isolates obtained from a sample collection in Thailand during 1999-2011 were sequenced for mutation analysis in rrs, rpsL and gidB. Genotypes of the isolates were identified using spoligotyping and large sequence polymorphisms. Mutations at codons 43 and 88 in rpsL represented 63.6% of the STR-resistant isolates and were mostly associated with Beijing strains. Mutations in rrs existed in 17.3% of the STR-resistant isolates; only 8.2% harboured resistance-associated mutations. Twenty-five different mutations were found in gidB, twelve of which are new. Eight gidB mutations were likely to contribute to STR resistance in ca. 14% of the resistant isolates; about one-half of the isolates also had a mutation in rrs or rpsL. Nearly all of the double mutants belonged to Beijing strains, whereas isolates carrying only STR-associated gidB mutation were non-Beijing strains. Three different alleles in gidB were also found, each specific to Beijing, East-African Indian and Euro-American lineages, respectively. Most of the STR-resistant isolates (80.9%) carried putative resistance-associated mutations in the analysed genes. Beijing strains were related not only to single resistance-associated mutations in rpsL or rrs but usually harboured a second mutation in gidB. Strains harbouring resistance-associated gidB mutations without rrs or rpsL mutations were more associated with non-Beijing isolates. Certain gidB mutations were also potential lineage markers.

Evaluating the Auto-MODS assay, a novel tool for tuberculosis diagnosis for use in resource-limited settings.

There is an urgent need for simple, rapid, and affordable diagnostic tests for tuberculosis (TB) to combat the great burden of the disease in developing countries. The microscopic observation drug susceptibility assay (MODS) is a promising tool to fill this need, but it is not widely used due to concerns regarding its biosafety and efficiency. This study evaluated the automated MODS (Auto-MODS), which operates on principles similar to those of MODS but with several key modifications, making it an appealing alternative to MODS in resource-limited settings. In the operational setting of Chiang Rai, Thailand, we compared the performance of Auto-MODS with the gold standard liquid culture method in Thailand, mycobacterial growth indicator tube (MGIT) 960 plus the SD Bioline TB Ag MPT64 test, in terms of accuracy and efficiency in differentiating TB and non-TB samples as well as distinguishing TB and multidrug-resistant (MDR) TB samples. Sputum samples from clinically diagnosed TB and non-TB subjects across 17 hospitals in Chiang Rai were consecutively collected from May 2011 to September 2012. A total of 360 samples were available for evaluation, of which 221 (61.4%) were positive and 139 (38.6%) were negative for mycobacterial cultures according to MGIT 960. Of the 221 true-positive samples, Auto-MODS identified 212 as positive and 9 as negative (sensitivity, 95.9%; 95% confidence interval [CI], 92.4% to 98.1%). Of the 139 true-negative samples, Auto-MODS identified 135 as negative and 4 as positive (specificity, 97.1%; 95% CI, 92.8% to 99.2%). The median time to culture positivity was 10 days, with an interquartile range of 8 to 13 days for Auto-MODS. Auto-MODS is an effective and cost-sensitive alternative diagnostic tool for TB diagnosis in resource-limited settings.

Genetic diversity and dynamic distribution of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in Thailand.

This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies.

Prospective evaluation of simply modified MODS assay: an effective tool for TB diagnosis and detection of MDR-TB.

BACKGROUND AND SETTING: Thailand is one of the highest tuberculosis (TB)-burdened countries. Chiang Rai, the northernmost province of Thailand has high tuberculosis and human immunodeficiency virus (HIV) prevalence and the laboratory workload for TB culture and drug susceptibility testing is increasing. OBJECTIVES: To evaluate the simply modified microscopic-observation drug-susceptibility assay (MODS) in the setting of a developing country. METHODS: In this cross-sectional diagnostic study, a total of 202 sputum samples of clinically diagnosed TB patients were used to test the performance of MODS assay in reference to gold standard BACTEC™ MGIT™ 960 liquid culture system and Ogawa solid culture. Sputum samples were collected from clinically diagnosed TB patients. Culture growth rate and time to culture positivity were compared among three methods. Performance of modified MODS assay was evaluated for detection of mycobacterium drug resistance in reference to MGIT antimicrobial susceptibility test (AST). RESULT: Median time to culture positivity by MODS, solid, and liquid culture were 12, 30, and 6 days respectively. Compared to the drug susceptibility test (DST) result of reference liquid culture, the sensitivity and specificity of MODS for detection of multidrug-resistant tuberculosis (MDR-TB) was 85.7% and 97.5% respectively. MODS assay has a positive predicative value of 80% and negative predictive value of 96.5% for isoniazid resistance, 70% and 100% for rifampicin resistance, and 66.7% and 99.1% for MDR-TB. CONCLUSION: MODS is a highly effective screening test for detection of MDR-TB.