Full-length 16S rDNA sequencing based on Oxford Nanopore Technologies revealed the association between gut-pharyngeal microbiota and tuberculosis in cynomolgus macaques.

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex (Mtbc), which develops from asymptomatic latent TB to active stages. The microbiome was purposed as a potential factor affecting TB pathogenesis, but the study was limited. The present study explored the association between gut-pharyngeal microbiome and TB stages in cynomolgus macaques using the full-length 16S rDNA amplicon sequencing based on Oxford Nanopore Technologies. The total of 71 macaques was divided into TB (-) control, TB (+) latent and TB (+) active groups. The differential abundance analysis showed that Haemophilus hemolyticus was decreased, while Prevotella species were increased in the pharyngeal microbiome of TB (+) macaques. In addition, Eubacterium coprostanoligenes in the gut was enriched in TB (+) macaques. Alteration of these bacteria might affect immune regulation and TB severity, but details of mechanisms should be further explored and validated. In summary, microbiota may be associated with host immune regulation and affect TB progression. The findings suggested the potential mechanisms of host-microbes interaction, which may improve the understanding of the role of microbiota and help develop therapeutics for TB in the future.

Advancing tuberculosis diagnosis and management in cynomolgus macaques using Xpert MTB/RIF ultra assay.

The detection and management of Mycobacterium tuberculosis complex (MTBC) infection, the causative agent of tuberculosis (TB), in macaques, including cynomolgus macaques (Macaca fascicularis), are of significant concern in research and regions where macaques coexist with humans or other animals. This study explored the utility of the Xpert MTB/RIF Ultra assay, a widely adopted molecular diagnostic tool to diagnose tuberculosis (TB) in humans, to detect DNA from the Mycobacterium tuberculosis complex in clinical samples obtained from cynomolgus macaques. This investigation involved a comprehensive comparative analysis, integrating established conventional diagnostic methodologies, assessing oropharyngeal-tracheal wash (PW) and buccal swab (BS) specimen types, and follow-up assessments at 3-month, 6-month, and 12-month intervals. Our results demonstrated that the Xpert MTB/RIF Ultra assay was able to detect MTBC in 12 of 316 clinical samples obtained from cynomolgus macaques, presenting a potential advantage over bacterial culture and chest radiographs. The Xpert MTB/RIF Ultra assay exhibited exceptional sensitivity (100%) at the animal level, successfully detecting all macaques positive for M. tuberculosis as confirmed by traditional culture methods. The use of PW samples revealed that 5 positive samples from 99 (5.1%) were recommended for testing, compared to 0 samples from 99 buccal swab (BS) samples (0.0%). In particular, the definitive diagnosis of TB was confirmed in three deceased macaques by MTB culture, which detected the presence of the bacterium in tissue autopsy. Our findings demonstrate that the implementation of the Xpert MTB/RIF Ultra assay, along with prompt isolation measures, effectively reduced active TB cases among cynomolgus macaques over a 12-month period. These findings highlight the advance of the Xpert MTB/RIF Ultra assay in TB diagnosis and its crucial role in preventing potential outbreaks in cynomolgus macaques. With its rapidity, high sensitivity, and specificity, the Xpert MTB/RIF Ultra assay can be highly suitable for use in reference laboratories to confirm TB disease and effectively interrupt TB transmission.

Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated "MKR superspreader", and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR's intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

Genomic evidence supporting the clonal expansion of extensively drug-resistant tuberculosis bacteria belonging to a rare proto-Beijing genotype.

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major public health issue in Thailand. The high prevalence of modern Beijing (Lineage 2.2.1) strains has been associated with multi- and extensively drug-resistant infections (MDR-, XDR-TB), complicating disease control. The impact of rarer proto-Beijing (L2.1) strains is less clear. In our study of thirty-seven L2.1 clinical isolates spanning thirteen years, we found a high prevalence of XDR-TB cases (32.4%). With ≤ 12 pairwise SNP distances, 43.2% of L2.1 patients belong to MDR-TB or XDR-TB transmission clusters suggesting a high level of clonal expansion across four Thai provinces. All XDR-TB (100%) were likely due to transmission rather than inadequate treatment. We found a 47 mutation signature and a partial deletion of the fadD14 gene in the circulating XDR-TB cluster, which can be used for surveillance of this rare and resilient M. tuberculosis strain-type that is causing increasing health burden. We also detected three novel deletion positions, a deletion of 1285 bp within desA3 (Rv3230c), large deletions in the plcB, plcA, and ppe38 gene which may play a role in the virulence, pathogenesis or evolution of the L2.1 strain-type.

Optimization of Culture Protocols to Isolate Leptospira spp. from Environmental Water, Field Investigation, and Identification of Factors Associated with the Presence of Leptospira spp. in the Environment.

The successful culture of Leptospira spp. from the environment is challenging. Here, we optimized the isolation of Leptospira spp. from water samples spiked with different species and initial concentrations of this organism. The time periods between water sampling and the isolation process were varied (0, 2, and 4 weeks). Bacterial cultures were observed under a microscope, and cultures were graded for cell density, weekly, for 12 weeks. Most pathogenic Leptospira spp. were difficult to culture under all conditions. All conditions of water samples spiked with novel species of Leptospira subclade P1 were culture positive within 2 weeks. For Leptospira subclade P2, storing samples for 2 weeks prior to isolation resulted in more successful isolation compared with isolation after other storage conditions. For subclade S1, all samples with initial bacterial concentrations of more than 103 colonies/mL, under all storage conditions, were successfully cultured. These results suggest that storing contaminated water samples for 2 to 4 weeks in the dark at an ambient temperature prior to culturing can improve the isolation of Leptospira spp. from the samples. We implemented this protocol and collected water samples from natural sources accessed by both humans and animals. Leptospira spp. was identified in 32% (35/109) of water samples. The animal species using a water source influenced the likelihood of water samples being contaminated with Leptospira spp. Cultures of Leptospira spp. from environmental samples can provide useful information for understanding the complex interactions between humans, animals and the environment in the transmission of leptospirosis.

Comparisons of whole-genome sequencing and phenotypic drug susceptibility testing for Mycobacterium tuberculosis causing MDR-TB and XDR-TB in Thailand.

Drug-resistant tuberculosis (TB) is a major public health problem. There is little information regarding the genotypic-phenotypic association of anti-TB drugs, especially for second-line drugs. This study compared phenotypic drug susceptibility testing (DST) with predictions based on whole-genome sequencing (WGS) data for 266 Mycobacterium tuberculosis isolates. Phenotypic DST used the standard proportional method. Clinical isolates of M. tuberculosis collected in Thailand between 1998 and 2013 comprised 51 drug-sensitive strains, six mono-resistant strains, two multiple-resistant strains, 88 multi-drug-resistant strains, 95 pre-extensively drug-resistant strains and 24 extensively drug-resistant strains. WGS analysis was performed using the computer programs PhyResSE and TB-Profiler. TB-Profiler had higher average concordance with phenotypic DST than PhyResSE for both first-line (91.96% vs. 91.4%) and second-line (79.67% vs. 78.20%) anti-TB drugs. The average sensitivity for all anti-TB drugs was also higher (83.13% vs. 72.08%) with slightly lower specificity (83.50% vs. 86.68%). Regardless of the program used, isoniazid, rifampicin and amikacin had the highest concordance with phenotypic DST (96.2%, 93.5% and 95.6%, respectively). Ethambutol, ethionamide and fluoroquinolones had the lowest concordance (87.34%, 81.44% and 73.85%, respectively). Concordance rates of ofloxacin (a second-generation fluoroquinolone), levofloxacin, moxifloxacin and gatifloxacin (third- and fourth-generation fluoroquinolones) were 91.79%, 76.62%, 72.64% and 57.35%, respectively. Discordance between phenotypic and WGS-based DSTs may be due, in part, to the choice of critical concentration and variable reproducibility of the phenotypic tests. It may also be due to limitations of the mutation databases (especially for the second-line drugs) and the analysis program used. Mutations related to fluoroquinolone resistance, especially the later generations, need to be identified.

Effect of point-of-care C-reactive protein testing on antibiotic prescription in febrile patients attending primary care in Thailand and Myanmar: an open-label, randomised, controlled trial.

BACKGROUND: In southeast Asia, antibiotic prescription in febrile patients attending primary care is common, and a probable contributor to the high burden of antimicrobial resistance. The objective of this trial was to explore whether C-reactive protein (CRP) testing at point of care could rationalise antibiotic prescription in primary care, comparing two proposed thresholds to classify CRP concentrations as low or high to guide antibiotic treatment. METHODS: We did a multicentre, open-label, randomised, controlled trial in participants aged at least 1 year with a documented fever or a chief complaint of fever (regardless of previous antibiotic intake and comorbidities other than malignancies) recruited from six public primary care units in Thailand and three primary care clinics and one outpatient department in Myanmar. Individuals were randomly assigned using a computer-based randomisation system at a ratio of 1:1:1 to either the control group or one of two CRP testing groups, which used thresholds of 20 mg/L (group A) or 40 mg/L CRP (group B) to guide antibiotic prescription. Health-care providers were masked to allocation between the two intervention groups but not to the control group. The primary outcome was the prescription of any antibiotic from day 0 to day 5 and the proportion of patients who were prescribed an antibiotic when CRP concentrations were above and below the 20 mg/L or 40 mg/L thresholds. The primary outcome was analysed in the intention-to-treat and per-protocol populations. The trial is registered with ClinicalTrials.gov, number NCT02758821, and is now completed. FINDINGS: Between June 8, 2016, and Aug 25, 2017, we recruited 2410 patients, of whom 803 patients were randomly assigned to CRP group A, 800 to CRP group B, and 807 to the control group. 598 patients in CRP group A, 593 in CRP group B, and 767 in the control group had follow-up data for both day 5 and day 14 and had been prescribed antibiotics (or not) in accordance with test results (per-protocol population). During the trial, 318 (39%) of 807 patients in the control group were prescribed an antibiotic by day 5, compared with 290 (36%) of 803 patients in CRP group A and 275 (34%) of 800 in CRP group B. The adjusted odds ratio (aOR) of 0·80 (95% CI 0·65-0·98) and risk difference of -5·0 percentage points (95% CI -9·7 to -0·3) between group B and the control group were significant, although lower than anticipated, whereas the reduction in prescribing in group A compared with the control group was not significant (aOR 0·86 [0·70-1·06]; risk difference -3·3 percentage points [-8·0 to 1·4]). Patients with high CRP concentrations in both intervention groups were more likely to be prescribed an antibiotic than in the control group (CRP ≥20 mg/L: group A vs control group, p<0·0001; CRP ≥40 mg/L: group B vs control group, p<0·0001), and those with low CRP concentrations were more likely to have an antibiotic withheld (CRP <20 mg/L: group A vs control group, p<0·0001; CRP <40 mg/L: group B vs control group, p<0·0001). 24 serious adverse events were recorded, consisting of 23 hospital admissions and one death, which occurred in CRP group A. Only one serious adverse event was thought to be possibly related to the study (a hospital admission in CRP group A). INTERPRETATION: In febrile patients attending primary care, testing for CRP at point of care with a threshold of 40 mg/L resulted in a modest but significant reduction in antibiotic prescribing, with patients with high CRP being more likely to be prescribed an antibiotic, and no evidence of a difference in clinical outcomes. This study extends the evidence base from lower-income settings supporting the use of CRP tests to rationalise antibiotic use in primary care patients with an acute febrile illness. A key limitation of this study is the individual rather than cluster randomised study design which might have resulted in contamination between the study groups, reducing the effect size of the intervention. FUNDING: Wellcome Trust Institutional Strategic Support Fund grant (105605/Z/14/Z) and Foundation for Innovative New Diagnostics (FIND) funding from the Australian Government.

Rapid screening drug susceptibility test in tuberculosis using sandwich electrochemical immunosensor.

Drug susceptibility testing (DST) for Mycobacterium tuberculosis currently faces multiple challenges, including lengthy ineffective standard methods, the expensive cost of molecular tests, and large bulky diagnostic machines. In this work, a disposable MPT64 sensor was developed to rapidly determine drug susceptibility (DS-TB) and multidrug resistant tuberculosis (MDR-TB) using an electrochemical sandwich-immunosensor for the detection of MPT64 as an indicator of Mycobacterium tuberculosis growth. Anti-MPT64 (as capture antibody; cAb) was immobilised on screen printed carbon electrodes to specifically bind with MPT64 target protein. A reporter probe of anti-MPT64 conjugated with horseradish peroxidase (HRP labeled rAb) then completed the sandwich immunocomplex. The current signals were received from the catalytic reaction between 3,3'-5,5'-tetramethylbenzidine (TMB), H2O2 and HRP labeled rAb using the chronoamperometric mode on a portable potentiostat. Increasing MPT64 concentration was taken as an indicator of growth, which was measured by the disposable MPT64 sensor. This sensor shows the possibility of determining DST by comparing the signals of DS-TB and MDR-TB growth in drug-free and drug-containing liquid medium. The time required to identify DS-TB and MDR-TB in pure culture MTB and leftover sputum sediments from patients are 3 days and 4-6 days, respectively. Therefore, using this sensor is significantly rapid and inexpensive and has the potential to be used for drug susceptibility testing of tuberculosis in middle to low income countries.

Presence of B. thailandensis and B. thailandensis expressing B. pseudomallei-like capsular polysaccharide in Thailand, and their associations with serological response to B. pseudomallei.

BACKGROUND: Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. B. thailandensis, some strains of which express a B. pseudomallei-like capsular polysaccharide (BTCV), is also commonly found in the environment in Southeast Asia but is considered non-pathogenic. The aim of the study was to determine the distribution of B. thailandensis and its capsular variant in Thailand and investigate whether its presence is associated with a serological response to B. pseudomallei. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the presence of B. pseudomallei and B. thailandensis in 61 rice fields in Northeast (n = 21), East (n = 19) and Central (n = 21) Thailand. We found BTCV in rice fields in East and Central but not Northeast Thailand. Fourteen fields were culture positive for B. pseudomallei alone, 8 for B. thailandensis alone, 11 for both B. pseudomallei and B. thailandensis, 6 for both B. thailandensis and BTCV, and 5 for B. pseudomallei, B. thailandensis and BTCV. Serological testing using the indirect hemagglutination assay (IHA) of 96 farmers who worked in the study fields demonstrated that farmers who worked in B. pseudomallei-positive fields had higher IHA titers than those who worked in B. pseudomallei-negative fields (median 1:40 [range: <1:10-1:640] vs. <1:10 [range: <1:10-1:320], p = 0.002). In a multivariable ordered logistic regression model, IHA titers were significantly associated with the presence of B. pseudomallei (aOR = 3.7; 95% CI 1.8-7.8, p = 0.001) but were not associated with presence of B. thailandensis (p = 0.32) or BTCV (p = 0.32). One sequence type (696) was identified for the 27 BTCV isolates tested. CONCLUSIONS/SIGNIFICANCE: This is the first report of BTCV in Thailand. The presence of B. pseudomallei and B. thailandensis in the same field was not uncommon. Our findings suggest that IHA positivity of healthy rice farmers in Thailand is associated with the presence of B. pseudomallei in rice fields rather than B. thailandensis or BTCV.

Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples.

BACKGROUND: Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples. METHODOLOGY/PRINCIPAL FINDINGS: First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 µL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei. CONCLUSIONS/SIGNIFICANCE: The LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis 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.

Novel DNA chip based on a modified DigiTag2 assay for high-throughput species identification and genotyping of Mycobacterium tuberculosis complex isolates.

A multipurpose high-throughput genotyping tool for the assessment of recent epidemiological data and evolutional pattern in Mycobacterium tuberculosis complex (MTBC) clinical isolates was developed in this study. To facilitate processing, 51 highly informative single nucleotide polymorphisms (SNPs) were selected for discriminating the clinically most relevant MTBC species and genotyping M. tuberculosis into its principle genetic groups (PGGs) and SNP cluster groups (SCGs). Because of the high flexibility of the DigiTag2 assay, the identical protocol and DNA array containing the identical set of probes were applied to the highly GC-rich mycobacterial genome. The specific primers with multiplex amplification and hybridization conditions based on the DigiTag2 principle were optimized and evaluated with 14 MTBC reference strains, 4 nontuberculous mycobacteria (NTM) isolates, and 322 characterized M. tuberculosis clinical isolates. The DNA chip that was developed revealed a 99.85% call rate, a 100% conversion rate, and 99.75% reproducibility. For the accuracy rate, 98.94% of positive calls were consistent with previous molecular characterizations. Our cost-effective technology was capable of simultaneously identifying the MTBC species and the genotypes of 96 M. tuberculosis clinical isolates within 6 h using only simple instruments, such as a thermal cycler, a hybridization oven, and a DNA chip scanner, and less technician skill was required than for other techniques. We demonstrate this approach's potential as a simple, flexible, and rapid tool for providing clearer information regarding circulating MTBC isolates.