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

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

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

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

Abstract 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

Utility of a Lateral Flow Immunoassay (LFI) to Detect <i>Burkholderia pseudomallei</i> in Soil Samples

<div><p>Background</p><p>Culture is the gold standard for the detection of environmental <i>B</i>. <i>pseudomallei</i>. 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 <i>B</i>. <i>pseudomallei</i> on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect <i>B</i>. <i>pseudomallei</i> capsular polysaccharide (CPS) in clinical samples as a tool to detect <i>B</i>. <i>pseudomallei</i> in environmental samples.</p><p>Methodology/Principal Findings</p><p>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 <i>B</i>. <i>pseudomallei</i> were spiked with <i>B</i>. <i>pseudomallei</i> 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 <i>B</i>. <i>pseudomallei</i> 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 <i>orf2</i>. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for <i>B</i>. <i>pseudomallei</i>, 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 <i>B</i>. <i>pseudomallei</i>, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for <i>B</i>. <i>pseudomallei</i>.</p><p>Conclusions/Significance</p><p>The LFI can be used to detect <i>B</i>. <i>pseudomallei</i> 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.</p></div

Venn diagram showing culture (blue), lateral flow immunoassay (LFI; yellow) and qPCR assay (red) results of 105 soil samples.

<p>Culture was performed according to the consensus guidelines for environmental sampling of <i>B</i>. <i>pseudomallei</i> developed by DEBWorP [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005204#pntd.0005204.ref011" target="_blank">11</a>], in which soil specimens were enriched in the enrichment broth for 2 days and the upper layer of enrichment broth on day 2 was streaked on agar plates and observed daily. For the LFI and qPCR assay, soil specimens were enriched in the broth for 7 days and the broth on day 7 was used for the tests.</p

Factors associated with indirect hemagglutination assay (IHA) results in 96 healthy rice farmers.

<p>Factors associated with indirect hemagglutination assay (IHA) results in 96 healthy rice farmers.</p

Map of the presence of <i>B</i>. <i>pseudomallei</i>, <i>B</i>. <i>thailandensis</i> and <i>B</i>. <i>thailandensis</i> expressing <i>B</i>. <i>pseudomallei</i>-like capsular polysaccharide (BTCV) in 61 rice fields in Northeast (n = 21), East (n = 19) and Central (n = 21) Thailand.

<p>(a) Map of Thailand. (b) Location of the 61 rice fields evaluated. Red, green and yellow pies represent rice fields that were culture positive and negative for <i>B</i>. <i>pseudomallei</i>, <i>B</i>. <i>thailandensis</i>, and BTCV, respectively. Province codes represent Burirum (NE1), Chaiyaphum (NE2), Khon Kaen (NE3), Udon Thani (NE4), Nong Bua Lam Phu (NE5), Loei (NE6) and Nakhon Ratchasima (NE7) in the Northeast, Chachoengsao (E1), Prachinburi (E2), Sa Kaeo (E3), Chanthaburi (E4), Chonburi (E5) and Rayong (E6) in the East, Phetchabun (C1), Phitsanulok (C2), Pathum Thani (C3), Saraburi (C4), Lopburi (C5), Nakhon Nayok (C6) and Bangkok (C7) in Central Thailand. ArcGis Version 10.2 (ESRI, Redlands, CA, USA) was used to map the sampled rice fields. The location of sampled rice fields was recorded by using the EpiCollect application (<a href="http://www.epicollect.net/" target="_blank">www.epicollect.net</a>, Imperial College, London).</p

Overlap between presence of <i>B</i>. <i>pseudomallei</i> (<i>B</i>. <i>ps</i>; red), <i>B</i>. <i>thailandensis</i> (<i>B</i>. <i>th</i>; green) and <i>B</i>. <i>thailandensis</i> expressing <i>B</i>. <i>pseudomallei</i>-like capsular polysaccharide (BTCV; yellow) in 61 sampled rice fields.

<p>Overlap between presence of <i>B</i>. <i>pseudomallei</i> (<i>B</i>. <i>ps</i>; red), <i>B</i>. <i>thailandensis</i> (<i>B</i>. <i>th</i>; green) and <i>B</i>. <i>thailandensis</i> expressing <i>B</i>. <i>pseudomallei</i>-like capsular polysaccharide (BTCV; yellow) in 61 sampled rice fields.</p

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

Summary: 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