An association study of NRAMP1, VDR, MBL and their interaction with the susceptibility to tuberculosis in a Chinese population

SummaryObjectivesTo investigate natural-resistance-associated macrophage protein 1 (NRAMP1), mannose-binding lectin (MBL), vitamin D receptor (VDR) gene polymorphisms and their interaction with susceptibility to pulmonary tuberculosis (PTB) in a Chinese population.MethodsA case-control study was conducted in PTB (n=151), age- and sex- matched healthy controls (HCs) (n=453). Genetic polymorphisms of NRAMP1 (INT4, D543NA and 3′UTR), MBL (HL, PQ, XY and AB) and VDR (FokI and Taq) were analyzed by using PCR-restriction fragment length polymorphism (RFLP) and PCR- single- strand conformation polymorphism (SSCP) techniques. Multifactor dimensionality reduction (MDR) analysis was carried out to assess the effects of the interaction between SNPs.ResultsThe distribution of NRAMP1- 3′UTR (TGTG/del), MBL- HL (H/L) and FokI (F/f) were significantly different between PTB patients and HCs (p<0.05). HPYA (OR: 1.88; 95% CI: 1.22-2.91), LPXA (OR: 3.17; 95% CI: 1.69- 5.96), LQYA (OR: 3.52; 95%CI: 1.50-8.23) and LPYB (OR: 12.37; 95%CI: 3.75- 40.85) of MBL were risk haplotypes for PTB. The TGTG- H- f (OR: 1.70; 95%CI: 1.10-2.62) and del- H-f (OR: 3.48; 95% CI: 1.45-8.37) of 3′UTR- HL- FokI were also high-risk haplotypes associated with tuberculosis.ConclusionsOur study suggests that genotypes of many polymorphic genes are associated with TB, it is necessary to further explore the mechanism of genotypes and gene-gene interaction in susceptibility to tuberculosis

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

New insights into the mechanism of action of pyrazinamide, implications for susceptibility testing, and future regimens*

Pyrazinamide (PZA) is included in the 2016 World Health Organization multidrug-resistant tuberculosis treatment guidelines and is a key component of most ongoing clinical trials investigating novel antibiotic combinations. PZA resistance is associated with worse tuberculosis treatment outcomes. Unfortunately, for such an important drug, phenotypic susceptibility testing is extremely challenging. The exacting bacterial growth conditions required to induce susceptibility to the drug reduce the accuracy of the susceptibility assay, even in experienced laboratories, and widespread testing is not performed. This situation is unacceptable for such a valuable and important drug. A more complete understanding of the mechanism of action of PZA would be expected to lead to improvements in this situation. Although the exact mechanism of action of PZA is not known yet, it is widely accepted that PZA is a prodrug requiring transformation to pyrazinoic acid, the active form, by the mycobacterial enzyme encoded by the pncA gene. Most clinical resistance indeed appears to be a result of a diverse range of mutations in this gene and sequencing of the pncA gene has been shown to have excellent predictive power for PZA resistance. The wider availably of pncA sequencing in combination with databases of the phenotypic implications of these mutations has helped make genetic testing for PZA resistance a practical proposition. For the past decades, it has been generally accepted that an extracellular low pH is required for PZA activity but work in our laboratory [1] and others [2] has recently challenged this assumption. Alternative bacterial stresses, apart from a reduced pH of the growth media (such as reduced temperature), can also induce a PZA-susceptible phenotype. The characterization of spontaneous in vitro-resistant pyrazinoic acid mutants selected under neutral pH conditions suggests a key role for the pantothenate/coenzyme A biosynthetic pathway. This has profound implications for the mechanism of action of PZA as well as potentially the bacterial population against which PZA is active in the host. These findings will be discussed as well as their implications for further research and the future of PZA susceptibility testing

The Relative Positioning of Genotyping and Phenotyping for Tuberculosis Resistance Screening in Two EU National Reference Laboratories in 2023.

The routine use of whole genome sequencing (WGS) as a reference typing technique for Mycobacterium tuberculosis epidemiology combined with the catalogued and extensive knowledge base of resistance-associated mutations means an initial susceptibility prediction can be derived from all cultured isolates in our laboratories based on WGS data alone. Preliminary work has confirmed, in our low-burden settings, these predictions are for first-line drugs, reproducible, robust with an accuracy similar to phenotypic drug susceptibility testing (pDST) and in many cases able to also predict the level of resistance (MIC). Routine screening for drug resistance by WGS results in approximately 80% of the isolates received being predicted as fully susceptible to the first-line drugs. Parallel testing with both WGS and pDST has demonstrated that routine pDST of genotypically fully susceptible isolates yields minimal additional information. Thus, rather than re-confirming all fully sensitive WGS-based predictions, we suggest that a more efficient use of available mycobacterial culture capacity in our setting is the development of a more extensive and detailed pDST targeted at any mono or multi-drug-resistant isolates identified by WGS screening. Phenotypic susceptibility retains a key role in the determination of an extended susceptibility profile for mono/multi-drugresistant isolates identified by WGS screening. The pDST information collected is also needed to support the development of future catalogues of resistance-associated mutations

The Epidemiological Significance and Temporal Stability of Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats-Based Method Applied to Mycobacterium tuberculosis in China

This study aimed to validate the epidemiological significance and temporal stability of Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) typing in a genetically and geographically diverse set of clinical isolates from patients diagnosed with pulmonary tuberculosis in China. Between 2010 and 2013, a total of 982 Mycobacterium tuberculosis isolates were collected from four population-based investigations in China. Apart from the currently applied 24-locus MIRU-VNTR, six additional hypervariable loci were analyzed in order to validate the MIRU-VNTR combinations in terms of their epidemiological links, clustering time span, and paired geographic distance. In vitro temporal stability was analyzed for both individual MIRU-VNTR loci, and for several combinations of loci. In the present study, four MIRU-VNTR combinations, including the hypervariable loci 3820, 3232, 2163a, and 4120, were evaluated. All of these combinations obtained a Hunter-Gaston discriminatory index (HGDI) value over 0.9900 with a reduced clustering proportion (from 32.0% to 25.6%). By comparing epidemiological links, clustering time span, and paired geographic distance, we found that the performances of the four MIRU-VNTR combinations were comparable to the insertion sequence 6110 restriction fragment length polymorphism (IS6110-RFLP), and significantly better than that of 24-locus MIRU-VNTR genotyping alone. The proportion of temporally stable loci ranged from 90.5% to 92.5% within the combined MIRU-VNTR genotyping, which is higher than IS6110-RFLP (85.4%). By adding four hypervariable loci to the standard 24-locus MIRU-VNTR genotyping, we obtained a high discriminatory power, stability and epidemiological significance. This algorithm could therefore be used to improve tuberculosis transmission surveillance and outbreak investigation in China

Seroprevalence of SARS-CoV-2 in Sweden, April 26 to May 9, 2021

A national point seroprevalence study of SARS-CoV-2 was conducted in Sweden in April–May 2021. In total, 2860 individuals 3 to 90 years old from a probability-based web panel were included. Results showed that an estimated 32.6% of the population in Sweden had detectable levels of antibodies, and among non-vaccinated 20.1% had detectable levels of antibodies. We tested for differences in seroprevalence between age groups and by sex and estimated seroprevalence among previously infected participants by time since reporting

Effect of iNOS inhibition on growth of clinical strains of <i>M</i>. <i>tuberculosis</i> in macrophages.

<p>Macrophages (RAW 264.7) were stimulated with IFN-γ/LPS and/or the iNOS inhibitor L-NMMA (1mM) or left untreated. Macrophages were infected with H37Rv (a), the INH-susceptible strains E3942 (b), CDC 1551 (c), the INH-resistant strains BTB 02–141 (d), and E1155 (e) at a MOI of 5 and bacterial numbers were determined by luminometry. Data are presented as mean fold change of the bacterial numbers on day 2 compared to day 0 (D2/D0) ± SEM, (n = 5–6). Significant differences are indicated with * (p≤0.05), ** (p≤0.01) or with *** (p≤0.001) as determined by one-way ANOVA with repeated measures.</p

Survival of <i>M</i>. <i>tuberculosis</i> exposed to DETA/NO and SIN-1.

<p>The bacterial strains were exposed to 0.1 mM DETA/NO (a) and 1 mM SIN-1 (b) and bacterial numbers were determined by luminometry after 4 days of incubation. Data are presented as mean bacterial survival (% of unexposed control) ± SEM, (n = 3). Significant differences from H37Rv are indicated with *(p≤0.05), or **(p≤0.01) as determined by one-way ANOVA with correction for multiple testing.</p

Exposure to pretomanid in broth cultures.

<p>The bacterial strains H37Rv and BTB 02–141 were exposed to increasing concentrations of PRT (0.032-1mg/L) or to INH (2mg/L) in broth The bacterial numbers were determined by luminometry directly after exposure (0h) and at 4h, 24h and 4 days later. Data are presented as mean bacterial survival (% of unexposed control) ± SEM (n = 4–5). Significant differences are indicated with * (p≤0.05) or ** (p≤0.01) as determined by two-way ANOVA.</p