Tuberculosis diagnostic networks: Moving beyond the laboratory to end tuberculosis in Africa

No abstract available

Universal Genotyping in Tuberculosis Control Program, New York City, 2001–2003

Real-time universal genotyping decreased unnecessary treatment

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

Stronger tuberculosis laboratory networks and services in Africa essential to ending tuberculosis

No abstract available

Rolling out Xpert MTB/RIF® for tuberculosis detection in HIV-positive populations: An opportunity for systems strengthening

Background: To eliminate preventable deaths, disease and suffering due to tuberculosis ,improved diagnostic capacity is critical. The Cepheid Xpert MTB/RIF® assay is recommended by the World Health Organization as the initial diagnostic test for people with suspected HIV associatedtuberculosis. However, despite high expectations, its scale-up in real-world settings has faced challenges, often due to the systems that support it. Opportunities for System Strengthening: In this commentary, we discuss needs and opportunities for systems strengthening to support widespread scale-up of Xpert MTB/RIF as they relate to each step within the tuberculosis diagnostic cascade, from finding presumptive patients, to collecting, transporting and testing sputum specimens, to reporting and receiving results, to initiating and monitoring treatment and, ultimately, to ensuring successful and timely treatment and cure. Investments in evidence-based interventions at each step along the cascade and within the system as a whole will augment not only the utility of Xpert MTB/RIF, but also the successful implementation of future diagnostic tests. Conclusion: Xpert MTB/RIF will only improve patient outcomes if optimally implemented within the context of strong tuberculosis programmes and systems. Roll-out of this technology to people living with HIV and others in resource-limited settings offers the opportunity to leverage current tuberculosis and HIV laboratory, diagnostic and programmatic investments, while also addressing challenges and strengthening coordination between laboratory systems, laboratory-programme interfaces, and tuberculosis-HIV programme interfaces. If successful, the benefits of this tool could extend beyond progress toward global End TB Strategy goals, to improve system-wide capacity for global disease detection and control

Injection of non- protein coding <i>Slc34a2a</i> RNA and <i>Slc34a2a</i> fragments interfere with zebrafish development.

<p>A) Schematic representation of a zebrafish head at 48 hpf; forebrain, blue; eyes, yellow; otic vesicles, green and cerebellum, red. Middle and left, wild type and <i>Slc34a2a</i>-FS injected embryo, respectively. Red arrows indicate the position of the cerebellum. B) Phenotypic quantification of <i>Slc34a2a</i> and <i>Slc34a2a</i>-FS injected embryos (364 <i>Slc34a2a</i>-FS injected embryos were assessed). C) Schematic representation of the fragments generated, even numbers represent sense orientation; uneven numbers, antisense orientation. The large black boxes represent exons comprised in the relevant fragments, the open boxes are exons that are not represented in the injected fragments. The small boxes in red indicate potential sites of hybridization of the injected fragments with an endogenous transcript on the opposite strand. D) Top view of 48 hpf embryos with the fragments (Frag) injected as indicated. E) Phenotypic assessment of injected embryos (90 or more per RNA). F) Eng2 stained embryos injected with the indicated fragments and the relevant controls. All the embryos were tested in parallel with the same solutions and under identical conditions to allow for a comparison of the relative intensities.</p

Ectopically expressed <i>Slc34a2a</i> sense-antisense transcripts cause a cerebellar phenotype in zebrafish embryos depending on RNA complementarity and Dicer

<div><p>Natural antisense transcripts (NATs) are complementary to protein coding genes and potentially regulate their expression. Despite widespread occurrence of NATs in the genomes of higher eukaryotes, their biological role and mechanism of action is poorly understood. Zebrafish embryos offer a unique model system to study sense-antisense transcript interplay at whole organism level. Here, we investigate putative antisense transcript-mediated mechanisms by ectopically co-expressing the complementary transcripts during early zebrafish development. In zebrafish the gene <i>Slc34a2a</i> (Na-phosphate transporter) is bi-directionally transcribed, the NAT predominantly during early development up to 48 hours after fertilization. Declining levels of the NAT, <i>Slc34a2a</i>(as), coincide with an increase of the sense transcript. At that time, sense and antisense transcripts co-localize in the endoderm at near equal amounts. Ectopic expression of the sense transcript during embryogenesis leads to specific failure to develop a cerebellum. The defect is RNA-mediated and dependent on sense-antisense complementarity. Overexpression of a <i>Slc34a2a</i> paralogue (Slc34a2b) or the NAT itself had no phenotypic consequences. Knockdown of Dicer rescued the brain defect suggesting that RNA interference is required to mediate the phenotype. Our results corroborate previous reports of <i>Slc34a2a</i>-related endo-siRNAs in two days old zebrafish embryos and emphasize the importance of coordinated expression of sense-antisense transcripts. Our findings suggest that RNAi is involved in gene regulation by certain natural antisense RNAs.</p></div

Injection of <i>Slc34a2a</i> RNA and related constructs into fertilized zebrafish eggs.

<p>A) ISH of wild type and injected embryos at 24 hpf. Horizontal labels at the top indicate the injected material, vertical labels, left, represent the probes used for ISH. B) RT-qPCR of injected zebrafish embryos; Slc34a2a, Slc34a2a(as) and Slc34a2b RNA was injected as indicated with the different colour from brown to blue and assayed after 10 and 24 hpf. The left group represents RT-qPCR reactions with Slc34a2a-specific primers; the middle group with Slc34a2a(as)-specific primers and the right group with Slc34a2b-specific primers. The values for non-injected controls are indicated with grey, transparent boxed.</p

Expression of <i>Slc34a2a</i> and related transcripts during zebrafish embryogenesis.

<p>(A) Schematic representation of the <i>Slc34a2a</i>, <i>Slc34a2a</i>(as) and <i>Rbpja</i> loci. The antisense transcript <i>Slc34a2a</i>(as) is depicted in red. (B) RT-qPCR analysis of <i>Slc34a2a</i>, <i>Slc34a2a</i>(as) and <i>Rbpja</i> transcripts including the paralog <i>Slc34a2b</i>. Based on negative controls using RNA as an input, the detection limit was set at a ΔCt of 12 which is in agreement with ISH results. (C) Demonstration of <i>Slc34a2a</i>, <i>Slc34a2a</i>(as), <i>Rbpja</i>, <i>Slc34a2b</i> and <i>Shh</i> (Sonic Hedgehog) transcripts at progressing stages of development by whole mount ISH.</p

Morpholino knockdown of <i>Slc34a2a</i>(as) and Dicer.

<p>A) RT-qPCR quantification of <i>Slc34a2a</i>, <i>Slc34a2a</i>(as) and <i>Slc34a2b</i> after splice site morpholino injection at 24 hpf. Wild type non injected controls, light blue bars; 5 ng splice-site MO injected embryos are in dark blue; 5 ng scrambled MO injected embryos are in blue. B) Phenotypic characterization of MO injected embryos at 24 and 48 hpf. The injected oligonucleotides and quantities are indicated below the bars. Phenotypic scaling was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178219#pone.0178219.g002" target="_blank">Fig 2</a>. C) Rescue of cerebellum development by Dicer knockdown. Phenotypic assessment of embryos injected with combinations of Dicer MO, p53 MO and <i>Slc34a2a</i>. D) 48 hpf zebrafish embryos injected with Dicer MO, p53 MO and <i>Slc34a2a</i> as indicated in the pictures. In the upper panel, heads with red arrows indicating the cerebellum are shown; the lower panel shows ISH of embryos with an Eng2 probe.</p