Genomic Sequencing from Sputum for Tuberculosis Disease Diagnosis, Lineage Determination, and Drug Susceptibility Prediction.

Universal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited, and targeted molecular assays are vulnerable to emerging resistance mutations. Improved protocols for direct-from-sputum Mycobacterium tuberculosis sequencing would accelerate access to comprehensive drug susceptibility testing and molecular typing. We assessed a thermo-protection buffer-based direct-from-sample M. tuberculosis whole-genome sequencing protocol. We prospectively analyzed 60 acid-fast bacilli smear-positive clinical sputum samples in India and Madagascar. A diversity of semiquantitative smear positivity-level samples were included. Sequencing was performed using Illumina and MinION (monoplex and multiplex) technologies. We measured the impact of bacterial inoculum and sequencing platforms on genomic read depth, drug susceptibility prediction performance, and typing accuracy. M. tuberculosis was identified by direct sputum sequencing in 45/51 samples using Illumina, 34/38 were identified using MinION-monoplex sequencing, and 20/24 were identified using MinION-multiplex sequencing. The fraction of M. tuberculosis reads from MinION sequencing was lower than from Illumina, but monoplexing grade 3+ samples on MinION produced higher read depth than Illumina (P < 0.05) and MinION multiplexing (P < 0.01). No significant differences in sensitivity and specificity of drug susceptibility predictions were seen across sequencing modalities or within each technology when stratified by smear grade. Illumina sequencing from sputum accurately identified 1/8 (rifampin) and 6/12 (isoniazid) resistant samples, compared to 2/3 (rifampin) and 3/6 (isoniazid) accurately identified with Nanopore monoplex. Lineage agreement levels between direct and culture-based sequencing were 85% (MinION-monoplex), 88% (Illumina), and 100% (MinION-multiplex). M. tuberculosis direct-from-sample whole-genome sequencing remains challenging. Improved and affordable sample treatment protocols are needed prior to clinical deployment.

Drug-Resistant Tuberculosis in Pet Ring-Tailed Lemur, Madagascar.

We diagnosed tuberculosis in an illegally wild-captured pet ring-tailed lemur manifesting lethargy, anorexia, and cervical lymphadenopathy. Whole-genome sequencing confirmed the Mycobacterium tuberculosis isolate belonged to lineage 3 and harbored streptomycin resistance. We recommend reverse zoonosis prevention and determination of whether lemurs are able to maintain M. tuberculosis infection.

Multidrug-resistant tuberculosis surveillance and cascade of care in Madagascar: a five-year (2012-2017) retrospective study.

BACKGROUND: In Madagascar, the multidrug-resistant tuberculosis (MDR-TB) surveillance programme was launched in late 2012 wherein previously treated TB cases and symptomatic MDR-TB contacts (hereafter called presumptive MDR-TB cases) undergo drug susceptibility testing. This retrospective review had per aim to provide an update on the national MDR-TB epidemiology, assess and enhance programmatic performance and assess Madagascar's MDR-TB cascade of care. METHODS: For 2012-2017, national TB control programme notification, clinical management data and reference laboratory data were gathered. The development and coverage of the surveillance programme, the MDR-TB epidemiology and programmatic performance indicators were assessed using descriptive, logistic and spatial statistical analyses. Data for 2017 was further used to map Madagascar's TB and MDR-TB cascade of care. RESULTS: The geographical coverage and diagnostic and referral capacities of the MDR-TB surveillance programme were gradually expanded whereas regional variations persist with regard to coverage, referral rates and sample referral delays. Overall, the rate of MDR-TB among presumptive MDR-TB cases remained relatively stable, ranging between 3.9% in 2013 and 4.4% in 2017. Most MDR-TB patients were lost in the second gap of the cascade pertaining to MDR-TB cases reaching diagnostic centres but failing to be accurately diagnosed (59.0%). This poor success in diagnosis of MDR-TB is due to both the current use of low-sensitivity smear microscopy as a first-line diagnostic assay for TB and the limited access to any form of drug susceptibility testing. Presumptive MDR-TB patients' sample referral took a mean delay of 28 days before testing. Seventy-five percent of diagnosed MDR-TB patients were appropriately initiated on treatment, and 33% reached long-term recurrence-free survival. CONCLUSIONS: An expansion of the coverage and strengthening of MDR-TB diagnostic and management capacities are indicated across all regions of Madagascar. With current limitations, the surveillance programme data is likely to underestimate the true MDR-TB burden in the country and an updated national MDR-TB prevalence survey is warranted. In absence of multiple drivers of an MDR-TB epidemic, including high MDR-TB rates, high HIV infection rates and inter-country migration, Madagascar is in a favourable starting position for MDR-TB control and elimination.

Evaluation of critical parameters in the hollow-fibre system for tuberculosis: A case study of moxifloxacin.

AIMS: The hollow­fibre system for tuberculosis (HFS­TB) is a preclinical model qualified by the European Medicines Agency to underpin the anti­TB drug development process. It can mimic in vivo pharmacokinetic (PK)­pharmacodynamic (PD) attributes of selected antimicrobials, which could feed into in silico models to inform the design of clinical trials. However, historical data and published protocols are insufficient and omit key information to allow experiments to be reproducible. Therefore, in this work, we aim to optimize and standardize various HFS­TB operational procedures. METHODS: First, we characterized bacterial growth dynamics with different types of hollow­fibre cartridges, Mycobacterium tuberculosis strains and media. Second, we mimicked a moxifloxacin PK profile within hollow­fibre cartridges, in order to check drug­fibres compatibility. Lastly, we mimicked the moxifloxacin total plasma PK profile in human after once daily oral dose of 400 mg to assess PK­PD after different sampling methods, strains, cartridge size and bacterial adaptation periods before drug infusion into the system. RESULTS: We found that final bacterial load inside the HFS­TB was contingent on the studied variables. Besides, we demonstrated that drug­fibres compatibility tests are critical preliminary HFS­TB assays, which need to be properly reported. Lastly, we uncovered that the sampling method and bacterial adaptation period before drug infusion significantly impact actual experimental conclusions. CONCLUSION: Our data contribute to the necessary standardization of HFS­TB experiments, draw attention to multiple aspects of this preclinical model that should be considered when reporting novel results and warn about critical parameters in the HFS­TB currently overlooked.

Concordance of targeted and whole genome sequencing for Mycobacterium tuberculosis genotypic drug susceptibility testing.

Targeted Next Generation Sequencing (tNGS) and Whole Genome Sequencing (WGS) are increasingly used for genotypic drug susceptibility testing (gDST) of Mycobacterium tuberculosis. Thirty-two multi-drugs resistant and 40 drug susceptible isolates from Madagascar were tested with Deeplex® Myc-TB and WGS using the Mykrobe analysis pipeline. Sixty-four of 72 (89 %) yielded concordant categorical gDST results for drugs tested by both assays. Mykrobe didn't detect pncA K96T, pncA Q141P, pncA H51P, pncA H82R, rrs C517T and rpsL K43R mutations, which were identified as minority variants in corresponding isolates by tNGS. One discrepancy (rrs C517T) was associated with insufficient sequencing depth on WGS. Deeplex® Myc-TB didn't detect inhA G-154A which isn't covered by the assay's amplification targets. Despite those targets being included in the Deeplex® Myc-TB assay, a pncA T47A and a deletion in gid were not identified in one isolate respectively. The evaluated WGS and tNGS gDST assays show high but imperfect concordance.

Complexities and benefits of adopting next-generation sequencing-based tuberculosis diagnostics: a qualitative study among stakeholders in low and high-income countries.

OBJECTIVES: To clarify perceived benefits, barriers and facilitators of Mycobacterium tuberculosis next-generation sequencing implementation in Madagascar and Canada, towards informing implementation of this diagnostic technology in public health agencies and clinical settings in and beyond these settings. DESIGN: This qualitative study involved conducting semistructured interviews with key stakeholders engaged with next-generation sequencing implementation in Madagascar and Canada. Team-based descriptive analysis supported by Nvivo V.12.0 was used to identify key themes. SETTING: The study was conducted with participants involved at the clinical, diagnostic and surveillance levels of tuberculosis (TB) management from Madagascar and Canada. PARTICIPANTS: Eighteen participants were interviewed (nine Madagascar and nine Canada) and included individuals purposively sampled based on involvement with TB surveillance, laboratory diagnosis and clinical management. RESULTS: The following five themes emerged in the analysis of Malagasy and Canadian interviews: (1) heterogeneity in experience with established TB diagnostics, (2) variable understanding of new sequencing-based diagnostics potential; (3) further evidence as being key to expand adoption; (4) ethical arguments and concerns; (5) operational and system-level considerations. CONCLUSION: There persists important lack of familiarity with TB next-generation sequencing (TB NGS) applications among stakeholders in Canada and Madagascar. This translates into skepticism on the evidence underlying its use and its true potential value added within global public health systems. If deployed, TB NGS testing should be integrated with clinical and surveillance programmes. Although this is perceived as a priority, leadership and funding responsibilities for this integration to happen remains unclear to clinical, laboratory and public health stakeholders.

Evaluation of Nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation: a genomic analysis.

BACKGROUND: Mycobacterium tuberculosis whole-genome sequencing (WGS) has been widely used for genotypic drug susceptibility testing (DST) and outbreak investigation. For both applications, Illumina technology is used by most public health laboratories; however, Nanopore technology developed by Oxford Nanopore Technologies has not been thoroughly evaluated. The aim of this study was to determine whether Nanopore sequencing data can provide equivalent information to Illumina for transmission clustering and genotypic DST for M tuberculosis. METHODS: In this genomic analysis, we analysed 151 M tuberculosis isolates from Madagascar, South Africa, and England, which were collected between 2011 and 2018, using phenotypic DST and matched Illumina and Nanopore data. Illumina sequencing was done with the MiSeq, HiSeq 2500, or NextSeq500 platforms and Nanopore sequencing was done on the MinION or GridION platforms. Using highly reliable PacBio sequencing assemblies and pairwise distance correlation between Nanopore and Illumina data, we optimise Nanopore variant filters for detecting single-nucleotide polymorphisms (SNPs; using BCFtools software). We then used those SNPs to compare transmission clusters identified by Nanopore with the currently used UK Health Security Agency Illumina pipeline (COMPASS). We compared Illumina and Nanopore WGS-based DST predictions using the Mykrobe software and mutation catalogue. FINDINGS: The Nanopore BCFtools pipeline identified SNPs with a median precision of 99·3% (IQR 99·1-99·6) and recall of 90·2% (88·1-94·2) compared with a precision of 99·6% (99·4-99·7) and recall of 91·9% (87·6-98·6) using the Illumina COMPASS pipeline. Using a threshold of 12 SNPs for putative transmission clusters, Illumina identified 98 isolates as unrelated and 53 as belonging to 19 distinct clusters (size range 2-7). Nanopore reproduced 15 out of 19 clusters perfectly; two clusters were merged into one cluster, one cluster had a single sample missing, and one cluster had an additional sample adjoined. Illumina-based clusters were also closely replicated using a five SNP threshold and clustering accuracy was maintained using mixed Illumina and Nanopore datasets. Genotyping resistance variants with Nanopore was highly concordant with Illumina, having zero discordant SNPs across more than 3000 SNPs and four insertions or deletions (indels), across 60 000 indels. INTERPRETATION: Illumina and Nanopore technologies can be used independently or together by public health laboratories performing M tuberculosis genotypic DST and outbreak investigations. As a result, clinical and public health institutions making decisions on which sequencing technology to adopt for tuberculosis can base the choice on cost (which varies by country), batching, and turnaround time. FUNDING: Academy for Medical Sciences, Oxford Wellcome Institutional Strategic Support Fund, and the Swiss South Africa Joint Research Award (Swiss National Science Foundation and South African National Research Foundation).

Different PPD-stimulated cytokine responses from patients infected with genetically distinct Mycobacterium tuberculosis complex lineages.

OBJECTIVES: The genetic diversity of Mycobacterium tuberculosis complex (MTBC) influences the immune response of the host, which may affect the immunodiagnostic tests and biomarker assessment studies used for tuberculosis (TB). This study aimed to determine whether the mycobacterial-antigen-stimulated cytokine responses vary with the genotype of the MTBC infecting the patient. METHODS: Eighty-one patients with confirmed active pulmonary TB were recruited, and MTBC clinical strains were isolated from their sputum for bacterial lineage single-nucleotide polymorphism typing. Whole blood was drawn from the patients to measure the purified protein derivative (PPD)-stimulated cytokine responses (GM-CSF, IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, TNF-α, IFN-α, IL-12, eotaxin, IL-13, IL-15, IL-17, MIP1-α, MIP1-ß, MCP1, IL1RA, IP10, IL2R, MIG) with the Luminex multiplex immunoassay. RESULTS: Of the 24 cytokines studied, three were produced differentially in whole blood dependent on the infecting lineage of MTBC. Decreased production of IL-17 was observed in patients infected with modern lineages compared with patients infected with ancestral lineages (P < 0.01), and production of IFN-γ and IL-2 was significantly decreased in patients infected with lineage 4 strains compared with patients infected with lineage 3 strains (P < 0.05). CONCLUSION: MTBC strains belonging to lineage 4 induced a decreased whole-blood PPD-stimulated pro-inflammatory cytokine response.

Detection of multidrug-resistant tuberculosis from stored DNA Samples: A multicenter study.

Background: In low-income countries, rapid detection of tuberculosis (TB) drug resistance is often restricted by the difficulties of transporting and storing sputum samples from remote health centers to the reference laboratories where molecular tests are available. The aim of this study was to evaluate the performance of four transport and storage systems for molecular detection of rifampicin (RIF) and isoniazid (INH) resistance. Methods: This was a multicenter study. Molecular detection of RIF and INH resistance was performed directly from smear-positive TB sputa spotted on a slide, FTA card, GenoCard, and ethanol using the Genotype MTBDRplus assay. The performance of the DNA extraction method from each storage support to detect drug resistance was assessed by calculating their sensitivity and specificity compared to the phenotypic method. Results: From all sites, the overall sensitivity and specificity for RIF-resistance detection was 88% and 85%, respectively, for slides, 86% and 92%, respectively, for GenoCard, 87% and 89%, respectively, for FTA card, and 88% and 92%, respectively, for ethanol. For INH-resistance detection, the overall sensitivity and specificity was 82% and 90%, respectively, for slides, 85% and 96%, respectively, for GenoCard, 86% and 92%, respectively, for FTA card, and 86% and 94%, respectively, for ethanol. Conclusion: Smear slides and filter cards showed to be very useful tools to facilitate DNA extraction from sputum samples with the potential to accelerate the detection of drug resistance in remote areas.

Exploring tuberculosis by molecular tests on DNA isolated from smear microscopy slides.

Tuberculosis (TB) is an infectious disease of global public health importance caused by Mycobacterium tuberculosis complex. The disease has worsened with the emergence of multidrug-resistant (MDR)-TB strains. The timely diagnosis and treatment of TB remains a key public health priority, and laboratories have a critical role in the rapid and accurate detection of TB and drug resistance. Molecular assays based on nucleic acid amplification techniques have been developed for the rapid, sensitive, and specific diagnosis of TB, with the ability to determine the drug sensitivity status. These molecular techniques are now available or are being implemented in developing countries. However, traditional microscopy and culture methods cannot yet be replaced; the molecular assays can be applied in parallel with these tests for the diagnosis of TB or for drug susceptibility testing. Performing such molecular tests is often restricted by constraints with regard to sputum sample storage and safe transportation from remote health centres to central laboratories. Since smear slides are performed routinely for the diagnosis of TB in most TB diagnostic laboratories, they are readily available and could be the ideal tool to transport sputum for further molecular tests. The aim of this review was to provide a comprehensive survey on the use of smear slides for both TB diagnosis and the molecular test approach. Based on the literature, stained smear microscopy slides can be a safe system for the transportation of sputum specimens from remote health centres to reference TB laboratories for further molecular TB or MDR-TB detection, and could help in the rapid diagnosis and therefore timely management of TB patients.

Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry.

A critical step in proteomic analyses comprises the implementation of a reliable cell lysis method with high yields of qualitative proteins. In Mycobacteria, the protein extraction step is often hampered by the thick waxy cell wall which is rich in mycolic acids. Harsh disruption techniques to release proteins from the cells are thus required. Here, we demonstrate an optimized protein extraction procedure for Mycobacterium tuberculosis (Mbt) that results in protein extracts that are useful for all currently used proteomics platforms, including gel and LC-MS based strategies. We compared the effectiveness of using both thiourea and urea and/or SDS and DTT in the solubilization buffer, in combination or not with sonication and/or bead beating. After some preliminary optimization steps on fast-growing Mbt-like organisms, namely Mycobacterium smegmatis and Mycobacterium fortuitum, the final protein extraction protocol was tested on M. tuberculosis. Based on the concentrations of the proteins recovered from each of the tested methods and on the quality of the extracted proteins as evaluated by SDS PAGE, we propose a lysis buffer that contains both thiourea and urea, in combination with two mechanical cell disruption methods: sonication and bead beating. The optimized protocol results in protein extracts that are useful in M. tuberculosis proteomics studies based on any proteomics strategy or platform.

Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis.

BACKGROUND: Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus. METHODS: Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing. RESULTS: For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%. CONCLUSION: The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.