Whole genome sequencing of M. tuberculosis for disease control in high-burden settings: study protocol for a cluster randomized controlled trial evaluating different community-wide intervention strategies in rural Madagascar.

BACKGROUND: Retrospective and descriptive molecular epidemiology studies have shown that Mycobacterium tuberculosis whole genome sequencing can identify outbreaks and disease transmission events with higher resolution than conventional epidemiological investigations. Those studies have strengthened our understanding of genomic polymorphisms correlating with person-to-person transmission and helped resolve putative transmission clusters. To date, systematic genomic surveillance programs implemented for M. tuberculosis were only implemented in low-incidence settings. The purpose of this study is to determine whether there is an impact of routine M. tuberculosis whole genome sequencing on tuberculosis case detection in a high-incidence setting. METHODS: A cluster randomized controlled trial will be performed. Forty-eight rural village groups (or Fokontany) in the Vohibato district of Madagascar will be randomized to one of three interventions arms. Arm 1 (standard of care) involves healthcare facility-based passive case detection with smear microscopy testing. Arm 2 (best practice) consists of active case finding and Xpert MTB/RIF Ultra PCR testing followed by household contact investigations. Arm 3 (novel intervention) includes the same interventions as arm 2, with addition of sputum culture and M. tuberculosis whole genome sequencing for all newly diagnosed cases. In arm 3, molecular suggested putative outbreaks are investigated, and additional TB suspects are appropriately tested. The intervention observational period will be 2 years. The primary outcome will be the number of detected cases/100,000/year in each arm after 1 year of intervention. DISCUSSION: This study is designed to determine whether there is an impact of prospective whole genome sequencing-based molecular typing on tuberculosis case detection in high-incidence settings. Investigating potential outbreaks and focusing active case finding in spatiotemporal settings where disease transmission is suggested by genomic typing is hypothesized to improve case detection in rural communities. TRIAL REGISTRATION: ClinicalTrials.gov NCT05406453 . Retrospectively registered on June 6, 2022.

An independent, multi-country head-to-head accuracy comparison of automated chest x-ray algorithms for the triage of pulmonary tuberculosis.

Background: Computer-aided detection (CAD) algorithms for automated chest X-ray (CXR) reading have been endorsed by the World Health Organization for tuberculosis (TB) triage, but independent, multi-country assessment and comparison of current products are needed to guide implementation. Methods: We conducted a head-to-head evaluation of five CAD algorithms for TB triage across seven countries. We included CXRs from adults who presented to outpatient facilities with at least two weeks of cough in India, Madagascar, the Philippines, South Africa, Tanzania, Uganda, and Vietnam. The participants completed a standard evaluation for pulmonary TB, including sputum collection for Xpert MTB/RIF Ultra and culture. Against a microbiological reference standard, we calculated and compared the accuracy overall, by country and key groups for five CAD algorithms: CAD4TB (Delft Imaging), INSIGHT CXR (Lunit), DrAid (Vinbrain), Genki (Deeptek), and qXR (qure.AI). We determined the area under the ROC curve (AUC) and if any CAD product could achieve the minimum target accuracy for a TB triage test (≥90% sensitivity and ≥70% specificity). We then applied country- and population-specific thresholds and recalculated accuracy to assess any improvement in performance. Results: Of 3,927 individuals included, the median age was 41 years (IQR 29-54), 12.9% were people living with HIV (PLWH), 8.2% living with diabetes, and 21.2% had a prior history of TB. The overall AUC ranged from 0.774-0.819, and specificity ranged from 64.8-73.8% at 90% sensitivity. CAD4TB had the highest overall accuracy (73.8% specific, 95% CI 72.2-75.4, at 90% sensitivity), although qXR and INSIGHT CXR also achieved the target 70% specificity. There was heterogeneity in accuracy by country, and females and PLWH had lower sensitivity while males and people with a history of TB had lower specificity. The performance remained stable regardless of diabetes status. When country- and population-specific thresholds were applied, at least one CAD product could achieve or approach the target accuracy for each country and sub-group, except for PLWH and those with a history of TB. Conclusions: Multiple CAD algorithms can achieve or exceed the minimum target accuracy for a TB triage test, with improvement when using setting- or population-specific thresholds. Further efforts are needed to integrate CAD into routine TB case detection programs in high-burden communities.

Solicited Cough Sound Analysis for Tuberculosis Triage Testing: The CODA TB DREAM Challenge Dataset.

Cough is a common and commonly ignored symptom of lung disease. Cough is often perceived as difficult to quantify, frequently self-limiting, and non-specific. However, cough has a central role in the clinical detection of many lung diseases including tuberculosis (TB), which remains the leading infectious disease killer worldwide. TB screening currently relies on self-reported cough which fails to meet the World Health Organization (WHO) accuracy targets for a TB triage test. Artificial intelligence (AI) models based on cough sound have been developed for several respiratory conditions, with limited work being done in TB. To support the development of an accurate, point-of-care cough-based triage tool for TB, we have compiled a large multi-country database of cough sounds from individuals being evaluated for TB. The dataset includes more than 700,000 cough sounds from 2,143 individuals with detailed demographic, clinical and microbiologic diagnostic information. We aim to empower researchers in the development of cough sound analysis models to improve TB diagnosis, where innovative approaches are critically needed to end this long-standing pandemic.

Tuberculosis in Lemurs and a Fossa at National Zoo, Madagascar, 2022.

We diagnosed Mycobacterium tuberculosis in captive lemurs and a fossa in Antananarivo, Madagascar. We noted clinical signs in the animals and found characteristic lesions during necropsy. The source of infection remains unknown. Our results illustrate the potential for reverse zoonotic infections and intraspecies transmission of tuberculosis in captive wildlife.

Impact of cobas PCR Media freezing on SARS-CoV-2 viral RNA integrity and whole genome sequencing analyses.

SARS-CoV-2 whole genome sequencing is a molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swabs and shipment to reference laboratories is usually required for sequencing. Cobas PCR Media transport medium facilitates high throughput SARS-CoV-2 RT-PCR analyses on cobas platforms. The manufacturer doesn't recommend freezing this transport medium because of risks of degrading molecular templates and impairing test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Sequencing performance, quality and results did not significantly differ between fresh and frozen samples (n = 10). Freezing of cobas PCR Media does not negatively affect SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories.

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.

Performance and impact of GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® assays on tuberculosis case detection in Madagascar.

BACKGROUND: Tuberculosis rapid molecular assays, including GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit®, are highly sensitive and specific. Such performance does not automatically translate in improved disease control and highly depends on their use, local epidemiology and the diagnostic algorithms they're implemented within. We evaluate the performance of both assays and assess their impact on additional cases notification when implemented within WHO recommended tuberculosis diagnostic algorithms in Madagascar. METHODS: Five hundred forty eight presumptive pulmonary tuberculosis patients were prospectively recruited between November 2013 and December 2014 in Antananarivo, Madagascar, a high TB incidence sub-Saharan African urban setting. Both molecular assays were evaluated as first line or add-on testing following negative smear microscopy. Based on locally defined assay performance characteristics we measure the impact of both assays and WHO-recommended diagnostic algorithms on additional tuberculosis case notifications. RESULTS: High sensitivity and specificity was confirmed for both GeneXpert MTB/RIF® (86.6% (95% CI 81.1-90.7%) and 97.4% (95% CI 94.9-98.8%)) and Loopamp MTBC Detection Kit® (84.6% (95% CI 78.9-89.0%) and 98.4% (95% CI 96.2-99.4%)). Implementation of GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® increased tuberculosis diagnostic algorithms sensitivity from 73.6% (95% CI 67.1-79.3%) up to 88.1% (95% CI 82.8-91.9%). This increase was highest when molecular assays were used as add-on testing following negative smear microscopy. As add-on testing, GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® respectively improved case detection by 23.8 and 21.2% (p < 0.05). CONCLUSION: Including GeneXpert MTB/RIF® or Loopamp MTBC Detection Kit® molecular assays for TB detection on sputum samples from presumptive TB cases can significantly increase case notification in TB diagnostic centers. The TB case detection rate is further increased when those tests are use as second-line follow-on testing following negative smear microscopy results. A country wide scale-up and digital integration of molecular-based TB diagnosis assays shows promises for TB control in Madagascar.

Antibiotic resistance prediction for Mycobacterium tuberculosis from genome sequence data with Mykrobe.

Two billion people are infected with Mycobacterium tuberculosis, leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, Mykrobe predictor, which provided offline species identification and drug resistance predictions for M. tuberculosis from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations.  Here we present a new tool, Mykrobe, which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. Mykrobe is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 M. tuberculosis Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 M. tuberculosis isolates. Using culture based DST as the reference, we estimate Mykrobe to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that Mykrobe gives concordant results with nanopore data.  We measure the ability of Mykrobe-based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.

Mycobacterium malmoense pulmonary infection in France: a case report.

BACKGROUND: Mycobacterium malmoense infections have frequently been reported in northern Europe since the late 1970s. Factors accounting for this geographically localized epidemiology remain poorly understood. CASE PRESENTATION: We report the case of a 54-year old man concomitantly diagnosed with non-small cell lung carcinoma and M. malmoense pulmonary infection. We present detailed clinical, microbiological and radiological elements strongly arguing for M. malmoense true pathogenicity. Since M. malmoense infection has rarely been reported in France, we also provide elements of the epidemiological investigation and a literature review of potential acquisition and transmission pathways of M. malmoense. We detail therapeutic interventions and subsequent favorable evolution. CONCLUSION: Mycobacterium malmoense is a recognized respiratory pathogen for which routes of infection need to be better investigated.

Human papillomavirus (HPV) DNA triage of women with atypical squamous cells of undetermined significance with cobas 4800 HPV and Hybrid Capture 2 tests for detection of high-grade lesions of the uterine cervix.

The triage of women with high-risk (HR) human papillomavirus (HPV)-positive smears for atypical squamous cells of undetermined significance (ASC-US) to colposcopy is now an integrated option in clinical guidelines. The performance of cobas 4800 HPV and that of Hybrid Capture 2 (HC2) for HR HPV DNA detection in cervical samples in PreservCyt were compared in 396 women referred to colposcopy for ASC-US. Of these, 316 did not have cervical intraepithelial neoplasia (CIN), 47 had CIN1, 29 had CIN2 or CIN3 (CIN2+), and 4 had CIN of unknown grade. HR HPV was detected in 129 (32.6%) and 149 (37.6%) samples with HC2 and cobas 4800 HPV, respectively (P = 0.15). The clinical sensitivities and specificities for detecting CIN2+ were 89.7% (95% confidence interval [CI], 72.8 to 97.2%) and 66.7% (95% CI, 61.7 to 71.3%) with cobas 4800 HPV and 93.1% (95% CI, 77.0 to 99.2%) and 72.2% (95% CI 67.4 to 76.5%) with HC2. The performance of cobas 4800 HPV was similar to that of HC2 for identifying women with ASC-US who would benefit the most from colposcopy.