Evaluation of cutting-edge diagnostic and treatment monitoring tools in a cohort of tuberculosis patients during the first month of treatment

Drug susceptibility testing (DST) is estimated to be performed only in 58% of previ-ously treated tuberculosis (TB) cases and in 12% of untreated cases. This figure emphasizes the need to evaluate the contribution of molecular-based methodologies for early detection of drug-resistant TB and implementing phenotypic susceptibility methods for drug resistance determination towards a personalized treatment. We evaluated the capacity of the Xpert MTB/RIF Ultra assay for the detection of M. tuberculosis complex (MTBc) DNA in clinical sam-ples of patients under therapy for pulmonary TB, through the analysis of respiratory samples taken at four timepoints: 0, 7, 14, and 28 days. The results were compared with those obtained from liquid cultures, used as gold standard. Minimum inhibitory concentration (MIC) deter-mination was used to ascertain drug resistance profiles. The results obtained with the Xpert Ultra throughout the first month of treatment shows a good performance, 97.6% sensitivity and 90% overall agreement (k=0.43), when compared with those obtained with liquid cultures. The performance of the test for the detection of MTBc DNA during the first month of treatment and according to the time points were: 95% sensitivity, visit 1 (day 0); 88%, visit 2 (day 7); 69%, visit 3 (day 14), and 69%, visit 4 (day 28). The isolates studied were susceptible to the four anti-TB drugs tested. No mutation was detected in the genes associated with resistance to these drugs. The results obtained showed that both methodologies complement each other: the Xpert Ultra offers early detection of MTBc DNA (detection of genetic material) and the culture allows performing DST to guide therapy and, at the same time, indicates how effective is being the treatment since, contrary to the Xpert Ultra, it detects viable M. tuberculosis. Together, they can improve TB diagnosis, treatment guidance and break the chain of transmission.Estima-se que os testes de susceptibilidade aos antibióticos (TSA) sejam realizados ape-nas em 58% dos casos de tuberculose (TB) previamente tratados e em 12% dos casos não tra-tados. Este número enfatiza a necessidade de avaliar a contribuição de metodologias de base molecular para a detecção precoce da tuberculose resistente aos antibióticos e a implementa-ção de métodos de susceptibilidade fenotípica para a determinação da resistência aos antibió-ticos, com vista a um tratamento personalizado. Avaliamos a capacidade do Xpert MTB/RIF Ultra para a detecção do ADN do complexo M. tuberculosis (MTBc) em amostras clínicas de doentes sob tratamento para TB pulmonar, através da análise de amostras respiratórias obti-das em quatro períodos temporais: 0, 7, 14, e 28 dias. Os resultados foram comparados com os obtidos a partir de culturas líquidas, utilizadas como referência. A determinação da concen-tração mínima inibitória (CMI) foi utilizada para determinar perfis de resistência aos antibió-ticos. Os resultados obtidos com o Xpert Ultra ao longo do primeiro mês de tratamento mos-tram um bom desempenho, 97.6% de sensibilidade e 90% de concordância geral (k=0,43), quando comparados com os resultados obtidos pela cultura líquida. A detecção do ADN de MTBc durante o primeiro mês de tratamento e de acordo com os quatro períodos temporais apresentam 95% de sensibilidade, visita 1 (dia 0); 88%, visita 2 (dia 7); 69%, visita 3 (dia 14), e 69%, visita 4 (dia 28). Os isolados estudados foram susceptíveis aos quatro antibióticos anti-TB testados. Não foi detectada nenhuma mutação nos genes associados à resistência a estes antibióticos. Os resultados obtidos mostraram que ambas as metodologias se complementam: o Xpert Ultra oferece a detecção precoce do ADN de MTBc (detecção de material genético) e a cultura permite realizar o TSA para orientar a terapia e, ao mesmo tempo, indica a eficácia do tratamento uma vez que, ao contrário do Xpert Ultra, detecta M. tuberculosis viável. Juntos, podem melhorar o diagnóstico da TB, a orientação do tratamento e quebrar a cadeia de trans-missão

Correlation of Xpert MTB/RIF with measures to assess Mycobacterium tuberculosis bacillary burden in high HIV burden areas of Southern Africa.

Traditionally, smear microscopy has been used as a point-of-care measure of bacillary burden in tuberculosis patients to inform infection control and contact tracing. Xpert MTB/RIF has the potential to replace smear. However, data to support the use of its quantitative output [cycle threshold (CT)] as an alternate point-of-care measure of bacillary burden are limited. This study assessed the correlation (Spearman's) between CT, smear, culture time-to-positivity (TTP), and clinical factors in patients with Xpert-positive sputum from Mozambique (n = 238) and South Africa (n = 462). Mean CT and smear grade correlated well (ρ0.72); compared to TTP and smear (ρ0.61); and mean CT and TTP (ρ0.50). In multivariate analyses, lower CT (higher bacillary load) was associated with negative HIV serostatus and low BMI. A smear positivity rule-out (95% sensitivity) CT cut-off of 28.0 was identified, with 54.1% specificity, 2.07 positive likelihood ratio, 0.09 negative likelihood ratio and 79.0% correctly classified. Cut-offs were higher for HIV positive compared to HIV negative individuals for any set sensitivity level. This study suggests Xpert CT values correlate well with smear, both in HIV positive and negative individuals, and that CT cut-offs might be broadly applicable to multiple settings. Studies to directly assess the association of CT with infectiousness are needed

Do Xpert MTB/RIF Cycle Threshold Values Provide Information about Patient Delays for Tuberculosis Diagnosis?

INTRODUCTION: Early diagnosis and initiation to appropriate treatment is vital for tuberculosis (TB) control. The XpertMTB/RIF (Xpert) assay offers rapid TB diagnosis and quantitative estimation of bacterial burden through Cycle threshold (Ct) values. We assessed whether the Xpert Ct value is associated with delayed TB diagnosis as a potential monitoring tool for TB control programme performance. MATERIALS AND METHODS: This analysis was nested in a prospective study under the routine TB surveillance procedures of the National TB Control Program in Manhica district, Maputo province, Mozambique. Presumptive TB patients were tested using smear microscopy and Xpert. We explored the association between Xpert Ct values and self-reported delay of Xpert-positive TB patients as recorded at the time of diagnosis enrolment. Patients with >60 days of TB symptoms were considered to have long delays. RESULTS: Of 1,483 presumptive TB cases, 580 were diagnosed as TB of whom 505 (87.0%) were due to pulmonary TB and 302 (94.1%) were Xpert positive. Ct values (range, 9.7-46.4) showed a multimodal distribution. The median (IQR) delay was 30 (30-45) days. Ct values showed no correlation with delay (R2 = 0.001, p = 0.621), nor any association with long delays: adjusted odds ratios (AOR) (95% confidence interval [CI]) comparing to >28 cycles 0.99 (0.50-1.96; p = 0.987) for 23-28 cycles, 0.93 (0.50-1.74; p = 0.828) for 16-22 cycles; and 1.05 (0.47-2.36; p = 0.897) for <16 cycles. Being HIV-negative (AOR [95% CI]), 2.05 (1.19-3.51, p = 0.009) and rural residence 1.74 (1.08-2.81, p = 0.023), were independent predictors of long delays. CONCLUSION: Xpert Ct values were not associated with patient delay for TB diagnosis and cannot be used as an indicator of TB control program performance

Point of care diagnostics for tuberculosis

The goals of the End TB strategy, which aims to achieve a 90% reduction in tuberculosis (TB) incidence and a 95% reduction in TB mortality by 2035, will not be achieved without new tools to fight TB. These include improved point of care (POC) diagnostic tests that are meant to be delivered at the most decentralised levels of care where the patients make the initial contact with the health system, as well as within the community. These tests should be able to be performed on an easily accessible sample and provide results in a timely manner, allowing a quick treatment turnaround time of a few minutes or hours (in a single clinical encounter), hence avoiding patient loss-to-follow-up. There have been exciting developments in recent years, including the WHO endorsement of Xpert MTB/RIF, Xpert MTB/RIF Ultra, loop-mediated isothermal amplification (TB-LAMP) and lateral flow lipoarabinomannan (LAM). However, these tests have limitations that must be overcome before they can be optimally applied at the POC. Furthermore, worrying short- to medium-term gaps exist in the POC diagnostic test development pipeline. Thus, not only is better implementation of existing tools and algorithms needed, but new research is required to develop new POC tests that allow the TB community to truly make an impact and find the ‘‘missed TB cases’’

Mortality and risk of tuberculosis among people living with HIV in whom TB was initially ruled out

Tuberculosis (TB) misdiagnosis remains a public health concern, especially among people living with HIV (PLHIV), given the high mortality associated with missed TB diagnoses. The main objective of this study was to describe the\xC2\xA0all-cause mortality, TB incidence rates and their\xC2\xA0associated risk factors in a cohort of PLHIV with presumptive TB in whom TB was initially ruled out. We retrospectively followed a cohort of PLHIV with presumptive TB over a 2\xC2\xA0year-period in a rural district in Southern Mozambique. During the study period 382 PLHIV were followed-up. Mortality rate was 6.8/100 person-years (PYs) (95% CI 5.2-9.2) and TB incidence rate was 5.4/100 PYs (95% CI 3.9-7.5). Thirty-six percent of deaths and 43% of TB incident cases occurred in the first 12\xC2\xA0months of the follow up. Mortality and TB incidence rates in the 2-year period after TB was initially ruled out was very high. The\xC2\xA0TB diagnostic work-up and linkage to HIV\xC2\xA0care should be strengthened to decrease TB burden and all-cause mortality among PLHIV with presumptive TB

IP-10 Kinetics in the First Week of Therapy are Strongly Associated with Bacteriological Confirmation of Tuberculosis Diagnosis in HIV-Infected Patients

Simple effective tools to monitor the long treatment of tuberculosis (TB) are lacking. Easily measured host derived biomarkers have been identified but need to be validated in larger studies and different population groups. Here we investigate the early response in IP-10 levels (between day 0 and day 7 of TB therapy) to identify bacteriological status at diagnosis among 127 HIV-infected patients starting TB treatment. All participants were then classified as responding or not responding to treatment blindly using a previously described IP-10 kinetic algorithm. There were 77 bacteriologically confirmed cases and 41 Xpert MTB/RIF® and culture negative cases. Most participants had a measurable decline in IP-10 during the first 7 days of therapy. Bacteriologically confirmed cases were more likely to have high IP-10 levels at D0 and had a steeper decline than clinically diagnosed cases (mean decline difference 2231 pg/dl, 95% CI: 897-3566, p = 0.0013). Bacteriologically confirmed cases were more likely to have a measurable decline in IP-10 at day 7 than clinically diagnosed cases (48/77 (62.3%) vs 13/41 (31.7%), p < 0.001). This study confirms the association between a decrease in IP-10 levels during the first week of treatment and a bacteriological confirmation at diagnosis in a large cohort of HIV positive patients

Aplicaciones de la secuenciación del genoma completo de Mycobacterium tuberculosis para la predicción de resistencias a antibióticos en muestras de Mozambique

Trabajo presentado a las XXVII Jornadas Internacionales sobre Tuberculosis celebradas en Barcelona del 13 al 14 de noviembre de 2023.Peer reviewe

Fine-grain population structure and transmission patterns of Mycobacterium tuberculosis in southern Mozambique, a high TB/HIV burden area

14 paginas, 5 figuras, 2 tablas.Genomic studies of the Mycobacterium tuberculosis complex (MTBC) might shed light on the dynamics of its transmission, especially in high-burden settings, where recent outbreaks are embedded in the complex natural history of the disease. To this end, we conducted a 1 year prospective surveillance-based study in Mozambique. We applied whole-genome sequencing (WGS) to 295 positive cultures. We fully characterized MTBC isolates by phylogenetics and dating evaluation, and carried out a molecular epidemiology analysis to investigate further associations with pre-defined transmission risk factors. The majority of strains (49.5%, 136/275) belonged to lineage (L) 4; 57.8 % of them (159/275) were in genomic transmission clusters (cut-off 5 SNPs), and a strikingly high proportion (45.5%) shared an identical genotype (0 SNP pairwise distance). We found two 'likely endemic' clades, comprising 67 strains, belonging to L1.2, which dated back to the late 19th century and were associated with recent spread among people living with human immunodeficiency virus (PLHIV). We describe for the first time the population structure of MTBC in our region, a high tuberculosis (TB)/HIV burden area. Clustering analysis revealed an unforeseen pattern of spread and high rates of progression to active TB, suggesting weaknesses in TB control activities. The long-term presence of local strains in Mozambique, which were responsible for large transmission among HIV/TB-coinfected patients, calls into question the role of HIV in TB transmission.This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programmes 101001038 (TB-RECONNECT), PID2019-104477RB-I00 from Ministerio de Economía y Competitividad (Spanish Government) (to I.C.). We acknowledge support from the Spanish Ministry of Science, Innovation and Universities through the ‘Centro de Excelencia Severo Ochoa 2019–2023’.Programme (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Programme. B.S receives a pre-doctoral fellowship from the Secretariat of Universities and Research, Ministry of Enterprise and Knowledge of the Government of Catalonia and co-funded by European Social Fund (AGAUR).Peer reviewe

Unravelling the population structure and transmission patterns of Mycobacterium tuberculosis in Mozambique, a high TB/HIV burden country

Genomic studies of Mycobacterium tuberculosis complex (MTBC) might shed light on the dynamics of its transmission, especially in high-burden settings, where recent outbreaks are embedded in the complex natural history of the disease. We applied Whole-genome sequencing (WGS) to characterize the local population of MTBC, unravel potential transmission links and evaluate associations with host and pathogen factors. Methods A one-year prospective study was conducted in Mozambique, a high HIV/TB burden country. WGS was applied to 295 positive cultures. We combined phylogenetic, geographical and clustering analysis, and investigated associations between risk factors of transmission. Findings A significant high proportion of strains were in recent transmission (45.5%). We fully characterized MTBC isolates by using phylogenetic approaches and dating evaluation. We found two likely endemic clades, comprised of 67 strains, belonging to L1.2, dating from the late XIX century and associated with recent spread among PLHIV. Interpretation Our results unveil the population structure of MTBC in our setting. The clustering analysis revealed an unexpected pattern of spread and high rates of progression, suggesting the failure of control measures. The long-term presence of local strains in Mozambique, which were responsible for large transmission among HIV/TB coinfected patients, hint at possible coevolution with sympatric host populations and challenge the role of HIV in TB transmission.Ministry of Enterprise and Knowledge (Government of Catalonia & European Social Fund, AGAUR fellowship); European Research Council (ERC) European Union’s Horizon 2020.N

Direct genotyping of Mycobacterium tuberculosis from Xpert® MTB/RIF remnants

Genotyping of Mycobacterium tuberculosis (MTB) isolates has markedly improved our knowledge of its transmission dynamics. MIRU-VNTR is considered the reference molecular tool for MTB fingerprinting. However, the dependence of this technique on cultured isolates means that we lack molecular epidemiology data from many settings where culture facilities have not been implemented. Efforts have been made to adapt the MIRU-VNTR procedure to direct analysis of clinical specimens, although implementation of these efforts has not proven successful. The large-scale roll-out of Xpert MTB/RIF (Xpert) technology, which is now in almost every TB-endemic country, including many where MTB is not cultured, provides us with a new opportunity to explore whether MTB genotyping could be performed from the remnants of the Xpert cartridge. We ran a pilot study in Mozambique in which the remnants of 24 positive Xpert assays for detection of MTB were used as template material for the 15-locus or the more discriminatory 24-locus MIRU-VNTR technique. MTB fingerprinting was possible in specimens with a high bacterial burden, according to the Xpert load categories, and within the first week after Xpert was performed. Given the wide availability, simple processing, and rapid reporting of results with Xpert, our findings suggest that MIRU-VNTR–based fingerprinting from remnants of Xpert could play a major role in extending MTB molecular epidemiology studies to settings where information on the transmission dynamics of this pathogen is lacking