Whole genome sequencing for drug-resistant tuberculosis management in South Africa: What gaps would this address and what are the challenges to implementation?

Global control of tuberculosis (TB) has been seriously impacted by the emergence and transmission of its drug-resistant forms. Delayed detection and incomplete characterisation of drug-resistant tuberculosis (DR-TB) contributes to morbidity and mortality, and to ongoing transmission of drug-resistant strains. Current culture-based and molecular diagnostic tools for TB present numerous disadvantages that could potentially lead to misdiagnosis, inappropriate treatment initiation and the amplification of drug resistance. The detection of drug-resistant tuberculosis (DR-TB) in South Africa relies on molecular diagnostic assays such as the Xpert MTB/RIF and line probe assays (MTBDRplus and MTBDRsl). However, these molecular assays are limited to detecting resistance to only a few first-line and second-line drugs. It is for this reason that next-generation sequencing (NGS) and bioinformatics pipelines have been developed for rapid detection of M. tuberculosis drug resistance, with the added advantage that sequence data could also have public health applications through understanding transmission patterns. This review highlights some of the challenges that are currently hampering the diagnosis and control of DR-TB in a high burden setting of the KwaZulu-Natal (KZN) province in South Africa. Shortfalls of current diagnostic techniques for DR-TB are discussed in detail and we also propose how these might be overcome with an accurate and rapid NGS system.

High incidence of tuberculosis in the first year of antiretroviral therapy in the Botswana National antiretroviral therapy programme between 2011 and 2015.

OBJECTIVE: Tuberculosis (TB) remains one of the leading causes of mortality and morbidity among people living with HIV. We sought to estimate the incidence of TB in a national database of HIV-infected patients receiving antiretroviral therapy (ART) in Botswana. DESIGN: A retrospective analysis of HIV-infected adult patients (≥18years) who initiated ART between 2011 and 2015 in the Botswana ART program. METHODS: Multivariable analysis using Cox regression included sex, age, viral load and CD4 T-cell counts. RESULTS: Of 45 729 patients, with a median follow-up of 1.7 years Q1 : Q3, 0.5, 3.1), 1791 patients developed TB over a median of 1.5 years (Q1 : Q3, 0.3, 3.1) of follow-up (incidence rate 1.9 per 100 person-years; 95% CI 1.8-2.0). At baseline, the median CD4 T-cell count was 272 cells/µl (Q1, Q3 146, 403). The risk of TB was greatest within the first year of ART (incidence rate 2.9 per 100 person-years; 95% CI 2.7-3.1) and in patients with CD4 T-cell counts below 50 cells/µl (incidence rate 8.3/100 person-years; 95% CI 7.1-9.7). Patients with viral loads above 10 000 copies/ml at 3 months post-ART initiation had two times higher risk of TB, hazard ratio 2.5 (95% CI 1.8-2.3). CONCLUSION: We report a high incidence of TB within the first year of ART and in patients with advanced immunodeficiency. Improved screening strategies and virologic monitoring during this early period on ART, coupled with TB preventive treatment, will reduce the burden of TB.

Detection of Second Line Drug Resistance among Drug Resistant Mycobacterium Tuberculosis Isolates in Botswana.

The emergence and transmission of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (M.tb) strains is a threat to global tuberculosis (TB) control. The early detection of drug resistance is critical for patient management. The aim of this study was to determine the proportion of isolates with additional second-line resistance among rifampicin and isoniazid resistant and MDR-TB isolates. A total of 66 M.tb isolates received at the National Tuberculosis Reference Laboratory between March 2012 and October 2013 with resistance to isoniazid, rifampicin or both were analyzed in this study. The genotypes of the M.tb isolates were determined by spoligotyping and second-line drug susceptibility testing was done using the Hain Genotype MTBDRsl line probe assay version 2.0. The treatment outcomes were defined according to the Botswana national and World Health Organization (WHO) guidelines. Of the 57 isolates analyzed, 33 (58%) were MDR-TB, 4 (7%) were additionally resistant to flouroquinolones and 3 (5%) were resistant to both fluoroquinolones and second-line injectable drugs. The most common fluoroquinolone resistance-conferring mutation detected was gyrA A90V. All XDR-TB cases remained smear or culture positive throughout the treatment. Our study findings indicate the importance of monitoring drug resistant TB cases to ensure rapid detection of second-line drug resistance.

Genetic diversity of Mycobacterium tuberculosis strains circulating in Botswana.

BACKGROUND: Molecular typing of Mycobacterium tuberculosis (M.tb) isolates can inform Tuberculosis (TB) control programs on the relative proportion of transmission driving the TB epidemic. There is limited data on the M. tb genotypes that are circulating in Botswana. The aim of this study was to generate baseline data on the genetic diversity of M.tb isolates circulating in the country. METHODS: A total of 461 M.tb isolates received at the Botswana National Tuberculosis Reference Laboratory between March 2012 and October 2013 were included in this study. Drug susceptibility testing was conducted using the BD BACTEC MGIT 960 System. M.tb strains were genotyped using spoligotyping and spoligotype patterns were compared with existing patterns in the SITVIT Web database. A subset of drug resistant isolates which formed spoligo clusters (n = 65) was additionally genotyped with 12-loci MIRU. Factors associated with drug resistance and clustering were evaluated using logistic regression. RESULTS: Of the 461 isolates genotyped, 458 showed 108 distinct spoligotype patterns. The predominant M.tb lineages were Lineage 4 (81.9%), Lineage 2 (9%) and Lineage 1 (7.2%). The predominant spoligotype families within Lineage 4 were LAM (33%), S (14%), T (16%), X (16%). Three hundred and ninety-two (86%) isolates could be grouped into 44 clusters (2-46 isolates per cluster); giving a clustering rate of 76%. We identified 173 (37.8%) drug resistant isolates, 48 (10.5%) of these were multi-drug resistant. MIRU typing of the drug resistant isolates allowed grouping of 46 isolates into 14 clusters, giving a clustering rate of 49.2%. There was no association between age, sex, treatment category, region and clustering. CONCLUSIONS: This study highlights the complexity of the TB epidemic in Botswana with multiple strains contributing to disease and provides baseline data on the population structure of M.tb strains in Botswana.

Correction: Immunological non-response and low hemoglobin levels are predictors of incident tuberculosis among HIV-infected individuals on Truvada-based therapy in Botswana.

[This corrects the article DOI: 10.1371/journal.pone.0192030.].

Immunological non-response and low hemoglobin levels are predictors of incident tuberculosis among HIV-infected individuals on Truvada-based therapy in Botswana.

BACKGROUND: There is a high burden of tuberculosis (TB) in HIV antiretroviral programmes in Africa. However, few studies have looked at predictors of incident TB while on Truvada-based combination antiretroviral therapy (cART) regimens. METHODS: We estimated TB incidence among individuals enrolled into an observational cohort evaluating the efficacy and tolerability of Truvada-based cART in Gaborone, Botswana between 2008 and 2011. We used Cox proportional hazards regressions to determine predictors of incident TB. RESULTS: Of 300 participants enrolled, 45 (15%) had a diagnosis of TB at baseline. During 428 person-years (py) of follow-up, the incidence rate of TB was 3.04/100py (95% CI, 1.69-5.06), with 60% of the cases occurring within 3 months of ART initiation. Incident cases had low baseline CD4+ T cell counts (153cells/mm3 [Q1, Q3: 82, 242]; p = 0.69) and hemoglobin levels (9.2g/dl [Q1, Q3: 8.5,10.1]; p<0.01). In univariate analysis, low BMI (HR = 0.73; 95% CI 0.58-0.91; p = 0.01) and hemoglobin levels <8 g/dl (HR = 10.84; 95%CI: 2.99-40.06; p<0.01) were risk factors for TB. Time to incident TB diagnosis was significantly reduced in patients with poor immunological recovery (p = 0.04). There was no association between baseline viral load and risk of TB (HR = 1.75; 95%CI: 0.70-4.37). CONCLUSION: Low hemoglobin levels prior to initiation of ART are significant predictors of incident tuberculosis. Therefore, there is potential utility of iron biomarkers to identify patients at risk of TB prior to initiation on ART. Furthermore, additional strategies are required for patients with poor immunological recovery to reduce excess risk of TB while on ART.

Mycobacterium tuberculosis Next-Generation Whole Genome Sequencing: Opportunities and Challenges.

Mycobacterium tuberculosis drug resistance is a threat to global tuberculosis (TB) control. Comprehensive and timely drug susceptibility determination is critical to inform appropriate treatment of drug-resistant tuberculosis (DR-TB). Phenotypic drug susceptibility testing (DST) is the gold standard for M. tuberculosis drug resistance determination. M. tuberculosis whole genome sequencing (WGS) has the potential to be a one-stop method for both comprehensive DST and epidemiological investigations. We discuss in this review the tremendous opportunities that next-generation WGS presents in terms of understanding the molecular epidemiology of tuberculosis and mechanisms of drug resistance. The potential clinical value and public health impact in the areas of DST for patient management and tracing of transmission chains for timely public health intervention are also discussed. We present the current challenges for the implementation of WGS in low and middle-income settings. WGS analysis has already been adapted routinely in laboratories to inform patient management and public health interventions in low burden high-income settings such as the United Kingdom. We predict that the technology will be adapted similarly in high burden settings where the impact on the epidemic will be greatest.

Plasma Cytokine Levels in Chronic Asymptomatic HIV-1 Subtype C Infection as an Indicator of Disease Progression in Botswana: A Retrospective Case Control Study.

HIV infects cells of the immune system causing immune activation and proliferation of immune cells, leading to alteration of production and activity of a number of cytokines. These changes in cytokine levels can affect the immune function, and have the potential to directly impact the course of HIV disease. We characterized plasma cytokine concentration profiles in HIV-1 subtype C chronically infected, antiretroviral therapy (ART)-naive participants to establish their influence on disease progression and viremia. Plasma levels of interleukin (IL)-1α, IL-7, IL-12p40, granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-γ were quantified in samples from 60 treatment-naive participants in the placebo arm of the completed Micronutrient-HIV disease progressions study, "Dikotlana" (2004-2009) in Gaborone, Botswana. Participants were stratified into progressors (P) and nonprogressors (NP) based on their rates of CD4(+) T cell depletion during the study period. Nonprogressors were those who had <1% CD4(+) T cell depletion at 24 months postenrollment. Progressors were defined as those with CD4(+) T cell depletion of >15% at 24 months postenrollment. Cytokine levels were compared between P and NP using the Mann-Whitney U-test. Logistic regression analysis was used to determine if cytokines predicted disease progression. Correlations of cytokines with CD4(+) T cell counts and viral loads were determined by the Spearman rank test. Median baseline CD4(+) T cell counts were 453 (Q1, Q3; 401, 592) and 479 (Q1, Q3; 401-592) for nonprogressors and progressors, respectively. Nonprogressors had a higher viral set point than progressors. IL-12p40 levels were significantly higher in the P than in NP at enrollment and 24 months (p < 0.05). Levels of IL-1α, IL-7, IFN-γ, and GM-CSF did not differ significantly between the two groups. Except for IL-12p40, which displayed an inverse correlation with CD4(+) T cell counts and a direct correlation with viral load, all other cytokines showed no correlations. IL-12p40 was found to be the most significant predictor of progression and its production was most likely driven by HIV replication products as evidenced by its direct correlation with viral load. In chronic HIV-1 subtype C infection, CD4(+) T cell counts and plasma cytokine levels may not necessarily evolve in parallel, suggesting the involvement of other factors in determining the rates of CD4(+) T cell depletion.

No clinically significant drug-resistance mutations in HIV-1 subtype C-infected women after discontinuation of NRTI-based or PI-based HAART for PMTCT in Botswana.

Risk of developing drug resistance after stopping antiretroviral regimens to prevent mother-to-child HIV-1 transmission is unknown. The Mma Bana Study randomized treatment-naive pregnant women with CD4 ≥200 cells per cubic millimeter to receive either abacavir/zidovudine/lamivudine [triple nucleoside reverse transcriptase inhibitor (NRTI) arm] or lopinavir/ritonavir/zidovudine/lamivudine [protease inhibitor (PI) arm]. Drugs were discontinued after 6 months of breastfeeding. One month after discontinuation, 29 NRTI arm samples and 25 PI arm samples were successfully genotyped. No clinically significant antiretroviral resistance mutations were detected. Eight minor resistance mutations were found among 11 (20%) women (3 from NRTI arm and 8 from PI arm), occurring at similar frequencies to those reported in HIV-1 subtype C treatment-naive cohorts.