Belén Saavedra

Background: We report the longitudinal analysis of QuantiFERON-TB Gold Plus (QFT-Plus) results from a clinical trial conducted in 3 countries with a high tuberculosis (TB)/human immunodeficiency virus (HIV) burden, comparing 3 different TB preventive treatment (TPT) regimens in people with HIV (PWH). Methods: PWH were enrolled in Ethiopia, Mozambique, and South Africa and randomized to (i) 1 course of weekly rifapentine-isoniazid for 3 months (3HP), (ii) weekly rifapentine-isoniazid for 3 months given annually for 2 years (p3HP), or (iii) daily isoniazid for 6 months (6H). QFT-Plus testing was performed at baseline, month 12, and month 24. Quantitative and qualitative results, and serial changes (conversions and reversions) were assessed. Results: From baseline to month 12, 13.0% (196/1502) of participants converted, with no differences between 3HP (13.1%) and 6H (12.9%). From month 12 to month 24, conversion occurred in 4.0% of participants receiving a second course of 3HP (p3HP arm), and in 9.4% of those not receiving a second course (3HP) (P = .110). Overall reversions occurred in 19.0% (178/935) of participants. Reversion rates were similar across study arms and timepoints. The risk of developing TB during the study period was higher among individuals who were QFT-Plus positive at baseline (adjusted hazard ratio, 1.66 [95% confidence interval, 1.41–1.89], P = .034). Conclusions: QFT-Plus conversions and reversions occurred frequently in PWH and did not differ by TPT regimen or between those who did and did not receive a second course of TPT. The high rate of QFT positivity, along with the large proportion of conversions despite TPT, highlights the complexity of interpreting serial interferon-gamma release assay results in PWH in high-transmission scenarios.

Culturing Mycobacterium tuberculosis (MTB) from tuberculosis cases is the basis for many research and clinical applications. Paradoxically, it is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. The alternative, culture-free sequencing from diagnostic samples, is a promising but challenging approach both to obtain and analyse the MTB genome from the complex sample. This study obtains high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics pipeline. Our approach reveals that 88% of variants called in culture-free sequencing analysis are false positives due to supplementary alignments, mostly in enriched-sputa samples. Overall, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants’ frequency (0.98). Our findings extrapolate to all publicly available data, thus demonstrating that in most cases culture accurately mirrors clinical samples.

Multidrug-resistant(MDR) tuberculosis in Southern Africa is of great concern, exacerbated by the spread of a clone harboring a mutation missed by Xpert Ultra. In Southern Mozambique, the presence of such mutation and rising cases of non-MDR isoniazid resistance highlights the need to ensure accurate detection of antimicrobial-resistance in the country.

In several low-income countries, the transport of sputa could take up to one week to reach the laboratories, resulting in increased contamination rates and a loss of growth. The aim of this study was to evaluate the effect of the OMNIgene-SPUTUM in preserving Mycobacterium tuberculosis on sputum samples simulating three hypothetical scenarios for conservation and/or decontamination: (1) sputum was mixed with OMN and conserved at room temperature for five days and then processed for culture (OMN); (2) sputum cultures followed the routine standing operating procedure at day 0 (STD); and (3) sputum samples were kept at room temperature for five days and mixed with the standard decontamination reagent (SDT5) and then processed for culture. The positivity rate based on smear microscopy was 36.4%, 29.1%, and 27.3% for STD, STD5, and OMN, respectively. The proportion of positive results by liquid culture (MGIT) was 39.1% (43/110) for STD, 26.4% (29/110) for STD5, and 20.0% for OMN (22/110). The overall concordance of liquid culture results was 51.8% (57/110): 37.3% (41/110) for negative results, 11.8% (13/110) for MTBC growth, and 2.7% (3/110) for contaminated results. The OMN arm showed better performance in solid culture than in liquid culture, with a notable reduction in contaminated results.

Mozambique is considered a high multidrug-resistant (MDR-TB) country by the World Health Organization (WHO) (3.7% new MDR-TB cases). The last national WHO survey was performed in 2008. Nowadays, South Africa and Eswatini are reporting an increase in MDR-TB cases heightened, in Eswatini, by the spread of a MDR-TB strain harbouring the rpoB _I491F mutation not detected by XpertUltra/RIF. Due to the concerning MDR-TB situation in its neighbouring countries, it is crucial to review the current resistance status in Mozambique. Our aim is to depict the current prevalence of drug-resistance to first and second-line drugs and track its trends since 2008. We analysed 312 Mycobacterium tuberculosis genomes from a population-based survey conducted in Manhiça in 2018 and looked for drug-resistance mutations included in the recently published WHO catalogue and compared with the prevalences reported in 2014 and 2008. We found that the overall resistance increased in 2014 (15.9%) and 2018 (14.4%) was slightly higher than the one reported in 2008. Although, new multidrug/rifampicin resistance cases remained consistent with the 2008 prevalence study (3.5%) indicating that MDR-TB is not spreading as rapidly as in neighbouring countries. Importantly, we detected a high isoniazid (INH) prevalence not associated with MDR-TB (4.2%, 5.5% and 7.6% in 2008, 2014 and 2018, p-value=0.03) suggesting that a sizeable number of cases are INH-resistant before starting treatment and not detected by XpertUltra/RIF. Fortunately, no mutations associated with second-line drug-resistance were found in the dataset. Our results show a stable drug-resistance situation in Manhiça with the need to monitor isoniazid resistance, and highlight the potential of WGS to be used in national surveys to expand our knowledge of drug-resistance prevalence throughout all Mozambican provinces.

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.

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. Funding: Ministry of Enterprise and Knowledge (Government of Catalonia & European Social Fund, AGAUR fellowship); European Research Council (ERC) European Union’s Horizon 2020.

Strengthening tuberculosis diagnosis is an international priority and the advocacy for multi-disease testing devices raises the possibility of improving laboratory efficiency. However, the advantages of centralized platforms might not translate into real improvements under operational conditions. This study aimed to evaluate the field use of the Abbott RealTime MTB (RT-MTB) and Xpert MTB/RIF assays, in a large cohort of HIV-positive and TB presumptive cases in Southern Mozambique. Over a 6-month period, 255 HIV-positive TB presumptive cases were consecutively recruited in the high TB/HIV burden district of Manhiça. The diagnostic performance of both assays was evaluated against two different reference standards: a microbiological gold standard (MGS) and a composite reference standard (CRS). Results from the primary analysis (MGS) showed improved sensitivity (Se) and reduced specificity (Sp) for the Abbott RT-MTB assay compared to the Xpert MTB/RIF (RT-MTB Se: 0.92 (95% CI: 0.75;0.99) vs Xpert Se: 0.73 (95% CI: 0.52;0.88) p value = 0.06; RT-MTB Sp: 0.80 (0.72;0.86) vs Xpert Sp: 0.96 (0.92;0.99) p value < 0.001). The lower specificity may be due to cross-reactivity with non-tuberculous mycobacteria (NTMs), the detection of non-viable MTBC, or the identification of true TB cases missed by the gold standard.

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 all-cause mortality, TB incidence rates and their associated 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 year-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 months of the follow up. Mortality and TB incidence rates in the 2-year period after TB was initially ruled out was very high. The TB diagnostic work-up and linkage to HIV care should be strengthened to decrease TB burden and all-cause mortality among PLHIV with presumptive TB.