Moumine Sanogo

Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

Background Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system. Methodology/Principal Findings We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes. Conclusions This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity.

Tuberculosis drug resistance in Bamako, Mali, from 2006 to 2014

BackgroundAlthough Drug resistance tuberculosis is not a new phenomenon, Mali remains one of the “blank” countries without systematic data.MethodsBetween 2006 and 2014, we enrolled pulmonary TB patients from local TB diagnostics centers and a university referral hospital in several observational cohort studies. These consecutive patients had first line drug susceptibility testing (DST) performed on their isolates. A subset of MDR was subsequently tested for second line drug resistance.ResultsA total of 1186 mycobacterial cultures were performed on samples from 522 patients, including 1105 sputa and 81 blood samples, yielding one or more Mycobacterium tuberculosis complex (Mtbc) positive cultures for 343 patients. Phenotypic DST was performed on 337 (98.3%) unique Mtbc isolates, of which 127 (37.7%) were resistant to at least one drug, including 75 (22.3%) with multidrug resistance (MDR). The overall prevalence of MDR-TB was 3.4% among new patients and 66.3% among retreatment patients. Second line DST was available for 38 (50.7%) of MDR patients and seven (18.4%) had resistance to either fluoroquinolones or second-line injectable drugs.ConclusionThe drug resistance levels, including MDR, found in this study are relatively high, likely related to the selected referral population. While worrisome, the numbers remained stable over the study period. These findings prompt a nationwide drug resistance survey, as well as continuous surveillance of all retreatment patients, which will provide more accurate results on countrywide drug resistance rates and ensure that MDR patients access appropriate second line treatment.

Stool microbiome reveals diverse bacterial ureases as confounders of oral urea breath testing for Helicobacter pylori and Mycobacterium tuberculosis in Bamako, Mali.

Detection of bacterial urease activity has been utilized successfully to diagnose Helicobacter pylori (H. pylori). While Mycobacterium tuberculosis (M. tuberculosis) also possesses an active urease, it is unknown whether detection of mycobacterial urease activity by oral urease breath test (UBT) can be exploited as a rapid point of care biomarker for tuberculosis (TB) in humans. We enrolled 34 individuals newly diagnosed with pulmonary TB and 46 healthy subjects in Bamako, Mali and performed oral UBT, mycobacterial sputum culture and H. pylori testing. Oral UBT had a sensitivity and specificity (95% CI) of 70% (46-88%) and 11% (3-26%), respectively, to diagnose culture-confirmed M. tuberculosis disease among patients without H. pylori, and 100% sensitivity (69-100%) and 11% specificity (3-26%) to diagnose H. pylori among patients without pulmonary TB. Stool microbiome analysis of controls without TB or H. pylori but with positive oral UBT detected high levels of non-H. pylori urease producing organisms, which likely explains the low specificity of oral UBT in this setting and in other reports of oral UBT studies in Africa.

Clinical characteristics of non-tuberculous mycobacterial pulmonary infections in Bamako, Mali

The global spread of non-tuberculous mycobacteria (NTM) may be due to HIV/AIDS and other environmental factors. The symptoms of NTM and tuberculosis (TB) disease are indistinguishable, but their treatments are different. Lack of research on the epidemiology of NTM infections has led to underestimation of its prevalence within TB endemic countries. This study was designed to determine the prevalence and clinical characteristics of pulmonary NTM in Bamako. A cross-sectional study which include 439 suspected cases of pulmonary TB. From 2006 to 2013 a total of 332 (76%) were confirmed to have sputum culture positive for mycobacteria. The prevalence of NTM infection was 9.3% of our study population and 12.3% of culture positive patients. The seroprevalence of HIV in NTM group was 17.1%. Patients who weighed <55 kg and had TB symptoms other than cough were also significantly more likely to have disease due to NTM as compared to those with TB disease who were significantly more likely to have cough and weigh more than 55 kg (OR 0.05 (CI 0.02-0.13) and OR 0.32 (CI 0.11-0.93) respectively). NTM disease burden in Bamako was substantial and diagnostic algorithms for pulmonary disease in TB endemic countries should consider the impact of NTM.

The most frequent Mycobacterium tuberculosis complex families in Mali (2006-2016) based on spoligotyping

Background: To identify strains of Mycobacterium tuberculosis complex (MTBc) circulating in Bamako region during the past 10 years. Methods: From 2006 to 2016, we conducted a cross-sectional study to identify with spoligotyping, clinical isolates from tuberculosis (TB)-infected patients at different stages of their treatments in Bamako, Mali. Results: Among the 904 suspected TB patients included in the study and thereafter tested in our BSL-3 laboratory, 492 (54.4%) had MTBc and therefore underwent spoligotyping. Overall, three subspecies, i.e., MTB T1 (31.9%) and MTB LAM10 (15.3%) from lineage 4 and M. africanum 2 (16.8%) from lineage 6 were the leading causes of TB in Bamako region during the past 10 years. Other spoligotypes such as MTB T3, MTB Haarlem 2, MTB EAI3, and MTB family 33 were also commonly seen from 2010 to 2016. Conclusion: This study showed a high genetic diversity of strains isolated in Bamako region and highlights that M. tuberculosis T1 strain was the most prevalent. Furthermore, the data indicate an increasing proportion of primary drug resistance overtime in Bamako.

Screening new tuberculosis patients in Mali for rifampicin resistance at 2 months

Objective/background: The recent call for universal drug susceptibility testing (DST) for all tuberculosis (TB) patients will be difficult to meet in settings where Xpert rollout is limited, such as low prevalence of HIV and Multi-drug Resistant Tuberculosis (MDR) settings. As recommended by World Health Organization (WHO) guidelines, the success of TB treatment is measured by Ziehl–Neelsen (ZN) microscopy or auramine–rhodamine fluorescent microscopy (FM) on sputum, in which conversion to negative smear at 2 months (M) is an important predictor of treatment success, defined as a negative smear at 5M. The sputum smear that fails to convert to negative at 5M are screened for rifampicin resistance. We tested in a prospective study whether an early screen for rifampicin resistance, based on FM results at 2M, could detect MDR patients early, rather than screening all patients with GeneXpert MTB/Rif at baseline. Methods: Between February 2015 and August 2016, we enrolled new TB patients in an IRB-approved prospective cohort study at four health centers in Bamako district. Fresh sputum samples were collected at 2M and 5M to measure FM smear conversion. Patients who failed to show a decline in FM positivity at 2M (moderate or many Acid Fast Bacilli (AFB)) had their sputum tested in GeneXpert to detect rifampicin resistance. Patients who had any AFB seen at 5M were also tested using GeneXpert. Results: Of the 570 patients who were enrolled in the study, 22 (3.8%) died and 27 (4.7%) were lost to follow-up. The prevalence of HIV and TB coinfection was 12.4%, and 65.6% of the patients were male. At 2M, 32 out of 429 patients still had moderate or many AFBs in FM, and were screened by Xpert, of whom 5 (15.6%) tested rifampicin-resistant and were referred for MDR treatment. Of the 310 patients who completed 5M of treatment, 35 (11.3%) met the definition of failure (few or moderate AFB in FM) and had their sputum tested in Xpert; moreover, four (11.4%) demonstrated rifampicin resistance. In total, 67 (21.6% of 310) patients were screened by Xpert, of whom nine were detected to have MDR (or 13.4% of those screened). Conclusion: Although we cannot exclude additional MDR patients having been missed by our screening strategy, our screening algorithm at 2M detected five out of nine MDR patients. Detecting patients at 2M allowed for earlier referral, and potentially less acquired drug resistance and lower mortality. This strategy may be advantageous while awaiting further rollout of Xpert machines that will permit universal DST.