Bassirou Diarra

Efflux Inhibition with Verapamil Potentiates Bedaquiline in Mycobacterium tuberculosis

ABSTRACT Drug efflux is an important resistance mechanism in Mycobacterium tuberculosis. We found that verapamil, an efflux inhibitor, profoundly decreases the MIC of bedaquiline and clofazimine to M. tuberculosis by 8- to 16-fold. This exquisite susceptibility was noted among drug-susceptible and drug-resistant clinical isolates. Thus, efflux inhibition is an important sensitizer of bedaquiline and clofazimine, and efflux may emerge as a resistance mechanism to these drugs.

Failure to Recognize Nontuberculous Mycobacteria Leads to Misdiagnosis of Chronic Pulmonary Tuberculosis

Background Nontuberculous mycobacterial (NTM) infections cause morbidity worldwide. They are difficult to diagnose in resource-limited regions, and most patients receive empiric treatment for tuberculosis (TB). Our objective here is to evaluate the potential impact of NTM diseases among patients treated presumptively for tuberculosis in Mali. Methods We re-evaluated sputum specimens among patients newly diagnosed with TB (naïve) and those previously treated for TB disease (chronic cases). Sputum microscopy, culture and Mycobacterium tuberculosis drug susceptibility testing were performed. Identification of strains was performed using molecular probes or sequencing of secA1 and/or 16S rRNA genes. Results Of 142 patients enrolled, 61 (43%) were clinically classified as chronic cases and 17 (12%) were infected with NTM. Eleven of the 142 (8%) patients had NTM disease alone (8 M. avium, 2 M. simiae and 1 M. palustre). All these 11 were from the chronic TB group, comprising 11/61 (18%) of that group and all were identified as candidates for second line treatment. The remaining 6/17 (35.30%) NTM infected patients had coinfection with M. tuberculosis and all 6 were from the TB treatment naïve group. These 6 were candidates for the standard first line treatment regimen of TB. M. avium was identified in 11 of the 142 (8%) patients, only 3/11 (27.27%) of whom were HIV positive. Conclusions NTM infections should be considered a cause of morbidity in TB endemic environments especially when managing chronic TB cases to limit morbidity and provide appropriate treatment.

Molecular strain typing of Mycobacterium tuberculosis complex in Bamako, Mali

OBJECTIVE To identify strains of Mycobacterium tuberculosis complex (MTC) circulating in Bamako and to examine the relationship between the strains and their drug susceptibility profiles. METHODS Between 2006 and 2010, we conducted a cross-sectional study using spoligotyping to identify strains of MTC recovered from 126 tuberculosis (TB) patients under treatment in Bamako, Mali. RESULT Three members of the MTC were isolated: M. tuberculosis (71.4%), M. africanum (27.8%) and M. bovis (0.8%). Of these, three strains were found to be the most prevalent: M. tuberculosis T1 (MTB T1; 38.9%), M. africanum F2 (MAF2; 26.2%) and M. tuberculosis Latin American and Mediterranean 10 (MTB LAM 10; 10.3%). MAF2 and MTB LAM 10 strains have a lower risk of multidrug resistance (MDR) than MTB T1 (respectively OR 0.1, 95%CI 0.03-0.4 and OR 0.1, 95%CI 0.01-0.8). Age ≥ 32 years (OR 1.4, 95%CI 0.4-3.9), negative human immunodeficiency virus status (OR 0.4, 95%CI 0.1-2.5) and male sex (OR 4, 95%CI 0.9-16.5) were not associated with MDR. The prevalence of MDR among treatment and retreatment failure patients was respectively 25% and 81.8% compared to new patients (2.9%). CONCLUSION This study indicates a low level of primary drug resistance in Bamako, affirms the importance of using correct drug regimens, and suggests that the MTB T1 strain may be associated with the development of resistance.

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.

Nontuberculous Mycobacteria Isolated from Tuberculosis Suspects in Ibadan, Nigeria.

In Nigeria, one of the highest tuberculosis (TB) burdened nations, sputum smear microscopy is routinely employed for TB diagnosis at Directly Observed Treatment Short-Course (DOTS) Centers. This diagnostic algorithm does not differentiate Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM). Between December 2008 and January 2009, consecutive patients diagnosed with TB were screened for inclusion at 10 DOTS centers in Ibadan, Nigeria. To verify Mycobacterium species in patients diagnosed, we cultured and identified mycobacterial isolates using PCR, line probe assay, and spoligotyping techniques. From 48 patients screened, 23 met the inclusion criteria for the study. All the 23 study patients had a positive culture. Overall, we identified 11/23 patients (48%) with MTC only, 9/23 (39%) with NTM only, and 3/23 (13%) with evidence of both MTC and NTM. Strains of MTC identified were Latin American Mediterranean (LAM) genotype (n = 12), M. africanum (n = 1), and the genotype family T (n = 1). Four M. avium-intracellulare-M. scrofulaceum complexes, one M. chelonae complex, one M. abscessus, and one M. intracellulare were identified. Our findings underscore the need to incorporate molecular techniques for more precise diagnosis of TB at DOTS centers to improve clinical outcomes and safe guard public health, particularly in TB endemic countries.

Half of rifampicin-resistant Mycobacterium tuberculosis complex isolated from tuberculosis patients in Sub-Saharan Africa have concomitant resistance to pyrazinamide

Background Besides inclusion in 1st line regimens against tuberculosis (TB), pyrazinamide (PZA) is used in 2nd line anti-TB regimens, including in the short regimen for multidrug-resistant TB (MDR-TB) patients. Guidelines and expert opinions are contradictory about inclusion of PZA in case of resistance. Moreover, drug susceptibility testing (DST) for PZA is not often applied in routine testing, and the prevalence of resistance is unknown in several regions, including in most African countries. Methods Six hundred and twenty-three culture isolates from rifampicin-resistant (RR) patients were collected in twelve Sub-Saharan African countries. Among those isolates, 71% were from patients included in the study on the Union short-course regimen for MDR-TB in Benin, Burkina Faso, Burundi, Cameroon, Central Africa Republic, the Democratic Republic of the Congo, Ivory Coast, Niger, and Rwanda PZA resistance, and the rest (29%) were consecutive isolates systematically stored from 2014–2015 in Mali, Rwanda, Senegal, and Togo. Besides national guidelines, the isolates were tested for PZA resistance through pncA gene sequencing. Results Over half of these RR-TB isolates (54%) showed a mutation in the pncA gene, with a significant heterogeneity between countries. Isolates with fluoroquinolone resistance (but not with injectable resistance or XDR) were more likely to have concurrent PZA resistance. The pattern of mutations in the pncA gene was quite diverse, although some isolates with an identical pattern of mutations in pncA and other drug-related genes were isolated from the same reference center, suggesting possible transmission of these strains. Conclusion Similar to findings in other regions, more than half of the patients having RR-TB in West and Central Africa present concomitant resistance to PZA. Further investigations are needed to understand the relation between resistance to PZA and resistance to fluoroquinolones, and whether continued use of PZA in the face of PZA resistance provides clinical benefit to the patients.

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.

Mycobacterium tuberculosis Beijing Strain, Bamako, Mali

To the Editor: Mycobacterium tuberculosis has >36 identified genotype families (1). Four genotypes cause 35% of documented cases of active tuberculosis (TB): Beijing (10%–11%), Latin American–Mediterranean (9.3%), Haarlem (7.5%), and the X clade (7%) (1,2) The Beijing clade strains, reported in 1995 from the People’s Republic of China, are widely recognized as highly pathogenic with a possible predilection for multidrug resistance (3). Predominant in Asia, these strains have been documented in other parts of the world (1,4,5). The virulence, propensity to become resistant, and distinct geographic distribution of the Beijing clade suggest it may have some adaptive advantage in producing disease in humans. Limited data suggest that its presence in Africa is low (2,4,5). In Bamako, Mali, 2 patients with active pulmonary TB came to the research clinic at Point G Hospital, affiliated with the University of Bamako Medical School, for recruitment under a US National Institute of Allergy and Infectious Diseases’ institutional review board–approved protocol. The first patient, a previously healthy 34-year-old man, sought treatment in March 2008. He had a 3-month history of fever, cough, shortness of breath, and left-sided chest pain; respiratory rate of 24/min; temperature of 36.8oC; and pulse rate of 68/min. He weighed 60 kg. His leukocyte count was 8,700 cells/μL, and he was positive for HIV-1 with a CD4+ T-cell count of 468 cells/μL. He reported contact with persons from other countries in Africa, China, and other parts of Asia. Chest radiograph showed a cavitary lesion on the left upper lobe and opacities throughout the left lung. Three sputum samples collected 3 days apart were digested and decontaminated with N-acetyl-L-cysteine, 4% NaOH; concentrated by high-speed centrifugation; stained with auramine-rhodamine; and evaluated by using fluorescent microscopy. The many acid-fast bacilli (AFB) seen were identified by using nucleic acid probes (AccuProbe, Gen-Probe, San Diego CA, USA). Antimycobacterial drug susceptibility was determined by using a manual indirect susceptibility test (mycobacterial growth indicator tube, AST SIRE System; BBL, Becton Dickinson, Franklin Lakes, NJ, USA) showed the isolate sensitive to isoniazid (0.1 μg/mL), rifampin (1.0 μg/mL), and ethambutol (3.5 μg/mL) but resistant to streptomycin (0.8 μg/mL). Spoligotyping using a commercially available kit (Spoligotyping Isogen Life Science, De Meern, the Netherlands) showed characteristics of the Beijing clade (Appendix Figure, panel A) (6). The patient began treatment with the standard first-line regimen of isoniazid, rifampin, pyrazinamide, and ethambutol fixed-dose combination (Svizera Laboratory, Mumbai, India) according to Malian National Guidelines. Follow-up sputum samples at 13 and 18 weeks of treatment were smear- and culture-negative for AFB. The second patient, a 28-year-old woman, sought treatment in July 2008. For 1 year, she had received first-line and retreatment regimens that failed to clear her sputum of AFB. She had begun second-line treatment for multidrug-resistant disease 2 days earlier. She had a history of fever, cough, and weight loss; temperature of 37.1oC; heart rate of 104 beats/min; respiratory rate of 24/min; and blood pressure of 90/60 Hg mm. She weighed 49 kg. Leukocyte count was 9,400 cells/μL. Serologic results for HIV-1 and -2 were negative. Chest radiograph showed a right apical cavitary lesion and a fibrotic lesion in the right middle lung field. She did not recall any exposure to TB. She worked as an assistant at a local telephone center. Two sputum samples, processed as described above, were positive for, and Gen-Probe testing confirmed, M. tuberculosis. According to antimycobacterial susceptibility testing, the strain was resistant to isoniazid (0.1 μg/mL), rifampin (1.0 μg/mL), ethambutol (3.5 μg/mL), and streptomycin (0.8 μg/mL). Spoligotyping confirmed the strain as Beijing clade, and restriction fragment length polymorphism (7) confirmed that it differed from that of patient 1 (Appendix Figure, panel B). The relevance of different genotypes, such as the Beijing clade, to disease progression is being studied. Evidence indicates the genotype may factor in transmission or pathogenesis. In a study in Cape Town, South Africa, disease produced by the Beijing clade increased exponentially over time, suggesting a possible pathogenic advantage; although most cases were drug susceptible, the likelihood of unsuccessful treatment was greater than for non-Beijing variants (8). Although the Beijing clade does not appear to have greater propensity than non-Beijing genotypes for acquiring resistance, certain variants within the group that become multidrug resistant may be more likely to acquire such resistance. Beijing strains particularly may tend to acquire resistance more easily than others under conditions of suboptimal treatment (9). In Cape Town during 2000–2003, the Beijing clade as a cause of disease in children increased from 13% to 33%, suggesting a selective advantage in transmissibility and disease production (10). These cases highlight the need to diagnose disease and resistance early and to begin appropriate treatment in TB-endemic countries. Knowledge of circulating strains and their resistance patterns is essential to developing effective programs to curtail the spread of TB within the country and the region; in this era of globalization, it is required for the successful control of TB worldwide.

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.