Development and clinical evaluation of a new multiplex PCR assay for a simultaneous diagnosis of tuberculous and nontuberculous mycobacteria.

BACKGROUND: The prevalence of non-tuberculous mycobacteria (NTM) has been increasing worldwide in both developed and developing countries. NTM infection is clinically indistinguishable from tuberculosis and therefore poses significant challenges in patient management, especially in patients chronically treated for pulmonary TB. In this study, we evaluated a new highly sensitive Multiplex MTB/NTM assay that can differentiate M. tuberculosis complex (MTBC) from all NTM, including the treatable and most common NTM, M. avium complex (MAC). METHODS: We developed and optimized a new open- Multiplex MTB/NTM assay with two gene-targets for MTBC (IS6110/senX3-regX3) and two targets for MAC (IS1311/DT1) with samples spiked with stored strains and testing 20 replicates. Patients with presumptive TB and NTM were enrolled at the Respiratory Disease Department of The University Teaching Hospital of Point G, in Mali. FINDINGS: In the development stage, the new assay showed a high analytic performance with 100% detections of MTBC and MAC at only 5 colony forming units (CFUs). Overall, without the treatment failure cases, the Multiplex assay and the Xpert showed a sensitivity, specificity, PPV and NPV of 83·3% [66·4-92·6], 96·6% [88·6-99·0], 92·5% [82·3-96·5] and 92·2% [82·7-96·5] and the Xpert had values of 96·7% [83·3-99·4], 80·0% [68·2-88·1], 70·7 [55·5-82·3] and 97·9% [89·3-99·6], respectively. The Multiplex assay successfully detected all (5/5) the MAC cases. INTERPRETATION: Our new Multiplex assay demonstrates better specificity than Xpert for all group studied, in addition to detecting potential NTM cases. The assay could therefore complement the widely used Xpert assay and enhance discrimination of TB and NTM infections. FUNDING: This work was supported by the National Institutes of Health (R03AI137674, U54EB027049, D43TW010350 and UM1AI069471) and Northwestern University's Institute for Global Health Catalyzer Fund.

Clinical risk factors associated with multidrug-resistant tuberculosis (MDR-TB) in Mali.

BACKGROUND: MDR-TB is a major threat to global TB control. In 2015, 580,000 were treated for MDR-TB worldwide. The worldwide roll-out of GeneXpert MTB/RIF® has improved diagnosis of MDR-TB; however, in many countries laboratories are unable to assess drug resistance and clinical predictors of MDR-TB could help target suspected patients. In this study, we aimed to determine the clinical factors associated with MDR-TB in Bamako, Mali. METHODS: We performed a cross-sectional study of 214 patients with presumed MDR-TB admitted to University of Bamako Teaching Hospital, Point-G between 2007 and 2016. We calculated crude and adjusted odds ratios for MDR-TB disease diagnosis using SPSS. RESULTS: We found that age ≤40years (OR=2.56. 95% CI: 1.44-4.55), two courses of prior TB treatment (OR=3.25, 95% CI: 1.44-7.30), TB treatment failure (OR=3.82, 95% CI 1.82-7.79), sputum microscopy with 3+ bacilli load (OR=1.98, 95% CI: 1.13-3.48) and a history of contact with a TB patient (OR=2.48, 95% CI: 1.11-5.50) were significantly associated with confirmation of MDR-TB disease. HIV was not a risk factor for MDR-TB (aOR=0.88, 95% CI: 0.34-1.94). CONCLUSION: We identified several risk factors that could be used to identify MDR-TB suspects and prioritize them for laboratory confirmation. Prospective studies are needed to understand factors associated with TB incidence and clinical outcomes of TB treatment and disease.

Plasma levels of Galectin-9 reflect disease severity in malaria infection.

BACKGROUND: Galectin-9 (Gal-9) is a ß-galactoside-binding lectin that interacts with sugar moieties on glycoproteins and glycolipids of cells and pathogens. Gal-9 is known as an immune modulator that induces cell death via interaction with T cell immunoglobulin and mucin domain-3 (Tim3), a co-inhibitory receptor, and it inhibits production of several pro-inflammatory cytokines (TNF, IL-6 and IL-1α) and enhances production of IL-10. To understand the immune pathology of malaria, the Gal-9 in plasma was measured. METHODS: Plasma samples and clinical parameters were obtained from 50 acute malaria cases (nine severe and 41 uncomplicated cases) from Thailand at three time points: day 0, day 7 and day 28. Gal-9 levels were determined by ELISA. A total of 38 species of cytokines and chemokines were measured using a BioPlex assay. RESULTS: Gal-9 levels were higher at day 0 compared to day 7 and day 28 (P < 0.0001). Gal-9 levels were also higher in severe malaria (SM) cases compared to uncomplicated (UM) cases at day 0 and day 7 (923 vs 617 pg/mL; P = 0.03, and 659 vs 348 pg/mL; P = 0.02 respectively). Median Gal-9 levels were higher in patients with blood urea nitrogen to creatinine ratio (BUN/creatinine) ≥20 (mg/dL) than in patients with BUN/creatinine <20 (mg/dL) at day 0 (817.3 vs 576.2 pg/mL, P = 0.007). Gal-9 was inversely significantly correlated with chloride levels in both SM and UM cases (r s = -0.73 and r s = -0.46, respectively). In both UM and SM cases, Gal-9 was significantly associated with pro- and anti-inflammatory cytokines and chemokines such as TNF, IL-6, IFN-α2, IFN-γ, IL-1Ra and IL-10. These correlations were observed at day 0 but disappeared at day 28. CONCLUSIONS: Gal-9 is released during acute malaria, and reflects its severity. This elevation of Gal-9 in acute malaria infection raises the possibility of its role in termination of the immune response by binding to Tim-3, a receptor of Gal-9.

Molecular identification of Mycobacterium bovis from cattle and human host in Mali: expanded genetic diversity.

BACKGROUND: Bovine tuberculosis (BTB) is a contagious, debilitating human and animal disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. The study objective were to estimate the frequency of BTB, examine genetic diversity of the M. bovis population in cattle from five regions in Mali and to determine whether M. bovis is involved in active tuberculosis (TB) in humans. Samples from suspected lesions on cattle at the slaughterhouses were collected. Mycobacterial smear, culture confirmation, and spoligotyping were used for diagnosis and species identification. Mycobacterium DNA from TB patients was spoligotyped to identify M. bovis. RESULTS: In total, 675 cattle have been examined for lesions in the five regions of Mali. Out of 675 cattle, 79 specimens presented lesions and then examined for the presence of M. bovis. Thus, 19 (24.1 %) were identified as M. bovis; eight (10.1 %) were non-tuberculous Mycobacterium (NTM). Nineteen spoligotype patterns were identified among 79 samples with five novel patterns. One case of M. bovis (spoligotype pattern SB0300) was identified among 67 TB patients. CONCLUSION: This study estimates a relatively true proportion of BTB in the regions of Mali and reveals new spoligotype patterns.

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.