Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda.

BACKGROUND: Bovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health. MATERIALS AND METHODS: A cross-sectional study employing a systematic random sampling of cattle (n = 300) with the collection of retropharyngeal lymph nodes and tonsils (n = 300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence. FINDINGS: Of the 300 sample pools, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. Mycobacterium bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistant (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle. CONCLUSIONS: This study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is present in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.

Predictors of Rifampicin-Resistant Tuberculosis Mortality among HIV-Coinfected Patients in Rwanda.

Tuberculosis (TB), including multidrug-resistant (MDR; i.e., resistant to at least rifampicin and isoniazid)/rifampicin-resistant (MDR/RR) TB, is the most important opportunistic infection among people living with HIV (PLHIV). In 2005, Rwanda launched the programmatic management of MDR/RR-TB. The shorter MDR/RR-TB treatment regimen (STR) has been implemented since 2014. We analyzed predictors of MDR/RR-TB mortality, including the effect of using the STR overall and among PLHIV. This retrospective study included data from patients diagnosed with RR-TB in Rwanda between July 2005 and December 2018. Multivariable logistic regression was used to assess predictors of mortality. Of 898 registered MDR/RR-TB patients, 861 (95.9%) were included in this analysis, of whom 360 (41.8%) were HIV coinfected. Overall, 86 (10%) patients died during MDR/RR-TB treatment. Mortality was higher among HIV-coinfected compared with HIV-negative TB patients (13.3% versus 7.6%). Among HIV-coinfected patients, patients aged ≥ 55 years (adjusted odds ratio = 5.89) and those with CD4 count ≤ 100 cells/mm3 (adjusted odds ratio = 3.77) had a higher likelihood of dying. Using either the standardized longer MDR/RR-TB treatment regimen or the STR was not correlated with mortality overall or among PLHIV. The STR was as effective as the long MDR/RR-TB regimen. In conclusion, older age and advanced HIV disease were strong predictors of MDR/RR-TB mortality. Therefore, special care for elderly and HIV-coinfected patients with ≤ 100 CD4 cells/mL might further reduce MDR/RR-TB mortality.

Case Report: Dynamics of Acquired Fluoroquinolone Resistance under Standardized Short-Course Treatment of Multidrug-Resistant Tuberculosis.

We report a case of acquired fluoroquinolone (FQ) resistance under short-course multidrug-resistant tuberculosis (MDR-TB) treatment. The patient was managed at Kabutare hospital, one of the two specialized MDR-TB clinics in Rwanda. A low dose of moxifloxacin was used in the first three critical months. Acquired resistance was identified at the ninth month of treatment, 3 months after stopping kanamycin in a strain initially susceptible only to FQs, kanamycin, and clofazimine. Fluoroquinolone resistance was detected in the same month by deep sequencing as routinely used second-line line probe assay and phenotypic drug susceptibility testing. High-dose FQ, preferably gatifloxacin, should be used to maximize effectiveness.

A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region.

The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle.

Prevalence and drivers of false-positive rifampicin-resistant Xpert MTB/RIF results: a prospective observational study in Rwanda.

BACKGROUND: The Xpert MTB/RIF (Xpert) assay is used globally to rapidly diagnose tuberculosis and resistance to rifampicin. We investigated the frequency and predictors of false-positive findings of rifampicin resistance with Xpert. METHODS: We did a prospective, observational study of individuals who were enrolled in a Rwandan nationwide diagnostic cohort study (DIAMA trial; NCT03303963). We included patients identified to have rifampicin resistance on initial Xpert testing. We did a repeat Xpert assay and used rpoB Sanger and deep sequencing alongside phenotypic drug susceptibility testing (pDST) to ascertain final rifampicin susceptibility status, with any (hetero)resistant result overriding. We used multivariable logistic regression to assess predictors of false rifampicin resistance on initial Xpert testing, adjusted for HIV status, tuberculosis treatment history, initial Xpert semi-quantitative bacillary load, and initial Xpert probe. FINDINGS: Between May 4, 2017, and April 30, 2019, 175 people were identified with rifampicin resistance at initial Xpert testing, of whom 154 (88%) underwent repeat Xpert assay. 54 (35%) patients were confirmed as rifampicin resistant on repeat testing and 100 (65%) were not confirmed with resistance. After further testing and sequencing, 121 (79%) of 154 patients had a final confirmed status for rifampicin susceptibility. 57 (47%) of 121 patients were confirmed to have a false rifampicin resistance result and 64 (53%) had true rifampicin resistance. A high pretest probability of rifampicin resistance did not decrease the odds of false rifampicin resistance (adjusted odds ratio [aOR] 6·0, 95% CI 1·0-35·0, for new tuberculosis patients vs patients who needed retreatment). Ten (16%) of the 64 patients with true rifampicin resistance did not have confirmed rifampicin resistance on repeat Xpert testing, of whom four had heteroresistance. Of 63 patients with a very low bacillary load on Xpert testing, 54 (86%) were falsely diagnosed with rifampicin-resistant tuberculosis. Having a very low bacillary load on Xpert testing was strongly associated with false rifampicin resistance at the initial Xpert assay (aOR 63·6, 95% CI 9·9-410·4). INTERPRETATION: The Xpert testing algorithm should include an assessment of bacillary load and retesting in case rifampicin resistance is detected on a paucibacillary sputum sample. Only when rifampicin resistance has been confirmed on repeat testing should multidrug-resistant tuberculosis treatment be started. When rifampicin resistance has not been confirmed on repeat testing, we propose that patients should be given first-line anti-tuberculosis drugs and monitored closely during treatment, including by baseline culture, pDST, and further Xpert testing. FUNDING: The European & Developing Countries Clinical Trials Partnership 2 programme, and Belgian Directorate General for Development Cooperation.

Highly successful treatment outcome of multidrug-resistant and genetic diversity of multidrug-resistant Mycobacterium tuberculosis strains in Rwanda.

OBJECTIVE: To determine prevalent MDR-TB genotypes and describe treatment outcome and bacteriology conversion in MDR-TB patients. METHODS: Review of laboratory records of 173 MDR-TB patients from all over Rwanda who initiated treatment under programmatic management of MDR-TB (PMDT) between 2014 and 2015. Fifty available archived isolates were genotyped by mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) genotyping. RESULT: Of the 170 patients whose outcome was known, 114 (66.3%) were cured and 36 (21%) completed the treatment, giving a successful outcome (cured and completed) of 150 (87.3%) patients. Of 20 MDR-TB patients with unfavourable treatment outcome, 18 died, one failed and one defaulted/stopped treatment. Of the 18 patients who died, 11 (61%) were HIV-coinfected. The treatment outcome was successful for 93.9% among HIV negative and 81.8% among HIV-coinfected patients (P = 0.02). Sputum smear conversion occurred in 3, 46, 57 and 78 patients before 2, 3, 4 and 6 months, respectively, with median time of sputum smear and culture conversion at 3 months. The 44 MDR-TB isolates with MIRU-VNTR result, showed high genetic diversity with low clustering rate (9.09%) and Uganda II being the most prevalent sub-family lineage detected in 68.2% of isolates. Beijing family was the least common genotype detected (2.3%, 1 isolate). CONCLUSION: The high success rates for MDR-TB treatment achieved in Rwanda were comparable to outcomes observed in resource-rich settings with HIV being an independent risk factor for poor treatment outcome. High genetic diversity and low clustering rate reported here suggest that reactivation of previous disease plays an important role in the transmission of MDR-TB in Rwanda.

OBJECTIF: Déterminer les génotypes prévalents de la TB-MDR et décrire les résultats du traitement et la conversion bactériologique chez les patients atteints de TB-MDR. MÉTHODES: Analyse des dossiers de laboratoire de 173 patients atteints de TB-MDR de l'ensemble du Rwanda qui ont débuté un traitement sous prise en charge programmatique de la TB-MDR (PMDT) entre 2014 et 2015. Cinquante isolats archivés disponibles ont été génotypés pour les unités répétitives intercalées de mycobactéries - nombre variable de tandems répétés (MIRU-VNTR). RÉSULTAT: Sur les 170 patients dont l'issue était connue, 114 (66,3%) étaient guéris et 36 (21%) avaient terminé le traitement, ce qui donne un résultat positif (guéri et complété) de 150 patients (87,3%). Sur 20 patients atteints de TB-MDR dont l'issue du traitement était défavorable, 18 sont décédés, un a eu un échec et le dernier a abandonné/arrêté le traitement. Sur les 18 patients décédés, 11 (61%) étaient coinfectés par le VIH. Le résultat du traitement a été positif pour 93,9% des personnes VIH négatives et 81,8% pour ceux coinfectées par le VIH (p = 0,02). La conversion des frottis d'expectoration est survenue chez 3, 46, 57 et 78 patients respectivement à 2, 3, 4 et 6 mois, avec une durée médiane entre le frottis d'expectoration et la conversion de culture de 3 mois. Les 44 isolats de TB-MDR avec un résultat MIRU-VNTR ont montré une diversité génétique élevée avec un faible taux de regroupement (9,09%) et la sous-famille de la lignée Ouganda II étant la plus prévalente détectée dans 68,2% des isolats. La famille Beijing était le génotype le moins fréquemment détecté (2,3%, 1 isolat). CONCLUSION: Les taux de succès élevés du traitement de la TB-MDR obtenus au Rwanda étaient comparables aux résultats observés dans les régions riches en ressources, le VIH étant un facteur de risque indépendant d'un mauvais résultat du traitement. La diversité génétique élevée et le faible taux de regroupement rapportés ici suggèrent que la réactivation d'une maladie antérieure joue un rôle important dans la transmission de la TB-MDR au Rwanda.

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.

Diagnostic performance of smear microscopy and incremental yield of Xpert in detection of pulmonary tuberculosis in Rwanda.

BACKGROUND: Tuberculosis control program of Rwanda is currently phasing in light emitting diode-fluorescent microscopy (LED-FM) as an alternative to Ziehl-Neelsen (ZN) smear microscopy. This, alongside the newly introduced Xpert (Cepheid, Sunnyvale, CA, USA) is expected to improve diagnosis of tuberculosis and detection of rifampicin resistance in patients at health facilities. We assessed the accuracy of smear microscopy and the incremental sensitivity of Xpert at tuberculosis laboratories in Rwanda. METHODS: This was a cross-sectional study involving four laboratories performing ZN and four laboratories performing LED-FM microscopy. The laboratories include four intermediate (ILs) and four peripheral (PLs) laboratories. After smear microscopy, the left-over of samples, of a single early-morning sputum from 648 participants, were tested using Xpert and mycobacterial culture as a reference standard. Sensitivity of each test was compared and the incremental sensitivity of Xpert after a negative smear was assessed. RESULTS: A total of 96 presumptive pulmonary tuberculosis participants were culture positive for M. tuberculosis. The overall sensitivity in PL of ZN was 55.1 % (40.2-69.3 %), LED-FM was 37 % (19.4-57.6 %) and Xpert was 77.6 % (66.6-86.4 %) whereas in ILs the same value for ZN was 58.3 % (27.7-84.8 %), LED-FM was 62.5 % (24.5-91.5 %) and Xpert was 90 (68.3-98.8 %). The sensitivity for all tests was significantly higher among HIV-negative individuals (all test p <0.05). The overall incremental sensitivity of Xpert over smear microscopy was 32.3 %; p < 0.0001. The incremental sensitivity of Xpert was statistically significant for both smear methods at PL (32.9 %; p = 0.001) but not at the ILs (30 %; p = 0.125) for both smear methods. CONCLUSIONS: Our study findings of the early implementation of the LED-FM did not reveal significant increment in sensitivity compared to the method being phased out (ZN). This study showed a significant incremental sensitivity for Xpert from both smear methods at peripheral centers where majority of TB patients are diagnosed. Overall our findings support the recommendation for Xpert as an initial diagnostic test in adults and children presumed to have TB.