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.
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.
Background: The World Health Organization-endorsed phenotypic and genotypic drug-susceptibility testing (gDST/pDST) assays for the detection of rifampicin-resistant (RR) tuberculosis (TB), may miss ...some clinically relevant rpoB mutants, including borderline mutations and mutations outside the gDST-targeted hotspot region. Sequencing of the full rpoB gene is considered the reference standard for rifampicin DST but is rarely available in RR-TB endemic settings and when done indirectly on cultured isolates may not represent the full spectrum of mutations. Hence, in most such settings, the diversity and trends of rpoB mutations remain largely unknown. Methods: This retrospective study included rpoB sequence data from a longitudinal collection of RR-TB isolates in Rwanda across 30 years (1991-2021). Results: Of 540 successfully sequenced isolates initially reported as RR-TB, 419 (77.6%) had a confirmed RR conferring mutation. The Ser450 Leu mutation was predominant throughout the study period. The Val170Phe mutation, not covered by rapid gDST assays, was observed in only four patients, three of whom were diagnosed by pDST. Along with the transition from pDST to rapid gDST, borderline RR-associated mutations, particularly Asp435Tyr, were detected more frequently. Borderline mutants were not associated with HIV status but presented lower odds of having rpoA-C compensatory mutations than other resistance-conferring mutations. Conclusion: Our analysis showed changes in the diversity of RR-TB conferring mutations throughout the study period that coincided with the switch of diagnostic tools to rapid gDST. The study highlights the importance of rapid molecular diagnostics reducing phenotypic bias in the detection of borderline rpoB mutations while vigilance for non-rifampicin resistance determinant region mutations is justified in any setting.
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.
IntroductionAn effective rifampicin-resistant tuberculosis (RR-TB) treatment regimen should include prevention of resistance amplification. While bedaquiline (BDQ) has been recommended in all-oral ...RR-TB treatment regimen since 2019, resistance is rising at alarming rates. This may be due to BDQ’s delayed bactericidal effect, which increases the risk of selecting for resistance to fluoroquinolones and/or BDQ in the first week of treatment when the bacterial load is highest. We aim to strengthen the first week of treatment with the injectable drug amikacin (AMK). To limit the ototoxicity risk while maximising the bactericidal effect, we will evaluate the safety of adding a 30 mg/kg AMK injection on the first and fourth day of treatment.Methods and analysisWe will conduct a single-arm clinical trial on 20 RR-TB patients nested within an operational study called ShoRRT (All oral Shorter Treatment Regimen for Drug resistant Tuberculosis). In addition to all-oral RR-TB treatment, patients will receive two doses of AMK. The primary safety endpoint is any grade 3–4 adverse event during the first 2 weeks of treatment related to the use of AMK. With a sample size of 20 patients, we will have at least 80% statistical power to support the alternative hypothesis, indicating that less than 14% of patients treated with AMK experience a grade 3–4 adverse event related to its use. Safety data obtained from this study will inform a larger multicountry study on using two high doses of AMK to prevent acquired resistance.Ethics and disseminationApproval was obtained from the ethics committee of Rwanda, Rwanda Food and Drug Authority, Universitair Ziekenhuis, the Institute of Tropical Medicine ethics review board. All participants will provide informed consent. Study results will be disseminated through peer-reviewed journals and conferences.Trial registration number NCT05555303.
Coordinated progress is urgently needed in four domains: redesigning the pharmaceutical innovation ecosystem and its policy environment to resolve the incoherence between market-driven approaches and ...public health needs; applying evidence-based and transparent prioritisation criteria in the national health systems; strengthening the regulatory and supply systems; and promoting financial health protection as part of universal health coverage. The inequitable access to COVID-19 vaccines uncovered multiple structural problems in the organisation, financing and governance of the medical research and development (R&D) and supply ecosystem,3 encompassing unclear demand, weak distribution systems, poor donation practices and corruption, historic inequalities in access to knowledge, training and technological capabilities, lack of technology sharing and transfer, limited local production, and the absence of public health perspective in intellectual property governance. ...efforts by the United Nations to come to a binding Research and Development treaty or convention to support collective public-health focused R&D and hold governments accountable regarding public funding of health-related R&D and commitments for access have not succeeded.11 In fact, equitable access to the latest generations of medicines is increasingly hampered by monopolies and high prices, even in wealthy countries (eg, cancer treatments and the newest gene-based and cell-based therapies), with governments unable (or unwilling) to leverage their power to negotiate or enforce fair pricing, alongside a lack of transparency on development and production costs.12 For example, the lack of transparency about the pricing of COVID-19 vaccines, and the associated power imbalance between governments seeking to procure vaccines for their population and pharmaceutical companies, has caused some blatant inequities, such as the South Africa government being charged more than some high-income countries.13 In the near future, this gap in access to essential medicines and vaccines could become even more pronounced, with the evolution to personalised medicine. The tension between control and access exacerbates the largely ignored North-South gap in access to adequate pain therapy for adults and children.16 Rabies vaccination is life-saving, and pre-exposure prophylaxis (PrEP) increases the likelihood of survival in bitten persons.
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.
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.
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.
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.
Abstract
Background
Since the roll-out of the Xpert MTB/RIF assay, continuous surveillance can provide an estimate of rifampicin-resistant TB (RR-TB) prevalence, provided high drug susceptibility ...testing (DST) coverage is achieved. We use national data from Rwanda to describe rifampicin DST coverage, estimate the prevalence of RR-TB and assess its predictors.
Methods
Routinely collected DST data were entered into an electronic TB case-based surveillance system. DST coverage was calculated among all bacteriologically confirmed pulmonary TB patients notified from 1 July 2019 to 30 June 2020 in Rwanda. The prevalence of RR-TB was estimated among those with DST results. Univariable and multivariable analysis was performed to explore predictors for RR TB.
Results
Among 4066 patients with bacteriologically confirmed pulmonary TB, rifampicin DST coverage was 95.6% (4066/4251). RR-TB was diagnosed in 73 patients. The prevalence of RR-TB was 1.4% (53/3659; 95% CI 1.09 to 1.89%) and 4.9% (20/406; 95% CI 3.03 to 7.51%) in new and previously treated TB cases, respectively. Predictors of RR-TB were: (1) living in Kigali City (adjusted OR aOR 1.65, 95% CI 1.03 to 2.65); (2) previous TB treatment (aOR 3.64, 95% CI 2.14 to 6.19); and (3) close contact with a known RR-TB patient (aOR 11.37, 95% CI 4.19 to 30.82).
Conclusions
High rifampicin DST coverage for routine reporting allowed Rwanda to estimate the RR-TB prevalence among new and previously treated patients.
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.
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.
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).
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.
The European & Developing Countries Clinical Trials Partnership 2 programme, and Belgian Directorate General for Development Cooperation.
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.
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.
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.
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.
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