Acquired rifampicin resistance during first TB treatment: magnitude, relative importance, risk factors and keys to control in low-income settings.

Background: The incidence of acquired rifampicin resistance (RIF-ADR; RR) during first-line treatment varies. Objectives: Compare clinically significant RIF-ADR versus primary and reinfection RR, between regimens (daily versus no rifampicin in the continuation phase; daily versus intermittent rifampicin in the continuation phase) and between rural Bangladesh and Kinshasa, Democratic Republic of Congo. Methods: From patients with treatment failure, relapse, or lost to follow-up, both the outcome and baseline sputum sample were prospectively collected for rpoB sequencing to determine whether RR was present in both samples (primary RR) or only at outcome (RIF-ADR or reinfection RR). Results: The most frequent cause of RR at outcome was primary RR (62.9%; 190/302). RIF-ADR was more frequent with the use of rifampicin throughout versus only in the intensive phase (difference: 3.1%; 95% CI: 0.2-6.0). The RIF-ADR rate was higher with intermittent versus daily rifampicin in the continuation phase (difference: 3.9%; 95% CI: 0.4-7.5). RIF-ADR after rifampicin-throughout treatment was higher when resistance to isoniazid was also found compared with isoniazid-susceptible TB. The estimated RIF-ADR rate was 0.5 per 1000 with daily rifampicin during the entire treatment. Reinfection RR was more frequent in Kinshasa than in Bangladesh (difference: 51.0%; 95% CI: 34.9-67.2). Conclusions: RR is less frequently created when rifampicin is used only during the intensive phase. Under control programme conditions, the RIF-ADR rate for the WHO 6 month rifampicin daily regimen was as low as in affluent settings. For RR-TB control, first-line regimens should be sturdy with optimal rifampicin protection. RIF-ADR prevention is most needed where isoniazid-polyresistance is high, (re)infection control where crowding is extreme.

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs.

Conventional molecular tests for detecting Mycobacterium tuberculosis complex (MTBC) drug resistance on clinical samples cover a limited set of mutations. Whole-genome sequencing (WGS) typically requires culture.Here, we evaluated the Deeplex Myc-TB targeted deep-sequencing assay for prediction of resistance to 13 anti-tuberculous drugs/drug classes, directly applicable on sputum.With MTBC DNA tests, the limit of detection was 100-1000 genome copies for fixed resistance mutations. Deeplex Myc-TB captured in silico 97.1-99.3% of resistance phenotypes correctly predicted by WGS from 3651 MTBC genomes. On 429 isolates, the assay predicted 92.2% of 2369 first- and second-line phenotypes, with a sensitivity of 95.3% and a specificity of 97.4%. 56 out of 69 (81.2%) residual discrepancies with phenotypic results involved pyrazinamide, ethambutol and ethionamide, and low-level rifampicin or isoniazid resistance mutations, all notoriously prone to phenotypic testing variability. Only two out of 91 (2.2%) resistance phenotypes undetected by Deeplex Myc-TB had known resistance-associated mutations by WGS analysis outside Deeplex Myc-TB targets. Phenotype predictions from Deeplex Myc-TB analysis directly on 109 sputa from a Djibouti survey matched those of MTBSeq/PhyResSE/Mykrobe, fed with WGS data from subsequent cultures, with a sensitivity of 93.5/98.5/93.1% and a specificity of 98.5/97.2/95.3%, respectively. Most residual discordances involved gene deletions/indels and 3-12% heteroresistant calls undetected by WGS analysis or natural pyrazinamide resistance of globally rare "Mycobacterium canettii" strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14 902 out of 19 422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free.Deeplex Myc-TB may enable fast, tailored tuberculosis treatment.

Xpert Mycobacterium tuberculosis/Rifampicin-Detected Rifampicin Resistance is a Suboptimal Surrogate for Multidrug-resistant Tuberculosis in Eastern Democratic Republic of the Congo: Diagnostic and Clinical Implications.

BACKGROUND: Rifampicin (RIF) resistance is highly correlated with isoniazid (INH) resistance and used as proxy for multidrug-resistant tuberculosis (MDR-TB). Using MTBDRplus as a comparator, we evaluated the predictive value of Xpert MTB/RIF (Xpert)-detected RIF resistance for MDR-TB in eastern Democratic Republic of the Congo (DRC). METHODS: We conducted a cross-sectional study involving data from new or retreatment pulmonary adult TB cases evaluated between July 2013 and December 2016. Separate, paired sputa for smear microscopy and MTBDRplus were collected. Xpert testing was performed subject to the availability of Xpert cartridges on sample remnants after microscopy. RESULTS: Among 353 patients, 193 (54.7%) were previously treated and 224 (63.5%) were MTBDRplus TB positive. Of the 224, 43 (19.2%) were RIF monoresistant, 11 (4.9%) were INH monoresistant, 53 (23.7%) had MDR-TB, and 117 (52.2%) were RIF and INH susceptible. Overall, among the 96 samples detected by MTBDRplus as RIF resistant, 53 (55.2%) had MDR-TB. Xpert testing was performed in 179 (50.7%) specimens; among these, 163 (91.1%) were TB positive and 73 (44.8%) RIF resistant. Only 45/73 (61.6%) Xpert-identified RIF-resistant isolates had concomitant MTBDRplus-detected INH resistance. Xpert had a sensitivity of 100.0% (95% CI, 92.1-100.0) for detecting RIF resistance but a positive-predictive value of only 61.6% (95% CI, 49.5-72.8) for MDR-TB. The most frequent mutations associated with RIF and INH resistance were S531L and S315T1, respectively. CONCLUSIONS: In this high-risk MDR-TB study population, Xpert had low positive-predictive value for the presence of MDR-TB. Comprehensive resistance testing for both INH and RIF should be performed in this setting.

Challenge to treat pre-extensively drug-resistant tuberculosis in a low-income country: A report of 12 cases.

Setting: Democratic Republic of the Congo is a high-burden TB country. Its capital, Kinshasa, reports annually about one-third of all MDR-TB cases in the country; thus, pre-XDRTB management is warranted. OBJECTIVES: To describe the main challenges in treating pre- XDR TB in this low resources setting and possible solutions. METHOD: This is a retrospective study of all pre-XDR TB patients diagnosed in Kinshasa in 2018. A personalized regimen was applied according to the clinical profile, drug availability, and the Drug susceptibility testing (DST). Treatment was administered by hospitalization during the intensive phase and in ambulatory care in the continuation phase except in emergencies. Monthly follow up included evaluating clinical and bacteriological features, renal and liver functions, QT interval on ECG, and audiometry for those under aminoglycosides. RESULTS: Among the 236 MDR-TB patients identified in 2018, 14 had pre-XDR. Two died before treatment initiation. Of the remaining 12. 75% were male, 50% were aged 25-44 years, 66.7% had previous anti-tuberculosis treatment, 75% had a body mass index < 18.5 kg/m2, and 1 patient was HIV positive. On radiography, all the patients had cavities. The median time from the diagnosis to treatment initiation was 48.5 days (range: 14-105). A favorable outcome occurred in 10 cases (83.3%), one patient died, and anotherwas lost to follow up. Nine (75%) patients reported adverse reactions, which were mild or moderate in 6 cases and severe in 2 cases. The severe reactions were psychosis (1 case) and ototoxicity (1 case). CONCLUSION: Successful pre-XDRTB treatment using the new strategy is possible even in a low-income country. The main challenges are diagnosis access, drug availability and follow-up laboratory facilities. These can be included in a global policy review by the NTP to ensure the sustainability of the strategies implemented.

Outcomes and adverse events of pre- and extensively drug-resistant tuberculosis patients in Kinshasa, Democratique Republic of the Congo: A retrospective cohort study.

BACKGROUND: Extensively drug-resistant tuberculosis (XDR TB) is a very serious form of tuberculosis that is burdened with a heavy mortality toll, especially before the advent of new TB drugs. The Democratic Republic of the Congo (DRC) is among the countries most affected by this new epidemic. METHODS: A retrospective analysis was performed of the records of all patients with pre- and extensively drug-resistant tuberculosis hospitalized from January 1, 2015 to December 31, 2017 and monitored for at least 6 months to one year after the end of their treatment in Kinshasa; an individualized therapeutic regimen with bedaquiline for 20 months was built for each patient. The adverse effects were systematically monitored. RESULTS: Of the 40 laboratory-confirmed patients, 32 (80%) patients started treatment, including 29 preXRB and 3 XDR TB patients. In the eligible group, 3 patients (9.4%) had HIV-TB coinfections. The therapeutic success rate was 53.2%, and the mortality rate was 46.8% (15/32); there were no relapses, failures or losses to follow-up. All coinfected HIV-TB patients died during treatment. The cumulative patient survival rate was 62.5% at 3 months, 53.1% at 6 months and 53.1% at 20 months. The most common adverse events were vomiting, Skin rash, anemia and peripheral neuropathy. CONCLUSION: The new anti-tuberculosis drugs are a real hope for the management of Drug Resistant tuberculosis patient and other new therapeutic combinations may improve favorable outcomes.

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, Predictors, and Successful Treatment Outcomes of Xpert MTB/RIF-identified Rifampicin-resistant Tuberculosis in Post-conflict Eastern Democratic Republic of the Congo, 2012-2017: A Retrospective Province-Wide Cohort Study.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) jeopardizes global TB control. The prevalence and predictors of Rifampicin-resistant (RR) TB, a proxy for MDR-TB, and the treatment outcomes with standard and shortened regimens have not been assessed in post-conflict regions, such as the South Kivu province in the eastern Democratic Republic of the Congo (DRC). We aimed to fill this knowledge gap and to inform the DRC National TB Program. METHODS: of adults and children evaluated for pulmonary TB by sputum smear microscopy and Xpert MTB/RIF (Xpert) from February 2012 to June 2017. Multivariable logistic regression, Kaplan-Meier estimates, and multivariable Cox regression were used to assess independent predictors of RR-TB and treatment failure/death. RESULTS: Of 1535 patients Xpert-positive for TB, 11% had RR-TB. Independent predictors of RR-TB were a positive sputum smear (adjusted odds ratio [aOR] 2.42, 95% confidence interval [CI] 1.63-3.59), retreatment of TB (aOR 4.92, 95% CI 2.31-10.45), and one or more prior TB episodes (aOR 1.77 per episode, 95% CI 1.01-3.10). Over 45% of RR-TB patients had no prior TB history or treatment. The median time from Xpert diagnosis to RR-TB treatment initiation was 12 days (interquartile range 3-60.2). Cures were achieved in 30/36 (83%) and 84/114 (74%) of patients on 9- vs 20/24-month MDR-TB regimens, respectively (P = .06). Predictors of treatment failure/death were the absence of directly observed therapy (DOT; adjusted hazard ratio [aHR] 2.77, 95% CI 1.2-6.66) and any serious adverse drug event (aHR 4.28, 95% CI 1.88-9.71). CONCLUSIONS: Favorable RR-TB cure rates are achievable in this post-conflict setting with a high RR-TB prevalence. An expanded Xpert scale-up; the prompt initiation of shorter, safer, highly effective MDR-TB regimens; and treatment adherence support are critically needed to optimize outcomes.

The relationship between transmission time and clustering methods in Mycobacterium tuberculosis epidemiology.

BACKGROUND: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. METHODS: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005-2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. FINDINGS: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. INTERPRETATION: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting.

Pseudo-outbreak of pre-extensively drug-resistant (Pre-XDR) tuberculosis in Kinshasa: collateral damage caused by false detection of fluoroquinolone resistance by GenoType MTBDRsl.

Fluoroquinolones are the core drugs for the management of multidrug-resistant tuberculosis (MDR-TB). Molecular drug susceptibility testing methods provide considerable advantages for scaling up programmatic management and surveillance of drug-resistant TB. We describe here the misidentification of fluoroquinolone resistance by the GenoType MTBDRsl (MTBDRsl) (Hain Lifescience GmbH, Nehren, Germany) line probe assay (LPA) encountered during a feasibility and validation study for the introduction of this rapid drug susceptibility test in Kinshasa, Democratic Republic of Congo. The double gyrA mutation 80Ala and 90Gly represented 57% of all fluoroquinolone mutations identified from MDR-TB patient sputum samples, as confirmed by DNA sequencing. This double mutation was previously found to be associated with susceptibility to fluoroquinolones, yet it leads to absent hybridization of a wild-type band in the MTBDRsl and is thus falsely scored as resistance. Our findings suggest that MTBDRsl results must be interpreted with caution when the interpretation is based solely on the absence of a wild-type band without confirmation by visualization of a mutant band. Performance of the MTBDRsl LPA might be improved by replacing the gyrA wild-type probes by additional probes specific for well-documented gyrA mutations that confer clinically relevant resistance.

Multi drug resistant tuberculosis in Mosango, a rural area in the Democratic Republic of Congo.

Multidrug Resistant Tuberculosis (MDR-TB) is a serious threat which jeopardizes the worldwide efforts to control TB. The Democratic Republic of Congo (DRC) is one of 27 countries with a high burden of MDR-TB. Data on the magnitude, trends, and the distribution of MDR-TB in DRC are scanty. Kinshasa, the capital city of DRC which accounts for 20% of all TB cases nationwide, is notifying more than 80% of all MDR suspects. We report here a cluster of MDR-TB cases that was investigated in the Mosango health district, in the Bandundu south Province, DRC in 2008. Phenotypic Drug Sensitivity Testing and DNA sequencing were performed on 18 sputum specimens collected from 4 MDR-TB suspects and 5 household contacts. Sequencing data confirmed that the 4 suspects were indeed Rifampicin resistant cases. Sequencing of the rpoB gene showed that 3 cases (patients A, B and D) had a single mutation encoding a substitution to 526Tyr, 531Trp and 526Leu respectively. Patient C had a double mutation encoding a change to 531Leu and 633Leu. Two of the investigated cases died within 4 months of a second-line treatment course. Results highlight the need to enhance adequate laboratory services within the country for both clinical as well as surveillance purposes.

[HIV seroprevalence among tuberculous patients in Kisangani in the Democratic Republic of Congo]. / Séroprévalence du VIH chez les tuberculeux à Kisangani en République Démocratique du Congo.

INTRODUCTION: HIV testing among TB patients is still low in DRC This study aimed to determine HIV infection prevalence among TB patients with positive microscopy (TPM+) in Kisangani city. METHODS: In the period of 15 h August to 31th December 2010, a cross-sectional study was conducted including pulmonary tuberculosis patients with positive microscopy (TPM+) from 15 to 49-years-old received in seven tuberculosis screening and treatment centers (STC). The Chi-square and Fisher's exact test were used to compare proportions and the Wilcoxon test to examine the relationships between quantitative variables with skewed distributions. RESULTS: HIV test has been proposed to 136 TPM+ patients. Among them, 118 (86.76%) agreed to be tested. 24 (20.3%) of them were HIV positive. HIV-positive patients were significantly older (P = 0.02), predominantly female (P < 0.001) unemployed (p = 0.01). There were more cases of TB treatment relapse (p < 0.01) and less knowledge of TB signs (p = 0.01) among HIV-positive patients. DISCUSSION: The Ministry of Health and its partners should extend to all STC provider-initiated HIV counceling and testing, ensure access to antiretroviral treatment for all HIV-positive TB patients and intensify TB screening in all patients living with HIV

Sequencing of the pncA gene in members of the Mycobacterium tuberculosis complex has important diagnostic applications: Identification of a species-specific pncA mutation in "Mycobacterium canettii" and the reliable and rapid predictor of pyrazinamide resistance.

Testing for susceptibility to pyrazinamide (PZA) and analysis of the pncA gene sequences of 423 Mycobacterium tuberculosis complex isolates have revealed a unique silent nucleotide substitution that enables the rapid identification of "M. canettii" (proposed name). Moreover, the lack of a defined mutation within the pncA gene strongly suggests that an alternative mechanism is responsible for PZA resistance. Our results indicate that DNA sequencing of the pncA gene has the potential to shorten the turnaround time and increase the accuracy of PZA susceptibility testing of the M. tuberculosis complex.