Rifampicin resistance conferring mutations among Mycobacterium tuberculosis strains in Rwanda.

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.

Strain diversity and gene mutations associated with presumptive multidrug-resistant Mycobacterium tuberculosis complex isolates in Northwest Ethiopia.

OBJECTIVES: In this study, we assessed the genetic diversity and gene mutations that confer resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolone (FQ), and second-line injectable (SLI) drugs in RIF-resistant (RR)/multidrug-resistant tuberculosis (MDR-TB) isolates in Northwest Ethiopia. METHODS: Spoligotyping was used to assign isolates to TB lineages (Ls), and Hain line probe assays were used to detect resistance to RIF, INH, and FQs, and SLIs. RESULTS: Among 130 analyzed strains, 68.5% were RR, and four major Mycobacterium tuberculosis complex lineages (L1, L3, L4, and L7) were identified with a predominance of the Euro-American L4 (72, 54.7%), while L7 genotypes were less common (3, 2.3%). Overall, the L4-T3-ETH (41, 32.0%), L3-CAS1-Delhi (29, 22.7%), and L3-CAS1-Killi (19, 14.8%) families were most common. Line probe analysis showed that among rpoB mutants, 65.2% were S450L, while 87.8% of katG mutants were S315T. Only three isolates showed mutation (c-15t) at the inhA gene, and no double mutation with katG and inhA genes was found. Six strains, two each of L1, L3, and L4, were resistant to FQs, having gyrA mutations (D94G, S91P), of which three isolates had additional resistance to SLI (rrs A1401G or C1402T mutations) including one isolate with low-level kanamycin (KAN) resistance. CONCLUSIONS: This study showed a predominance of L4-T3-ETH, L3-CAS1-Delhi, and L3-CAS1-Killi families, with a high rate of rpoB_S450L and katG_S315T mutations and a low proportion of gyrA and rrs mutations. L7 was less frequently observed in this study. Further investigations are, therefore, needed to understand L7 and other lineages with undefined mutations.

Evaluation of two short standardised regimens for the treatment of rifampicin-resistant tuberculosis (STREAM stage 2): an open-label, multicentre, randomised, non-inferiority trial.

BACKGROUND: The STREAM stage 1 trial showed that a 9-month regimen for the treatment of rifampicin-resistant tuberculosis was non-inferior to the 20-month 2011 WHO-recommended regimen. In STREAM stage 2, we aimed to compare two bedaquiline-containing regimens with the 9-month STREAM stage 1 regimen. METHODS: We did a randomised, phase 3, non-inferiority trial in 13 hospital clinics in seven countries, in individuals aged 15 years or older with rifampicin-resistant tuberculosis without fluoroquinolone or aminoglycoside resistance. Participants were randomly assigned 1:2:2:2 to the 2011 WHO regimen (terminated early), a 9-month control regimen, a 9-month oral regimen with bedaquiline (primary comparison), or a 6-month regimen with bedaquiline and 8 weeks of second-line injectable. Randomisations were stratified by site, HIV status, and CD4 count. Participants and clinicians were aware of treatment-group assignments, but laboratory staff were masked. The primary outcome was favourable status (negative cultures for Mycobacterium tuberculosis without a preceding unfavourable outcome) at 76 weeks; any death, bacteriological failure or recurrence, and major treatment change were considered unfavourable outcomes. All comparisons used groups of participants randomly assigned concurrently. For non-inferiority to be shown, the upper boundary of the 95% CI should be less than 10% in both modified intention-to-treat (mITT) and per-protocol analyses, with prespecified tests for superiority done if non-inferiority was shown. This trial is registered with ISRCTN, ISRCTN18148631. FINDINGS: Between March 28, 2016, and Jan 28, 2020, 1436 participants were screened and 588 were randomly assigned. Of 517 participants in the mITT population, 133 (71%) of 187 on the control regimen and 162 (83%) of 196 on the oral regimen had a favourable outcome: a difference of 11·0% (95% CI 2·9-19·0), adjusted for HIV status and randomisation protocol (p<0·0001 for non-inferiority). By 76 weeks, 108 (53%) of 202 participants on the control regimen and 106 (50%) of 211 allocated to the oral regimen had an adverse event of grade 3 or 4; five (2%) participants on the control regimen and seven (3%) on the oral regimen had died. Hearing loss (Brock grade 3 or 4) was more frequent in participants on the control regimen than in those on the oral regimen (18 [9%] vs four [2%], p=0·0015). Of 134 participants in the mITT population who were allocated to the 6-month regimen, 122 (91%) had a favourable outcome compared with 87 (69%) of 127 participants randomly assigned concurrently to the control regimen (adjusted difference 22·2%, 95% CI 13·1-31·2); six (4%) of 143 participants on the 6-month regimen had grade 3 or 4 hearing loss. INTERPRETATION: Both bedaquiline-containing regimens, a 9-month oral regimen and a 6-month regimen with 8 weeks of second-line injectable, had superior efficacy compared with a 9-month injectable-containing regimen, with fewer cases of hearing loss. FUNDING: USAID and Janssen Research & Development.

Keeping up with the guidelines: design changes to the STREAM stage 2 randomised controlled non-inferiority trial for rifampicin-resistant tuberculosis.

Results from the STREAM stage 1 trial showed that a 9-month regimen for patients with rifampicin-resistant tuberculosis was non-inferior to the 20-month regimen recommended by the 2011 WHO treatment guidelines. Similar levels of severe adverse events were reported on both regimens suggesting the need for further research to optimise treatment. Stage 2 of STREAM evaluates two additional short-course regimens, both of which include bedaquiline. Throughout stage 2 of STREAM, new drug choices and a rapidly changing treatment landscape have necessitated changes to the trial's design to ensure it remains ethical and relevant. This paper describes changes to the trial design to ensure that stage 2 continues to answer important questions. These changes include the early closure to recruitment of two trial arms and an adjustment to the definition of the primary endpoint. If the STREAM experimental regimens are shown to be non-inferior or superior to the stage 1 study regimen, this would represent an important contribution to evidence about potentially more tolerable and more efficacious MDR-TB regimens, and a welcome advance for patients with rifampicin-resistant tuberculosis and tuberculosis control programmes globally.Trial registration: ISRCTN ISRCTN18148631 . Registered 10 February 2016.

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.

Multidrug-resistant tuberculosis control in Rwanda overcomes a successful clone that causes most disease over a quarter century.

SUMMARY BACKGROUND: Multidrug-resistant (MDR) tuberculosis (TB) poses an important challenge in TB management and control. Rifampicin resistance (RR) is a solid surrogate marker of MDR-TB. We investigated the RR-TB clustering rates, bacterial population dynamics to infer transmission dynamics, and the impact of changes to patient management on these dynamics over 27 years in Rwanda. METHODS: We analysed whole genome sequences of a longitudinal collection of nationwide RR-TB isolates. The collection covered three important periods: before programmatic management of MDR-TB (PMDT; 1991-2005), the early PMDT phase (2006-2013), in which rifampicin drug-susceptibility testing (DST) was offered to retreatment patients only, and the consolidated phase (2014-2018), in which all bacteriologically confirmed TB patients had rifampicin DST done mostly via Xpert MTB/RIF assay. We constructed clusters based on a 5 SNP cut-off and resistance conferring SNPs. We used Bayesian modelling for dating and population size estimations, TransPhylo to estimate the number of secondary cases infected by each patient, and multivariable logistic regression to assess predictors of being infected by the dominant clone. RESULTS: Of 308 baseline RR-TB isolates considered for transmission analysis, the clustering analysis grouped 259 (84.1%) isolates into 13 clusters. Within these clusters, a single dominant clone was discovered containing 213 isolates (82.2% of clustered and 69.1% of all RR-TB), which we named the "Rwanda Rifampicin-Resistant clone" (R3clone). R3clone isolates belonged to Ugandan sub-lineage 4.6.1.2 and its rifampicin and isoniazid resistance were conferred by the Ser450Leu mutation in rpoB and Ser315Thr in katG genes, respectively. All R3clone isolates had Pro481Thr, a putative compensatory mutation in the rpoC gene that likely restored its fitness. The R3clone was estimated to first arise in 1987 and its population size increased exponentially through the 1990s', reaching maximum size (∼84%) in early 2000 s', with a declining trend since 2014. Indeed, the highest proportion of R3clone (129/157; 82·2%, 95%CI: 75·3-87·8%) occurred between 2000 and 13, declining to 64·4% (95%CI: 55·1-73·0%) from 2014 onward. We showed that patients with R3clone detected after an unsuccessful category 2 treatment were more likely to generate secondary cases than patients with R3clone detected after an unsuccessful category 1 treatment regimen. CONCLUSIONS: RR-TB in Rwanda is largely transmitted. Xpert MTB/RIF assay as first diagnostic test avoids unnecessary rounds of rifampicin-based TB treatment, thus preventing ongoing transmission of the dominant R3clone. As PMDT was intensified and all TB patients accessed rifampicin-resistance testing, the nationwide R3clone burden declined. To our knowledge, our findings provide the first evidence supporting the impact of universal DST on the transmission of RR-TB.

Rifampicin susceptibility discordance between Xpert MTB/RIF G4 and Xpert Ultra before MDRT-TB treatment initiation: A case report from Uganda.

Tuberculosis (TB) resistance to rifampicin, the most powerful drug leads to increase in mortality. Globally, half a million new patients develop such resistant TB each year, coupled with both inappropriate diagnosis and treatment initiation. We report a case of rifampicin resistant Mycobacterium tuberculosis whose rifampicin resistance was missed by Xpert MTB/RIF Assay G4 but detected by the Xpert MTB/RIF Ultra assay at different time points leading to increased delays for MDR-TB treatment initiation at Mulago Hospital, Kampala, Uganda. Our case report compels greater urgency in accelerating the transition to the newer assay, Ultra, to benefit from higher sensitivity of rifampicin resistance detection.

Discordance of the Repeat GeneXpert MTB/RIF Test for Rifampicin Resistance Detection Among Patients Initiating MDR-TB Treatment in Uganda.

BACKGROUND: The Global Laboratory Initiative (GLI) guidelines recommend repeat for GeneXpertMTB/RIF (XpertMTB/RIF) in patients with a low pretest probability of rifampicin resistance (RR). METHODS: This was a cross-sectional study using results of sputum specimens collected from participants screened for the STREAM 2 trial. We recruited all patients with XpertMTB/RIF RR-TB detected who were referred for RR/multidrug-resistant (MDR) TB treatment initiation at Mulago National Referral Hospital, Kampala, between September 2017 and October 2019. At baseline, smear microscopy, repeat XpertMTB/RIF, Xpert Ultra, and MTBDRplus assays were done on sputum specimens. Culture-based drug susceptibility testing (DST) was performed on discordant specimens. We analyzed the prevalence and factors associated with discordance between initial and repeat XpertMTB/RIF RR and false XpertMTB/RIF RR. False XpertMTB/RIF RR was defined as no RR detected by any of Xpert Ultra, LPA, or culture DST (reference comparator). RESULTS: A total of 126/130 patients had repeat XpertMTB/RIF results, of whom 97 (77.0%) had M. tuberculosis detected, 81 (83.5%) had RR detected, and 1 (1.0%) had RR indeterminate. The prevalence of discordant XpertMTB/RIF RR was 15/96 (15.6%), whereas false XpertMTB/RIF RR prevalence was 10/96 (10.4%).Low-bacillary load sputum specimens were more likely to have discordant XpertMTB/RIF RR and false XpertMTB/RIF RR results (adjusted odds ratio [aOR], 0.04; 95% CI, 0.00-0.37; P = .01; aOR, 0.02; 95% CI, 0.01-0.35; P = .01, respectively). CONCLUSIONS: Our findings show a high false-positive rifampicin resistance rate in low-TB burden patients, which calls for repeat testing in order to prevent unnecessary prescription of anti-MDR-TB therapy.

Genetic diversity of the Mycobacterium tuberculosis complex strains from newly diagnosed tuberculosis patients in Northwest Ethiopia reveals a predominance of East-African-Indian and Euro-American lineages.

OBJECTIVES: This study described the population structure of M. tuberculosis complex (MTBc) strains among patients with pulmonary or lymph node tuberculosis (TB) in Northwest Ethiopia and tested the performance of culture isolation and MPT64-based speciation for Lineage 7 (L7). METHODS: Patients were recruited between April 2017 and June 2019 in North Gondar, Ethiopia. The MPT64 assay was used to confirm MTBc, and spoligotyping was used to characterize mycobacterial lineages. Line probe assay (LPA) was used to detect resistance to rifampicin and isoniazid. RESULTS: Among 274 MTBc genotyped isolates, there were five MTBc lineages: L1-L4 and L7 were identified, with predominant East-African-Indian (L3) (53.6%) and Euro-American (L4) (40.1%) strains, and low prevalence (2.6%) of Ethiopia L7. The genotypes were similarly distributed between pulmonary and lymph node TB, and all lineages were equally isolated by culture and recognized as MTBc by the MPT64 assay. Additionally, LPA showed that 259 (94.5%) MTBc were susceptible to both rifampicin and isoniazid, and one (0.4%) was multi-drug resistant (resistant to both rifampicin and isoniazid). CONCLUSION: These findings show that TB in North Gondar, Ethiopia, is mainly caused by L3 and L4 strains, with low rates of L7, confirmed as MTBc by MPT64 assay and with limited resistance to rifampicin and isoniazid.

A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination.

Criteria defining bedaquiline resistance for tuberculosis have been proposed addressing an emerging concern. We evaluated bedaquiline phenotypic drug susceptibility testing (pDST) criteria using drug-resistant tuberculosis clinical isolates tested at five reference laboratories. Isolates were tested at the proposed bedaquiline MGIT960 and 7H11 agar proportion (AP) critical concentrations and also at higher dilutions. The epidemiological cutoff value for the broth microdilution (BMD) plates (frozen and dry) was investigated. Sanger sequencing was performed (atpE and Rv0678 genes) for any isolate testing resistant. The composite reference standard (CRS) defined susceptibility or resistance as is if all pDST methods agreed. If the pDST result was discordant, sequencing results were used for final classification. Geographically diverse and bedaquiline-unexposed isolates were tested (n = 495). The epidemiological cutoff value for BMD was confirmed to be 0.12 µg/ml. The majority of isolates were determined to be susceptible by all methods (467/495; 94.3%), and 28 were determined to be resistant by at least one method; 4 of these were determined to be resistant by all methods. Of the 28 resistant isolates, 12 harbored Rv0678 mutations exclusively. Isolates with insertions/deletions were more likely to be determined to be resistant by more than one method (5/7) compared to isolates with a single nucleotide polymorphism (1/5). Applying the CRS to 24 discordant pDST, BMD dry correctly detected most (15/24; 63%), followed by MGIT960 and BMD frozen (13/24; 61%) and lastly AP (12/24; 50%). Applying the CRS, the prevalence of bedaquiline resistance was 2.2% and ranged from 1.4 to 3.4%, depending on the method used. All methods performed well for bedaquiline susceptibility determination; however, resistance detected should be investigated by a second, alternative method.

Multidrug-resistant patients receiving treatment in Niger who are infected with M. tuberculosis Cameroon family convert faster in smear and culture than those with M. tuberculosis Ghana family.

In this study, we analyzed the M. tuberculosis complex (MTBc) population structure among multidrug-resistant TB (MDR-TB) patients in Niger and tested whether the Cameroon family displayed a slower response to MDR-TB treatment. We genotyped baseline clinical isolates that had been collected from pulmonary MDR-TB patients recruited consecutively between 2008 and 2016 in Niger. Spoligotyping was used to analyze the genetic diversity of mycobacterial lineages, and Kaplan Meier's analysis to compare treatment outcomes. A total of 222 MTBc isolates were genotyped; 204 (91,9%) were identified as the Euro-American L4 lineage, with the Ghana family (106, 47,4%) and the Cameroon family (63, 28,4%) being predominant. Patients infected by Cameroon family isolates 61(96,8%) showed faster conversion (log-rank p < 0.01) than those infected with Ghana family isolates (91,5%), and were more likely to experience favorable outcome (adjusted odds ratio [aOR] 4.4; 95%CI 1.1-17.9]; p = 0.015). We found no association between MTBc families and second-line drug resistance profiles (p > 0.05). Our findings show that MDR-TB in Niger is caused by major spoligotypes of the Euro-American L4; with more rapid smear and culture conversion in patients infected with the Cameroon family. These first insights may alert clinicians that slow conversion may be associated with the type of infecting strain.

Validation of Bedaquiline Phenotypic Drug Susceptibility Testing Methods and Breakpoints: a Multilaboratory, Multicountry Study.

Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical Mycobacterium tuberculosis isolates as susceptible/resistant to the drug. Three methods were used: Middlebrook 7H11 agar proportion (AP) assay, broth microdilution (BMD) assay, and mycobacterial growth indicator tube (MGIT) assay. Each of the five laboratories tested the 40-isolate (20 unique isolates, duplicated) EQA panel at three time points. The study validated the sensitivity and specificity of a bedaquiline MIC susceptibility breakpoint of 0.12 µg/ml for the BMD method and WHO interim CCs of 1 µg/ml for MGIT and 0.25 µg/ml for the 7H11 AP methods. Categorical agreements between observed and expected results and sensitivities/specificities for correctly identifying an isolate as susceptible/resistant were highest at the 0.25, 0.12, and 1 µg/ml bedaquiline concentrations for the AP method, BMD (frozen or dry plates), and MGIT960, respectively. At these concentrations, the very major error rates for erroneously categorizing an isolate as susceptible when it was resistant were the lowest and within CLSI guidelines. The most highly reproducible bedaquiline DST methods were MGIT960 and BMD using dry plates. These findings validate the use of standardized DST methodologies and interpretative criteria to facilitate routine phenotypic bedaquiline DST and to monitor the emergence of bedaquiline resistance.

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.

Variable ability of rapid tests to detect Mycobacterium tuberculosis rpoB mutations conferring phenotypically occult rifampicin resistance.

We compared the ability of commercial and non-commercial, phenotypic and genotypic rapid drug susceptibility tests (DSTs) to detect rifampicin resistance (RR)-conferring 'disputed' mutations frequently missed by Mycobacterium Growth Indicator Tube (MGIT), namely L430P, D435Y, L452P, and I491F. Strains with mutation S450L served as positive control while wild-types were used as negative control. Of the 38 mutant strains, 5.7% were classified as RR by MGIT, 16.2% by Trek Sensititre MYCOTB MIC plate, 19.4% by resazurin microtiter plate assay (REMA), 50.0% by nitrate reductase assay (NRA), and 62.2% by microscopic observation direct susceptibility testing (MODS). Reducing MGIT rifampicin concentration to 0.5 µg/ml, and/or increasing incubation time, enhanced detection of disputed mutations from 5.7% to at least 65.7%, particularly for mutation I491F (from 0.0 to 75.0%). Compared with MGIT at standard pre-set time with 0.25 µg/ml ECOFF as breakpoint, we found a statistically significant increase in the ability of MGIT to resolve disputed mutants and WT strains at extended incubation period of 15 and 21 days, with 0.5 µg/ml and 1 µg/ml ECOFF respectively. MODS detected 75.0% of the I491F strains and NRA 62.5%, while it was predictably missed by all molecular assays. Xpert MTB/RIF, Xpert Ultra, and GenoscholarTB-NTM + MDRTB detected all mutations within the 81 bp RR determining region. Only GenoType MTBDRplus version 2 missed mutation L430P in 2 of 11 strains. Phenotypic and genotypic DSTs varied greatly in detecting occult rifampicin resistance. None of these methods detected all disputed mutations without misclassifying wild-type strains.

A Trial of a Shorter Regimen for Rifampin-Resistant Tuberculosis.

BACKGROUND: Cohort studies in Bangladesh showed promising cure rates among patients with multidrug-resistant tuberculosis who received existing drugs in regimens shorter than that recommended by the World Health Organization (WHO) in 2011. METHODS: We conducted a phase 3 noninferiority trial in participants with rifampin-resistant tuberculosis that was susceptible to fluoroquinolones and aminoglycosides. Participants were randomly assigned, in a 2:1 ratio, to receive a short regimen (9 to 11 months) that included high-dose moxifloxacin or a long regimen (20 months) that followed the 2011 WHO guidelines. The primary efficacy outcome was a favorable status at 132 weeks, defined by cultures negative for Mycobacterium tuberculosis at 132 weeks and at a previous occasion, with no intervening positive culture or previous unfavorable outcome. An upper 95% confidence limit for the between-group difference in favorable status that was 10 percentage points or less was used to determine noninferiority. RESULTS: Of 424 participants who underwent randomization, 383 were included in the modified intention-to-treat population. Favorable status was reported in 79.8% of participants in the long-regimen group and in 78.8% of those in the short-regimen group - a difference, with adjustment for human immunodeficiency virus status, of 1.0 percentage point (95% confidence interval [CI], -7.5 to 9.5) (P = 0.02 for noninferiority). The results with respect to noninferiority were consistent among the 321 participants in the per-protocol population (adjusted difference, -0.7 percentage points; 95% CI, -10.5 to 9.1). An adverse event of grade 3 or higher occurred in 45.4% of participants in the long-regimen group and in 48.2% in the short-regimen group. Prolongation of either the QT interval or the corrected QT interval (calculated with Fridericia's formula) to 500 msec occurred in 11.0% of participants in the short-regimen group, as compared with 6.4% in the long-regimen group (P = 0.14); because of the greater incidence in the short-regimen group, participants were closely monitored and some received medication adjustments. Death occurred in 8.5% of participants in the short-regimen group and in 6.4% in the long-regimen group, and acquired resistance to fluoroquinolones or aminoglycosides occurred in 3.3% and 2.3%, respectively. CONCLUSIONS: In persons with rifampin-resistant tuberculosis that was susceptible to fluoroquinolones and aminoglycosides, a short regimen was noninferior to a long regimen with respect to the primary efficacy outcome and was similar to the long regimen in terms of safety. (Funded by the U.S. Agency for International Development and others; Current Controlled Trials number, ISRCTN78372190; ClinicalTrials.gov number, NCT02409290.).

Potential Application of Digitally Linked Tuberculosis Diagnostics for Real-Time Surveillance of Drug-Resistant Tuberculosis Transmission: Validation and Analysis of Test Results.

BACKGROUND: Tuberculosis (TB) is the highest-mortality infectious disease in the world and the main cause of death related to antimicrobial resistance, yet its surveillance is still paper-based. Rifampicin-resistant TB (RR-TB) is an urgent public health crisis. The World Health Organization has, since 2010, endorsed a series of rapid diagnostic tests (RDTs) that enable rapid detection of drug-resistant strains and produce large volumes of data. In parallel, most high-burden countries have adopted connectivity solutions that allow linking of diagnostics, real-time capture, and shared repository of these test results. However, these connected diagnostics and readily available test results are not used to their full capacity, as we have yet to capitalize on fully understanding the relationship between test results and specific rpoB mutations to elucidate its potential application to real-time surveillance. OBJECTIVE: We aimed to validate and analyze RDT data in detail, and propose the potential use of connected diagnostics and associated test results for real-time evaluation of RR-TB transmission. METHODS: We selected 107 RR-TB strains harboring 34 unique rpoB mutations, including 30 within the rifampicin resistance-determining region (RRDR), from the Belgian Coordinated Collections of Microorganisms, Antwerp, Belgium. We subjected these strains to Xpert MTB/RIF, GenoType MTBDRplus v2.0, and Genoscholar NTM + MDRTB II, the results of which were validated against the strains' available rpoB gene sequences. We determined the reproducibility of the results, analyzed and visualized the probe reactions, and proposed these for potential use in evaluating transmission. RESULTS: The RDT probe reactions detected most RRDR mutations tested, although we found a few critical discrepancies between observed results and manufacturers' claims. Based on published frequencies of probe reactions and RRDR mutations, we found specific probe reactions with high potential use in transmission studies: Xpert MTB/RIF probes A, Bdelayed, C, and Edelayed; Genotype MTBDRplus v2.0 WT2, WT5, and WT6; and Genoscholar NTM + MDRTB II S1 and S3. Inspection of probe reactions of disputed mutations may potentially resolve discordance between genotypic and phenotypic test results. CONCLUSIONS: We propose a novel approach for potential real-time detection of RR-TB transmission through fully using digitally linked TB diagnostics and shared repository of test results. To our knowledge, this is the first pragmatic and scalable work in response to the consensus of world-renowned TB experts in 2016 on the potential of diagnostic connectivity to accelerate efforts to eliminate TB. This is evidenced by the ability of our proposed approach to facilitate comparison of probe reactions between different RDTs used in the same setting. Integrating this proposed approach as a plug-in module to a connectivity platform will increase usefulness of connected TB diagnostics for RR-TB outbreak detection through real-time investigation of suspected RR-TB transmission cases based on epidemiologic linking.

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.

Bedaquiline susceptibility testing of Mycobacterium tuberculosis in an automated liquid culture system.

OBJECTIVES: The objective of this study was to evaluate the performance of the BACTEC MGIT960 system to test the susceptibility to bedaquiline for Mycobacterium tuberculosis complex. METHODS: We determined the quality control (QC) range of bedaquiline using the M. tuberculosis H37Rv reference strain and the epidemiological cut-off (ECOFF) in MGIT960 and on Middlebrook 7H11 agar (M7H11) using 47 strains from bedaquiline treatment-naive patients. The accuracy of MGIT960 was evaluated versus M7H11 using 74 'probably susceptible to bedaquiline' and 18 'probably resistant to bedaquiline' strains. Repeatability and reproducibility of MGIT960 were assessed using five strains showing different resistance levels. RESULTS: The QC range for the H37Rv strain was between 0.125 and 0.50 mg/L. The WT MIC distribution ranged from ≤0.03 to 1.00 mg/L in MGIT960 and from ≤0.008 to 0.25 mg/L on M7H11 with suggested ECOFFs of 1.00 and 0.25 mg/L, respectively. Applying these ECOFFs, the probably susceptible and probably resistant strains were distinguishable by both methods, albeit with only a 2-fold increased MIC for one of the resistant strains compared with the ECOFF. Intermethod agreement to classify the isolates was excellent (100%). All replicates in the repeatability and reproducibility experiments fell within the normal range. CONCLUSIONS: The MGIT960 system proved to be highly stable, reproducible and accurate relative to the M7H11 agar method for determining the bedaquiline MIC. The small margin between the suggested ECOFF and the lowest MIC for the mutant strains risks making both methods prone to discordant results. Further validation in clinical settings linked to treatment outcome data is needed.