Bedaquiline resistance in patients with drug-resistant tuberculosis in Cape Town, South Africa: a retrospective longitudinal cohort study.

BACKGROUND: Bedaquiline is a life-saving tuberculosis drug undergoing global scale-up. People at risk of weak tuberculosis drug regimens are a priority for novel drug access despite the potential source of Mycobacterium tuberculosis-resistant strains. We aimed to characterise bedaquiline resistance in individuals who had sustained culture positivity during bedaquiline-based treatment. METHODS: We did a retrospective longitudinal cohort study of adults (aged ≥18 years) with culture-positive pulmonary tuberculosis who received at least 4 months of a bedaquiline-containing regimen from 12 drug-resistant tuberculosis treatment facilities in Cape Town, South Africa, between Jan 20, 2016, and Nov 20, 2017. Sputum was programmatically collected at baseline (ie, before bedaquiline initiation) and each month to monitor treatment response per the national algorithm. The last available isolate from the sputum collected at or after 4 months of bedaquiline was designated the follow-up isolate. Phenotypic drug susceptibility testing for bedaquiline was done on baseline and follow-up isolates in MGIT960 media (WHO-recommended critical concentration of 1 µg/mL). Targeted deep sequencing for Rv0678, atpE, and pepQ, as well as whole-genome sequencing were also done. FINDINGS: In total, 40 (31%) of 129 patients from an estimated pool were eligible for this study. Overall, three (8%) of 38 patients assessable by phenotypic drug susceptibility testing for bedaquiline had primary resistance, 18 (47%) gained resistance (acquired or reinfection), and 17 (45%) were susceptible at both baseline and follow-up. Several Rv0678 and pepQ single-nucleotide polymorphisms and indels were associated with resistance. Although variants occurred in Rv0676c and Rv1979c, these variants were not associated with resistance. Targeted deep sequencing detected low-level variants undetected by whole-genome sequencing; however, none were in genes without variants already detected by whole-genome sequencing. Patients with baseline fluoroquinolone resistance, clofazimine exposure, and four or less effective drugs were more likely to have bedaquiline-resistant gain. Resistance gain was primarily due to acquisition; however, some reinfection by resistant strains occurred. INTERPRETATION: Bedaquiline-resistance gain, for which we identified risk factors, was common in these programmatically treated patients with sustained culture positivity. Our study highlights risks associated with implementing life-saving new drugs and shows evidence of bedaquiline-resistance transmission. Routine drug susceptibility testing should urgently accompany scale-up of new drugs; however, rapid drug susceptibility testing for bedaquiline remains challenging given the diversity of variants observed. FUNDING: Doris Duke Charitable Foundation, US National Institute of Allergy and Infectious Diseases, South African Medical Research Council, National Research Foundation, Research Foundation Flanders, Stellenbosch University Faculty of Medicine Health Sciences, South African National Research Foundation, Swiss National Science Foundation, and Wellcome Trust.

Xpert MTB/RIF Ultra on contaminated liquid cultures for tuberculosis and rifampicin-resistance detection: a diagnostic accuracy evaluation.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is a widely used rapid front-line tuberculosis and rifampicin-susceptibility testing. Mycobacterium Growth Indicator Tube (MGIT) 960 liquid culture is used as an adjunct but is vulnerable to contamination. We aimed to assess whether Ultra can be used on to-be-discarded contaminated cultures. METHODS: We stored contaminated MGIT960 tubes (growth-positive, acid-fast bacilli [AFB]-negative) originally inoculated at a high-volume laboratory in Cape Town, South Africa, to diagnose patients with presumptive pulmonary tuberculosis. Patients who had no positive tuberculosis results (smear, Ultra, or culture) at contamination detection and had another, later specimen submitted within 3 months of the contaminated specimen were selected. We evaluated the sensitivity and specificity of Ultra on contaminated growth from the first culture for tuberculosis (next-available non-contaminated culture result reference standard) and rifampicin resistance (vs MTBDRplus on a later isolate). We calculated potential time-to-diagnosis improvements and also evaluated the immunochromatographic MPT64 TBc assay. FINDINGS: Between June 1 and Aug 31, 2019, 36 684 specimens from 26 929 patients were processed for diagnostic culture. 2402 (7%) cultures from 2186 patients were contaminated. 1068 (49%) of 2186 patients had no other specimen submitted. After 319 exclusions, there were 799 people with at least one repeat specimen submitted; of these, we included in our study 246 patients (31%) with a culture-positive repeat specimen and 429 patients (54%) with a culture-negative repeat specimen. 124 patients (16%) with a culture-contaminated repeat specimen were excluded. When Ultra was done on the initial contaminated growth, sensitivity was 89% (95% CI 84-94) for tuberculosis and 95% (75-100) for rifampicin-resistance detection, and specificity was 95% (90-98) for tuberculosis and 98% (93-100) for rifampicin-resistance detection. If our approach were used the day after contamination detection, the time to tuberculosis detection would improve by a median of 23 days (IQR 13-45) and provide a result in many patients who had none. MPT64 TBc had a sensitivity of 5% (95% CI 0-25). INTERPRETATION: Ultra on AFB-negative growth from contaminated MGIT960 tubes had high sensitivity and specificity, approximating WHO criteria for sputum test target product performance and exceeding drug susceptibility testing. Our approach could mitigate negative effects of culture contamination, especially when repeat specimens are not submitted. FUNDING: The European & Developing Countries Clinical Trials Partnership, National Institutes of Health.

Non-actionable Results, Accuracy, and Effect of First- and Second-line Line Probe Assays for Diagnosing Drug-Resistant Tuberculosis, Including on Smear-Negative Specimens, in a High-Volume Laboratory.

BACKGROUND: Rapid tuberculosis (TB) drug susceptibility testing (DST) is crucial. Genotype MTBDRsl is a widely deployed World Health Organization (WHO)-endorsed assay. Programmatic performance data, including non-actionable results from smear-negative sputum, are scarce. METHODS: Sputa from Xpert MTB/RIF individuals (n = 951) were routinely-tested using Genotype MTBDRplus and MTBDRsl (both version 2). Phenotypic DST was the second-line drug reference standard. Discrepant results underwent Sanger sequencing. FINDINGS: 89% (849 of 951) of individuals were culture-positive (56%, 476 of 849 smear-negative). MTBDRplus had at least 1 nonactionable result (control and/or TB-detection bands absent or invalid, precluding resistance reporting) in 19% (92 of 476) of smear-negatives; for MTBDRsl, 40% (171 of 427) were nonactionable (28%, 120 of 427 false-negative TB; 17%, 51 of 427 indeterminate). In smear-negatives, MTBDRsl sensitivity for fluoroquinolones was 84% (95% confidence interval, 67%-93), 81% (54%-95%) for second-line injectable drugs, and 57% (28%-82%) for both. Specificities were 93% (89%-98%), 88% (81%-93%), and 97% (91%-99%), respectively. Twenty-three percent (172 of 746) of Xpert rifampicin-resistant specimens were MTBDRplus isoniazid-susceptible. Days-to-second-line-susceptibility reporting with the programmatic advent of MTBDRsl improved (6 [5-7] vs 37 [35-46]; P < .001). CONCLUSIONS: MTBDRsl did not generate a result in 4 of 10 smear-negatives, resulting in substantial missed resistance. However, if MTBDRsl generates an actionable result, that is accurate in ruling-in resistance. Isoniazid DST remains crucial. This study provides real-world, direct, second-line susceptibility testing performance data on non-actionable results (that, if unaccounted for, cause an overestimation of test utility), accuracy, and care cascade impact.

Spatial heterogeneity of extensively drug resistant-tuberculosis in Western Cape Province, South Africa.

Tuberculosis (TB) remains a leading infectious disease killer globally. Treatment outcomes are especially poor among people with extensively drug-resistant (XDR) TB, until recently defined as rifampicin-resistant (RR) TB with resistance to an aminoglycoside (amikacin) and a fluoroquinolone (ofloxacin). We used laboratory TB test results from Western Cape province, South Africa between 2012 and 2015 to identify XDR-TB and pre-XDR-TB (RR-TB with resistance to one second-line drug) spatial hotspots. We mapped the percentage and count of individuals with RR-TB that had XDR-TB and pre-XDR-TB across the province and in Cape Town, as well as amikacin-resistant and ofloxacin-resistant TB. We found the percentage of pre-XDR-TB and the count of XDR-TB/pre-XDR-TB highly heterogeneous with geographic hotspots within RR-TB high burden areas, and found hotspots in both percentage and count of amikacin-resistant and ofloxacin-resistant TB. The spatial distribution of percentage ofloxacin-resistant TB hotspots was similar to XDR-TB hotspots, suggesting that fluoroquinolone-resistace is often the first step to additional resistance. Our work shows that interventions used to reduce XDR-TB incidence may need to be targeted within spatial locations of RR-TB, and further research is required to understand underlying drivers of XDR-TB transmission in these locations.

Frequent Suboptimal Thermocycler Ramp Rate Usage Negatively Impacts GenoType MTBDRsl VER 2.0 Performance for Second-Line Drug-Resistant Tuberculosis Diagnosis.

Strengthening second-line drug-resistant tuberculosis (TB) detection is a priority. GenoType MTBDRplus VER 2.0 performance is reduced with non-recommended ramp rate usage (temperature change speed between PCR cycles); however, ramp rate's effect on GenoType MTBDRsl VER 2.0 (MTBDRsl) performance, is unknown. Fifty-two Xpert MTB/RIF Ultra-positive rifampicin-resistant smear-negative sputa and a Mycobacterium tuberculosis dilution series were tested at a manufacturer-recommended (2.2°C/second) or suboptimal (4.0°C/second) ramp rate. M. tuberculosis-complex-DNA positivity, indeterminates, fluoroquinolone- and second-line injectable-resistance accuracy, banding differences, and, separately, inter-reader variability were assessed. Five (39%) of 13 re-surveyed laboratories did not use the manufacturer-recommended ramp rate. On sputum, 2.2°C/second improved indeterminates versus 4.0°C/second (0 of 52 versus 7 of 51; P = 0.006), incorrect drug-class diagnostic calls (0 of 104 versus 6 of 102; P = 0.013), and incorrect banding calls (0 of 1300 versus 54 of 1275; P < 0.001). Similarly, 2.2°C/second improved valid results [(52 of 52 versus 41 of 51; +21% (P = 0.001)] and banding call inter-reader variability [34 of 1300 (3%) versus 52 of 1300 (4%); P = 0.030]. At the suboptimal ramp rate, false-resistance and false-susceptible calls resulted from wild-type band absence rather than mutant band appearance, resulting in misclassification of moxifloxacin resistance level from high-to-low. Suboptimal ramp rate contributes to poor MTBDRsl performance. Laboratories must ensure that the manufacturer-recommended ramp rate is used.

Diagnostic accuracy of the FluoroType MTB and MTBDR VER 2.0 assays for the centralized high-throughput detection of Mycobacterium tuberculosis complex DNA and isoniazid and rifampicin resistance.

OBJECTIVES: To evaluate the accuracy of two new molecular diagnostic tests for the detection of drug-resistant tuberculosis, the FluoroType MTB and MTBDR VER 2.0 assays, in combination with manual and automated DNA extraction methods. METHODS: Sputa from 360 Xpert Ultra Mycobacterium tuberculosis complex (MTBC)-positive patients and 250 Xpert Ultra MTBC-negative patients were tested. GenoType MTBDRplus served as reference for MTBC and drug resistance detection. Sanger sequencing was used to resolve discrepancies. RESULTS: FluoroType MTB VER 2.0 showed similar MTBC sensitivity compared with FluoroType MTBDR VER 2.0 (manual DNA extraction: 91.6% (294/321) versus 89.8% (291/324); p 0.4); automated DNA extraction: 92.1% (305/331) versus 87.7% (291/332); p 0.05)). FluoroType MTBDR VER2.0 showed comparable diagnostic accuracy to FluoroType MTBDR VER1.0 as previously reported for the detection of MTBC and rifampicin and isoniazid resistance. CONCLUSIONS: The FluoroType MTB and MTBDR VER 2.0 assays together with an automated DNA extraction and PCR set-up platform may improve laboratory operational efficiency for the diagnosis of MTBC and resistance to rifampicin and isoniazid and show promise for the implementation in a centralized molecular drug susceptibility testing model.

Decentralized Care for Rifampin-Resistant Tuberculosis, Western Cape, South Africa.

In 2011, South Africa implemented a policy to decentralize treatment for rifampin-resistant tuberculosis (TB) to reduce durations of hospitalization and enable local treatment. We assessed policy implementation in Western Cape Province, where services expanded from 6 specialized TB hospitals to 406 facilities, by analyzing National Health Laboratory Service data on TB during 2012-2015. We calculated the percentage of patients who visited a TB hospital <1 year after rifampin-resistant TB diagnosis, the median duration of their hospitalizations, and the total distance between facilities visited. We assessed temporal changes with linear regression and stratified results by location. Of 2,878 patients, 65% were from Cape Town. In Cape Town, 29% visited a TB hospital; elsewhere, 68% visited a TB hospital. We found that hospitalizations and travel distances were shorter in Cape Town than in the surrounding areas.

Xpert MTB/RIF Ultra and Xpert MTB/RIF for diagnosis of tuberculosis in an HIV-endemic setting with a high burden of previous tuberculosis: a two-cohort diagnostic accuracy study.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is a new test for tuberculosis undergoing global roll-out. We assessed the performance of Ultra compared with Xpert MTB/RIF (Xpert) in an HIV-endemic setting where previous tuberculosis is frequent and current test performance is suboptimal. METHODS: In this two-cohort diagnostic accuracy study, we used sputum samples from patients in South Africa to evaluate the accuracy of Ultra and Xpert against a single culture reference standard. For the first cohort (cohort A), we recruited adults (aged ≥18 years) with symptoms of presumptive tuberculosis at Scottsdene clinic in Cape Town, South Africa. We collected three sputum samples from each patient in cohort A, two at the first visit of which one was tested using Xpert and the other was tested using culture, and one sample the next morning which was tested using Ultra. In a separate cohort of patients with presumptive tuberculosis and recent previous tuberculosis (≤2 years) who had submitted sputum samples to the National Health Laboratory Services (cohort B), decontaminated sediments were, after processing, randomly allocated (1:1) for testing with Ultra or Xpert. For both cohorts we calculated the sensitivity and specificity of Ultra and Xpert and evaluated the effects of different methods of interpreting Ultra trace results. FINDINGS: Between Feb 6, 2016, and Feb 2, 2018, we recruited 302 people into cohort A, all of whom provided sputum samples and 239 were included in the head-to-head analyses of Ultra and Xpert. For cohort B, we collected sputum samples from eligible patients who had submitted samples between Dec 6, 2016, and Dec 21, 2017, to give a cohort of 831 samples, of which 352 were eligible for inclusion in analyses and randomly assigned to Ultra (n=173) or Xpert (n=179). In cohort A, Ultra gave more non-actionable results (not positive or negative) than did Xpert (28 [10%] 275 vs 14 [5%] 301; p=0·011). In the head-to-head analysis, in smear-negative patients, sensitivity of Ultra was 80% (95% CI 64-90) and of Xpert was 73% (57-85; p=0·45). Overall, specificity of Ultra was lower than that of Xpert (90% [84-94] vs 99% [95-100]; p=0·001). In cohort B, overall sensitivity was 92% (81-98) for Xpert versus 86% (73-95; p=0·36) for Ultra and overall specificity was 69% (60-77) for Ultra versus 84% (78-91; p=0·005) for Xpert. Ultra specificity estimates improved after reclassification of results with the lowest Ultra-positive semiquantitation category (trace) to negative (15% [8-22]). In cohort A, the positive predictive value (PPV) for Ultra was 78% (67-87) and for Xpert was 96% (87-99; p=0·004); in cohort B, the PPV for Ultra was 50% (43-57) and for Xpert was 70% (61-78; p=0·014). Ultra PPV estimates in previously treated patients were low: at 15% tuberculosis prevalence, half of Ultra-positive patients with presumptive tuberculosis would be culture negative, increasing to approximately 70% in patients with recent previous tuberculosis. In cohort B, 21 (28%) of 76 samples that were Ultra positive were rifampicin indeterminate (all trace) and, like cohort A, most were culture negative (19 [90%] of 21). INTERPRETATION: In a setting with a high burden of previous tuberculosis, Ultra generated more non-actionable results and had diminished specificity compared with Xpert. In patients with recent previous tuberculosis, a quarter of Ultra-positive samples were indeterminate for rifampicin resistance and culture negative, suggesting that additional drug-resistance testing will probably be unsuccessful. Our data have implications for the handling of Ultra-positive results in patients with previous tuberculosis in high burden settings. FUNDING: South African Medical Research Council, the EDCTP2 program, and the Faculty of Medicine and Health Sciences, Stellenbosch University.

Extract from used Xpert MTB/RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk.

Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra's predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on  CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >"low semi-quantitation") detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10-3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.

Using routinely collected laboratory data to identify high rifampicin-resistant tuberculosis burden communities in the Western Cape Province, South Africa: A retrospective spatiotemporal analysis.

BACKGROUND: South Africa has the highest tuberculosis incidence globally (781/100,000), with an estimated 4.3% of cases being rifampicin resistant (RR). Control and elimination strategies will require detailed spatial information to understand where drug-resistant tuberculosis exists and why it persists in those communities. We demonstrate a method to enable drug-resistant tuberculosis monitoring by identifying high-burden communities in the Western Cape Province using routinely collected laboratory data. METHODS AND FINDINGS: We retrospectively identified cases of microbiologically confirmed tuberculosis and RR-tuberculosis from all biological samples submitted for tuberculosis testing (n = 2,219,891) to the Western Cape National Health Laboratory Services (NHLS) between January 1, 2008, and June 30, 2013. Because the NHLS database lacks unique patient identifiers, we performed a series of record-linking processes to match specimen records to individual patients. We counted an individual as having a single disease episode if their positive samples came from within two years of each other. Cases were aggregated by clinic location (n = 302) to estimate the percentage of tuberculosis cases with rifampicin resistance per clinic. We used inverse distance weighting (IDW) to produce heatmaps of the RR-tuberculosis percentage across the province. Regression was used to estimate annual changes in the RR-tuberculosis percentage by clinic, and estimated average size and direction of change was mapped. We identified 799,779 individuals who had specimens submitted from mappable clinics for testing, of whom 222,735 (27.8%) had microbiologically confirmed tuberculosis. The study population was 43% female, the median age was 36 years (IQR 27-44), and 10,255 (4.6%, 95% CI: 4.6-4.7) cases had documented rifampicin resistance. Among individuals with microbiologically confirmed tuberculosis, 8,947 (4.0%) had more than one disease episode during the study period. The percentage of tuberculosis cases with rifampicin resistance documented among these individuals was 11.4% (95% CI: 10.7-12.0). Overall, the percentage of tuberculosis cases that were RR-tuberculosis was spatially heterogeneous, ranging from 0% to 25% across the province. Our maps reveal significant yearly fluctuations in RR-tuberculosis percentages at several locations. Additionally, the directions of change over time in RR-tuberculosis percentage were not uniform. The main limitation of this study is the lack of unique patient identifiers in the NHLS database, rendering findings to be estimates reliant on the accuracy of the person-matching algorithm. CONCLUSIONS: Our maps reveal striking spatial and temporal heterogeneity in RR-tuberculosis percentages across this province. We demonstrate the potential to monitor RR-tuberculosis spatially and temporally with routinely collected laboratory data, enabling improved resource targeting and more rapid locally appropriate interventions.

Diagnostic Accuracy and Utility of FluoroType MTBDR, a New Molecular Assay for Multidrug-Resistant Tuberculosis.

Most cases of multidrug-resistant (MDR) tuberculosis (TB) are never diagnosed (328,300 of the ∼490,000 cases in 2016 were missed). The Xpert MTB/RIF assay detects resistance only to rifampin, despite ∼20% of rifampin-resistant cases being susceptible to isoniazid (a critical first-line drug). Consequently, many countries require further testing with the GenoType MTBDRplus assay. However, MTBDRplus is not recommended for use on smear-negative specimens, and thus, many specimens require culture-based drug susceptibility testing. Furthermore, MTBDRplus requires specialized expertise, lengthy hands-on time, and significant laboratory infrastructure and interpretation is not automated. To address these gaps, we evaluated the accuracy of the FluoroType MTBDR (FluoroType) assay. Sputa from 244 smear-positive and 204 smear-negative patients with presumptive TB (Xpert MTB positive, n = 343) were tested. Culture and MTBDRplus on isolates served as reference standards (for active TB and MDR-TB, respectively). Sanger sequencing and MTBDRplus, both of which were performed on sputa, were used to resolve discrepancies. The sensitivity of FluoroType for the detection of M. tuberculosis complex was 98% (95% confidence interval [CI], 95 to 99%) and 92% (95% CI, 84 to 96%) for smear-positive and smear-negative specimens, respectively (232/237 versus 90/98 specimens; P < 0.009). The sensitivity and specificity for smear-negative specimens were 100% and 97%, respectively, for rifampin resistance; 100% and 98%, respectively, for isoniazid resistance; and 100% and 100%, respectively, for MDR-TB. FluoroType identified 98%, 97%, and 97% of the rpoB, katG, and inhA promoter mutations, respectively. FluoroType has excellent sensitivity with sputa equivalent to that of MTBDRplus with the isolates and can provide rapid drug susceptibility testing for rifampin and isoniazid. In addition, the capacity of FluoroType to simultaneously identify virtually all mutations in the rpoB, katG, and inhA promoter may be useful for individualized treatment regimens.

Implications of Failure to Routinely Diagnose Resistance to Second-Line Drugs in Patients With Rifampicin-Resistant Tuberculosis on Xpert MTB/RIF: A Multisite Observational Study.

BACKGROUND.: Xpert MTB/RIF (Xpert) detects rifampicin-resistant tuberculosis (RR-tuberculosis), enabling physicians to rapidly initiate a World Health Organization-recommended 5-drug regimen while awaiting second-line drug-susceptibility test (DST) results. We quantified the second-line DST results time and proportion of patients potentially placed on suboptimal therapy. METHODS.: We included RR-tuberculosis patients detected using Xpert at the South African National Health Laboratory Services (NHLS) of the Western Cape between November 2011 and June 2013 and at Eastern Cape, Free State, and Gauteng NHLS between November 2012 and December 2013. We calculated time from specimen collection to phenotypic second-line DST results. We identified isoniazid and ethionamide resistance mutations on line probe assay and performed pyrazinamide sequencing. RESULTS.: Among 1332 RR-tuberculosis patients, only 44.7% (596) had second-line DST for both fluoroquinolones and second-line injectable: 55.8% (466 of 835) in the Western Cape and 26.2% (130 of 497) in the other provinces. Patients with smear negative disease and age ≤10 years were less likely to have a result (risk ratio [RR] = 0.72; 95% CI, 0.64-0.81 and RR = 0.49; 95% CI, 0.26-0.79). Median time to second-line DST was 53 days (range, 8-259). Of the 252 patients with complete second-line DST, 101 (40.1%) potentially initiated a suboptimal regimen: 46.8% in the Western Cape and 25.3% in the other provinces. CONCLUSIONS.: Many South Africans diagnosed with RR-tuberculosis by Xpert initiate a suboptimal regimen, with information to adjust therapy available in half of all patients after a median 7 weeks. Algorithm completion and time delays remain challenging.

Improved method for collection of sputum for tuberculosis testing to ensure adequate sample volumes for molecular diagnostic testing.

The quality and quantity of sputum collected has an important impact on the laboratory diagnosis of pulmonary TB. We conducted a pilot study to assess a new collection cups for the collection of sputum for the diagnosis of pulmonary tuberculosis. The pilot study utilized the standard collection cup in South Africa demonstrating a mean collection volume of 2.86±2.36SDml for 198 samples; 19% of the specimens contained <1ml and 12% contained >5ml. We designed and tested two novel sputum cups with a narrow bottom section and clear minimum and maximum markings to allow patients and clinicians to know whether sufficient sputum volume has been produced. The cups differed in their shape and manufacturing approach. The two options also support different mixing approaches being considered for a highly sensitive companion TB-screening assay being developed at Northwestern University (XtracTB assay). Sputum was collected from 102 patients at Nolungile Youth Centre, Khayelitsha, Cape Town, South Africa for a total of 204 samples. The mean volumes collected from the two cups were 2.70±0.88SDml and 2.88±0.89SDml. While the mean volumes of current and novel cups are similar, the volume ranges collected with the novel cups were narrower, and 98% of the specimen volumes were within the target range. Only 4 samples contained >5ml, but none were >6ml, and none of the specimens contained <1ml. The number of coughs that produced the samples, patient HIV and TB status plus qualitative descriptions of the sputum specimens were also evaluated.

Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study.

BACKGROUND: The emergence of programmatically incurable tuberculosis threatens to destabilise control efforts. The aim of this study was to collect prospective patient-level data to inform treatment and containment strategies. METHODS: In a prospective cohort study, 273 South African patients with extensively drug-resistant tuberculosis, or resistance beyond extensively drug-resistant tuberculosis, were followed up over a period of 6 years. Transmission dynamics, infectiousness, and drug susceptibility were analysed in a subset of patients from the Western Cape using whole-genome sequencing (WGS; n=149), a cough aerosol sampling system (CASS; n=26), and phenotypic testing for 18 drugs (n=179). FINDINGS: Between Oct 1, 2008, and Oct 31, 2012, we enrolled and followed up 273 patients for a median of 20·3 months (IQR 9·6-27·8). 203 (74%) had programmatically incurable tuberculosis and unfavourable outcomes (treatment failure, relapse, default, or death despite treatment with a regimen based on capreomycin, aminosalicylic acid, or both). 172 (63%) patients were discharged home, of whom 104 (60%) had an unfavourable outcome. 54 (31%) home-discharged patients had failed treatment, with a median time to death after discharge of 9·9 months (IQR 4·2-17·4). 35 (20%) home-discharged cases were smear-positive at discharge. Using CASS, six (23%) of 26 home-discharged cases with data available expectorated infectious culture-positive cough aerosols in the respirable range (<5 µm), and most reported inter-person contact with suboptimal protective mask usage. WGS identified 17 (19%) of the 90 patients (with available sequence data) that were discharged home before the diagnosis of 20 downstream cases of extensively drug-resistant tuberculosis with almost identical sequencing profiles suggestive of community-based transmission (five or fewer single nucleotide polymorphisms different and with identical resistance-encoding mutations for 14 drugs). 11 (55%) of these downstream cases had HIV co-infection and ten (50%) had died by the end of the study. 22 (56%) of 39 isolates in patients discharged home after treatment failure were resistant to eight or more drugs. However, five (16%) of 31 isolates were susceptible to rifabutin and more than 90% were likely to be sensitive to linezolid, bedaquiline, and delamanid. INTERPRETATION: More than half of the patients with programmatically incurable tuberculosis were discharged into the community where they remained for an average of 16 months, were at risk of expectorating infectious cough aerosols, and posed a threat of transmission of extensively drug-resistant tuberculosis. Urgent action, including appropriate containment strategies, is needed to address this situation. Access to delamanid, bedaquiline, linezolid, and rifabutin, when appropriate, must be accelerated along with comprehensive drug susceptibility testing. FUNDING: UK Medical Research Council, South African Medical Research Council, South African National Research Foundation, European & Developing Countries Clinical Trials Partnership, Oppenheimer Foundation, Newton Fund, Biotechnology and Biological Sciences Research Council, King Abdullah University of Science & Technology.

Prevalence of pyrazinamide resistance across the spectrum of drug resistant phenotypes of Mycobacterium tuberculosis.

Pyrazinamide resistance is largely unknown in the spectrum of drug resistant phenotypes. We summarize data on PZA resistance in clinical isolates from South Africa. PZA DST should be performed when considering its inclusion in treatment of patients with rifampicin-resistant TB or MDR-TB.