ABSTRACT This study evaluated thin-layer agar (TLA) as a faster alternative for both indirect minimum inhibitory concentration (MIC) determination of bedaquiline (BDQ) from culture isolates and direct drug-susceptibility testing (DST) from sputum samples. Indirect BDQ-MIC results from TLA were compared to the established 7H11 solid DST. Direct-TLA-DST performance was assessed using 143 baseline sputum samples from rifampicin-resistant TB cases. Direct-TLA tested susceptibility to rifampicin, isoniazid, levofloxacin, and BDQ, with results compared to Löwenstein-Jensen (LJ) and MGIT. For indirect BDQ-MIC determination, TLA accurately identified H37Rv MICs within the WHO control range (0.015–0.12μg/mL). Optimal results were obtained with standard incubation and day 7 reading of the plates. TLA correctly classified all wild-type clinical strains as BDQ-susceptible and detected 9 out of 10 BDQ-resistant strains with elevated MICs. Direct-TLA-DST detected MTB in 53.8% of samples, compared to 55.9% on LJ and 69.4% in MGIT. Uninterpretable results due to contamination or medium drying were low (4.9%). The median time to result was 15 days for smear-positive and 22 days for smear-negative samples, faster than WHO-endorsed methods. Sensitivity was 100% for rifampicin and 87.8% for isoniazid, with a specificity of 100% for all drugs except isoniazid (96.2%). No BDQ nor levofloxacin resistance was detected, thus direct TLA sensitivity could not be assessed. In conclusion, direct-TLA-DST offers a reliable and faster alternative to conventional DST methods for BDQ and other anti-TB drugs. Essentially, this technique can be operated at BioSafety Level 2, allowing decentralization of pDST for managing drug-resistant TB in settings with limited laboratory infrastructure. IMPORTANCE This paper addresses the critical need for faster direct drug-susceptibility testing (DST) on sputum, especially for bedaquiline (BDQ), which is a key drug in treating drug-resistant TB. Currently, there is a lack of rapid, reliable methods for direct BDQ testing from sputum samples, limiting timely and accurate treatment decisions and monitoring. By demonstrating the potential of thin-layer agar (TLA) for direct BDQ-MIC determination, this study offers a promising solution that could significantly improve patient care.

BACKGROUND:Pre-extensively drug-resistant (pre-XDR) tuberculosis (ie, multidrug-resistant or rifampicin-resistant with additional resistance to any fluoroquinolone) is difficult to treat. endTB-Q aimed to evaluate the efficacy and safety of bedaquiline, delamanid, linezolid, and clofazimine (BDLC) compared with the standard of care for patients with pre-XDR tuberculosis. METHODS:This open-label, multicentre, stratified, non-inferiority, randomised, controlled, phase 3 trial was conducted in ten hospitals in India, Kazakhstan, Lesotho, Pakistan, Peru, and Viet Nam. Participants aged 15 years or older who had pulmonary tuberculosis with resistance to rifampicin and fluoroquinolones were included. Participants were randomly assigned (2:1) to the BDLC group (all-oral bedaquiline 400 mg once per day for 2 weeks followed by 200 mg three times per week, delamanid 100 mg twice per day, linezolid 600 mg once per day for 16 weeks and then either 300 mg once per day or 600 mg three times per week, and clofazimine 100 mg once per day) or the control group (individualised WHO-recommended longer standard of care). Randomisation was stratified by country and baseline disease extent. BDLC was administered for 39 weeks (9-month regimen) for extensive disease and 24 weeks (6-month regimen) for limited disease and extended to 9 months for those with a positive culture at 8 weeks or later or a missing 8-week culture result. Site staff and participants were not masked, whereas investigators and laboratory staff were masked to treatment assignment. The primary endpoint was favourable outcome (two consecutive, negative cultures including one between weeks 65 and 73; or favourable bacteriological, radiological, and clinical evolution) at week 73 after randomisation in the modified intention-to-treat (mITT) and per-protocol populations. We report the risk differences adjusted for stratification variables, with a non-inferiority margin of -12%. This trial is registered with ClinicalTrials.gov, NCT03896685. FINDINGS:Between April 4, 2020, and March 28, 2023, 1030 individuals were screened and 324 (31%) were randomly assigned (219 to the BDLC group and 105 to the control group). 114 (46%) participants were female and 133 (54%) were male. Median age was 30·5 years (IQR 21·6-43·0). 157 (64%) participants had extensive disease at baseline. In the BDLC group, 47 (29%) of 163 were assigned to receive the 6-month regimen and 116 (71%) the 9-month regimen. The core regimen of BDLC plus one or more other drugs was used for 76 (91%) of 84 participants in the control group. At week 73, favourable outcome was reached by 141 (87%) participants in the BDLC group versus 75 (89%) in the control group in the mITT population (adjusted risk difference 0·2% [95% CI -9·1 to 9·5]; pnon-inferiority=0·0051) and by 138 (88%) of 157 versus 71 (93%) of 76 in the per-protocol population (adjusted risk difference -3·5% [-12·8 to 5·9]; pnon-inferiority=0·037). Overall non-inferiority was not shown. 145 (68%) of 213 participants in the BDLC group and 77 (73%) of 105 in the control group had at least one grade 3 or higher adverse event, with eight (4%) and two (2%) all-cause deaths by week 73, respectively. INTERPRETATION:The shortened BDLC strategy was not non-inferior to the control. Accumulating evidence suggests that this patient population might require longer, reinforced regimens. FUNDING:Unitaid, Médecins Sans Frontières, Partners In Health, Interactive Research and Development, Ramón Areces Foundation, the Jung Foundation for Science and Research, Research Foundation-Flanders. TRANSLATIONS:For the Hindi, Marathi, Spanish, Vietnamese, Russian, Urdu and French translations of the abstract see Supplementary Materials section.

Rifampicin plays a pivotal role in the first-line treatment of tuberculosis.Mutations in the rpoB gene confer varying levels of rifampicin resistance (1).Historically, phenotypic drug susceptibility testing

Background Treatment for fluoroquinolone-resistant multidrug-resistant/rifampicin-resistant tuberculosis (pre-XDR TB) often lasts longer than treatment for less resistant strains, yields worse efficacy results, and causes substantial toxicity. The newer anti-tuberculosis drugs, bedaquiline and delamanid, and repurposed drugs clofazimine and linezolid, show great promise for combination in shorter, less-toxic, and effective regimens. To date, there has been no randomized, internally and concurrently controlled trial of a shorter, all-oral regimen comprising these newer and repurposed drugs sufficiently powered to produce results for pre-XDR TB patients. Methods endTB-Q is a phase III, multi-country, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of a treatment strategy for patients with pre-XDR TB. Study participants are randomized 2:1 to experimental or control arms, respectively. The experimental arm contains bedaquiline, linezolid, clofazimine, and delamanid. The control comprises the contemporaneous WHO standard of care for pre-XDR TB. Experimental arm duration is determined by a composite of smear microscopy and chest radiographic imaging at baseline and re-evaluated at 6 months using sputum culture results: participants with less extensive disease receive 6 months and participants with more extensive disease receive 9 months of treatment. Randomization is stratified by country and by participant extent-of-TB-disease phenotype defined according to screening/baseline characteristics. Study participation lasts up to 104 weeks post randomization. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 324 participants across 2 arms affords at least 80% power to show the non-inferiority, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per-protocol populations. Discussion This internally controlled study of shortened treatment for pre-XDR TB will provide urgently needed data and evidence for clinical and policy decision-making around the treatment of pre-XDR TB with a four-drug, all-oral, shortened regimen. Trial registration ClinicalTrials.Gov NCT03896685. Registered on 1 April 2018; the record was last updated for study protocol version 4.3 on 17 March 2023.

Background Development of rapid biomarker-based tests that can diagnose tuberculosis using non-sputum samples is a priority for tuberculosis control. We aimed to compare the diagnostic accuracy of the novel Fujifilm SILVAMP TB LAM (FujiLAM) assay with the WHO-recommended Alere Determine TB-LAM Ag test (AlereLAM) using urine samples from HIV-positive patients. Methods We did a diagnostic accuracy study at five outpatient public health facilities in Uganda, Kenya, Mozambique, and South Africa. Eligible patients were ambulatory HIV-positive individuals (aged >= 15 years) with symptoms of tuberculosis irrespective of their CD4 T-cell count (group 1), and asymptomatic patients with advanced HIV disease (CD4 count < 200 cells per mu L, or HIV clinical stage 3 or 4; group 2). All participants underwent clinical examination, chest x-ray, and blood sampling, and were requested to provide a fresh urine sample, and two sputum samples. FujiLAM and AlereLAM urine assays, Xpert MTB/RIF Ultra assay on sputum or urine, sputum culture for Mycobacterium tuberculosis, and CD4 count were systematically carried out for all patients. Sensitivity and specificity of FujiLAM and AlereLAM were evaluated against microbiological and composite reference standards. Findings Between Aug 24, 2020 and Sept 21, 2021, 1575 patients (823 [52.3%] women) were included in the study: 1031 patients in group 1 and 544 patients in group 2. Tuberculosis was microbiologically confirmed in 96 (9 & BULL;4%) of 1022 patients in group 1 and 18 (3.3%) of 542 patients in group 2. Using the microbiological reference standard, FujiLAM sensitivity was 60% (95% CI 51-69) and AlereLAM sensitivity was 40% (31-49; p < 0.001). Among patients with CD4 counts of less than 200 cells per mu L, FujiLAM sensitivity was 69% (57-79) and AlereLAM sensitivity was 52% (40-64; p=0.0218). Among patients with CD4 counts of 200 cells per mu L or higher, FujiLAM sensitivity was 47% (34-61) and AlereLAM sensitivity was 24% (14-38; p=0.0116). Using the microbiological reference standard, FujiLAM specificity was 87% (95% CI 85-89) and AlereLAM specificity was 86% (95 CI 84-88; p=0.941). FujiLAM sensitivity varied by lot number from 48% (34-62) to 76% (57-89) and specificity from 77% (72-81) to 98% (93-99). Interpretation Next-generation, higher sensitivity urine-lipoarabinomannan assays are potentially promising tests that allow rapid tuberculosis diagnosis at the point of care for HIV-positive patients. However, the variability in accuracy between FujiLAM lot numbers needs to be addressed before clinical use. Copyright (c) 2022 The Author(s). Published by Elsevier Ltd.

Background Non-sputum-based diagnostic approaches are crucial in children at high risk of disseminated tuberculosis (TB) who cannot expectorate sputum. We evaluated the diagnostic accuracy of stool Xpert MTB/RIF and urine AlereLAM tests in this group of children. Methods Hospitalised children with presumptive TB and either age <2 years, HIV-positive or with severe malnutrition were enrolled in a diagnostic cohort. At enrolment, we attempted to collect two urine, two stool and two respiratory samples. Urine and stool were tested with AlereLAM and Xpert MTB/RIF, respectively. Respiratory samples were tested with Xpert MTB/RIF and mycobacterial culture. Both a microbiological and a composite clinical reference standard were used. Results The study analysed 219 children; median age 16.4 months, 72 (32.9%) HIV-positive and 184 (84.4%) severely malnourished. 12 (5.5%) and 58 (28.5%) children had confirmed and unconfirmed TB, respectively. Stool and urine were collected in 21,9 (100%) and 216 (98.6%) children, respectively. Against the microbiological reference standard, the sensitivity and specificity of stool Xpert MTB/RIF was 50.0% (6/12, 95% CI 21.1-78.9%) and 99.1% (198/200, 95% 96.4-99.9%), while that of urine AlereLAM was 50.0% (6/12, 95% 21.1-78.9%) and 74.6% (147/197, 95% 67.9-80.5%), respectively. Against the composite reference standard, sensitivity was reduced to 11.4% (8/70) for stool and 26.2% (17/68) for urine, with no major difference by age group (<2 and >= 2 years) or HIV status. Conclusions The Xpert MTB/RIF assay has excellent specificity on stool, but sensitivity is suboptimal. Urine AIereLAM is compromised by poor sensitivity and specificity in children.

Inappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.https://bit.ly/3i43wb6

Background: Phenotypic drug-susceptibility testing (pDST), which relies on growth inhibition in the drug-containing media, remains a challenge for fastidious Mycobacterium tuberculosis complex (MTBc) isolates due to insufficient growth on the growth controls (GC). Middlebrook 7H11 (M7H11) medium contains casein hydrolysate, which may favor the growth of such strains. Method: In this study, we tested whether M7H11 reduces invalid results due to insufficient growth on the GCs and the turnaround time (TAT) of pDST for MTBc compared to Middlebrook 7H10 (M7H10) without affecting the accuracy of the pDST results and how it differs between rifampicin- and isoniazid-susceptible non multi-drug resistant (non-MDR), MDR and MDR with additional resistance to fluoroquinolones (Pre-XDR) MTBc isolates. We compared the proportions of invalid pDST results due to lack of growth on the GCs, TATs of valid parallel drug-susceptibility testings as an indicator of speed of MTBc growth, and colony-forming unit (CFU) count on the most diluted GC of the parallel pDSTs after equal incubation periods as an indicator of growth abundance on M7H11 and M7H10. We also analyzed the agreement between the pDST results of the same drug or drugs in the same drug class, tested in parallel on both media. Results: For MDR and pre-XDR isolates, relative to M7H10, M7H11 significantly reduced the occurrence of invalid pDST results due to insufficient growth on the GCs (odds ratio [OR] = ∞ [95% confidence interval (CI) 1.9–∞], P = 0.004 for MDR, OR = ∞ [95% CI 3.3–∞], P = 0.0001 for pre-XDR) and the TAT of pDSTs (OR = 17 [95% CI 2.6–710.4], P = 0.0001 for MDR, OR = 9.3 [95% CI 4.0–26.5], P < 0.0001 for pre-XDR). The growth abundance of MTBc on M7H11 was significantly higher compared to M7H10 (17 CFU on M7H10 vs. 28 on M7H11), irrespective of drug-resistance profiles. The agreement between the pDST results between the two media was high (Cohen's k > 0.98). Conclusion: Our study findings suggest that M7H11 is preferred over M7H10 for pDSTs of MTBc isolates.

Abstract Background Treatment of multidrug- and rifampin-resistant tuberculosis (MDR/RR-TB) is expensive, labour-intensive, and associated with substantial adverse events and poor outcomes. While most MDR/RR-TB patients do not receive treatment, many who do are treated for 18 months or more. A shorter all-oral regimen is currently recommended for only a sub-set of MDR/RR-TB. Its use is only conditionally recommended because of very low-quality evidence underpinning the recommendation. Novel combinations of newer and repurposed drugs bring hope in the fight against MDR/RR-TB, but their use has not been optimized in all-oral, shorter regimens. This has greatly limited their impact on the burden of disease. There is, therefore, dire need for high-quality evidence on the performance of new, shortened, injectable-sparing regimens for MDR-TB which can be adapted to individual patients and different settings. Methods endTB is a phase III, pragmatic, multi-country, adaptive, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of shorter treatment regimens containing new drugs for patients with fluoroquinolone-susceptible, rifampin-resistant tuberculosis. Study participants are randomized to either the control arm, based on the current standard of care for MDR/RR-TB, or to one of five 39-week multi-drug regimens containing newly approved and repurposed drugs. Study participation in all arms lasts at least 73 and up to 104 weeks post-randomization. Randomization is response-adapted using interim Bayesian analysis of efficacy endpoints. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 750 patients across 6 arms affords at least 80% power to detect the non-inferiority of at least 1 (and up to 3) experimental regimens, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per protocol populations. Discussion The lack of a safe and effective regimen that can be used in all patients is a major obstacle to delivering appropriate treatment to all patients with active MDR/RR-TB. Identifying multiple shorter, safe, and effective regimens has the potential to greatly reduce the burden of this deadly disease worldwide. Trial registration ClinicalTrials.gov Identifier NCT02754765. Registered on 28 April 2016; the record was last updated for study protocol version 3.3, on 27 August 2019.

Xpert MTB/RIF rapidly detects resistance to rifampicin (RR); however, this test misses I491F-RR conferring the rpoB mutation, common in southern Africa. In addition, Xpert MTB/RIF does not distinguish between viable and dead Mycobacterium tuberculosis (MTB).

29 BACKGROUND: Xpert®MTB/RIF rapidly detects resistance to rifampicin (RR), however this 30 test misses the I491F-RR conferring rpoB mutation, common in Southern Africa. In addition, 31 Xpert®MTB/RIF does not distinguish between viable and dead Mycobacterium tuberculosis 32 (MTB). 33 OBJECTIVE: To investigate the ability of thin layer agar (TLA) direct drug-susceptibility testing 34 (DST) to detect MTB and its drug-resistance profiles in field conditions in Eswatini. 35 DESIGN: Consecutive samples were tested in parallel with Xpert®MTB/RIF and TLA for 36 rifampicin (1.0 μg/ml) and ofloxacin (2.0 μg/ml). TLA results were compared at the Reference 37 Laboratory in Antwerp with indirect DST on Löwenstein-Jensen or 7H11 solid media and 38 additional phenotypic and genotypic testing to resolve discordance. 39 RESULTS: TLA showed a positivity rate for MTB detection of 7.1% versus 10.0% for 40 Xpert®MTB/RIF. Of a total of 4547 samples included in the study, 200 isolates were available 41 for comparison to the composite reference. Within a median of 18.4 days, TLA detected RR 42 with 93.0% sensitivity (CI-77.4-98.0) and 99.4% specificity (CI 96.7-99.9), versus 62.5% (CI 43 42.7-78.8) and 99.3% (CI 96.2-99.9) for Xpert®MTB/RIF. Eight isolates, 28.6% of all RR 44 confirmed isolates, carried the I491F mutation, all detected by TLA. TLA also correctly 45 identified 183 of the 184 ofloxacin-S isolates (99.5% specificity, CI 97.0-99.9). 46 CONCLUSIONS: In field conditions, TLA rapidly detects RR, and in this specific setting 47 contributed to detection of additional RR patients over Xpert®MTB/RIF, mainly but not 48 exclusively due to I491F. TLA also accurately excluded fluoroquinolones resistance. 49 50 on A ril 7, 2021 by gest httpaac.asm .rg/ D ow nladed fom Background 53 In 2019, globally about 10 million people developed tuberculosis (TB), and half a million 54 people developed TB resistant to rifampicin (RR-TB) [1]. Even if rifampicin (RMP) drug55 susceptibility testing (DST) coverage at TB diagnosis increased to 61%, a considerable number 56 of patients with undetected RR-TB are still treated with an ineffective rifampicin-based 57 treatment regimen. These patients are at high risk of treatment failure, and continue 58 spreading RR-TB[1]. 59 To enhance RR-detection, the End TB strategy recommends to improve case detection and 60 DST coverage, also with the use of molecular techniques. The tests used most commonly to 61 detect drug resistance are Xpert®MTB/RIF (Cepheid, USA), which simultaneously detects 62 Mycobacterium tuberculosis complex (MTB) and RR, and line probe assays (LPA) such as 63 Genotype MTBDRplus, (Hain, Lifescience, Germany), for isoniazid (INH) and RMP, and 64 MTBDRsl for fluoroquinolone (FQ) and second-line injectables [2]. The roll-out of 65 Xpert®MTB/RIF, also in peripheral laboratories, substantially decreased diagnostic delay 66 [3][4]. However, these rapid molecular techniques miss specific rpoB mutants at positions 67 outside the RR determining region of the rpoB gene (RRDR), which are associated with 68 equally poor treatment outcomes as ‘common’ rpoB mutants [5]. One example is the I491F 69 RR-conferring mutation, among the so called disputed mutations [6], which in Eswatini 70 accounts for 56% of all RR-TB cases [7], reason of grave concern [8][9]. Furthermore, I491F 71 is in most of the cases tested as false rifampicin sensitive (RS) in MGIT phenotypic DST (pDST) 72 and is only detected by sequencing of the entire rpoB gene or slow pDST on solid medium, 73 such as Löwenstein-Jensen (LJ) [9][10][11]. Isolates with these mutations are also partially 74 missed by other non-commercial methods as microscopic observation direct susceptibility 75 testing (MODS), with a reported sensitivity of 75% [12]. 76 on A ril 7, 2021 by gest httpaac.asm .rg/ D ow nladed fom TLA has previously been described as an affordable method to detect MTB [13][14]. This 77 technique has limited costs [15] and can provide results considerably faster than indirect 78 pDST methods. Direct Thin Layer Agar DST (referred as TLA below) is not among the non79 commercial methods recommended by WHO [16]. However, when TLA is used for 80 simultaneous MTB and drug resistance detection, clinical samples are inoculated 81 immediately after decontamination on the medium with and without antibiotics [17] 82 avoiding the intermediate step of MTB isolation, which eliminates the need for high-level 83 biohazard containment such as needed for indirect pDST [18]. 84 These characteristics make this test suitable for low resource settings [19], although studies 85 on its applicability in routine practice are still limited. In settings where the prevalence of 86 I491F mutation is low, TLA has shown similar overall performance compared to indirect 87 MGIT-DST in terms of sensitivity, specificity and turnaround time [17][15]. However, the 88 ability of TLA to detect RR due to mutations outside the RRDR is unknown. 89 The primary objective of this study was to investigate the use of TLA as direct pDST test for 90 RR-TB detection in a peripheral laboratory, applied to smear-positive (Sm+) and smear91 negative (Sm-) sputum samples in a setting with high prevalence of the rpoB I149F mutation. 92 In addition, we describe the test performance for detection of MTB and resistance to 93 ofloxacin (OFX), as indicator for FQ resistance, key class of drug in the treatment of RR-TB. 94 DST results were evaluated in comparison to a composite reference standard that included 95 genotypic plus phenotypic testing. 96 Materials and Methods 97 Study patients and test flow 98 The study was conducted in the Nhlangano TB laboratory (NTBL), Eswatini, by Médecins Sans 99 Frontières-Switzerland and the Institute of Tropical Medicine (ITM), Antwerp, Belgium. The 100 on A ril 7, 2021 by gest httpaac.asm .rg/ D ow nladed fom catchment area included 22 health clinics and 3 health centres (including Nhlangano) at up 101 to 90 km of distance (map in Figure 1), from where samples were sent to NTBL in cold chain 102 (2-8°C) without preservatives. 103 We included all samples collected from consecutive patients older than 15 years, who 104 presented signs of presumptive TB, did not start TB treatment in the previous month, and 105 consented to participate in this study. As per routine practice, patients were asked to 106 produce two good quality sputum specimens (labelled as sample A and sample B) in 50 ml 107 sterile screw-cap containers and, when collected at the clinics, sent at the health centres, 108 where sample A was tested by Xpert®MTB/RIF following Cepheid procedures. Any patients 109 Xpert®MTB/RIF positive for TB on sample A, regardless of the RMP result, had sample B sent 110 to the National TB Reference Laboratory (NTBRL) in Mbabane for routine testing with liquid 111 culture and pDST. In this case, sample C was collected for the purpose of this study. All 112 leftovers from sample A, sample B, and sample C, if collected, were decontaminated and 113 tested for the study with TLA, fluorescence microscopy (FM), Xpert®MTB/RIF and LJ culture 114 at the NTBL. For any positive TLA plate, inoculated with either sample A, B or C, the 115 corresponding LJ isolate was sent to ITM for extended phenotypic and genotypic testing. In 116 case the LJ culture was contaminated or remained negative, an LJ subculture from the growth 117 of the corresponding TLA plate was shipped. 118 Laboratory methods at NTBL 119 We performed TLA using 4-quadrant polystyrene plates prepared at NTBL. The medium 120 contained 7H11 agar supplemented with a broad-spectrum antibiotic mixture to suppress 121 contamination, as previously described [15][17]. TLA plates included: drug-free growth 122 control (GC), p-Nitrobenzoic acid (PNB) (500 μg/ml), RMP (1.0 μg/ml) and OFX (2.0 μg/ml). 123 The processed sputum sediment was resuspended with 2 ml phosphate buffer (PBS) and two 124 on A ril 7, 2021 by gest httpaac.asm .rg/ D ow nladed fom drops were inoculated per each well. 125 During incubation at 5% CO2, plates were read at day 5, 7 10, 13, 15, 20, 25, 30, 35, 40, as 126 previously described [15] with few modifications. In addition, the drug-containing wells were 127 read on the day of GC positivity and on the next scheduled reading. 128 Plates were reported as positive for non-tuberculous mycobacteria (NTM) in case of 129 mycobacterial growth in the quadrant containing PNB, while no or poor growth ( 3 colonies) 130 on PNB with a positive GC compartment was considered positive for MTB [20]. Any growth 131 on PNB was tested with the MPT64Ag test (SD Bioline). 132 Laboratory methods at the reference laboratory 133 Upon receipt of isolates at ITM, fresh subcultures were made on LJ medium, and indirect134 DST was performed using the proportion method on LJ for RMP (40 μg/ml) and on 7H11 for 135 OFX (2.0 μg/ml). Indirect solid DSTs were read blindly with respect to TLA. 136 All isolates with a discordant RMP result between any two of three tests (Xpert®MTB/RIF, 137 TLA or indirect-DST) were further tested by MTBDRplus, had pDST done in MGIT (RMP 1.0 138 μg/ml), and the minimal inhibitory concentration (MIC) for RMP was determined on LJ (10139 160 μl/ml). All isolates showing RR on any test and 100 isolates showing RS on all tests were 140 investigated by Sanger sequencing of the rpoB target at ITM and/or by whole genome 141 sequencing (WGS) performed at the Borstel Research Center (Germany), as previously 142 described [21][22]. To constitute a composite reference standard for RMP resistance, MIC 143 prevailed on pDST, and resistance found on any of the genotypic tests overrode any 144 phenotypic result. 145 In case of discordance between TLA and indirect-DST for OFX, isolates were investigated by 146 LPA MTBDRsl and sequencing (target genes and/or WGS). As composite reference standard, 147 any resistance related to high confidence mutation found on any of the genotypic DST 148 on A ril 7, 2021 by gest httpaac.as

Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. Methods We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017). Results MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. Conclusion The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Xpert MTB/RIF (Xpert) and culture are the most reliable methods for tuberculosis diagnosis but are still poorly accessible in many low-resource countries. We aimed to assess the effects of OMNIgene Sputum (OM-S) and ethanol in preserving sputum for Xpert and OM-S for mycobacterial growth indicator tube (MGIT) testing over periods of 15 and 8 days, respectively. Sputum samples were collected from newly diagnosed smear-positive patients.

Empirical treatment of tuberculosis (TB) may be necessary in patients with negative or no Xpert MTB/RIF results. In a context with access to Xpert, we assessed mortality in the 6 months after the initial TB consultation among HIV-positive and HIV-negative patients who received empirical TB treatment or TB treatment based on bacteriological confirmation and we compared it with the mortality among those who did not receive TB treatment.

Determine-TB LAM assay is a urine point-of-care test useful for TB diagnosis in HIV-positive patients. We assessed the incremental diagnostic yield of adding LAM to algorithms based on clinical signs, sputum smear-microscopy, chest X-ray and Xpert MTB/RIF in HIV-positive patients with symptoms of pulmonary TB (PTB).Prospective observational cohort of ambulatory (either severely ill or CD4<200cells/μl or with Body Mass Index<17Kg/m2) and hospitalized symptomatic HIV-positive adults in Kenya. Incremental diagnostic yield of adding LAM was the difference in the proportion of confirmed TB patients (positive Xpert or MTB culture) diagnosed by the algorithm with LAM compared to the algorithm without LAM. The multivariable mortality model was adjusted for age, sex, clinical severity, BMI, CD4, ART initiation, LAM result and TB confirmation.Among 474 patients included, 44.1% were severely ill, 69.6% had CD4<200cells/μl, 59.9% had initiated ART, 23.2% could not produce sputum. LAM, smear-microscopy, Xpert and culture in sputum were positive in 39.0% (185/474), 21.6% (76/352), 29.1% (102/350) and 39.7% (92/232) of the patients tested, respectively. Of 156 patients with confirmed TB, 65.4% were LAM positive. Of those classified as non-TB, 84.0% were LAM negative. Adding LAM increased the diagnostic yield of the algorithms by 36.6%, from 47.4% (95%CI:39.4-55.6) to 84.0% (95%CI:77.3-89.4%), when using clinical signs and X-ray; by 19.9%, from 62.2% (95%CI:54.1-69.8) to 82.1% (95%CI:75.1-87.7), when using clinical signs and microscopy; and by 13.4%, from 74.4% (95%CI:66.8-81.0) to 87.8% (95%CI:81.6-92.5), when using clinical signs and Xpert. LAM positive patients had an increased risk of 2-months mortality (aOR:2.7; 95%CI:1.5-4.9).LAM should be included in TB diagnostic algorithms in parallel to microscopy or Xpert request for HIV-positive patients either ambulatory (severely ill or CD4<200cells/μl) or hospitalized. LAM allows same day treatment initiation in patients at higher risk of death and in those not able to produce sputum.

Prevalence of nontuberculous mycobacteria (NTM) disease is poorly documented in countries with high prevalence of tuberculosis (TB). We describe prevalence, risk factors, and TB program implications for NTM isolates and disease in Cambodia. A prospective cohort of 1,183 patients with presumptive TB underwent epidemiologic, clinical, radiologic, and microbiologic evaluation, including >12-months of follow-up for patients with NTM isolates. Prevalence of NTM isolates was 10.8% and of disease was 0.9%; 217 (18.3%) patients had TB. Of 197 smear-positive patients, 171 (86.8%) had TB confirmed (167 by culture and 4 by Xpert MTB/RIF assay only) and 11 (5.6%) had NTM isolates. HIV infection and past TB were independently associated with having NTM isolates. Improved detection of NTM isolates in Cambodia might require more systematic use of mycobacterial culture and the use of Xpert MTB/RIF to confirm smear-positive TB cases, especially in patients with HIV infection or a history of TB.

Molecular resistance testing fails to explain all fluoroquinolone resistance, with a continued need for a suitable rapid phenotypic drug susceptibility testing method.To evaluate the optimal method for phenotypic fluoroquinolone susceptibility testing.Using Lowenstein-Jensen medium, Middlebrook 7H11 agar, BACTEC-MGIT 960 and the resazurin microtitre plate assay, we determined susceptibility to fluoroquinolones in Mycobacterium tuberculosis and investigated cross-resistance between ofloxacin, levofloxacin, moxifloxacin and gatifloxacin. We compared MICs of all four fluoroquinolones for 91 strains on Lowenstein-Jensen (as the gold standard) with their MICs in resazurin plates, and with ofloxacin susceptibility at a single concentration in MGIT and on 7H11 agar, in addition to sequencing of the gyrAB genes.Applying a cut-off of 2 mg/L ofloxacin, 1 mg/L levofloxacin and 0.5 mg/L moxifloxacin and gatifloxacin in all methods, some discordance between solid medium and MGIT methods was observed, yet this tended to be explained by MICs around the cut-off. The high discordance between Lowenstein-Jensen (LJ) and resazurin plates suggests that the currently applied cut-offs for all fluoroquinolones in the resazurin method should decrease and minor changes in colour (from blue to purple) be considered as meaningful. High-level resistance in all assays to all drugs correlated well with the presence of gyrA mutations, in support of recent findings that fluoroquinolone resistance should be tested at different concentrations, as patients with lower levels of resistance may continue to benefit from high-dose fluoroquinolone-based therapy.

BACKGROUND: Molecular techniques rapidly detect resistance to rifampicin (RMP) and isoniazid (INH), but do not eliminate the need for culture-based drug susceptibility testing (DST) against other drugs. The thin-layer agar (TLA) test, a non-commercial direct DST method, has demonstrated good performance for INH and RMP; however, evidence is still limited, and its applicability for DST of ofloxacin (OFX) and kanamycin (KM) is unknown.DESIGN: We compared 279 TLA DST results with those of MGIT for INH and RMP, and 280 results for OFX and KM with those of the 7H11 agar proportion method, obtained from 320 smear-positive samples from 165 Georgian TB patients. Discrepancies were solved by comparison with a composite reference standard. The prevalence of multidrug-resistant tuberculosis (TB) was 30 of 164 patients (18.3%), 2 (6.7%) of whom had extensively drug-resistant TB.RESULTS: TLA showed 94.7%, 98.2%, 100% and 78.9% sensitivity, respectively, for INH, RMP, OFX and KM, with 100% specificity. Average time to results was 7 days in TLA, 23 in MGIT and 49 for 7H11 agar.CONCLUSIONS: In low-resource settings, TLA can be applied for the rapid detection of resistance to INH, RMP and fluoroquinolones. Further studies are necessary to improve sensitivity to ICM and further assess its performance for OFX and other drugs and its applicability in field conditions.

Background The Xpert® MTB/RIF (Xpert) is an automated molecular test for simultaneous detection of tuberculosis (TB) and rifampicin resistance, recommended by the World Health Organization as the preferred diagnostic method for individuals presumed to have multi-drug resistant TB (MDR-TB) or HIV-associated TB. We describe the performance of Xpert and key lessons learned during two years of implementation under routine conditions in 33 projects located in 18 countries supported by Médecins Sans Frontières across varied geographic, epidemiological and clinical settings. Methods Xpert was used following three strategies: the first being as the initial test, with microscopy in parallel, for all presumptive TB cases; the second being only for patients at risk of MDR-TB, or with HIV- associated TB, or presumptive paediatric TB; and the third being as the initial test for these high-risk patients plus as an add-on test to microscopy in others. Routine laboratory data were collected, using laboratory registers. Qualitative data such as logistic aspects, human resources, and tool acceptance were collected using a questionnaire. Findings In total, 52,863 samples underwent Xpert testing from April 2011 to December 2012. The average MTB detection rate was 18.5%, 22.3%, and 11.6% for the three different strategies respectively. Analysis of the results on samples tested in parallel showed that using Xpert as add-on test to microscopy would have increased laboratory TB confirmation by 49.7%, versus 42.3% for Xpert replacing microscopy. The main limitation of the test was the high rate of inconclusive results, which correlated with factors such as defective modules, cartridge version (G3 vs. G4) and staff experience. Operational and logistical hurdles included infrastructure renovation, basic computer training, regular instrument troubleshooting and maintenance, all of which required substantial and continuous support. Conclusion The implementation of Xpert was feasible and significantly increased TB detection compared to microscopy, despite the high rate of inconclusive results. Xpert implementation was accompanied by considerable operational and logistical challenges. To further decentralize diagnosis, simpler, low-cost TB technologies well-suited to low-resource settings are still urgently needed.

SETTING:Despite the widespread introduction of Xpert(®) MTB/RIF in developing countries, reports of its use and value in routine conditions remain limited.OBJECTIVE:To describe Xpert results in relation to microscopy, treatment initiation, cost and workload under routine conditions at four sites in Cambodia, Georgia, Kenya and Swaziland.DESIGN:Laboratory and clinical information on presumed TB patients were obtained from routine registers over a period of at least 6 months between March and November 2012.RESULTS:Among the 6086 presumed TB patients included in the analysis, Xpert testing increased the number of biologically confirmed cases by 15% to 67% compared to microscopy. Up to 12% of the initial Xpert results were inconclusive. Between 56% and 83% of patients were started on treatment based on microscopy and/or Xpert results, with median delays of 1-16 days. Rifampicin resistance was detected in 3-19% of Xpert-positive patients.CONCLUSION:Despite the additional numbers of cases detected by Xpert compared to microscopy, large proportions of patients are still started on treatment empirically in routine practice. Patient and specimen flow should be optimised to reduce delays in treatment initiation. Simple, non-sputum-based point-of-care tests with high sensitivity are needed to improve TB diagnosis and management.