Specific gyrA gene mutations predict poor treatment outcome in MDR-TB.

OBJECTIVES: Mutations in the gyrase genes cause fluoroquinolone resistance in Mycobacterium tuberculosis. However, the predictive value of these markers for clinical outcomes in patients with MDR-TB is unknown to date. The objective of this study was to determine molecular markers and breakpoints predicting second-line treatment outcomes in M. tuberculosis patients treated with fourth-generation fluoroquinolones. METHODS: We analysed treatment outcome data in relation to the gyrA and gyrB sequences and MICs of ofloxacin, gatifloxacin and moxifloxacin for pretreatment M. tuberculosis isolates from 181 MDR-TB patients in Bangladesh whose isolates were susceptible to injectable drugs. RESULTS: The gyrA 90Val, 94Gly and 94Ala mutations were most frequent, with the highest resistance levels for 94Gly mutants. Increased pretreatment resistance levels (>2 mg/L), related to specific mutations, were associated with lower cure percentages, with no cure in patients whose isolates were resistant to gatifloxacin at 4 mg/L. Any gyrA 94 mutation, except 94Ala, predicted a significantly lower proportion of cure compared with all other gyrA mutations taken together (all non-94 mutants + 94Ala) [OR = 4.3 (95% CI 1.4-13.0)]. The difference in treatment outcome was not explained by resistance to the other drugs. CONCLUSIONS: Our study suggests that gyrA mutations at position 94, other than Ala, predict high-level resistance to gatifloxacin and moxifloxacin, as well as poor treatment outcome, in MDR-TB patients in whom an injectable agent is still effective.

Gatifloxacin is superior to levofloxacin and moxifloxacin in shorter treatment regimens for multidrug-resistant TB.

SETTING: Data were collected from patients starting one of the shorter treatment regimens (STRs) for multidrug-resistant tuberculosis (MDR-TB) in Bangladesh, Niger or Cameroon.OBJECTIVE: To estimate the effect of either a gatifloxacin (GFX), moxifloxacin (MFX) or levofloxacin (LVX) based STR on bacteriological effectiveness.DESIGN: Retrospective study of prospectively collected data.RESULTS: Among 1530 patients, bacteriological effectiveness was 96.7% overall. Stratified by treatment with a GFX-, LVX- or MFX-based regimen effectiveness was respectively 97.5%, 95.5% and 94.7%. Compared to those on a GFX-based regimen, the estimated summary odds ratio of having an adverse outcome was more than double (OR 2.05, 95% CI 1.09-3.90) in patients treated with either an LVX-based or MFX-based regimen. After adjusting for initial resistance, patients treated with an LVX-based regimen and MFX-based regimen had respectively a 4.5- and 8.4-fold times larger odds of an adverse bacteriological outcome. None among 859 patients at risk treated with a GFX-based compared to at least 4 of 228 among those on an MFX-based regimen acquired fluoroquinolone resistance.CONCLUSION: GFX-based regimens had superior bacteriological effectiveness than MFX-based or LVX-based regimens. As GFX is currently unavailable in most MDR-TB programs, its reintroduction should be prioritised.

Twenty years of rifampicin resistance surveillance in Bangladesh: periodic vs. continuous monitoring.

OBJECTIVE: To analyse 20 years of tuberculosis (TB) drug resistance surveillance, comparing conventional periodic random drug resistance surveys with continuous monitoring, in Damien Foundation-supported districts of Bangladesh. DESIGN: Retrospective study of data on TB drug resistance from five periodic surveys among newly registered patients vs. continuous monitoring of retreatment patients from 1996 to 2016. RESULTS: Periodic surveys and continuous monitoring showed similar trends in rifampicin (RMP) resistance; with all smear-positives registered as denominator, prevalence in new cases was found to be at approximately the same level as incidence in retreatment cases. Changes in trends observed using continuous monitoring preceded those detected in periodic surveys by a few years. The accurate interpretation of trend changes requires detailed knowledge of changes in treatment regimens, referral criteria, testing methods and operational factors. CONCLUSION: Low rates of resistance to RMP, isoniazid and the fluoroquinolones were maintained over the two decades, indicating excellent TB programme performance, including highly active standard first- and second-line treatment regimens. Continuous monitoring is feasible, but requires rigorous application of referral guidelines and data maintenance. Contrary to random surveys, continuous monitoring provides early indications of programme performance, essential for individual patient management, and is more efficient and cost-effective.

Early and rapid microscopy-based diagnosis of true treatment failure and MDR-TB.

SETTING: Damien Foundation tuberculosis (TB) control project in Bangladesh. OBJECTIVE: Early diagnosis of true TB treatment failure and multidrug resistance (MDR) for more efficient DOTS-Plus. DESIGN: Prospective comparison of performance on smear-positive sputum of fluorescein diacetate (FDA) vital staining vs. culture, and of slide drug susceptibility testing (slide DST) vs. the Löwenstein-Jensen (LJ) proportion method. RESULTS: FDA reached 92% positive and 97% negative predictive value directly on fresh sputum, but only 94% and 62%, respectively, on transported smears. Accuracy on washed cetylpyridinium chloride transported sputum was similar to that on fresh sputum. Slide DST on fresh smear-positive sputum failed less often than LJ DST, with 96% accurate results for rifampicin and MDR-TB diagnosis. Good results were obtained for isoniazid (90% accuracy), but not for ethambutol or streptomycin. CONCLUSIONS: We can confirm that FDA staining allows rapid screening for viable acid-fast bacilli and true treatment failure in delayed smear converters or smear-defined failures, while slide DST assures fast and accurate confirmation of MDR-TB in selected populations. The tests can be applied safely in resource-poor settings. Their successive use could be an efficient strategy for screening and an early start on standardised regimens of DOTS-Plus candidates.

Extension of the intensive phase reduces unfavourable outcomes with the 8-month thioacetazone regimen.

SETTING: Damien Foundation tuberculosis (TB) control projects in Bangladesh. OBJECTIVE: To assess the effectiveness of a 1-month extension of the intensive phase for smear-positives at 2 months of an 8-month regimen with a continuation phase consisting of isoniazid (INH) and thioacetazone (Th). DESIGN: A prospective study of two cohorts of newly registered smear-positive cases, with extension of the intensive phase for the control cohort, but not for the study cohort. Culture and drug susceptibility testing (DST) of smear-defined failures and relapses and of random samples of new cases. RESULTS: Among 8230 study patients (86.7% 2-month conversion) and 7206 controls (83.4% conversion), smear-defined failure or relapse outcome was 3.0% for 2-month smear-negatives vs. 3.1% for 2-month smear-positives with extension (non-significant, NS), and 8.2% for 2-month smear-positives with no extension (P < 0.00001). Culture-confirmed failure and relapse reached 1.9% in 2-month smear-negatives and 1.6% (NS) in 2-month smear-positives with vs. 3.7% (P < 0.001) in 2-month smear-positives with no extension. The relative risk (RR) of non-extension in 2-month smear-positives was 2.4 (cultures) to 2.7 (smears). The same RR and borderline significance was found for non-extension of patients with pan-susceptible strains. CONCLUSIONS: Extension of the intensive phase considerably reduces failures and relapses with a weaker regimen in patients smear-positive at 2 months. Its effectiveness may vary with extent of initial drug resistance vs. power of the regimen.

Performance of variations of carbolfuchsin staining of sputum smears for AFB under field conditions.

SETTING: A field project in Bangladesh. OBJECTIVE: To compare the effectiveness of commonly used carbolfuchsin staining variations. DESIGN: Routine hot Ziehl-Neelsen (ZN) 1% basic fuchsin staining for 15 min in 75 field clinics. Blind reading of duplicate smears stained by ZN 1% vs. 0.3% basic fuchsin applied for 5 min, or by ZN 1% 5 min vs. Kinyoun cold staining. Rechecking of discordant series. RESULTS: For comparable numbers of false positives, sensitivity was significantly lower with Kinyoun than with ZN 1% 5 min (85.6% vs. 93.0%, P < 0.001). Sensitivity with ZN 1% 5 min was not significantly higher than with 0.3% 5 min staining (89.9% vs. 86.5%). Routine examination using 1% 15 min ZN identified more positives than any of the study techniques. CONCLUSIONS: Kinyoun cold staining sensitivity was unsatisfactory in field clinics. The sensitivity of the WHO/IUATLD recommended 0.3% fuchsin for 5 min was not significantly different from the original 1% ZN for 5 min, but 1% 15 min hot staining might be superior. A reduced fuchsin concentration together with a short staining time may leave too narrow a margin for error. TB programmes using hot ZN with a concentrated stain or longer staining time should not be urged to change.

Disputed rpoB mutations can frequently cause important rifampicin resistance among new tuberculosis patients.

SETTING: Greater Mymensingh area, Bangladesh. OBJECTIVES: To document among new tuberculosis (TB) patients the proportions and treatment outcomes of silent, non-disputed and disputed (generally missed by rapid drug susceptibility testing [DST]) rpoB mutations, and their detection by commercial molecular assays. DESIGN: Retrospective analysis of rpoB sequences from randomly selected ethanol-preserved diagnostic sputum samples; comparison of sequencing with conventional DST results and standard first-line treatment outcome; retesting of samples with mutations using the Xpert MTB/RIF and GenoType MTBDRplus assays. RESULTS: Of 1091 samples, 5.8% failed amplification, and six contained other mycobacteria. In 2005 and 2010, respectively 2/500 (0.4%) and 11/522 (2.1%) amplicons showed non-silent mutations. At least 7/13 of these belonged to the disputed group, with 5/7 patients suffering adverse treatment outcome. One silent mutation went undetected by commercial assays. Following routine DST indications, only three cases with a non-silent mutation were eventually detected. CONCLUSIONS: Disputed rpoB mutations may be responsible for the majority of rifampicin (RMP) resistance among new cases, and lead to adverse outcomes of first-line treatment. Silent mutations do not necessarily cause Xpert or line-probe assay false RMP-resistant results. Molecular RMP DST could greatly simplify resistance surveillance, in addition to offering the best prospects for early and accurate individual diagnosis.

Anti-tuberculosis drug resistance in Bangladesh: reflections from the first nationwide survey.

OBJECTIVE: To determine the prevalence of tuberculosis (TB) drug resistance in Bangladesh. DESIGN: Weighted cluster sampling among smear-positive cases, and standard culture and drug susceptibility testing on solid medium were used. RESULTS: Of 1480 patients enrolled during 2011, 12 falsified multidrug-resistant TB (MDR-TB) patients were excluded. Analysis included 1340 cases (90.5% of those enrolled) with valid results and known treatment antecedents. Of 1049 new cases, 12.3% (95%CI 9.3-16.1) had strains resistant to any of the first-line drugs tested, and 1.4% (95%CI 0.7-2.5) were MDR-TB. Among the 291 previously treated cases, this was respectively 43.2% (95%CI 37.1-49.5) and 28.5% (95%CI 23.5-34.1). History of previous anti-tuberculosis treatment was the only predictive factor for first-line drug resistance (OR 34.9). Among the MDR-TB patients, 19.2% (95%CI 11.3-30.5; exclusively previously treated) also showed resistance to ofloxacin. Resistance to kanamycin was not detected. CONCLUSION: Although MDR-TB prevalence was relatively low, transmission of MDR-TB may be increasing in Bangladesh. MDR-TB with fluoroquinolone resistance is rapidly rising. Integrating the private sector should be made high priority given the excessive proportion of MDR-TB retreatment cases in large cities. TB control programmes and donors should avoid applying undue pressure towards meeting global targets, which can lead to corruption of data even in national surveys.

Scanty AFB smears: what's in a name?

SETTING: A tuberculosis control project in Bangladesh. OBJECTIVE: To document the frequency and diagnostic value of smears with scanty acid-fast bacilli (AFB) (IUATLD/WHO scale, < 10/100 high power fields), and to assess the appropriateness of the current positivity threshold. DESIGN: Analysis of databases of laboratory registers, patient records and the diagnostic yield of sputum collection strategies. RESULTS: Scanty smears constituted about 10% of suspect and almost 50% of follow-up smears. In suspect series, 10% of scanty 1-9/100 were not confirmed by another positive or scanty AFB sputum, compared to 7.5% of results at the current cut-off value of 10/100. Considering such results as positive by adopting a lower cut-off as low as the 1/100 used in the ATS scale added 1.5% false positives at the most. In return, the gain in confirmed positive cases was up to 10%, and that in positive results exceeded the incremental yield of the third diagnostic sputum. Significance of scanty follow-up smears at the end of the intensive phase was suggested by their association with treatment failure and unfavourable outcome overall. CONCLUSIONS: Scanty results (IUATLD/WHO scale) are not rare and should not be ignored. Adoption of a considerably lower positivity threshold would be appropriate in control programmes where basic conditions for reliable AFB microscopy, including regular quality assessment, are present.

Drug resistance monitoring: combined rates may be the best indicator of programme performance.

SETTING: Greater Mymensingh District, Bangladesh. OBJECTIVES: To determine changes in prevalence of drug resistance of Mycobacterium tuberculosis under DOTS. DESIGN: Drug susceptibility testing of systematic samples of M. tuberculosis isolated from all sputum smear-positive cases newly registered in sentinel centres during 1995 and 2001. Continuous monitoring of retreatment registrations and resistance of strains from relapse and failure cases. RESULTS: Of 942 strains from the new cases in 2001, 10.8% showed resistance to any drug, 6.2% to isoniazid, 0.4% to rifampicin (all of them multidrug-resistant, MDR), 7.1% to streptomycin, and 1.0% to ethambutol. Corresponding rates for 99 strains from previously treated cases were 32%, 20%, 3%, 20% and 2%, respectively. Although most rates of resistance had decreased since 1995, increased streptomycin resistance was the only significant change when new and previously treated cases were considered separately. However, combined resistance for any drug, isoniazid, rifampicin and MDR had decreased significantly. CONCLUSION: As suggested by monitoring of resistance in failure and relapse cases and by routine programme reports, drug resistance had decreased. Combined resistance demonstrated changes between periodic surveys better than its subgroups, and may be a more reliable and comprehensive indicator. However, continuous monitoring of the pool of resistant retreatment cases is a more efficient strategy.

Successful '9-month Bangladesh regimen' for multidrug-resistant tuberculosis among over 500 consecutive patients.

SETTING: Tuberculosis (TB) program, Damien Foundation Projects, Bangladesh. OBJECTIVE: To summarize the outcome and its determinants of the first treatment for multidrug-resistant TB using a standardized regimen consisting of a minimum 9 months. DESIGN: This was a prospective, observational study of a gatifloxacin (GFX) based directly observed regimen, mainly with initial hospitalization. The 4-month intensive phase was extended until sputum smear conversion. Patients were monitored using culture for up to 2 years after treatment completion. RESULTS: Of the 515 patients who met the study inclusion criteria and were successively enrolled from 2005 to 2011, 84.4% had a bacteriologically favorable outcome. Due to extensive disease with delayed sputum conversion, only half of the patients completed treatment within 9 months; however, 95% were able to complete treatment within 12 months. Eleven patients failed or relapsed, and 93.1% of the 435 patients who were successfully treated completed at least 12 months post-treatment follow-up. The strongest risk factor for a bacteriologically unfavorable outcome was high-level fluoroquinolone (FQ) resistance, particularly when compounded by initial pyrazinamide (PZA) resistance. Low-level FQ resistance had no unfavorable effect on treatment outcome. Amplification of drug resistance occurred only once, in a patient strain that was initially only susceptible to kanamycin and clofazimine. CONCLUSION: The excellent outcome of the Bangladesh regimen was largely maintained. Bacteriological treatment failures and relapses were rare, except among patients with high-level GFX resistance, notably in the presence of PZA resistance.

An operational study comparing microscopes and staining variations for tuberculosis LED FM.

SETTING: Tuberculosis control projects, Damien Foundation Bangladesh. OBJECTIVES: To compare transmitted fluorescence (Olympus CX21™/FRAEN FluoLED™) with epi-fluorescence (Zeiss Primostar iLED™) light-emitting diode microscopes (LED-FM) and various auramine staining and destaining/counterstaining techniques for the detection of acid-fast bacilli. DESIGN: Multicentre blinded reading of routine smears on both types of microscopes using different staining techniques in multiple phases. LED-FM rechecking of discordant series with and without restaining to calculate operating characteristics. RESULTS: Among 64 874 smears, both instruments detected 9.6% positives. Compared to the standard technique, the stronger auramine-O formulation did not perform better. Thiazine red counterstaining tended to yield more false-positive as well as false-negative errors. Combined destaining/counterstaining (sensitivity 93%, positive predictive value [PPV] 98%) proved significantly less effective. Both destaining with 1% hydrochloric acid (HCl) and 10% alcohol and the standard 0.5% HCl and 70-95% alcohol were equally accurate (sensitivity 95-96%, PPV 99%). The sturdiness of the microscopes in field conditions was sub-optimal: only 5/16 instruments did not break down. CONCLUSIONS: Both microscopes performed equally well. The standard staining technique is as good as the more complicated and expensive variations. A destaining solution containing only 10% alcohol works perfectly well. The inferior quality of LED-FM microscope components is an obstacle to FM expansion.

Transmission of multidrug-resistant and extensively drug-resistant tuberculosis in rural Bangladesh: lessons learnt.

We report community transmission of multidrug-resistant (MDR-) and extensively drug-resistant tuberculosis (XDR-TB) documented by fingerprinting, with secondary cases appearing over a period of 10 years. The index case failed MDR-TB treatment, with amplification to XDR-TB after refusing treatment when first diagnosed and developing pre-XDR-TB on private treatment. Some of the first MDR-TB patients were not started on appropriate treatment due to delayed diagnosis or to excessively rigid application of National TB Programme guidelines. Early presumptive MDR- and XDR-TB diagnosis and removal of barriers, such as obligatory hospitalisation, could have stopped this trend of resistance amplification and transmission.

Nous décrivons la transmission dans la collectivité de la tuberculose à germes multirésistants (TB-MDR) et ultrarésistants (TB-XDR) documentées par les empreintes digitales, ainsi que les cas secondaires apparaissant au cours d'une période de 10 ans. Dans le cas index, on a vu échouer le traitement de la TB-MDR et le passage à une TB-XDR après refus du traitement lors du premier diagnostic et l'apparition d'une pré-TB-XDR au cours d'un traitement dans le secteur privé. Certains des premiers patients TB-MDR n'ont pas été mis sous traitement approprié en raison des délais du diagnostic ou d'une application trop rigide des directives du Programme national de la tuberculose. Un diagnostic précoce de la suspicion de la TB-MDR ou de la TB-XDR et l'élimination des barrières comme l'hospitalisation obligatoire auraient pu arrêter cette tendance vers l'amplification et la transmission de la résistance.

Se comunica la presencia de transmisión de tuberculosis multidrogoresistente (TB-MDR) y extremadamente drogorresistente (TB-XDR), confirmada mediante las huellas genéticas, con aparición de casos secundarios en un período de 10 años. El caso inicial de TB-MDR presentó un fracaso terapéutico con progresión hacia una forma TB-XDR, tras haber rechazado el tratamiento en el momento del primer diagnóstico y recibido luego un tratamiento en el sector privado durante el cual la enfermedad evolucionó hacia una TB-pre-XDR. Algunos de los pacientes que contrajeron la TB-MDR no iniciaron un tratamiento adecuado debido a los retrasos en el diagnóstico o a una aplicación demasiado estricta de las normas del Programa Nacional contra la Tuberculosis. Habría sido posible interrumpir esta tendencia de amplificación de la resistencia y transmisión continua si se hubiese establecido un diagnóstico presuntivo precoz de TB-MDR o -XDR y eliminado los obstáculos como la hospitalización obligatoria.

Extension of the intensive phase reduces relapse but not failure in a regimen with rifampicin throughout.

SETTING: Damien Foundation tuberculosis (TB) control projects in Bangladesh. OBJECTIVE: To assess the effectiveness of extending the intensive phase (P1) of treatment by 1 month for patients who are smear-positive after 2 months of a 6-month regimen containing rifampicin (RMP) throughout. DESIGN: Prospective operational study randomising P1 extension for new smear-positive cases with any number of acid-fast bacilli in the 2-month smear (2M+). Smear-defined failures and relapses underwent culture and drug susceptibility testing in addition to DNA sequencing of the rpoB gene before and after treatment. RESULTS: Of 16,708 patients evaluated, 12,967 were smear-negative at 2 months (2M-); 1871 and 1870 2M+ were randomised to no extension or extension. Respectively 0.3% (95%CI 0.2-0.4), 1.2% (95%CI 0.7-1.8) and 2.0% (95%CI 1.4-2.8) smear- and culture-positive failures, and 1.2% (95%CI 1.0-1.4), 2.6% (95%CI 1.9-3.4) and 0.9% (95%CI 0.5-1.4) relapses were detected. Extension significantly reversed the relative risk (RR) of relapse of 2M+ vs. 2M- patients from 2.2 (95%CI 1.6-3.0) to 0.7 (95%CI 0.4-1.2). The RR for failure remained high, at 7.3 (95%CI 4.7-11.5) with and 4.2 (95%CI 2.5-7.2) without extension. More multi-drug resistance was found after extension, but acquired RMP resistance was similar in all arms. The fair sensitivity of the 2-month smear for failure or relapse (40%) was offset by a very low positive predictive value (3%). CONCLUSIONS: Extension of P1 is very inefficient with this 6-month regimen. Operational research should define appropriate algorithms allowing an earlier switch to the next higher regimen for those in need, using follow-up smears for screening.

Hydrochloric vs. sulphuric acid in water for Ziehl-Neelsen staining of acid-fast bacilli.

SETTING: Damien Foundation Bangladesh tuberculosis (TB) control projects. OBJECTIVES: To compare 25% sulphuric acid in water (H(2)SO(4)) with hydrochloric acid in water (HCl) to differentiate acid-fast bacilli in sputum smears stained with 1% carbolfuchsin. DESIGN: For 1 year, all 158 microscopy laboratories used either H(2)SO(4) or 3%/6%/10% HCl for their routine work, alternating monthly between H(2)SO(4) and HCl. Each month a sample of five smears per laboratory was rechecked blind. After recording qualitative staining aspects, all sample smears were restained before rechecking, using H(2)SO(4) for destaining. RESULTS: A total of 368,059 H(2)SO(4) and 335,436 HCl smears were routinely read, yielding 7.2% positive or scanty results in both groups. Of these, 9492 were rechecked. There was no difference in false-negatives detected (0.66%, 95%CI 0.44-0.95 for H(2)SO(4) vs. 0.68%, 95%CI 0.46-0.98 for HCl), but apparently there were more false-positives with H(2)SO(4) (2.12%, 95%CI 0.92-4.14 vs. 0.28%, 95%CI 0.00-1.54, P = 0.05). Qualitatively, only 3% HCl yielded significantly inferior differentiation results. CONCLUSIONS: HCl 6-10% in water can be recommended for Ziehl-Neelsen destaining above H(2)SO(4). Diluting is easier and safer, and it may cause less confusion with false-positives during rechecking, including a restaining step.

Methylene blue is a good background stain for tuberculosis light-emitting diode fluorescence microscopy.

SETTING: Damien Foundation Bangladesh tuberculosis (TB) control projects. OBJECTIVES: To compare blue ink, potassium permanganate and methylene blue background staining for transmitted light-emitting diode (LED) TB fluorescence microscopy (FM). DESIGN: Auramine smears made in triplicate from Ziehl-Neelsen (ZN) acid-fast bacilli (AFB) positive or negative sputum and stained with one of the background variations were read blind by LED FM. Reference laboratory rechecking of discordant series was used before and after auramine restaining as the gold standard. RESULTS: Of 1977 series evaluated, 991 (50.1%) were made from ZN-positive specimens. There were 919, 942 and 958 FM true-positives with blue ink, permanganate and methylene blue counterstaining, against respectively 12, 12 and 16 false-positives. Methylene blue counterstaining was more sensitive (95.6%, 95%CI 94.2-96.8) than blue ink or permanganate (92.7%, 95%CI 90.9-94.3 and 93.6%, 95%CI 91.9-95.0; respectively P < 0.01 and < 0.05). No AFB could be found in 85% and 18% of 180 discordant series without and with restaining. CONCLUSIONS: Methylene blue is at least equivalent to potassium permanganate counterstaining for FM using blue LED transmitted excitation and is cheaper than blue ink. Restaining of all smears prior to first re-reading may be unavoidable for blinded rechecking of auramine-stained smears for external quality assessment.