First-line tuberculosis treatment with double-dose rifampicin is well tolerated.

OBJECTIVE: To compare the occurrence of unfavourable treatment and safety outcomes of double-dose rifampicin (RMP; 20 mg/kg/d, intervention) with standard dose (10 mg/kg/d, control) in a first-line tuberculosis (TB) treatment regimen for smear-positive TB patients in Bangladesh.DESIGN: This was a randomised clinical trial. The primary efficacy and safety endpoints were the occurrence of an unfavourable treatment outcome (death, failure, relapse or loss to follow-up) and the occurrence of any serious drug-related adverse event (SAE).RESULTS: In primary efficacy analysis, among 343 control and 347 intervention patients, respectively 15.5% and 11.8% had an unfavourable outcome. In safety analysis, among 349 intervention and 352 control patients, respectively 4.3% and 2.6% experienced an SAE. These differences were not significant. There was a significantly lower occurrence of SAEs, explained by a lower occurrence of hepatic toxicity, in a RMP double-dosed but erroneously HZE (isoniazid+pyrazinamide+ethambutol) under-dosed subgroup.CONCLUSIONS: Our findings show that there is no statistically significant difference in terms of efficacy and safety between standard and double-dose RMP. An accidental finding (related to dosage levels of the standard regimen) suggests that high-dose RMP is potentially a lesser cause of hepatotoxicity. Larger trials with more power, or trials with at least a triple-dose might be needed to clearly see the effect of high-dose RMP on unfavourable outcomes.

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.

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.

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.

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.

Fluorescein diacetate vital staining allows earlier diagnosis of rifampicin-resistant tuberculosis.

SETTING: Damien Foundation Project, Bangladesh. OBJECTIVE: To evaluate sputum smear fluorescein diacetate (FDA) vital staining to predict culture-defined failure and rifampicin (RMP) resistance. DESIGN: A retrospective, operational study. RESULTS: A total of 1633 episodes of auramine smear-defined late conversion and failure could be evaluated (respectively 640 and 584 on first treatment and 185 and 224 on retreatment). Negative FDA was 95% predictive of negative culture in patients on first treatment, while its positive predictive value was around 95% during retreatment. The predictive value of a positive (not scanty) result for RMP resistance or environmental non-tuberculous mycobacteria (NTM) was at least 90%, except in late converters on first-line treatment; a negative result was over 95% exclusive of the same except in retreatment failures. FDA correctly identified 88-98% of all RMP resistance. CONCLUSIONS: FDA staining increased the proportion of tuberculosis patients put on second-line treatment without receiving the standard first-line retreatment regimen. In our setting, with excellent microscopy, late case presentation and low resistance prevalence, it proved indispensable for efficient culture and referrals of early suspects for rapid drug susceptibility testing (DST). In other settings with low prevalence of NTM and difficult access to accurate and rapid DST, FDA-positive failures might even be considered for immediate start of second-line treatment.

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.

Performance and acceptability of the FluoLED Easy module for tuberculosis fluorescence microscopy.

SETTING: Tuberculosis (TB) reference laboratory in Bangkok, Thailand, and two health centres in Dar es Salaam, Tanzania. OBJECTIVES: To assess the performance and user-friendliness of a light-emitting diode (LED) module (FluoLED Easy) for TB fluorescence microscopy (FM). DESIGN: Equivalence study vs. conventional FM in Bangkok using blinded re-reading; routine detection in the health centres in Dar es Salaam compared to Ziehl-Neelsen (ZN) over 2 years, with rechecking of FM smears. RESULTS: For 461 smears re-read, 99.1% concordance with conventional FM was obtained. FluoLED introduction caused a lasting increase in detection in the routine of each of the health centres by on average 20%. Blinded rechecking failed due to unreliable registration. Onsite rechecking of a convenience sample showed absence of false-positive results in one centre and confusion with artefacts that could have been avoided by more training in the other. LED FM was highly appreciated, with both laboratories refusing to revert to ZN as originally intended. CONCLUSIONS: A simple microscope with a FluoLED module can yield results equivalent to those of conventional FM. Low cost, technical appropriateness and excellent acceptance justify its use in low-income settings, contrary to classical systems. LED FM can lead to increased sensitivity, but for optimal yield good training and quality assurance remain essential requirements.

Ziehl-Neelsen staining: theory and practice.

The information provided in the guidelines of the World Health Organization and the International Union Against Tuberculosis and Lung Disease for Ziehl-Neelsen staining is not practical on a number of points. The advice given here is meant to supplement the guidelines. It is based on experiments on and field experience of basic fuchsin stain and staining solutions.