Drug Susceptibility Testing Of Tuberculosis Research Articles

Changing prevalence and resistance patterns in children with drug-resistant tuberculosis in Mumbai

Background: The prevalence of drug-resistant (DR) tuberculosis (TB) in children is increasing. Although, in India, multi-drug-resistant (MDR) TB rates have been relatively stable, the number of children with pre-extensively drug-resistant and extensively drug-resistant (XDR) TB is increasing.Aim: To determine whether the prevalence of DR TB in children in Mumbai is changing and to study the evolving patterns of resistance.Methods: A retrospective study was undertaken in 1311 paediatric patients referred between April 2007 and March 2013 to the Paediatric TB clinic at B. J. Wadia Hospital for Children, Mumbai. Children were defined as having DR TB on the basis of drug susceptibility testing (DST) of Mycobacterium tuberculosis grown on culture of body fluids (in the case of extra pulmonary TB) or from gastric lavage/bronchi-alveolar lavage/sputum in patients with pulmonary TB or from DST of the contacts. The prevalence of DR TB was calculated and the type of DR was evaluated yearly and in the pre-2010 and post-2010 eras.Results: The overall prevalence of DR TB was 86 (6.6%) with an increase from 23 (5.6%) patients pre-2010 to 63 (7%) post-2010 (P = 0.40). Nine (10.4%) patients were diagnosed on the basis of contact with a parent with DR TB. Overall fluoroquinolone resistance increased from 9 (39.1%) pre-2010 to 59 (93.7%) post-2010 (P = 0.0001): moxifloxacin resistance increased from 2 (8.7%) to 29 (46%) (P = 0.0018) and ofloxacin resistance increased from 7 (30.4%) to 30 (47.6%) (P = 0.14). Ethionamide resistance also increased from 6 (26.1%) to 31 (49.2%) (P = 0.04), aminoglycoside resistance was one (4.3%) pre-2010 and 12 (19%) post-2010 (P = 0.17) and resistance remained virtually the same for both amikacin [0 pre-2010 and 6 (9.5%) after 2010] and kanamycin [one (4.3%) pre- and 6 (9.5%) post-2010]. Of the first-line drugs, resistance remained the same for isoniazid [23 (100%) to 61 (96.8%)], rifampicin [22 (95.7%) to 51 (80.9%),P = 0.17], pyrazinamide [15 (65.2%) to 35 (55.6%), P = 0.47], ethambutol [14 (60.9%) to 38 (60.3%), P = 1.00] and streptomycin [19 (82.6%) to 50 (79.4%), P = 1.00]. Resistance to PAS remained unchanged [2 (8.7%) to 5 (7.9%), P = 1.00].Conclusion: There is increasing resistance to second-line anti-tuberculosis (ATT) drugs, particularly flouroquinolones and ethionamide. Hence, there is an urgent need to avoid the use of ATT drugs for non-tuberculous infection and to increase surveillance for DR TB in adults as MDR TB in children is usually through contact with an adult with infectious MDR TB.

High prevalence of primary drug resistance in children with intrathoracic tuberculosis in India

Background:Drug susceptibility testing (DST) of Mycobacterium tuberculosis (Mtb) isolates is crucial for the effective treatment of tuberculosis. Data on DST patterns in Mtb isolates in childhood tuberculosis are scanty.Aims:To determine drug resistance patterns in Mtb isolates from a paediatric TB cohort in North India.Methods:403 children aged 6 months to14 year with probable intrathoracic tuberculosis were enrolled prospectively. All were treatment-naïve. 802 ambulatory-induced sputa (IS) and 787 gastric aspirate (GA) samples were cultured in BACTEC-MGIT960 system, and DST of the Mtb isolates was undertaken using the automated BACTEC-MGIT960 SIRE kit.Results:Of the 403 children, 147 (36.4%) were culture-confirmed: 132 (89.8%) isolates were Mtb and 15 (10.2%) non-tuberculous mycobacteria (NTM). Five Mtb isolates were contaminated and the remaining 127 were subjected to in-vitro drug susceptibility testing against streptomycin, isoniazid, rifampicin and ethambutol. Twenty-six (20.47%) isolates were resistant to one or more drugs, seven (5.5%) were resistant to rifampicin singly or in combination, and 11 (8.7%) were resistant to isoniazid singly or in combination. Mono-resistance to isoniazid, rifampicin, streptomycin and ethambutol was detected in four (3.1%), one (0.8%), four (3.1%) and two (1.6%), respectively. Five children (3.9%) had MDR-TB; 101 (79.9%) children had Mtb isolates which were sensitive to all four drugs.Conclusions:The rifampicin and isoniazid resistance rates were much higher than those in the adult TB population in India.

A Low Cost/Low Power Open Source Sensor System for Automated Tuberculosis Drug Susceptibility Testing.

In this research an open source, low power sensor node was developed to check the growth of mycobacteria in a culture bottle with a nitrate reductase assay method for a drug susceptibility test. The sensor system reports the temperature and color sensor output frequency change of the culture bottle when the device is triggered. After the culture process is finished, a nitrite ion detecting solution based on a commercial nitrite ion detection kit is injected into the culture bottle by a syringe pump to check bacterial growth by the formation of a pigment by the reaction between the solution and the color sensor. Sensor status and NRA results are broadcasted via a Bluetooth low energy beacon. An Android application was developed to collect the broadcasted data, classify the status of cultured samples from multiple devices, and visualize the data for the end users, circumventing the need to examine each culture bottle manually during a long culture period. The authors expect that usage of the developed sensor will decrease the cost and required labor for handling large amounts of patient samples in local health centers in developing countries. All 3D-printerable hardware parts, a circuit diagram, and software are available online.

Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country.

With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) M. tuberculosis isolates were multidrug-resistant. Twenty-four isolates yielded discrepant DST results. For rifampicin, isoniazid and streptomycin, 96%, 97% and 93% of isolates, respectively, were susceptible or resistant by all four methods, whereas for ethambutol, this agreement was observed for only 76% of isolates (P <0.05 for rifampicin or isoniazid or streptomycin versus ethambutol). Occurrence of rare mutations in three isolates that confer low-level resistance caused lower agreement for rifampicin among the four methods (kappa coefficient (κ) range, 0.84 to 0.95). For isoniazid, there was perfect agreement among phenotypic methods and molecular methods (κ, 1.00) but lower agreement between phenotypic and molecular methods. Three isolates were detected as polydrug-resistant by MGIT 960 system but as multidrug-resistant by DNA sequence-based method. The agreement was higher for streptomycin among the two phenotypic methods (κ, 0.97) while targeted sequencing yielded lower agreement (κ range, 0.86 to 0.89). The discrepancy for ethambutol resulted largely due to lower concordance of MGIT 960 results (κ range, 0.53 to 0.64). The MGIT 960 system is an accurate method for DST of M. tuberculosis against isoniazid and streptomycin while the results of rifampicin susceptibility should be complemented with DNA sequencing-based method when the suspicion for resistance is high. The possibility of false susceptibility to ethambutol with MGIT 960 system suggests that molecular or other phenotypic methods may be more useful when accurate ethambutol susceptibility results are warranted.

Detection of drug resistance in Mycobacterium tuberculosis.

Detection of drug resistance in Mycobacterium tuberculosis.

Direct tuberculosis drug susceptibility testing: time-saving and cost-effective in detecting MDR-TB.

Recent advances have made molecular diagnosis of tuberculosis (TB) and drug susceptibility testing (DST) possible, but the high costs involved present a huge challenge. The refinement and improvement of affordable methods therefore remain a priority. Conventional indirect DST is inexpensive and reliable, but time-consuming. A direct DST method for the direct testing of sputum samples without culture has been developed to reduce the time required for DST, but there have been conflicting results. Direct and indirect DST against isoniazid and rifampicin were performed on 208 sputum smear-positive specimens, 186 from newly diagnosed patients and 22 from previously treated patients; respectively 169 and 180 of the direct and indirect DST results were reportable. In comparison with indirect DST, direct DST resulted in a saving of on average 10.5 days. The time to direct DST results was inversely correlated with the number of acid-fast bacilli in the sputum samples. Direct DST is highly sensitive, reliable, cost-effective and time-saving in comparison with indirect DST.

Early Detection of Drug Susceptibility Test for Mycobacterium Tuberculosis by Slide DST Method Using Two Media- Middle Brook 7H9 Broth and Human Blood Media

Early Detection of Drug Susceptibility Test for Mycobacterium Tuberculosis by Slide DST Method Using Two Media- Middle Brook 7H9 Broth and Human Blood Media

Pulmonary Tuberculosis Diagnosis: Where We Are?

In recent years, in spite of medical advancement, tuberculosis (TB) remains a worldwide health problem. Although many laboratory methods have been developed to expedite the diagnosis of TB, delays in diagnosis remain a major problem in the clinical practice. Because of the slow growth rate of the causative agent Mycobacterium tuberculosis, isolation, identification, and drug susceptibility testing of this organism and other clinically important mycobacteria can take several weeks or longer. During the past several years, many methods have been developed for direct detection, species identification, and drug susceptibility testing of TB. A good understanding of the effectiveness and practical limitations of these methods is important to improve diagnosis. This review summarizes the currently-used advances in nonmolecular and molecular diagnostics.

Evaluating Shared Laboratory Services: Detecting Mycobacterium Tuberculosis Complex and Drug Resistance Using Molecular and Culture-Based Methods.

We explored sharing nucleic acid amplification testing (NAAT) for detection of Mycobacterium tuberculosis complex (MTBC) and molecular and phenotypic drug susceptibility testing between two state public health tuberculosis (TB) laboratories, and evaluated turnaround times and cost-effectiveness. From September 1, 2012, to May 30, 2013, the Wisconsin State Laboratory of Hygiene (Wisconsin Lab) submitted specimens to the Microbial Diseases Laboratory of the California Department of Public Health (California Lab) for NAAT and molecular drug susceptibility testing (MDST) by pyrosequencing, and culture-based TB drug susceptibility testing by the BACTEC(TM) MGIT(TM) 960 system. A total of 182 specimens were referred to the California Lab, and 47 TB cases and 12 drug-resistant cases were identified. The average time for specimen transport was two days, which included one day for processing and packaging in the submitting laboratory. The average turnaround time for NAAT was 0.3 days at the Wisconsin Lab and 3.8 days at the California Lab, including time for specimen transport. Turnaround time for culture-based drug susceptibility testing increased by a median of 16 days when specimens were sent to the California Lab, but MDST results were reported in fewer than four days, a median of 22 days sooner than any culture-based drug susceptibility testing results. This study revealed advantages and disadvantages associated with sharing services, and identified opportunities for improvement. Referral of specimens resulted in longer turnaround times for mycobacteriology test results and additional costs for transporting specimens. However, specialized testing such as pyrosequencing may not be available in low TB incidence areas, and these rapid results can have positive effects on patient management and TB control.

Evaluation of use of line probe assay on smear-positive direct specimen from extra-pulmonary tuberculosis site

BackgroundLine probe assay (LPA) is used for first-line drug susceptibility testing (DST) of smear-positive pulmonary tuberculosis (TB) patients. For extra-pulmonary (EP) and smear-negative TB patients, the samples are inoculated in culture and isolates of Mycobacterium tuberculosis (MTB) are tested on LPA. This results in considerable delay and loses the benefit of rapid diagnostics. In the present study, smear-positive EP specimens were tested directly on LPA and their results were compared with LPA conducted on culture isolates of same specimens. MethodAll EP specimens received from different parts of Gujarat State in 2014 were subjected to ZN smear microscopy and inoculated on liquid culture. Smear-positive samples were directly tested with LPA. Simultaneously, culture isolates of MTB were also subjected to LPA. Results of LPA conducted on both direct specimen and culture isolates were compared. ResultOf 391 extra-pulmonary specimens, 177 were smear positive and tested directly on LPA. Simultaneously, 88 were culture positive and their isolates were tested on LPA. With LPA on direct specimen, 127 (32%) had valid results with median time to diagnose rifampicin resistance of 5 days (IQR 2-7). In comparison, 88 (23%) specimens had valid results with culture isolates tested on LPA and with longer turnaround time (18–40 days). Among 51 samples, with valid LPA results both on direct samples and isolates, 50 (98%) had concordance for drug resistance pattern. ConclusionThere is advantage in testing extra-pulmonary smear-positive samples directly on LPA and the results would also be available rapidly.

Reduced turn-around time for Mycobacterium tuberculosis drug susceptibility testing with a proportional agar microplate assay

Multidrug-resistant tuberculosis is a major issue worldwide; however, accessibility to drug susceptibility testing (DST) is still limited in developing countries, owing to high costs and complexity. We developed a proportion method on 12-well microplates for DST. The assay reduced the time to results to <12 days and <10 days when bacterial growth was checked with the naked eye or a microscope, respectively. Comparison with the Canetti–Grosset method showed that the results of the two assays almost overlapped (kappa index 0.98 (95% CI 0.91–1.00) for isoniazid, rifampicin, streptomycin; and kappa index 0.92 (95% CI 0.85–0.99) for ethambutol). The sequencing of genes involved in drug resistance showed similar level of phenotype–genotype agreement between techniques. Finally, measurement of the MICs of rifampicin and ethambutol suggests that the currently used critical ethambutol concentration should be revised, and that the current molecular drug susceptibility tests for rifampicin need to be re-evaluated, as in vitro rifampicin-sensitive isolates could harbour drug resistance-associated mutation(s).

Cost-effectiveness of improvements in diagnosis and treatment accessibility for tuberculosis control in India

Inaccurate diagnosis and inaccessibility of care undercut the effectiveness of high-quality anti-tuberculosis treatment and select for resistance. Rapid diagnostic systems, such as Xpert(®) MTB/RIF for tuberculosis (TB) diagnosis and drug susceptibility testing (DST), and programs that provide high-quality DOTS anti-tuberculosis treatment to patients in the unregulated private sector (public-private mix [PPM]), may help address these challenges, albeit at increased cost. We extended a microsimulation model of TB in India calibrated to demographic, epidemiologic, and care trends to evaluate 1) replacing DST with Xpert; 2) replacing microscopy and culture with Xpert to diagnose multidrug-resistant TB (MDR-TB) and non-MDR-TB; 3) implementing nationwide PPM; and combinations of (3) with (1) or (2). PPM (assuming costs of $38/person) and Xpert improved health and increase costs relative to the status quo. PPM alone or with Xpert cost <1 gross domestic product/capita per quality-adjusted life-year gained relative to the next best intervention, and dominated Xpert interventions excluding PPM. While both PPM and Xpert are promising tools for combatting TB in India, PPM should be prioritized over Xpert, as private sector engagement is more cost-effective than Xpert alone and, if sufficient resources are available, would substantially increase the value of Xpert if both interventions are implemented together.

Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples

Aims and objectivesCurrent methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) are either costly or slow. As the prevalence of multidrug-resistant (MDR) strains increases, the need for fast, reliable, and inexpensive methods is obvious. This study evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct DST of MTB directly from clinical sputum samples. MethodsA total of 111 sputa with positive microscopy results for acid-fast bacilli (AFB) with more than 10 AFB per high-power field were used in the study. The samples were decontaminated using the modified Petroff method. The NRA results were compared with the reference indirect proportion method. ResultsThe sensitivity and the specificity of the direct NRA were 90% and 97.3%, 92.6% and 98.2%, 52.9% and 100%, and 28.6% and 100% for rifampin, isoniazid, streptomycin, and ethambutol, respectively. The results were in most cases available in 28days (84.3%). ConclusionsThe direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.

A Novel Reporter Phage To Detect Tuberculosis and Rifampin Resistance in a High-HIV-Burden Population.

Improved diagnostics and drug susceptibility testing for Mycobacterium tuberculosis are urgently needed. We developed a more powerful mycobacteriophage (Φ(2)GFP10) with a fluorescent reporter. Fluorescence-activated cell sorting (FACS) allows for rapid enumeration of metabolically active bacilli after phage infection. We compared the reporter phage assay to GeneXpert MTB/RIF for detection of M. tuberculosis and rifampin (RIF) resistance in sputum. Patients suspected to have tuberculosis were prospectively enrolled in Durban, South Africa. Sputum was incubated with Φ(2)GFP10, in the presence and absence of RIF, and bacilli were enumerated using FACS. Sensitivity and specificity were compared to those of GeneXpert MTB/RIF with an M. tuberculosis culture as the reference standard. A total of 158 patients were prospectively enrolled. Overall sensitivity for M. tuberculosis was 95.90% (95% confidence interval (CI), 90.69% to 98.64%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). In acid-fast bacillus (AFB)-negative sputum, sensitivity was 88.89% (95% CI, 73.92% to 96.82%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). Sensitivity for RIF-resistant M. tuberculosis in AFB-negative sputum was 90.00% (95% CI, 55.46% to 98.34%), and specificity was 91.94% (95% CI, 82.16% to 97.30%). Compared to GeneXpert, the reporter phage was more sensitive in AFB smear-negative sputum, but specificity was lower. The Φ(2)GFP10 reporter phage showed high sensitivity for detection of M. tuberculosis and RIF resistance, including in AFB-negative sputum, and has the potential to improve phenotypic testing for complex drug resistance, paucibacillary sputum, response to treatment, and detection of mixed infection in clinical specimens.

Clinical and epidemiological profiles of individuals with drug-resistant tuberculosis.

Drug-resistant tuberculosis (TB) is a growing global threat. Approximately 450,000 people developed multidrug-resistant TB worldwide in 2012 and an estimated 170,000 people died from the disease. This paper describes the sociodemographic, clinical-epidemiological and bacteriological aspects of TB and correlates these features with the distribution of anti-TB drug resistance. Mycobacterium tuberculosis (MT) cultures and drug susceptibility testing were performed according to the BACTEC MGIT 960 method. The results demonstrated that MT strains from individuals who received treatment for TB and people who were infected with human immunodeficiency virus were more resistant to TB drugs compared to other individuals (p < 0.05). Approximately half of the individuals received supervised treatment, but most drug-resistant cases were positive for pulmonary TB and exhibited positive acid-fast bacilli smears, which are complicating factors for TB control programs. Primary healthcare is the ideal level for early disease detection, but tertiary healthcare is the most common entry point for patients into the system. These factors require special attention from healthcare managers and professionals to effectively control and monitor the spread of TB drug-resistant cases.

Potential market for novel tuberculosis diagnostics: worth the investment?

The potential available market (PAM) for new diagnostics for tuberculosis that meet the specifications of the high-priority target product profiles (TPPs) is currently unknown. We estimated the PAM in 2020 in 4 high-burden countries (South Africa, Brazil, China, and India) for tests that meet the specifications outlined in the TPPs. The yearly PAM was estimated for the most likely application of each TPP. In 2020 the PAM for all 4 countries together was estimated to be (1) 12M tests/year with a value of 48M-71M USD for a sputum smear-replacement test; (2) 16M tests/year with a value of 65M-97M USD for a biomarker test; (3) 18M tests/year with a value of 18M-35M USD for a triage test; (4) 12M tests/year with a value of 59M-2238M USD for a tuberculosis detection plus drug susceptibility test (DST) all-in-one or 1.5M tests/year for a DST that follows a positive tuberculosis detection test with a corresponding value of 75M-121M for both tuberculosis detection and DST. Although there is a considerable potential market for novel tuberculosis diagnostics that fit the specification of the TPPs in the 4 high-burden countries, the actual market for an individual product remains uncertain.

Utility of GenoType MTBDRPlus Assay for Direct Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis from Probable Tuberculous Meningitis Patients

Aims: To evaluate GenoType ® MTBDRplus line probe assay as a diagnostic tool for detection of Mycobacterium tuberculosis and drug susceptibility testing from cerebrospinal fluid of probable tuberculous meningitis patients. Study Design: A prospective, double blind study. Place and Duration of study: Dept. of Microbiology and Neurology, Institute of Human Behavior Original Research Article Gupta et al.; BJMMR, 8(9): 772-780, 2015; Article no.BJMMR.2015.505 773 and Allied sciences, Delhi, India between February 2014 to October 2014. Methodology: Cerebrospinal fluid collected from 107 probable meningitis patients with diagnostic score >10 were subjected to smear microscopy, automated liquid culture (BACTEC MGIT 960) and Polymerase chain reaction (IS6110). All the samples were also subjected to GenoType ® MTBDRplus line probe assay for detecting M. tuberculosis and drug susceptibility. Drug susceptibility testing of all the M. tuberculosis isolates was done by BACTEC MGIT 960 and GenoType ® MTBDRplus line probe assay. Results: The sensitivity, specificity of the assay for M. tuberculosis detection was 49.5%, 100% against clinical diagnosis as reference standard and 68.9%, 100% against definitive diagnosis as reference standard. A diagnostic accuracy of 56.8% (kappa 0.22), 75% (kappa 0.46), were seen in patients with probable and confirmed diagnosis respectively. The drug susceptibility results for Isoniazid and Rifampicin could be delineated in only 39.2% of patients. Conclusion: This assay proved to have better sensitivity, diagnostic accuracy than smear microscopy and automated liquid culture for early detection of M. tuberculosis from probable tuberculous meningitis patients and has comparable sensitivity to culture (39.2%) for detection of drug susceptibility (though on different isolates). Rapid turnaround time and user friendliness makes it an acceptable assay for simultaneous early detection of M. tuberculosis and its drug susceptibility for better patient management.

COMPARISON OF CONVENTIONAL METHOD, RADIOMETRIC METHOD AND METHOD USING OXIDATION-REDUCTION DYE FOR DETECTION OF MULTI DRUG RESISTANT TUBERCULOSIS

Today Tuberculosis (TB) has become the most important communicable disease in the world. The emergence of multidrug resistant (MDR) TB has become the main threat to TB treatment and control programs. Rapid detection of TB is critical for the effective treatment of patients. Recently, a method using the Oxidation - Reduction dye has been proposed for drug susceptibility testing of Mycobacterium tuberculosis (M.tuberculosis). In the present study, we have compared Drug Susceptibility Testing (DST) by conventional method, Radiometricmethod like BACTEC460TB (B460TB) System and Oxidation-Reduction Assay such as Microplate Alamar Blue Assay (MABA) for detection of MDR TB. Total 60 clinical isolates of M.tuberculosis were included in the study. The results obtained by MABA for DST of M.tuberculosis were compared with those obtained by B460TB system and Conventional method. DST results obtained by MABA showed good agreement with results obtained by B460TBsystem and Conventional method for Isoniazid and Rifampicin. In our study specificity and sensitivity for susceptible and resistant strain was found to be 98% and 100% for Rifampicin respectively and 100% and 95% for Isoniazid respectively for MABA.MABA appears to be a reliable method for the rapid and simultaneous detection of MDR-TB and DST of M. tuberculosis. It is simple and inexpensive method for DST of M.Tuberculosis with minimal biohazard risk. Article DOI: https://dx.doi.org/10.20319/lijshls.2015.s11.7289 This work is licensed under the Creative Commons Attribution-Non-commercial 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA.

Drug resistance among extrapulmonary TB patients: Six years experience from a supranational reference laboratory.

Background & objectives:There is limited information available about the drug resistance patterns in extrapulmonary tuberculosis (EPTB), especially from high burden countries. This may be due to difficulty in obtaining extrapulmonary specimens and limited facilities for drug susceptibility testing. This study was undertaken to review and report the first and second-line anti-TB drug susceptibility patterns in extrapulmonary specimens received at the National Institute for Research in Tuberculosis (NIRT), Chennai, India, between 2005 and 2012.Methods:Extrapulmonary specimens received from referring hospitals were decontaminated and cultured using standard procedures. Drug susceptibility testing (DST) for Mycobacterium tuberculosis was done by absolute concentration or resistance ratio methods for the first and the second line anti-TB drugs.Results:Between 2005 and 2012, of the 1295 extrapulmonary specimens, 189 grew M. tuberculosis, 37 (19%) cases were multidrug resistant (MDR) while one was extensively drug resistant (XDR). Specimen-wise MDR prevalence was found to be: CSF-10 per cent, urine-6 per cent, fluids and aspirates-27 per cent, pus-23 per cent, lymph nodes-19 per cent. Resistance to isoniazid and ethionamide was found to be high (31 and 38%, respectively).Interpretation & conclusions:Drug resistance including MDR-TB was observed in a significant proportion of extrapulmonary specimens referred for DST. Access to culture and DST for extrapulmonary specimens should be expanded. Guidelines for MDR-TB management should have explicit sections on extra-pulmonary tuberculosis and training on laboratory techniques is urgently required.

Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples

Aims and objectivesCurrent methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) are either costly or slow. As the prevalence of multidrug-resistant (MDR) strains increases, the need for fast, reliable, and inexpensive methods is obvious. This study evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct DST of MTB directly from clinical sputum samples. MethodsA total of 111 sputa with positive microscopy results for acid-fast bacilli (AFB) with more than 10 AFB per high-power field were used in the study. The samples were decontaminated using the modified Petroff method. The NRA results were compared with the reference indirect proportion method. ResultsThe sensitivity and the specificity of the direct NRA were 90% and 97.3%, 92.6% and 98.2%, 52.9% and 100%, and 28.6% and 100% for rifampin, isoniazid, streptomycin, and ethambutol, respectively. The results were in most cases available in 28days (84.3%). ConclusionsThe direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.