Evaluating the Auto-MODS assay, a novel tool for tuberculosis diagnosis for use in resource-limited settings.

There is an urgent need for simple, rapid, and affordable diagnostic tests for tuberculosis (TB) to combat the great burden of the disease in developing countries. The microscopic observation drug susceptibility assay (MODS) is a promising tool to fill this need, but it is not widely used due to concerns regarding its biosafety and efficiency. This study evaluated the automated MODS (Auto-MODS), which operates on principles similar to those of MODS but with several key modifications, making it an appealing alternative to MODS in resource-limited settings. In the operational setting of Chiang Rai, Thailand, we compared the performance of Auto-MODS with the gold standard liquid culture method in Thailand, mycobacterial growth indicator tube (MGIT) 960 plus the SD Bioline TB Ag MPT64 test, in terms of accuracy and efficiency in differentiating TB and non-TB samples as well as distinguishing TB and multidrug-resistant (MDR) TB samples. Sputum samples from clinically diagnosed TB and non-TB subjects across 17 hospitals in Chiang Rai were consecutively collected from May 2011 to September 2012. A total of 360 samples were available for evaluation, of which 221 (61.4%) were positive and 139 (38.6%) were negative for mycobacterial cultures according to MGIT 960. Of the 221 true-positive samples, Auto-MODS identified 212 as positive and 9 as negative (sensitivity, 95.9%; 95% confidence interval [CI], 92.4% to 98.1%). Of the 139 true-negative samples, Auto-MODS identified 135 as negative and 4 as positive (specificity, 97.1%; 95% CI, 92.8% to 99.2%). The median time to culture positivity was 10 days, with an interquartile range of 8 to 13 days for Auto-MODS. Auto-MODS is an effective and cost-sensitive alternative diagnostic tool for TB diagnosis in resource-limited settings.

Evaluation of the BD Bactec MGIT 320 system for detection of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis.

Two different laboratories evaluated growth and detection of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis by the BD Bactec MGIT 320 using the BD Bactec MGIT 960 (BD Diagnostics, Sparks, MD) as a reference method. Out of 359 processed sputum specimens for detection of mycobacteria, 99.7% were in agreement between the MGIT 320 and MGIT 960. Streptomycin (STR), isoniazid (INH), rifampin (RIF), ethambutol (EMB) (collectively known as SIRE), and pyrazinamide (PZA) drug susceptibility testing was performed on 89 clinical strains, prepared from both liquid and solid inocula. The results of SIRE and PZA were 100% reproducible between the two instruments tested at both laboratories.

Multidrug-resistant TB and HIV in Thailand: overlapping, but not independently associated, risk factors.

The HIV and multi-drug resistant tuberculosis (MDR-TB) epidemics are closely linked. In Thailand as part of a sentinel surveillance system, we collected data prospectively about pulmonary TB cases treated in public clinics. A subset of HIV-infected TB patients identified through this system had additional data collected for a research study. We conducted multivariate analysis to identify factors associated with MDR-TB. Of 10,428 TB patients, 2,376 (23%) were HIV-infected; 145 (1%) had MDR-TB. Of the MDR-TB cases, 52 (37%) were HIV-infected. Independent risk factors for MDR-TB included age 18-29 years old, male sex, and previous TB treatment, but not HIV infection. Among new patients, having an injection drug use history was a risk factor for MDR-TB. Of 539 HIV-infected TB patients in the research study, MDR-TB was diagnosed in 19 (4%); the only significant risk factors were previous TB treatment and previous hepatitis. In Thailand, HIV is common among MDR-TB patients, but is not an independent risk factor for MDR-TB. Populations at high risk for HIV-young adults, men, injection drug users - should be prioritized for drug susceptibility testing.

Multi-drug resistant TB and HIV in Thailand: overlapping, but not independently associated risk factors.

The HIV and multi-drug resistant tuberculosis (MDR-TB) epidemics are closely linked. In Thailand as part of a sentinel surveillance system, we collected data prospectively about pulmonary TB cases treated in public clinics. A subset of HIV-infected TB patients identified through this system had additional data collected for a research study. We conducted multivariate analysis to identify factors associated with MDR-TB. Of 10,428 TB patients, 2,376 (23%) were HIV-infected; 145 (1%) had MDR-TB. Of the MDR-TB cases, 52 (37%) were HIV-infected. Independent risk factors for MDR-TB included age 18-29 years old, male sex, and previous TB treatment, but not HIV infection. Among new patients, having an injection drug use history was a risk factor for MDR-TB. Of 539 HIV-infected TB patients in the research study, MDR-TB was diagnosed in 19 (4%); the only significant risk factors were previous TB treatment and previous hepatitis. In Thailand, HIV is common among MDR-TB patients, but is not an independent risk factor for MDR-TB. Populations at high risk for HIV-young adults, men, injection drug users - should be prioritized for drug susceptibility testing.

TB, HIV-associated TB and multidrug-resistant TB on Thailand's border with Myanmar, 2006-2007.

OBJECTIVE: To measure the burden and improve management of tuberculosis (TB), HIV-associated TB and MDR TB in Tak Province, Thailand, which borders Myanmar. METHODS: From September 2006 to August 2007, we collected uniform data about TB cases and enhanced human immunodeficiency virus (HIV) counselling and testing. We provided mycobacterial culture and drug-susceptibility testing in public or non-governmental organization facilities. Patients were classified by nationality and, for non-Thais, by migration status. RESULTS: Of 1662 TB cases in the 12-month period, 1087 (65%) occurred in non-Thais. Of non-Thais, 415 (38%) lived in Myanmar but crossed the border for healthcare. HIV infection was diagnosed in 18% of Thais compared with 12% of non-Thais (P < 0.01); HIV status was unknown for 22% of Thais and 27% of non-Thais (P = 0.02). Overall, multidrug-resistant (MDR) TB was diagnosed in 27 patients, 19 (70%) in non-Thais. Among TB cases never previously treated for TB, no MDR cases were diagnosed in Thais or in Myanmar refugees, but six cases were diagnosed in migrants from Myanmar. CONCLUSIONS: In Thailand, TB, HIV-associated TB and MDR TB in migrants from Myanmar are important public health problems; they need to be resolved in both the countries.

Comparison of microplate hybridization with gel electrophoresis and dot blot hybridization for the rapid detection of Mycobacterium tuberculosis PCR products.

A microplate ELISA hybridization assay has been developed for the detection of the IS6110 PCR products of M. tuberculosis from sputum specimens. In this study, its efficacy was evaluated by comparison with agarose gel electrophoresis (AGE) and dot blot hybridization (DBH), with culture results as the 'gold standard'. The assay was used with 190 sputum samples: the PCR results detected by ELISA and AGE showed close agreement, with sensitivity, specificity and accuracy of 90%, 100% and 96% respectively. The same values for DBH were 92%, 98% and 96% respectively. The validities of these methods were not statistically significantly different (p>0.05). The agreement rates of PCR product detection by AGE comparing with DBH and ELISA were 0.964 and 0.964 respectively, while that of DBH and ELISA was 1.0 by Kappa analysis. The overall agreement was not statistically significantly different (p>0.05). Use of DBH or ELISA hybridization increased the sensitivity of detection by AGE 10-fold from 10 pg to 1 pg of purified DNA per reaction; ie from about 30 to about 3 organisms. The amount of PCR product detected by ELISA was only one half of that detected by the other methods; the total assay time of ELISA following the PCR was 4 hours. In conclusion, the microplate hybridization assay may replace AGE and DBH for the detection of the PCR products of M. tuberculosis because of its sensitivity, specificity and accuracy. Additional advantages of the microplate assay over AGE and DBH include rapidity, ease of use, greater safety, cost effectiveness and greater objectivity in the reading of results; the technique is suitable for use in epidemiological studies for the analysis of a large number of samples.

Direct identification of Mycobacterium tuberculosis from sputum on Ziehl-Neelsen acid fast stained slides by use of silica-based filter combined with polymerase chain reaction assay.

This paper describes a method for isolation of deoxyribonucleic acid (DNA) from Ziehl-Neelsen stained sputum smears on glass slides; and isolated DNA was used for the IS6110 polymerase chain reaction (PCR)-based identification of M. tuberculosis. A total of 221 samples from newly diagnosed suspected tuberculosis cases were first examined by microscopic examination. For DNA extraction by silica-based filter, a home-made modified spin column gave the efficacy as did the nucleospin tissue reagent kit and therefore was selected for PCR template preparation. The extracted DNA was amplified by the IS6110 PCR using a primer pair that amplifies a 377-bp target, and the product was analyzed by agarose gel electrophoresis with confirmation by Southern blot hybridization. In comparison with culture, PCR with template prepared by the silica based filter showed overall sensitivity and specificity of 91.7 and 100 per cent, respectively. This study used the over one year and less than one year slides samples to study the effect of storage time. In the more than one year storage group, PCR assay gave a sensitivity and specificity of 83.3 and 100 per cent, respectively. In conclusion, the applicability of the PCR directly to DNA extracted from Ziehl-Neelsen stained smears could become a valuable alternative approach for rapid identification of M. tuberculosis, and could be used to evaluate quality of the control of local laboratories in tuberculosis (TB) screening and solve the problem of specimen transportation. In addition, the method could be used in retrospective studies involving a wide range of PCR-based analyses, such as detection of rifampicin resistant gene in multidrug-resistant tuberculosis (MDR-TB) study.

Drug-resistant tuberculosis among prisoners of three prisons in Bangkok and the vicinity.

The purpose of this study was to determine the prevalence of drug-resistant tuberculosis and some factors associated with drug resistance among prisoners of three prisons in Bangkok and the vicinity. Susceptibility testing to four first-line antituberculous drugs was performed on 165 M. tuberculosis strains isolated from prisoners of three prisons including Klongprem Central (KC) prison, Bangkwang Central (BC) prison and the Correctional Institution (CI) for Male Drug Addicts. Of 165 smear positive tuberculosis (TB) cases with drugs susceptibility results, resistance to one or more drugs was 49.7 per cent. Resistance to one, two, three, and four drugs was 20.0, 13.3, 4.2 and 12.1 per cent, respectively. Multidrug resistant tuberculosis (MDR-TB) was 18.8 per cent. Patients classified as primary and acquired drug resistant were 6.7 and 50.0 per cent. The primary drug resistance to one or more drugs among prisoners at KC, BC and CI were 42.5, 36.4 and 53.9 per cent, respectively and MDR-TB were 8.2, 3.0, and 7.7 per cent, respectively. Of several factors analyzed in the present study, only a history of previous TB treatment was significantly associated with drug resistance (p < 0.05). In conclusion, the results indicate the high prevalence of drug-resistant tuberculosis and the seriousness of the TB problem in prisons. The public health sector and prison authorities should work in close collaboration and co-ordination to continue improving TB case detection. Directly Observed Treatment Short course (DOTS) is highly recommended. Moreover, discharged prisoners with tuberculosis should be appropriately referred to hospitals or TB control centers.

Prospective evaluation of simply modified MODS assay: an effective tool for TB diagnosis and detection of MDR-TB.

BACKGROUND AND SETTING: Thailand is one of the highest tuberculosis (TB)-burdened countries. Chiang Rai, the northernmost province of Thailand has high tuberculosis and human immunodeficiency virus (HIV) prevalence and the laboratory workload for TB culture and drug susceptibility testing is increasing. OBJECTIVES: To evaluate the simply modified microscopic-observation drug-susceptibility assay (MODS) in the setting of a developing country. METHODS: In this cross-sectional diagnostic study, a total of 202 sputum samples of clinically diagnosed TB patients were used to test the performance of MODS assay in reference to gold standard BACTEC™ MGIT™ 960 liquid culture system and Ogawa solid culture. Sputum samples were collected from clinically diagnosed TB patients. Culture growth rate and time to culture positivity were compared among three methods. Performance of modified MODS assay was evaluated for detection of mycobacterium drug resistance in reference to MGIT antimicrobial susceptibility test (AST). RESULT: Median time to culture positivity by MODS, solid, and liquid culture were 12, 30, and 6 days respectively. Compared to the drug susceptibility test (DST) result of reference liquid culture, the sensitivity and specificity of MODS for detection of multidrug-resistant tuberculosis (MDR-TB) was 85.7% and 97.5% respectively. MODS assay has a positive predicative value of 80% and negative predictive value of 96.5% for isoniazid resistance, 70% and 100% for rifampicin resistance, and 66.7% and 99.1% for MDR-TB. CONCLUSION: MODS is a highly effective screening test for detection of MDR-TB.

Tuberculosis in Thailand: epidemiology and program performance, 2001-2005.

BACKGROUND: The World Health Organization (WHO) recommends a package of services branded 'DOTS' (directly observed treatment, short course) to help countries detect at least 70% of all infectious tuberculosis (TB) cases and cure 85% of detected cases. We analyzed the epidemiology of TB and the national TB program (NTP) performance for the first 5 years of DOTS implementation in Thailand. METHODS: We reviewed data routinely collected through the NTP from 2001 to 2005 and data from special projects conducted by the NTP from 2001 to 2006. RESULTS: In 2005, the TB notification rate was 94 per 100,000 persons. Using the WHO estimated incidence as the denominator, the case detection rate was 76% for smear-positive cases in 2005. From 2002 to 2005, the notification rate declined 2% for smear-positive cases. In 2005, 68% of smear-positive patients were successfully treated; from 2001 to 2005, treatment success never exceeded 75%. Separate surveys conducted from 2002 to 2006 found that 13-17% of TB cases were HIV-infected. The estimated prevalence of multidrug-resistant TB in new patients increased from 1% in 2002 to 1.7% in 2006. CONCLUSIONS: Since DOTS implementation, Thailand has exceeded the international TB case detection target, but has remained well below the treatment success target. The large discrepancy between case finding and treatment success rates indicates that actions are urgently needed to reduce TB morbidity and prevent drug-resistant TB.

Evaluating the potential impact of the new Global Plan to Stop TB: Thailand, 2004-2005.

OBJECTIVE: WHO's new Global Plan to Stop TB 2006-2015 advises countries with a high burden of tuberculosis (TB) to expand case-finding in the private sector as well as services for patients with HIV and multidrug-resistant TB (MDR-TB). The objective of this study was to evaluate these strategies in Thailand using data from the Thailand TB Active Surveillance Network, a demonstration project begun in 2004. METHODS: In October 2004, we began contacting public and private health-care facilities monthly to record data about people diagnosed with TB, assist with patient care, provide HIV counselling and testing, and obtain sputum samples for culture and susceptibility testing. The catchment area included 3.6 million people in four provinces. We compared results from October 2004-September 2005 (referred to as 2005) to baseline data from October 2002-September 2003 (referred to as 2003). FINDINGS: In 2005, we ascertained 5841 TB cases (164/100 000), including 2320 new smear-positive cases (65/100 000). Compared with routine passive surveillance in 2003, active surveillance increased reporting of all TB cases by 19% and of new smear-positive cases by 13%. Private facilities diagnosed 634 (11%) of all TB cases. In 2005, 1392 (24%) cases were known to be HIV positive. The proportion of cases with an unknown HIV status decreased from 66% (3226/4904) in 2003 to 23% (1329/5841) in 2005 (P< 0.01). Of 4656 pulmonary cases, mycobacterial culture was performed in 3024 (65%) and MDR-TB diagnosed in 60 (1%). CONCLUSION: In Thailand, piloting the new WHO strategy increased case-finding and collaboration with the private sector, and improved HIV services for TB patients and the diagnosis of MDR-TB. Further analysis of treatment outcomes and costs is needed to assess this programme's impact and cost effectiveness.