The first insight into Mycobacterium tuberculosis complex isolates in the lower northern region in Thailand.

BACKGROUND: Tuberculosis (TB) remains an important infectious disease and different genotypes have been reported. This study aimed to investigate the genetic diversity and molecular epidemiology of TB in the lower northern region of Thailand, where genotyping data are limited. METHODS: A total of 159 Mycobacterium tuberculosis complex (MTBC) isolates from this region were genotyped by spoligotyping and the major spoligotypes were further subdivided by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method. RESULTS: Spoligotyping identified 34 types and classified them into 14 clusters. East African-Indian (EAI) groups were the most frequent (44.7%), followed by Beijing (36.5%), with a higher prevalence of drug resistance. By 15-loci MIRU-VNTR typing, the major groups of the Beijing and EAI2_NTB were further differentiated into 44 and 21 subtypes forming 9 and 5 subclusters with cluster rates of 0.26 and 0.44, respectively. The Hunter-Gaston Discriminatory Index among the Beijing and EAI2_NTB groups were 0.987 and 0.931, respectively, indicating high diversity. CONCLUSIONS: This is the first look at the MTBC genotypes in the lower northern region of Thailand, which could aid in understanding the distribution and potential spread of MTBC and Mycobacterium bovis in the target region to support TB control in Thailand.

Clinical Performance of the Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for the Diagnosis of COVID-19 in a Thai Community Hospital at the Thailand-Myanmar Border.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) continues to be a global health threat and is a public health issue in Thailand and other countries. The extensive cross-border between Thailand and Myanmar is considered to be at a potentially high risk for COVID-19 distribution in this region. In this instance, simple and cost-effective tests for rapid and early detection of COVID-19 would be useful for effective patient management and control of the disease. METHODS: This study was conducted at Mae Sot Hospital on the border of Thailand-Myanmar to evaluate the diagnostic performance of a simple colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay developed recently for the rapid detection of SARS-CoV-2. Nasopharyngeal specimens were routinely collected and processed through automated nucleic acid extraction followed by real-time reverse transcription-polymerase chain reaction (rRT-PCR) using the Molaccu® COVID-19 Detection Kit. The RT-LAMP assay was further performed on remnant RNA samples, and the visual results were compared to those of rRT-PCR as a reference. RESULTS: Of the 727 samples tested, the RT-LAMP assay could detect 322 out of 374 samples positive for SARS-CoV-2 by rRT-PCR with 100% (n = 353/353) negative agreement. The comparative analysis demonstrated the overall accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP at 92.85% (n = 675/727, 95% CI: 90.73-94.61), 86.10% (n = 322/374, 95% CI: 82.17-89.44), 100% (n = 353/353, 95% CI: 98.96-100), 100% (n = 322/322, 95% CI: 98.86-100), and 87.16% (n = 353/405, 95% CI: 84.06-89.73), respectively. CONCLUSION: This RT-LAMP assay showed good diagnostic performance in the hospital setting. It can increase laboratory capacity for rapid SARS-CoV-2 testing and has the potential for use as an alternative or a backup assay at the point of need, especially where alternatives are unavailable for any reason, such as a decline in COVID-19 cases.

QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-tube assays for detecting latent tuberculosis infection in Thai healthcare workers.

Detecting latent tuberculosis infection (LTBI) is important, especially in high-risk populations including healthcare workers (HCWs). QuantiFERON-TB Gold Plus (QFT-Plus) is a new version of the interferon-gamma release assays (IGRAs) to replace the QuantiFERON-TB Gold In-tube (QFT-GIT). However, data on the use of QFT-Plus for LTBI detection in high TB-burden countries are limited. This study was conducted in a TB-endemic setting in Thailand. HCWs were enrolled in the study and underwent both tests during the annual health screening. The testing results were compared and the concordance was determined. Of 102 HCWs, 11 (10.78%) were positive according to both tests, and 15 (14.71%) were positive according to QFT-Plus. The overall agreement between assays was 96.08%, with Cohen's kappa coefficient (k) at 0.82. All four discordant results occurred with QFT-GIT negative and QFT-Plus positive. The comparison between QFT-GIT and QFT-Plus based on each antigen tube (TB1 or TB2) exhibited similar concordance with 99.02% and 95.10% agreement, respectively. The intra-comparison between TB1 and TB2 of QFT-Plus also showed good concordance at 96.08%. Among this group of HCWs, the LTBI prevalence of any positive results in both tests was low. Overall, the study showed good agreement between QFT-Plus and QFT-GIT (k = 0.82) with a minimal difference, suggesting similar assay performance to that mainly carried out in TB-low incidence countries. The results support the use of QFT-Plus for detecting LTBI in a format similar to QFT-GIT.

Molecular Characterization of Mutations in Isoniazid- and Rifampicin-Resistant Mycobacterium tuberculosis Isolated in Thailand.

The control of drug-resistant tuberculosis (TB) is a major challenge. The frequency and mutation characteristics indicate the efficiency of molecular tests for the rapid detection of TB drug resistance. This study examined the existence of katG and inhA mutations for isoniazid (INH) resistance and rpoB mutations for rifampicin (RFP) resistance. In total, 178 drug-resistant Mycobacterium tuberculosis (MTB) isolates were analyzed. Mutations in katG encoding and inhA regulatory regions were detected in 136/168 (81.0%) and 29/168 (17.3%), respectively, with the most prominent mutation of Ser315Thr substitution in katG in 126/168 (75.0%), and -15 C to T substitution in the regulatory region of the inhA (26/168; 15.5%). Two distinct katG mutations (Tyr337Cys, 1003InsG) were identified. Of 125 RFP-resistant isolates, 118 (94.4%) carried mutations affecting the 81-bp RFP resistance-determining region, with the most commonly affected codons 450, 445, and 435 identified in 74 (59.2%), 26 (20.8%), and 12 (9.6%) isolates, respectively. Genetic mutations were highly associated with phenotypic INH and RFP resistance, and the majority shared similarities with those reported in previous studies in Thailand and other Asian countries. These data are useful for guiding the use and improvement of molecular tests for TB drug resistance.

QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-tube assays for detecting latent tuberculosis infection in Thai healthcare workers

ABSTRACT Detecting latent tuberculosis infection (LTBI) is important, especially in high-risk populations including healthcare workers (HCWs). QuantiFERON-TB Gold Plus (QFT-Plus) is a new version of the interferon-gamma release assays (IGRAs) to replace the QuantiFERON-TB Gold In-tube (QFT-GIT). However, data on the use of QFT-Plus for LTBI detection in high TB-burden countries are limited. This study was conducted in a TB-endemic setting in Thailand. HCWs were enrolled in the study and underwent both tests during the annual health screening. The testing results were compared and the concordance was determined. Of 102 HCWs, 11 (10.78%) were positive according to both tests, and 15 (14.71%) were positive according to QFT-Plus. The overall agreement between assays was 96.08%, with Cohen's kappa coefficient (k) at 0.82. All four discordant results occurred with QFT-GIT negative and QFT-Plus positive. The comparison between QFT-GIT and QFT-Plus based on each antigen tube (TB1 or TB2) exhibited similar concordance with 99.02% and 95.10% agreement, respectively. The intra-comparison between TB1 and TB2 of QFT-Plus also showed good concordance at 96.08%. Among this group of HCWs, the LTBI prevalence of any positive results in both tests was low. Overall, the study showed good agreement between QFT-Plus and QFT-GIT (k = 0.82) with a minimal difference, suggesting similar assay performance to that mainly carried out in TB-low incidence countries. The results support the use of QFT-Plus for detecting LTBI in a format similar to QFT-GIT.

Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand.

Loop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.

Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand

ABSTRACT Loop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.

Putative extensive and pre-extensive drug resistant-tuberculosis associated with unusual genotypes on the Thailand-Myanmar border.

Extensive drug-resistant tuberculosis (XDR-TB) is highly life threatening and its diagnosis is usually difficult and time-consuming. Here we present the first two cases of XDR and pre-XDR-TB diagnosed in 2018 on the Thailand-Myanmar border, more specifically in Tak province. Rapid detection of XDR-TB was performed by loop-mediated isothermal amplification (LAMP), Xpert MTB/RIF, and line probe assays. Mutation analyses targeting rpoB, katG, inhA, gyrA and rrs genes showed an association with drug-resistant phenotypes, except for rifampicin resistance. Spoligotyping revealed uncommon Beijing and T2 genotypes and the analysis of M. tuberculosis interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) showed the presence of more polymorphisms. This report highlights the importance of the early detection of drug-resistant tuberculosis by molecular tests followed by phenotyping assays. Based on the up-to-date definition of XDR- and pre-XDR-TB, the susceptibility testing for bedaquiline and linezolid is required and the two reported cases may correspond to putative XDR-TB.

T-SPOT®.TB test and clinical risk scoring for diagnosis of latent tuberculosis infection among Thai healthcare workers.

BACKGROUND: Screening for latent tuberculosis infection (LTBI) is important to identify healthcare workers (HCWs) benefiting from preventive therapy. Interferon-gamma release assays (IGRAs) are sensitive and specific tests for LTBI diagnosis. However, in settings where IGRAs are not available, clinical risk assessment may be used as an alternative to diagnose LTBI. METHODS: A cross-sectional study was conducted among HCWs of a tertiary-care university hospital in Thailand. All HCWs underwent T-SPOT®.TB test (T-SPOT) and assessment of LTBI clinical risks. Clinical risks associated with T-SPOT positivity were determined by multivariable logistic regression analysis and were given scores accordingly. The performance of the clinical risk scoring was evaluated in comparison to T-SPOT. RESULTS: Among 140 enrolled HCWs, 125 (89%) were females, the median age was 27 years and 23 (16%) had T-SPOT positivity. Independent factors associated with T-SPOT positivity were age ≥30 years (adjusted odds ratio [aOR] 3.95; P = 0.002), working duration ≥60 months (aOR 3.75, P = 0.004) and frequency of TB contact ≥6 times (aOR 8.83, P = 0.005). The study's clinical risk scoring had the area under the curve by receiver operating curve analysis of 0.76 (P < 0.001) using T-SPOT positivity as a reference standard. The score of ≥3 had the best performance in diagnosing LTBI with sensitivity, specificity, positive predictive value and negative predictive value of 70%, 71%, 32% and 92%, respectively. CONCLUSIONS: In this setting where LTBI was prevalent among HCWs but IGRAs are not widely available, the clinical risk scoring may be used as an alternative to diagnose LTBI in HCWs.

The performance of an in-house loop-mediated isothermal amplification for the rapid detection of Mycobacterium tuberculosis in sputum samples in comparison with Xpert MTB/RIF, microscopy and culture.

Simple, low-cost and effective diagnostic tests for tuberculosis (TB) are needed especially in TB-high burden settings. The present study evaluated the performance of an in-house loop-mediated isothermal amplification (LAMP) for diagnosing TB by comparing it to Xpert MTB/RIF, microscopy and culture. In Thailand, a total of 204 excess sputum samples volume after the processing of cultures were used for Mycobacterium tuberculosis (MTB) detection by Xpert MTB/RIF and LAMP. Based on culture results as the gold standard, the overall sensitivity of LAMP and Xpert MTB/RIF were 82.1% (126/153; 95% confidential interval [CI]: 75.4-88.98%) and 86.9 % (133/153; 95% CI: 80.5-90.8%) respectively, and the specificity of both tests was 100% (51/51; 95% CI: 93.0-100.0%). In comparison with Xpert MTB/RIF, the sensitivity and specificity of LAMP were 94.7% (126/133; 95% CI: 89.5-97.9%), and 100.0% (73/73; 95% CI: 94.9-100.0%), respectively. The average threshold cycle (Ct) of Xpert MTB/RIF detection for positive and negative LAMP results was statistically different, of 18.4 and 27.0, respectively (p < 0.05). In comparison with the acid-fast staining technique, and analyzing LAMP and Xpert MTB/RIF in smear-negative/culture-positive specimens, there was an increase of the detection rate by 47.7% (21/44) and 54.6% (24/44). The diagnostic sensitivity and specificity of LAMP appeared to be comparable to those of Xpert MTB/RIF. We claim that this LAMP has potential to provide a sensitive diagnostic test for the rapid TB diagnosis. It allowed a fast detection of MTB before the cultures and it could be used in resource-limited laboratory settings.

Comparison of Loop-Mediated Isothermal Amplification, Microscopy, Culture, and PCR for Diagnosis of Pulmonary Tuberculosis.

The diagnosis of tuberculosis (TB) in endemic countries is challenging due to high caseloads and limited resources. A simple and cost-effective diagnostic test for the rapid detection of Mycobacterium tuberculosis (M. tuberculosis) in clinical specimens is crucially needed. We evaluated the performance of an in-house assay based on loop-mediated isothermal amplification (LAMP) targeting the M. tuberculosis 16S ribosomal RNA (rRNA) gene for the diagnosis of TB in Thailand. A total of 252 sputum samples from suspected cases of pulmonary TB were analyzed. The sensitivity of LAMP was 99.04% (103/104; 95% confidence interval [CI]: 94.76-9.98%) and 72.73% (16/22; 95% CI: 49.78-89.27%) for smear-positive and smear-negative samples with TB-culture positivity, respectively. LAMP detected 20.69% (24/116) of TB culture negative samples but all those were positive by conventional polymerase chain reaction (PCR). The sensitivity of LAMP was higher than that of sputum microscopy while the performance of LAMP was similar to PCR. None of the samples positive for non-tuberculous mycobacteria by culture and PCR were positive by LAMP. Compared to TB culture, the positive predictive value (PPV), negative predictive value (NPV), and kappa coefficient of LAMP were 83.22%, 88.33%, and 0.75 respectively. Based on the diagnostic performance, we propose that LAMP would be suitable as a potential diagnostic test for rapid TB diagnosis in resource-limited laboratory settings.

Loop-Mediated Isothermal Amplification for Rapid Identification of Mycobacterium tuberculosis in Comparison with Immunochromatographic SD Bioline MPT64 Rapid® in a High Burden Setting.

Loop-mediated isothermal amplification (LAMP) was assessed for rapid identification of Mycobacterium tuberculosis complex (MTC) in comparison with an immunochromatographic test (ICT) using SD Bioline Ag MPT64 Rapid®. One hundred and fifty-one MGIT cultures positive for acid-fast bacilli were tested for MTC. DNA was extracted from a small portion of culture samples by heat lysis and subjected to LAMP analysis. Of these, 144 were positive and 5 were negative by both tests. One culture that was ICT negative but was LAMP positive was confirmed to have a mutation in the mpt64 gene. The agreement was 98.68% (95% confidence interval [CI]: 94.80-99.77), and the kappa value was 0.83% (95% CI: 0.59-1.00). Good correlation results suggested that LAMP assay is a reliable molecular test for rapid identification of MTC and is practical for use in resource-limited, high burden settings.

QuantiFERON-TB Gold In-Tube test in active tuberculosis patients and healthy adults.

Interferon-gamma (IFN-γ) release assays have improved latent tuberculosis (TB) detection and have been considered promising for the diagnosis of TB disease. However, diagnosis efficacy data is limited in high burden countries. The aim of this study was to determine the diagnostic potential of the QuantiFERON-TB Gold In-Tube (QFT-GIT) test for the diagnosis of active TB in an endemic setting for TB. A cross-sectional study was conducted in a group of 102 Thai patients with clinical symptoms and chest x-ray findings suggesting of active pulmonary TB and a group of 112 healthy adults. Testing was carried out using sputum microscopy, mycobacterial culture and QFT-GIT test. Of these patients, QFT-GIT was positive in 73 (71.57%), negative in 27 (26.47%), and undetermined in 2 (1.96%) cases. Among healthy controls, QFT-GIT was positive in 18 (16.07%), negative in 93 (83.04%), and undetermined in 1 (0.89%) person. Based on TB culture results, the sensitivity of QFTGIT for diagnosing active TB was 84.21% (95% confidence interval (CI); 72.13-92.52). The positive and negative predictive values were 65.75% (95% CI; 59.26-71.70) and 66.67% (95% CI; 49.94-80.04), respectively. The median IFN-γ level in culture-confirmed TB patients was 3.91 compared to 0.03 IU/mL of the healthy group. QFT-GIT appears to be a useful indirect test for TB diagnosis in Thailand and its use is recommended in association with clinical and radiological assessments for identifying active or latent TB.

QuantiFERON-TB Gold In-Tube Test for Tuberculosis Prevention in HIV-Infected Patients.

Optimal testing strategies for diagnosing latent tuberculosis infection and the administration of isoniazid preventive therapy (IPT) remain uncertain among human immunodeficiency virus (HIV)-infected patients. A 4-year prospective study was conducted among Thai HIV-infected patients who underwent simultaneous tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube Test (QFT-IT) at care entry. Based on baseline test results, patients were categorized into the following 4 groups: i) QFT-IT-positive, TST-reactive; ii) QFT-IT-positive, TST-non-reactive; iii) QFT-IT-negative, TST-reactive; and iv) QFT-IT-negative, TST-non-reactive. The QFT-IT-positive patients were offered 9-month IPT and were QFT-IT tested annually. Of the 150 enrolled patients, 8, 12, 16, and 114 patients were assigned to groups 1, 2, 3, and 4, respectively. Sixteen of 19 QFT-IT-positive patients (84%) completed IPT. The incidence of tuberculosis was significantly higher in patients who declined IPT than in those underwent treatment (11.11 vs. 0 case/100 patient-year; P ＜ 0.001). Among the 16 patients completing IPT, 11 (69%) and 2 (12%) had QFT-IT reversion at 1 and 2 years after IPT, respectively. The remaining 3 (19%) did not demonstrate any reversion, and their baseline interferon-γ (IFN-γ) levels were above 1.2 IU/mL. Initial QFT-IT-guided IPT was effective in preventing tuberculosis. Serial QFT-IT for evaluating IPT effectiveness had limitations because of delayed or lack of reversion, especially for patients with high baseline IFN-γ levels.

Tuberculin Skin Test and QuantiFERON(®)-TB Gold In-Tube Test for Diagnosing Latent Tuberculosis Infection among Thai Healthcare Workers.

A cross-sectional study was conducted on the performance of the tuberculin skin test (TST) and QuantiFERON(®)-TB Gold In-Tube test (QFT-IT) for detecting latent tuberculosis infection among Thai healthcare workers (HCWs). Each HCW underwent both the TST and QFT-IT during the annual health screening. Among the 260 HCWs enrolled, the median age was 30 years (range 19-60 years), 92% were women, 64% were nurses and nurse assistants, 78% were Bacillus Calmette Guérin vaccinated, and 37% had previously taken the TST. Correlation between TST reaction size and the interferon-γ level was weak (r = 0.29; P < 0.001). Of the HCWs, 38% and 20% had a reactive TST and a positive QFT-IT, respectively. Using QFT-IT positivity as a standard for latent tuberculosis diagnosis, the cut-off for TST reactivity with the best performance was ≥13 mm with a sensitivity, specificity, false positivity, and false negativity of 71%, 70%, 30%, and 29%, respectively (area under the curve 0.73; P < 0.001). The independent factor associated with a false reactive TST was a previous TST (adjusted odds ratio 1.83; P = 0.04). Our findings suggest that the QFT-IT may be the preferred test among HCWs with previous TST. In settings where the QFT-IT is not available, appropriate cut-offs for TST reactivity should be evaluated for use among HCWs.

Tuberculin skin test and QuantiFERON-TB Gold In-tube Test for latent tuberculosis in Thai HIV-infected adults.

BACKGROUND AND OBJECTIVE: Limited data exist for the performance of QuantiFERON-TB Gold In-tube Test (QFT-IT) in comparison to tuberculin skin test (TST) for detecting latent tuberculosis (LTB) in patients with human immunodeficiency virus (HIV) infection from tuberculosis (TB)-endemic Asia-Pacific countries. METHODS: A cohort study of Thai HIV-infected patients without history of TB or LTB treatment was conducted from March 2012 through March 2013. Each patient underwent simultaneous TST and QFT-IT. RESULTS: Among the 150 enrolled subjects, the median age was 40 years (range 17-65), 53% were male, and the median CD4 count was 367 cells/µL (range 8-1290). Reactive TST and positive QFT-IT were 16% and 13%, respectively, with low concordance between tests (kappa = 0.26); correlation between TST reaction size and level of interferon-γ was moderate (r = 0.34). Independent factors associated with discordant results were long-term smoking (adjusted odds ratio (aOR) 5.74; P = 0.002) for TST-reactive, QFT-IT-negative subjects, and age greater than 52 years (aOR 5.56; P = 0.02) and female gender (aOR 4.40; P = 0.04) for TST non-reactive, QFT-IT-positive subjects. The level of agreement between both tests improved when using a TST cut-off of ≥ 10 mm (kappa = 0.39). CONCLUSIONS: In our setting where QFT-IT is available but has limited use due to cost, TST with a cut-off of 10 mm for reactivity should be the initial LTB test. HIV-infected women and persons older than 52 years with non-reactive TST and long-term smokers with reactive TST may benefit from subsequent QFT-IT.

Applicability of in-house loop-mediated isothermal amplification for rapid identification of Mycobacterium tuberculosis complex grown on solid media.

A simple, rapid, and low-cost identification method is required in tuberculosis high-burden countries. We report the applicability of in-house loop-mediated isothermal amplification (LAMP) targeting 16S ribosomal RNA for the rapid identification of Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media. Eighty acid-fast staining-positive clinical isolates were selected and used to evaluate the LAMP assay in comparison with polymerase chain reaction and conventional culture-based tests. The LAMP assay identified 60 M. tuberculosis isolates from 80 clinical isolates using simple heat-extracted DNA directly from the colony suspension. The results were in complete agreement with those obtained using the other methods, and the utility of the direct LAMP assay from a colony was demonstrated. The LAMP assay appears to be a practical and low-cost method that can be used for the rapid identification of M. tuberculosis isolates and suitable for endemic low-resource settings.

Rapid identification of Mycobacterium tuberculosis in BACTEC MGIT960 cultures by in-house loop-medicated isothermal amplification.

Definitive diagnosis of tuberculosis (TB) by conventional culture, followed by bacterial identification based on biochemical tests is time-consuming and tedious. Simple loop-mediated isothermal amplification (LAMP) specific for Mycobacterium tuberculosis complex, targeting the M. tuberculosis 16S ribosomal RNA gene, termed TB-LAMP, was evaluated as an alternative for rapid culture confirmation. TB-LAMP was assessed for its ability to detect M. tuberculosis complex in BACTEC MGIT 960-positive cultures. Of the 103 cultures evaluated, 100 were identified to contain M. tuberculosis complex by TB-LAMP and had concordant results with standard biochemical tests of niacin accumulation, nitrate reductase, lack of heat-stable catalase, and susceptibility to para-nitrobenzoic acid. These results indicate that TB-LAMP in combination with BACTEC MGIT 960 is a specific, reliable, and technically feasible method for rapid and accurate identification of M. tuberculosis complex.

SNP genotypes of Mycobacterium leprae isolates in Thailand and their combination with rpoT and TTC genotyping for analysis of leprosy distribution and transmission.

Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.