Diagnostic performance of whole-genome sequencing for identifying drug-resistant TB in Thailand.

BACKGROUND: Whole-genome sequencing (WGS) is a promising tool for the detection of drug-resistant TB (DR-TB). To date, there have been few comparisons of diagnostic performance of WGS and phenotypic drug susceptibility testing (DST) in DR-TB.METHODS: We compared drug resistance-conferring mutations identified by WGS analysis using TB-Profiler and Mykrobe with phenotypic DST profiles based on the Löwenstein-Jensen proportion method using drug-resistant Mycobacterium tuberculosis (n = 537) isolates from across Thailand. Based on available phenotypic DST results, diagnostic performance was analysed for resistance against isoniazid, rifampicin, ethambutol (EMB), streptomycin, ethionamide (ETH), kanamycin, capreomycin (CPM), para-aminosalicylic acid, ofloxacin and levofloxacin.RESULTS: High agreement between the two methods was observed for most drugs (>91%), except EMB (57%, 95% CI 53-61) and ETH (70%, 95% CI 66-74). Also, low specificity was observed for EMB (49%, 95% CI 44-54) and ETH (66%, 95% CI 61-71). Sensitivity was high for most drugs (range 83-98%), except CPM (77%, 95% CI 59-88).CONCLUSION: Low agreement between WGS and phenotypic tests for drug resistance was found for EMB and ETH. The current genomic database is insufficient for the identification of CPM resistance. Challenges remain for routine usage of WGS-based DST, especially for second-line anti-TB drugs.

Prevalence and associated risk factors of drug-resistant tuberculosis in Thailand: results from the fifth national anti-tuberculosis drug resistance survey.

OBJECTIVE: To assess the prevalence and risk factors of drug-resistant tuberculosis (TB), the fifth national anti-TB drug resistance survey was conducted in Thailand. METHODS: A cross-sectional study was conducted by stratified cluster sampling with probability proportional to size of TB cases from public health facilities in 100 clusters throughout Thailand from August 2017 to August 2018. Susceptibility testing of TB isolates to first- and second-line anti-TB drugs was performed on Löwenstein-Jensen medium using the indirect proportion method. Multiple imputation was done for handling missing data using Stata 16. The proportion of TB cases with drug resistance was determined. The odds ratio was used to evaluate risk factors associated with drug-resistant TB. RESULTS: Among 1501 new TB and 69 previously treated TB cases, 14.0% [95% confidence interval (CI): 12.1-16.1] and 33.4% (95% CI: 23.6-44.8), respectively, had resistance to any anti-TB drug. Multidrug-resistant TB accounted for 0.8% (95% CI: 0.5-1.4) of new TB cases and 13.0% (95% CI: 6.5-24.4) of previously treated TB cases. Drug-resistant TB was associated with prior TB treatment [odds ratio (OR), 2.9; 95% CI: 1.6-5.0], age at 45-54 years (OR, 1.6; 95% CI: 1.0-2.4), male (OR, 1.5; 95% CI: 1.0-2.1) and human immunodeficiency virus (HIV) infection (OR, 1.6; 95% CI: 1.0-2.4). CONCLUSIONS: The burden of drug-resistant TB remains high in Thailand. Intensified prevention and control measures should be implemented to reduce the risks of drug-resistant TB in high-risk groups previously treated, especially individuals of late middle age, males and those with coinfection of TB and HIV.

Detection of tuberculosis in cynomolgus macaques (Macaca fascicularis) using a supplementary Monkey Interferon Gamma Releasing Assay (mIGRA).

Cynomolgus monkeys (Macaca fascicularis; MF) are commonly used as nonhuman primate models for pharmaceutical product testing. In their habitat range, monkeys have close contact with humans, allowing the possibility of bidirectional transmission of tuberculosis (TB) between the two species. Although the intradermal tuberculin skin test (TST) is used for TB detection in MF, it has limitations. Herein, we established the mIGRA, combining human QuantiFERON-TB Gold-Plus and monkey IFN-γ ELISApro systems, and used it to investigate 39 captive MF who were cage-mates or lived in cages located near a monkey who died from the naturally TB infection. During a 12-month period of study, 14 (36%), 10 (26%), and 8 (21%) monkeys showed TB-positive results using the mIGRA, the TST, and TB culture, respectively. Among the 14 mIGRA-positive monkeys, 8 (57.1%) were TST-positive and 7 (50%) were culture-positive, indicating early TB detection in the latent and active TB stages with the mIGRA. Interestingly, 3 (37.5%) of the TST-negative monkeys were culture-positive. Our study showed that the mIGRA offers many advantages, including high sensitivity and high throughput, and it requires only one on-site visit to the animals. The assay may be used as a supplementary tool for TB screening in MF.

Indo-Oceanic Mycobacterium tuberculosis strains from Thailand associated with higher mortality.

SETTING: This study was conducted among tuberculosis (TB) patients in a highly endemic Thai province.OBJECTIVE: To evaluate the association between different Mycobacterium tuberculosis lineages and clinical characteristics, especially mortality.DESIGN: We enrolled 1,304 TB patients registered from 2002-2011 with culture isolates whose lineages were identified by specific regions of deletion. Data on mortality within 1 year of follow-up were extracted from the registration system and hospital records. Mortality-associated risk factors, including bacterial lineages, as independent variables were analysed using Cox regression models.RESULTS: Of 1,304 isolates, 521 (40.0%) and 582 (44.6%) belonged to Indo-Oceanic and East-Asian lineages, respectively. Indo-Oceanic strains significantly increased the mortality risk compared with East-Asian strains (adjusted hazard ratio [aHR] 1.42, 95%CI 1.02-1.99) or modern lineages (aHR 1.49, 95%CI 1.08-2.06) in the 172 patients who died within 1 year after TB diagnosis. The former also caused significantly higher mortality than modern lineages among patients who died within 6 months after TB diagnosis (aHR 1.62, 95%CI 1.12-2.35). No significant association was found between drug resistance and death.CONCLUSION: In Thailand, the Indo-Oceanic lineage of M. tuberculosis increased mortality risk compared with modern lineages or the East-Asian lineage, the latter being considered highly virulent in previous studies.

Second-line drug susceptibilities of multidrug-resistant tuberculosis strains isolated in Thailand: an update.

The increasing incidence of multidrug-resistant tuberculosis (MDR-TB) and the emergence of extensively drug-resistant tuberculosis (XDR-TB) globally hamper the successful treatment and effective control of TB. Information on second-line drug susceptibility, which is of utmost importance for patient care, is still limited. This study demonstrates the susceptibilities of 1447 strains of MDR-TB, including 58 XDR-TB strains, isolated from Siriraj Hospital, Bangkok, Thailand, to aminoglycosides, fluoroquinolones, ethionamide (ETH), para-aminosalicylic acid (PAS) and linezolid. Results revealed that 93-94% of the MDR-TB strains were susceptible to aminoglycosides, 85-98% to fluoroquinolones, 78% to ETH, 85% to PAS and 99% to linezolid.

Draft Genome Sequence of the Mycobacterium tuberculosis Strain 43-16836, Belonging to the Indo-Oceanic Lineage, Isolated From Tuberculous Meningitis in Thailand.

We present the draft genome sequence of Mycobacterium tuberculosis strain 43-16836, belonging to the Indo-Oceanic lineage, isolated from a tuberculous meningitis patient in Thailand. The genome is 4,381,942 bp long with 4,316 protein-coding genes and contains new single nucleotide polymorphisms (SNPs), including SNPs of genes that may encode cell wall components and possibly influence virulence.

Genotypic analysis of genes associated with isoniazid and ethionamide resistance in MDR-TB isolates from Thailand.

Nucleotide sequences of genes conferring isoniazid resistance (katG, inhA, oxyR-ahpC and ndh) and ethionamide resistance (ethA) in 160 drug-resistant Mycobacterium tuberculosis clinical isolates from Thailand were analysed. Mutations in the katG gene were found in 129 isolates, predominantly at codon 315, which was mutated in 127 isolates. Twenty-two isolates had mutations in the inhA promoter and coding region. Mutations in the oxyR-ahpC intergenic region and in ndh were detected in four and one isolate(s), respectively. Of 24 ethionamide-resistant isolates, 13 had mutations in the ethA gene. However, these mutations were dispersed along the entire gene, with no codon predominating significantly.

Genome-wide SNP-based linkage analysis of tuberculosis in Thais.

Tuberculosis, a potentially fatal infectious disease, affects millions of individuals annually worldwide. Human protective immunity that contains tuberculosis after infection has not been clearly defined. To gain insight into host genetic factors, nonparametric linkage analysis was performed using high-throughput microarray-based single nucleotide polymorphism (SNP) genotyping platform, a GeneChip array comprised 59 860 bi-allelic markers, in 93 Thai families with multiple siblings, 195 individuals affected with tuberculosis. Genotyping revealed a region on chromosome 5q showing suggestive evidence of linkage with tuberculosis (Z(lr) statistics=3.01, logarithm of odds (LOD) score=2.29, empirical P-value=0.0005), and two candidate regions on chromosomes 17p and 20p by an ordered subset analysis using minimum age at onset of tuberculosis as the covariate (maximum LOD score=2.57 and 3.33, permutation P-value=0.0187 and 0.0183, respectively). These results imply a new evidence of genetic risk factors for tuberculosis in the Asian population. The significance of these ordered subset results supports a clinicopathological concept that immunological impairment in the disease differs between young and old tuberculosis patients. The linkage information from a specific ethnicity may provide unique candidate regions for the identification of the susceptibility genes and further help elucidate the immunopathogenesis of tuberculosis.

Prevalence and manifestations of tuberculosis in renal transplant recipients: a single-center experience in Thailand.

This retrospective review presented the prevalence and manifestations of tuberculosis among renal transplant recipients in our center between 1987 and mid 2007. The prevalence of tuberculosis was 5/151 (3.3%) recipients with a median age of 49 years (range = 38-55). The median time of diagnosis after transplantation was 23 months (range = 1-47). All five patients had pulmonary tuberculosis. None developed extrapulmonary infection. Presenting symptoms were fever (60%), productive cough (80%), weight loss (40%), and hemoptysis (20%). One patient had non-parathyroid-related hypercalcemia. Cyclosporine dosage needed to be increased in all patients. Two subjects who experienced side effects of hepatitis and/or jaundice from rifampicin were switched to second-line drugs. Infection with Mycobacterial tuberculosis is a not uncommon problem in renal transplant recipients especially in endemic areas. Tuberculosis must be excluded for immunosuppressed patients with clinical or radiological suspicion.

Distribution of rpoB mutations among multidrug-resistant Mycobacterium tuberculosis (MDRTB) strains from Thailand and development of a rapid method for mutation detection.

Since rifampicin resistance is a surrogate marker for multidrug-resistant Mycobacterium tuberculosis (MDRTB), the present study aimed to investigate rpoB mutations conferring rifampicin resistance in M. tuberculosis strains from Thailand, and to develop a rapid, inexpensive and simple PCR-based method for rapid detection of MDRTB. Overall, 267 M. tuberculosis isolates, including 143 MDRTB isolates, were investigated. Isolates of the Beijing strain predominated among the MDRTB isolates (79.1%), but accounted for only 45.5% of the susceptible isolates. Mutations in the rpoB gene were found most commonly at codons 531, 526 and 516 (58%, 25.2% and 9.1%, respectively). A multiplex allele-specific PCR was developed and tested with 216 clinical isolates. In comparison with the proportion method, the method showed 94.2% sensitivity and 100% specificity, and had a 100% positive predictive value and a 95% negative predictive value, which suggested that this method could be useful for screening for MDRTB, particularly in resource-limited countries.

Restriction fragment length polymorphism study of nationwide samples of Mycobacterium tuberculosis in Thailand, 1997-1998.

SETTING: During 1997-1998, a national anti-tuberculosis drug resistance survey was conducted in Thailand as a part of a global project. OBJECTIVE: To evaluate the IS6110 hybridisation patterns and the level of clustering, which was expected to be low due to the short duration of the sample collection. DESIGN: Eight hundred and twenty-eight bacterial isolates were available for fingerprinting by standard IS6110 hybridisation. RESULTS: The restriction fragment length polymorphism patterns varied with geographic locations, ages of the patients, and resistance to rifampicin and streptomycin. The Beijing strain was more common among younger patients, and their prevalence appeared to decrease with the distance from Bangkok, while the opposite was true for the single-banded isolates. Excluding isolates containing five or less copies of IS6110, 26.4% were clustered. Clustering was more common among females. The clustered isolates were sometimes from different provinces and, if resistant to drugs, usually possessed different resistance profiles. CONCLUSIONS: The results question the validity of inferring recent transmission from the clustering of IS6110 hybridisation patterns in some settings in Thailand. The level of recent transmission in a nationwide study in a country with a high incidence of tuberculosis should be evaluated with caution.

Second-line drug susceptibilities of Thai multidrug-resistant Mycobacterium tuberculosis isolates.

The emergence of multidrug-resistant tuberculosis (MDR-TB) is increasing and is exacerbated by the human immunodeficiency virus (HIV) epidemic. The standard short-course regimen used for the treatment of tuberculosis is likely to be ineffective against MDR-TB, leading to the need for second-line drugs. In such situations, drug susceptibility testing (DST) is necessary to select an appropriate treatment regimen. In this study, DST of 99 MDR-TB strains isolated in Thailand was performed using a drug-impregnated disc method. The results showed that 94.95% of the strains were susceptible to amikacin and kanamycin, 90.91% to ciprofloxacin and ofloxacin, 85.86% to para-aminosalicylic acid, and 78.79% to ethionamide.

Development and evaluation of a one-tube seminested PCR assay for the detection and identification of Penicillium marneffei.

A one-tube seminested polymerase chain reaction (PCR) assay was developed to detect and identify Penicillium marneffei DNA coding for 18S rRNA both from purified DNA and from clinical samples. DNA from 120 strains of organisms and 19 blood samples from AIDS patients was amplified with F3, CPL1 and PM primers. Under optimized conditions, these primers detected 100% specifically amplified products of 251 and 331 bp from all P. marneffei DNA preparations (47 strains) and from two blood samples of AIDS patients suspected to suffer from penicilliosis marneffei. The assay was sensitive to detect as little as 10 pg purified DNA, which is equivalent to 250 cells. This PCR assay might be useful as an alternative test, if a rapid diagnosis of penicilliosis marneffei is needed.

Comparative evaluation of Candi Select test and conventional methods for identification of Candida albicans in routine clinical isolates.

The Candi Select test (Sanofi Diagnostics, Pasteur, Marnes-La-Coquette, France) is a new yeast-selective medium for the identification of Candida albicans in the clinical laboratory. The performance of this test was compared with the conventional methods of chlamydospore formation, germ tube formation and carbohydrate fermentation. Four hundred and twenty clinical yeast isolates from 412 fresh clinical specimens, including 283 C. albicans, 59 C. tropicalis, 39 Trichosporon spp., 19 C. glabrata, 11 Cryptococcus neoformans and 9 other yeasts, were evaluated. Colonies of C. albicans produced a blue-green colour on the Candi Select media which could be distinguished from the other yeasts with the naked eye within 24-48 h. The sensitivity and specificity of the Candi Select test for the identification of C. albicans were 99.65% and 97.08%, respectively. The blue-green colonies of C. albicans were easy to identify and recognize in mixed cultures and did not need detailed microscopic examination.

Rapid and simple MTT method for rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis.

The MTT method for rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis was developed by using bacterial suspension prepared from colonies on solid media. The MTT tube assay in 1 ml Middlebrook 7H9 broth was completed within 4 days for rifampicin (RMP) and within 7 days for isoniazid (INH). When MTT assay results with 279 M. tuberculosis clinical isolates were compared with those of the conventional proportion method on Löwenstein-Jensen medium, high specificity and sensitivity values of 100% and 94.1%, respectively, for RMP susceptibility testing, and 99.5% and 89.2%, respectively, for INH susceptibility testing were obtained. The accuracy of the MTT method for RMP and INH was > 0.97 concordance with the proportion method. The MTT method is simple, inexpensive and rapid. The high level of agreement with the conventional proportion method suggests a potential to rapidly detect drug-resistant M. tuberculosis in developing countries, as only basic microbiological equipment is need.

Antibody detection for the diagnosis of tuberculous pleuritis.

SETTING: University Hospital, Bangkok, Thailand. OBJECTIVE: To evaluate the diagnostic value of antibody detection in serum and in pleural effusion as a marker of tuberculous pleuritis (TBP). DESIGN: Cross-sectional study. MATERIALS AND METHODS: One hundred and fifty-five patients with pleural effusion who underwent diagnostic evaluation at Siriraj Hospital between March 1999 and May 2000 were recruited. Samples of pleural fluid were examined biochemically, cytologically and microbiologically. Pathological examination of pleural tissue was also performed. The diagnosis of TBP or other diagnosis was made by either pathological finding or culture result. Immunochromatographic tuberculosis (ICT-TB) tests for antibody detection were then performed using the stored serum samples and effusions from those patients with a final definite diagnosis. This test detects antibodies to five secreted antigens of Mycobacterium tuberculosis, including the 38 kDa antigen. RESULTS: We investigated 67 patients with TBP, 44 with malignant pleural effusions, seven with transudates and one with cryptococcal pleuritis. The combined ICT-TB serum and effusion tests were positive in 34/67 TBP and 22/52 non-TBP patients. The sensitivity, specificity, positive predictive value and negative predictive value of the ICT-TB test were 50.7, 57.7, 60.7 and 47.6%, respectively. In 11 TBP patients with human immunodeficiency virus (HIV) co-infection, the sensitivity of the ICT-TB test was 45.6%. There was no correlation between the test positivity and culture result or duration of disease. CONCLUSIONS: The diagnostic value of antibody detection in TBP is modest in an area with intermediate prevalence of tuberculosis, independently of HIV serological status.

Detection of Mycobacterium tuberculosis from sputum specimens using one-tube nested PCR.

One-tube nested PCR was developed for diagnosis of pulmonary tuberculosis using sequences based on thel6SrRNA gene. The usage of primers 16SOL, 16SOR, 16SIL and 16SIR with optimized conditions could detect 555 bp DNA band from 21 species, 41 strains of mycobacteria and one isolate of Nocardia asteroides. It also revealed a specific 306 bp DNA band from 59 strains of M. tuberculosis complex. Cross amplification was observed in M. marinum, M. ulcerans and a few isolates of M. fortuitum complex. The developed method could detect as little as 100 fg of M. tuberculosis DNA. The PCR mixtures could be stored at 0 degrees C for 2 months or at -20 degrees C for at least 20 months without decrease in sensitivity. Using one-tube nested PCR for detection of M. tuberculosis compared with acid fast staining and culture results from 153 sputum specimens revealed 88.6% sensitivity and 89.2% specificity in smear positive specimens and 93.2% sensitivity and 85.0% specificity in culture positive specimens.

Penicilliosis-associated hemophagocytic syndrome in a human immunodeficiency virus-infected child: the first case report in children.

Infection-associated hemophagocytic syndrome (IAHS) has been found in many systemic infectious conditions with a high mortality rate. Disseminated Penicillium marneffei infection is a common opportunistic condition among HIV-infected patients in many regions in Southeast Asia. We report the first case of IAHS caused by penicilliosis in an HIV-infected child who presented with cytopenias and recovered promptly after antifungal and intravenous immunoglobulin therapy.

Penicillium marneffei: types and drug susceptibility.

The PCR fingerprints of 30 Penicillium marneffei isolates from Chiang Rai in Northern Thailand and Bangkok in central Thailand were studied through use of single-nucleotide primers (GACA)4 and the phage M13 core sequence. Discrimination of fingerprint patterns was based on differences in the number of major bands. The P. marneffei isolates were divided into four types, i.e., A, B, C, and D. Type A was found in two isolates from Chiang Rai (6.7%). Types B and C respectively were found in two (6.7%) and one (3.3%) isolates from Bangkok. The predominate type D (83.3%) was found in isolates obtained from Chiang Rai and Bangkok. The PCR fingerprinting method was found to be useful for the epidemiological study of P. marneffei, a dimorphic opportunistic fungus and an emerging pathogen in the HIV pandemic. In vitro drug susceptibility testing by broth macrodilution to four antifungal agents against the yeast form of P. marneffei was performed. The MIC ranges for amphotericin B, fluconazole, itraconazole, and ketoconazole were 0.125-0.5, 4.0-8.0, < 0.032, and < 0.125 microgram/ml respectively.