High rate of reinfection and possible transmission of Mycobacterium avium complex in Northeast Thailand.

The Mycobacterium avium complex (MAC) includes two main species of non-tuberculous mycobacteria (NTM), M. avium and Mycobacterium intracellulare. These can cause serious disease, especially in immunocompromised patients. Little information is available concerning genetic diversity of NTM. We used multilocus sequence typing (MLST) based on a highly discriminative gene set to analyze MAC serially isolated from patients to determine the rate of MAC reinfection. Genomic DNA was sequenced from 49 MAC isolates (15 cases comprised of 11 true infections and 4 instances of colonization). More than half of the MAC isolates tested were found to be multidrug resistant. The discriminatory power was assessed of 24 house-keeping genes (fusA, atpD, pheT, glnA, topA, secA, argH, glpK, murC, cya, pta, rrl, rrs, hsp65, rpoB, 16S-23S rRNA ITS, recF, lipT, pepB, gnd, aspB, groEL, sodA and est) previously used for genotyping of MAC and other NTM. Seven genes (fusA, secA, rpoB, hsp65, 16S rRNA, 23S rRNA, 16S-23S rRNA ITS) had a discriminatory power index higher than 0.9 and were included in the optimized set that we used. This set was significantly better for genotyping and diagnosis of MAC than previously used 4-gene, 5-gene and 9-gene sets. MLST using our 7-gene set indicated that the rate of reinfection was 54.55% (6/11 cases). Persistent infections (n = 5 cases, 45.45%) were found. A changing of clone in the same patient was found in 1/4 (25%) of the colonization cases. Two small clusters of possible MAC transmission between humans were found. Our study demonstrated that the high frequency of apparent treatment failure of MAC might be artefactual, as a consequence of a high rate of MAC reinfection in Thai population. Our useful highly discriminative gene set for MAC species and clonal strain analysis could be further applied for the diagnosis and patient management.

Assessment of antimycobacterial activities of pure compounds extracted from Thai medicinal plants against clarithromycin-resistant Mycobacterium abscessus.

BACKGROUND: Infection with Mycobacterium abscessus is usually chronic and is associated with clarithromycin resistance. Increasing drug resistance is a major public-health problem and has led to the search for new antimycobacterial agents. We evaluated the antimycobacterial activity, toxicity, and synergistic effects of several plant secondary metabolites against M. abscessus. METHODS: Twenty-three compounds were evaluated for antimycobacterial activity against thirty M. abscessus clinical isolates by broth microdilution to determine their minimum inhibitory concentration (MIC) values. Toxicity was evaluated using red and white blood cells (RBCs and WBCs). The compounds were used in combination with clarithromycin to investigate the possibility of synergistic activity. RESULTS: Five out of twenty-three compounds (RL008, RL009, RL011, RL012 and RL013) exhibited interesting antimycobacterial activity against M. abscessus, with MIC values ranging from <1 to >128 µg/mL. These extracts did not induce hemolytic effect on RBCs and displayed low toxicity against WBCs. The five least-toxic compounds were tested for synergism with clarithromycin against seven isolates with inducible clarithromycin resistance and seven with acquired clarithromycin resistance. The best synergistic results against these isolates were observed for RL008 and RL009 (8/14 isolates; 57%). CONCLUSIONS: This study demonstrated antimycobacterial and synergistic activities of pure compounds extracted from medicinal plants against clarithromycin-resistant M. abscessus. This synergistic action, together with clarithromycin, may be effective for treating infections and should be further studied for the development of novel antimicrobial agents.

Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria.

Mixed infection with multiple species of nontuberculous mycobacteria (NTM) is difficult to identify and to treat. Current conventional molecular-based methods for identifying mixed infections are limited due to low specificity. Here, we evaluated the utility of whole-genome sequencing (WGS) analysis to detect and identify mixed NTM infections. Analytical tools used included PubMLST, MetaPhlAn3, Kraken2, Mykrobe-Predictor and analysis of heterozygous SNP frequencies. The ability of each to identify mixed infections of NTM species was compared. Sensitivity was tested using 101 samples (sequence sets) including 100 in-silico simulated mixed samples with various proportions of known NTM species and one sample of known mixed NTM species from a public database. Single-species NTM control samples (155 WGS samples from public databases and 15 samples from simulated reads) were tested for specificity. Kraken2 exhibited 100% sensitivity and 98.23% specificity for detection and identification of mixed NTM species with accurate estimation of relative abundance of each species in the mixture. PubMLST (99% and 96.47%) and MetaPhlAn3 (95.04% and 83.52%) had slightly lower sensitivity and specificity. Mykrobe-Predictor had the lowest sensitivity (57.42%). Analysis of read frequencies supporting single nucleotide polymorphisms (SNPs) could not detect mixed NTM samples. Clinical NTM samples (n = 16), suspected on the basis of a 16S-23S rRNA gene sequence-based line-probe assay (LPA) to contain more than one NTM species, were investigated using WGS-analysis tools. This identified only a small proportion (37.5%, 6/16 samples) of the samples as mixed infections and exhibited only partial agreement with LPA results. LPAs seem to be inadequate for detecting mixed NTM species infection. This study demonstrated that WGS-analysis tools can be used for diagnosis of mixed infections with different species of NTM.

Differentiation between persistent infection/colonization and re-infection/re-colonization of Mycobacterium abscessus isolated from patients in Northeast Thailand.

Mycobacterium abscessus can cause true infection or be present in the host as a harmless colonist. The ability of M. abscessus to cause disease and develop drug resistance is known to have a genetic basis. We aimed to differentiate between persistent infection and reinfection using multilocus sequence typing (MLST) and to study the genetic diversity of M. abscessus relative to multi-organ infection and drug resistance in Northeast Thailand. DNA was extracted from 62 M. abscessus isolates (24 cases). The following genes were sequenced: argH, cya, glpK, gnd, murC, pta, purH and rpoB. Drug susceptibility tests were performed using broth microdilution. Subspecies classification and phylogeny were determined. Among the 24 cases (62 isolates), 19 cases (49 isolates) were of true NTM infection and 5 cases (13 isolates) examples of colonization. Two subspecies, M. abscessus subsp. massiliense (12 cases, 32 isolates) and M. abscessus subsp. abscessus (12 cases, 30 isolates) were identified. The major sequence type (ST) was ST227. Two clonal groups among patients were found; clonal cluster I (5 cases, 8 isolates) and clonal cluster II (2 cases, 4 isolates) but no epidemiological link was apparent. Reinfection (2 cases with different clones of M. abscessus strains; >9 SNPs different) and persistent infection (14 cases with the same clone; <6 SNPs) were distinguished based on a phylogeny. Based on these SNP cutoff values, 3 cases of persistent colonization (same strain through time) and 2 cases of re-colonization (different strains through time) were identified. M. abscessus subsp. abscessus was significantly associated with clarithromycin resistance (p < .001) and multi-organ infection (p = .03). Molecular epidemiology based on MLST can be used to differentiate between reinfection vs persistent infection, persistent colonization vs re-colonization. ST227 was the main epidemic strain in Northeast Thailand.

Analysis of drug-susceptibility patterns and gene sequences associated with clarithromycin and amikacin resistance in serial Mycobacterium abscessus isolates from clinical specimens from Northeast Thailand.

Mycobacterium abscessus is an important infectious agent highly associated with drug resistance and treatment failure. We investigated the drug resistance situation of M. abscessus in Northeast Thailand and the possible genetic basis for this. Sixty-eight M. abscessus clinical isolates were obtained from 26 patients at Srinagarind Hospital during 2012-2016. Drug susceptibility tests and sequencing of erm(41), rrl and rrs genes were performed. Mycobacterium abscessus was resistant to 11/15 antibiotics (nearly 100% resistance in each case). Partial susceptibility to four antibiotics was found (amikacin, tigecycline, clarithromycin and linezolid). Non-massiliense subspecies were significantly associated with clarithromycin resistance (p<0.0001) whereas massiliense subspecies were associated with tigecycline resistance (p = 0.028). Inducible clarithromycin resistance was seen in 22/68 (32.35%) isolates: 21 of these isolates (95.45%) belonged to non-massiliense subspecies and resistance was explicable by the T28C mutation in erm(41). Inducible clarithromycin resistance was found in one isolate of the massiliense subspecies. Acquired clarithromycin resistance explicable by the A2271G/C mutation of rrl was seen in only 7/16 (43.75%) of strains. Inducible and acquired resistance mechanisms can be interchangeable during the course of infection. Rrs mutations were not associated with amikacin resistance in our study. Antibiotic resistance in subspecies of M. abscessus was reported from Northeast Thailand. Known resistance-associated mutations cannot explain all of the resistance patterns observed.

Epidemiology of and risk factors for extrapulmonary nontuberculous mycobacterial infections in Northeast Thailand.

BACKGROUND: Nontuberculous mycobacterial (NTM) infection is increasing worldwide. Current epidemiological data and knowledge of risk factors for this disease are limited. We investigated the trends in and risk of NTM infection in Northeast Thailand during 2012-2016. METHODS: Patient demographics, infection site(s), and underlying disease or conditions from 530 suspected cases of NTM infections were retrieved from medical records, reviewed and analyzed. A diagnosis of true NTM infection was accepted in 150 cases. Risk factor analyses were done for extrapulmonary NTM infections compared to pulmonary NTM infections and for Mycobacterium abscessus compared to members of the Mycobacterium avium complex (MAC). Trend analysis among NTM species causing NTM infections was performed. RESULTS: The most common species of NTMs causing extrapulmonary (n = 114) and pulmonary (n = 36) NTM infections in Northeast Thailand were M. abscessus (25.4% of extrapulmonary infected cases and 27.8% of pulmonary cases) followed by MAC (14.9% of extrapulmonary and 13.9% of pulmonary cases). Presence of anti-IFN-γ autoantibodies was the major risk factor for extrapulmonary (odds ratio (OR) = 20.75, 95%CI [2.70-159.24]) compared to pulmonary NTM infection. M. abscessus infection was less likely (OR = 0.17; 95%CI [0.04-0.80]) to be found in patients with HIV infection than was MAC infection. The prevalence of NTM infection, especially M. abscessus, in Northeast Thailand has recently increased. Extrapulmonary NTM and complicated NTM infections have increased in concordance with the recent trend of increasing frequency of anti-IFN-γ autoantibodies in the population. CONCLUSIONS: M. abscessus was the commonest NTM pathogen followed by MAC. The prevalence of NTM infections and anti-IFN-γ are showing an upward trend. Autoimmune disease due to anti-IFN-γ is the major risk factor for extrapulmonary NTM infection in Northeast Thailand.

DIAGNOSTIC TEST OF SPUTUM GENEXPERT MTB/RIF FOR SMEAR NEGATIVE PULMONARY TUBERCULOSIS.

The objective of this study was to evaluate the performance of the Gene-Xpert MTB/RIF sputum test for diagnosing pulmonary tuberculosis (TB) among patients sputum acid-fast bacillus (AFB) smear negative results in Thailand, a country with a high prevalence of pulmonary tuberculosis. We studied 151 patients who presented to Srinagarind Hospital, Khon Kaen, Thailand with a 2 week or more history of fever and/or cough and an abnormal chest radiograph between 2010 and 2014; these patients had at least 2 negative sputum AFB smear results. Of these, 76 were diagnosed as having either confirmed or probable pulmonary TB: the 32 confirmed cases were those with a positive sputum culture for Mycobacterium tuberculosis (MTB) and the 44 probable case were those with clinical and radiographic findings consistent with TB and who had a response to anti-TB therapy. Seventy-five cases were diagnosed as not having pulmonary TB. Of the 32 patients with a positive sputum culture for MTB, 26 had a positive GeneXpert MTB/RIF sputum test. Compared to sputum culture for MTB the GeneXpert MTB/ RIF test gave a sensitivity of 83.9% (95% CI: 66.3-94.5) and a specificity of 92.1% (95% CI: 83.6-97), a positive predictive value (PPV) of 81.3% (95% CI: 63.6-92.8) and a negative predictive value (NPV) of 93.3% (95% CI: 85.1-97.8). The GeneXpert MTB/RIF test had a fair sensitivity and specificity for diagnosing smear negative pulmonary TB. It may be useful for diagnosing pulmonary TB in patients with a negative sputum AFB smear. The assay is faster than culture and can detect rifampicin resistant strains of MTB.