Heteroresistance of Mycobacterium tuberculosis in the Sputum Detected by Droplet Digital PCR.

Heteroresistance in MTB refers to the presence of distinct subpopulations of bacteria with varying levels of antibiotic susceptibility within a population. Multidrug-resistant and rifampicin-resistant TB are serious global health concerns. In this study, we aimed to determine the prevalence of heteroresistance in MTB from sputum samples of new TB cases using Droplet Digital PCR mutation detection assays for katG and rpoB genes, which are commonly associated with resistance to isoniazid and rifampicin, respectively. We found that out of 79 samples, 9 (11.4%) exhibited mutations in katG and rpoB genes. INH mono-resistant TB, RIF mono-resistant TB, and MDR-TB samples constituted 1.3%, 6.3%, and 3.8% of new TB cases, respectively. Heteroresistance in katG, rpoB, and both genes were found in 2.5%, 5%, and 2.5% of total cases, respectively. Our results suggest that these mutations may have arisen spontaneously, as the patients had not yet received anti-TB drugs. ddPCR is a valuable tool for the early detection and management of DR-TB, as it can detect both mutant and wild-type strains in a population, enabling the detection of heteroresistance and MDR-TB. Overall, our findings highlight the importance of early detection and management of DR-TB for effective TB control (in katG, rpoB, and katG/rpoB).

Relationship between Helicobacter Pylori Infection and the Risk of Esophageal Cancer in Thailand.

OBJECTIVE: Esophageal cancer (EC) is a multifactorial disease and a leading cause of mortality. Epidemiological and molecular studies have provided evidence that Helicobacter pylori (H. pylori) infection is an important cause of gastric carcinogenesis and thus, may be related to EC. However, esophagus H. pylori infection in Thai patients with newly diagnosed EC has not been reported. Moreover, the evidence of the association with H. pylori to EC is controversial. This study investigated the possible association between H. pylori infection with a virulence gene and EC in Thailand. METHODS: A case-control study was conducted that involved 105 newly diagnosed EC patients and 108 healthy controls. The prevalence of H. pylori infection detected in formalin-fixed, paraffin-embedded EC tissue in esophageal biopsy specimens from the subjects was measured using real-time PCR. All the data were collected in face to face interviews using a structured questionnaire. Multivariable unconditional logistic regression was used to calculate and analyses the odds ratios (ORs) of the data. RESULTS: A significant association was found between H. pylori infection and EC (p < 0.001, 95% CI:3.11-10.48). H. pylori-positive subjects had a 2.76 times higher risk of developing ESCC. Moreover, the H. pylori-positive subjects who were CagA-positive had slightly higher ORs and statistically significant risk factors. CONCLUSIONS: H. pylori infection was found to be associated with a risk of EC in Thailand, and among the H. pylori-positive subjects who were CagA-positive had a higher risk factor of ESCC but not of EAC.

Detection of Mycobacterium tuberculosis Complex in Sputum Samples Using Droplet Digital PCR Targeting mpt64.

Tuberculosis (TB) is one of the top 10 causes of death worldwide. It is challenging to find methods of diagnosis of active pulmonary TB that are sensitive enough to detect cases for proper treatment before unintentional transmission. Droplet digital PCR (ddPCR) is a highly sensitive method to detect genetic material of pathogens, but it has rarely been used for diagnosis of TB. This study compared the sensitivity of ddPCR with that of GeneXpert and AFB smear microscopy in 180 leftover sputum samples from patients suspected of having TB on the basis of clinical symptoms and radiography. Absolute quantification of copy numbers of MTB-specific genes was possible using ddPCR targeting the mpt64 gene. Among the 180 samples, 41.1% were diagnosed as having TB using ddPCR. The sensitivities of AFB smear microscopy, GeneXpert and ddPCR were 41.9%, 82.4% and 100%, respectively. AFB smear microscopy and GeneXpert both had a specificity of 100%, and the specificity of ddPCR was 95.3%. The accuracy of ddPCR (97.2%) is higher than that of GeneXpert (92.7%). This robust ddPCR system could potentially be used as a method for early diagnosis of TB.

Whole-Genome Sequencing and Drug-Susceptibility Analysis of Serial Mycobacterium abscessus Isolates from Thai Patients.

Mycobacterium abscessus is an important pathogen that can cause serious human diseases and is difficult to treat due to antibiotic resistance. In this study, we analyzed, using whole-genome sequence (WGS) data, M. abscessus strains serially isolated from patients at various time intervals. We undertook genetic diversity analysis between subspecies, mutation-rate estimation and identification of drug-resistant mutations with minimum inhibitory concentration (MIC) analysis. Clonal isolates of M. abscessus:­subsp. abscessus (MAB) and subsp. massiliense (MMAS)­causing persistent infection through time, differed by 0−7 and 0−14 SNPs, respectively, despite being isolated 1 to 659 days apart. Two cases caused by MMAS differed by ≥102 SNPs at 350 days apart and were regarded as examples of reinfection. Isolates collected ≤7 days apart exhibited a high mutation rate (133.83 ± 0.00 SNPs/genome (5 Mb)/year for MMAS and 127.75 SNPs/genome (5 Mb)/year for MAB). Mutation rates declined in a time-dependent manner in both subspecies. Based on isolates collected > 180 days apart, MMAS had a significantly higher average mutation rate than MAB (2.89 ± 1.02 versus 0.82 ± 0.83 SNPs/genome (5 Mb)/year, (p = 0.01), respectively). All well-known drug-resistance mutations were found to be strongly associated with high MIC levels for clarithromycin and ciprofloxacin. No known mutations were identified for strains resistant to linezolid and amikacin. MAB strains in the study were susceptible to amikacin, while most MMAS strains were susceptible to clarithromycin, amikacin and linezolid. No hetero-resistance was found in the strains analyzed. Our study reports the genetic diversity and mutation rate of M. abscessus between the two major subspecies and confirms the drug resistance-associated mutations. Information about drug-resistance and associated mutations can be applied in diagnosis and patient management.

The relationship between P16INK4A and TP53 promoter methylation and the risk and prognosis in patients with oesophageal cancer in Thailand.

DNA methylation can regulate the expression of tumour suppressor genes P16 and TP53, environmental factors, which are both important factors related to an increased risk and prognosis of oesophageal cancer (EC). However, the association between these two genes methylation status, as well as the effects of gene-environment interactions, EC risk remains unclear. A Hospital-based case-control study data were collected from 105 new EC cases and 108 controls. Promoter methylation status was investigated for P16 and TP53 genes using methylation-specific polymerase (MSP) chain reaction methods with SYBR green. Logistic and Cox regression models were used to analyse the association of P16 and TP53 promotor methylation status with EC risk and prognosis, respectively. Our results suggest P16, TP53 methylation significantly increased the risk of EC (OR = 5.24, 95% CI: 2.57-10.66, P < 0.001; OR = 3.38, 95% CI: 1.17-6.67, P < 0.001, respectively). In addition, P16 and TP53 promoter methylation status and the combined effects between environmental factors and its methylations in tissue were correlated with the EC risk and prognosis of EC patients. As a new biomarker, the methylation of P16 and TP53 can serve as a potential predictive biomarker of EC.

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.

Novel mutations detected from drug resistant Mycobacterium tuberculosis isolated from North East of Thailand.

The emergence of drug-resistant tuberculosis is a major global public health threat. Thailand is one of the top 14 countries with high tuberculosis and multi-drug resistant tuberculosis rates. Immediate detection of drug-resistant tuberculosis is necessary to reduce mortality and morbidity by effectively providing treatment to ameliorate the formation of resistant strains. Limited data exist of mutation profiles in Northeastern Thailand. Here, 65 drug-resistant Mycobacterium tuberculosis isolates were used to detect mutations by polymerase chain reaction (PCR) and DNA sequencing. In the katG gene, mutations were occurred in 47 (79.7%) among 59 isoniazid resistant samples. For rpoB gene, 31 (96.9%) were observed as mutations in 32 rifampicin resistant isolates. Of 47 katG mutation samples, 45 (95.7%) had mutations in katG315 codon and 2 (4.3%) showed novel mutations at katG365 with amino acid substitution of CCG-CGG (Pro-Arg). Moreover, out of 31 rpoB mutation isolates, the codon positions rpoB516, rpoB526, rpoB531 and rpoB533 were 3 (9.7%), 8 (25.8%), 11 (35.5%) and 1 (3.2%), respectively. Seven isolates of double point mutation were found [rpoB516, 526; 1 (3.2%) and rpoB516, 531; 6 (19.4%)]. In addition, 1 (3.2%) sample had triple point mutation at codon positions rpoB516, 526 and 531. Common and novel mutation codons of the rpoB and katG genes were generated. Although DNA sequencing showed high accuracy, conventional PCR could be applied as an initial marker for screening drug-resistant Mycobacterium tuberculosis isolates in limit resources region. Mutations reported here should be considered when developing new molecular diagnostic methods for implementation in Northeastern Thailand.

Association between infection with Campylobacter species, poor oral health and environmental risk factors on esophageal cancer: a hospital-based case-control study in Thailand.

BACKGROUND: Previous studies have shown the association between Campylobacter species infection and that environmental factors, poor oral hygiene in particular, are linked to an increased risk of esophageal cancer (EC). However, no study has reported on these factors in Thailand. Thus, this study's objective was to evaluate the impact of the relationship between Campylobacter infection and environmental factors on EC incidence in the population of Thailand. METHODS: Data from a case-control study were collected from 105 newly diagnosed EC cases and 105 controls recruited from 2007 to 2017. Infection with Campylobacter spp. was detected in the formalin-fixed paraffin-embedded (FFPE) tissue of EC taken from gastroesophageal biopsy specimens obtained from the participants, and evaluated using TaqMan® real-time PCR. Multivariable logistic regression was performed to calculate the odds ratios (ORs) and perform data analysis. RESULTS: Smoking, alcohol use, a family history of cancer, history of gastroesophageal reflux disease, poor oral hygiene and Campylobacter spp. infection were shown to be significant risk factors for EC (p < 0.05). The combination of poor oral hygiene and infection with Campylobacter spp. constituted significant risk for EC (p < 0.001). In addition, the risk of EC in subjects co-infected with C. rectus and C. concisus that practiced poor oral hygiene was even higher and was significant (ORadj = 4.7; 95% CI 2.41-9.98; p = 0.003). CONCLUSIONS: In Thailand, the major risk factors for EC are smoking status, alcohol drinking, family history of cancer, GERD, poor oral hygiene and Campylobacter spp. infection. This study found Campylobacter spp. prevalence to be associated with EC and appears to be enhanced by poor oral hygiene, suggesting that a combination of poor oral hygiene and Campylobacter species infection may together act as an important etiological risk factor for EC.

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.

Proteomic analysis of drug-susceptible and multidrug-resistant nonreplicating Beijing strains of Mycobacterium tuberculosis cultured in vitro.

The existence of latent tuberculosis infection (LTBI) is one of the main obstacles hindering eradication of tuberculosis (TB). To better understand molecular mechanisms and explore biomarkers for the pathogen during LTBI, we cultured strains of Mycobacterium tuberculosis (Mtb) under stress conditions, mimicking those in the host granuloma intracellular environment, to induce entry into the non-replicating persistence stage. The stresses included hypoxia, low pH (5.0), iron deprivation (100 µM of 2, 2'-dipyridyl) and nutrient starvation (10% M7H9 medium). Three Mtb strains were studied: two clinical isolates (drug-susceptible Beijing (BJ) and multidrug-resistant Beijing (MDR-BJ) strains) and the reference laboratory strain, H37Rv. We investigated the proteomics profiles of these strains cultured in stressful conditions and then validated the findings by transcriptional analysis. NarJ (respiratory nitrate reductase delta chain) was significantly up-regulated at the protein level and the mRNA level in all three Mtb strains. The narJ gene is a member of the narGHJI operon encoding all nitrate reductase subunits, which play a role in nitrate metabolism during the adaptation of Mtb to stressful intracellular environments and the subsequent establishment of latent TB. The identification of up-regulated mRNAs and proteins of Mtb under stress conditions could assist development of biomarkers, drug targets and vaccine antigens.

Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing.

The exploration of vaginal microbiota by using next-generation sequencing (NGS) of 16S ribosomal RNA (rRNA) gene is widely used. Up to now, different hypervariable regions have been selected to study vaginal microbiota by NGS and there is no standard method for analysis. The study aimed to characterize vaginal microbiota from clinical samples using NGS targeting the 16S rRNA gene and to determine the performance of individual and concatenated hypervariable region sequences to generate the taxonomic profiles of the vaginal microbiota. Fifty-one vaginal DNA samples were subjected to 16S rRNA gene NGS based on the Ion Torrent PGM platform with the use of two primer sets spanning seven hypervariable regions of the 16S rRNA gene. Our analysis revealed that the predominant bacterial genera were Lactobacillus, Gardnerella and Atopobium, which accounted for 78%, 14% and 2%, respectively, of sequences from all vaginal bacterial genera. At the species level, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae accounted for 72%, 10% and 6%, respectively, of the bacterial cells present. Analyses using the V3 region generally indicated the highest bacterial diversity followed by the V6-V7 and V4 regions, while the V9 region gave the lowest bacterial resolution. NGS based on the 16S rRNA gene can give comprehensive estimates of the diversity of vaginal bacterial communities. Selection of sequences from appropriate hypervariable regions is necessary to provide reliable information on bacterial community diversity.

Genetic diversity of Mycobacterium tuberculosis using 24-locus MIRU-VNTR typing and Spoligotyping in Upper Myanmar.

INTRODUCTION: MIRU-VNTR typing and Spoligotyping are the useful molecular tools for TB epidemiology study. Information regarding genetic diversity and tuberculosis (TB) transmission in Upper Myanmar only is scares. METHODOLOGY: We determined the genetic diversity of Mycobacterium tuberculosis (Mtb) and TB transmission from Upper Myanmar TB Reference Laboratory, Mandalay Region, including Mandalay (72), Shan (22), Magway (15), Sagaing (13), Nay Pyi Taw (8), Kachin (7), Chin (2) and Kayah (1). One hundred and forty Mtb isolates were genotyped using 24-locus MIRU-VNTR typing and spoligotyping. Lineage classification and TB transmission analysis were performed. RESULTS: 24-locus MIRU-VNTR typing identified 135 unique profiles and two clusters compared to 35 spoligotyping profiles which contained 12 clusters and 23 unique isolates, Beijing (n=100, 71.4%) was found to be prominent lineage by combine two methods. The expected proportion attributable to recent transmission based on clustering rate was 2.1%. One cluster case was more likely to be in MDR patient. CONCLUSIONS: Our findings showed Beijing genotypes were dominant in Upper Myanmar. The usage and analysis of 24-locus MIRU-VNTR typing might prove useful for our broader understanding of TB outbreaks and epidemiology than spoligotyping. The genotypic pattern of this combined method suggests that the lower transmission rate may be due to a higher possibility of reactivation cases in Upper Myanmar.

24-locus MIRU-VNTR and Spoligotyping analysis of drug-resistant Mycobacterium tuberculosis strains isolated from Northeastern Thailand.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB) infection, remains a global health problem with increased concerns due to drug-resistant tuberculosis. However, molecular genotyping profiles may give insight of the transmission of TB in a particular region. The present study aimed to characterize the genetic diversity of drug-resistant MTB and evaluate primer sets applied for the epidemiological study of circulating MTB in Northeastern Thailand. A total of 92 MTB isolates, resistant to rifampicin and/or isoniazid, were collected from the Office of Disease Prevention and Control between 2013 and 2016. All isolates were genotyped by 24-locus MIRU-VNTR typing combined with spoligotyping. We also analyzed the distributions of drug susceptibility pattern and demographic data among different genotypes. In comparison with different loci sets, discriminatory power based on 12, 15, 24 standard primers were investigated. Eighty-six particular profiles were found; among the patterns, two clusters were produced in 8 strains. East African Indians (EAI) were the most prevalent strains (33 isolates, 35.87%) followed by Beijing (30 isolates, 32.61%), with 23 unknown isolates strains also found. The HGDI based on combination of 24 loci analysis and spoligotyping was 0.9962. The number of tandem repeat generated was highly discriminant (HGDI>0.6) at locus 580 (0.66), 960 (0.67), 2163b (0.73), 2165 (0.62), 2461 (0.68) 3690 (0.73) and 4052 (0.79), respectively. In contrast, the diversity at locus 154 and 2059 was not revealed. The results emphasized that 24-locus MIRU-VNTR and spoligotyping could be useful for epidemiological surveillance of drug-resistant MTB in this region. At a given allelic diversity, 7 primer sets containing MIRU04, MIRU10, QUB2163b, ETRA, ETRB, Mtub39 and QUB26 may be considered for screening the VNTR patterns. In addition, this study gathered both demographics and genotypic data within the same investigation for further tuberculosis prevention and control.

Genome Sequence of Lactobacillus fermentum 47-7, a Good In Vitro Probiotic Strain Isolated from a Healthy Thai Infant.

We report the genome sequence of Lactobacillus fermentum 47-7, a good in vitro probiotic strain isolated from an infant. Its genome size is 1.83 Mb, it is assembled from 180 contigs, and it consists of 1,636 protein-coding genes, 15 rRNAs, 57 tRNAs, and 4 noncoding RNAs. This genome sequence will be useful for a variety of applications.

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.

Characterization of vaginal microbiota in Thai women.

BACKGROUND: The vaginal microbiota (VMB) plays a key role in women's reproductive health. VMB composition varies with ethnicity, making it necessary to characterize the VMB of the target population before interventions to maintain and/or improve the vaginal health are undertaken. Information on the VMB of Thai women is currently unavailable. We therefore characterized the VMB in normal Thai women. METHODS: Vaginal samples derived from 25 Thai women were subjected to 16S rRNA gene next-generation sequencing (NGS) on the Ion Torrent PGM platform. RESULTS: Two groups of VMB were detected, lactobacilli-dominated (LD) and non-lactobacilli dominated (NLD) groups. Lactobacillus iners was the most common species found in the LD group while Gardnerella vaginalis followed by Atopobium vaginae and Pseudumonas stutzeri were commonly found in the NLD group. CONCLUSIONS: The VMB patterns present in normal Thai women is essential information to further determine the factors associated with VMB patterns in vaginal health and disease and to develop proper management of reproductive health of Thai women.

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.

Detection and genotyping of Helicobacter pylori in saliva versus stool samples from asymptomatic individuals in Northeastern Thailand reveals intra-host tissue-specific H. pylori subtypes.

BACKGROUND: Two-thirds of the world's population is thought to be infected by Helicobacter pylori. Although most people infected with H. pylori are asymptomatic, this pathogen is associated with several gastric pathologies including cancer. The risk factors for colonization are still unclear and the genetic diversity within individual hosts has never been clearly investigated. RESULT: This study determined the prevalence of, and explored risk factors for, H. pylori infection directly from paired saliva (n = 110) and stool (n = 110) samples from asymptomatic persons in Northeast Thailand. Samples were subjected to indirect immunofluorescence assay (IFA), 16S rRNA-based real-time PCR and vacA-based semi-nested PCR. Partial vacA gene sequences of H. pylori were compared between saliva and stool samples. The overall prevalence of H. pylori infection in our asymptomatic study population was 64%. Age, gender, occupation and frequency of brushing teeth were not found to be associated with H. pylori colonization. The vacA gene was successfully sequenced from both saliva and stool samples of 12 individuals. For seven of these individuals, saliva and stool sequences fell into different clusters on a phylogenetic tree, indicating intra-host genetic variation of H. pylori. CONCLUSION: This study reports a high prevalence of H. pylori infection in asymptomatic persons in this region of Thailand and demonstrates that genotypes (vacA gene sequences) of H. pylori may differ between the oral cavity and intestinal tract.

Chemokines additional to IFN-γ can be used to differentiate among Mycobacterium tuberculosis infection possibilities and provide evidence of an early clearance phenotype.

Current diagnostic tests for tuberculosis (TB) remain limited in their ability to discriminate between active TB (ATB) and latent TB infection (LTBI). Early clearance (EC) of TB by individuals exposed to Mycobacterium tuberculosis is a debated phenomenon for which evidence is lacking. We measured and compared secreted chemokines in the plasma fraction from 48 ATB, 38 LTBI, 162 presumed EC and 39 healthy controls (HC) using the QuantiFERON®-TB Gold In-Tube assay. Single chemokine markers were limited in their ability to discriminate between ATB and LTBI: IFN-γ showed 16.7% sensitivity; CCL2 showed moderate sensitivity (70.8%) and specificity (74.4%); CXCL10 showed high sensitivity (87.5%) and specificity (78.9%). Compared to IFN-γ alone, IFN-γ combined with CXCL10 significantly improved (p < 0.001) the sensitivity and specificity to discriminate between ATB and HC (97.9% sensitivity and 94.9% specificity) and between ATB and LTBI (89.6% sensitivity and 71.1% specificity). Levels of CCL2 were very significantly lower (p < 0.0001) in EC compared to HC groups and hence CCL2 is a useful marker for EC. This study demonstrated the potential application of profiling using multiple chemokines for differentiating among the various M. tuberculosis infection possibilities. We also present evidence to support the EC phenomenon based on the decrease of CCL2 levels.

DETECTION OF HELICOBACTER PYLORI AND VIRULENCE-ASSOCIATED GENES IN SALIVA SAMPLES OF ASYMPTOMATIC PERSONS IN NORTHEAST THAILAND.

The aims of the study were to develop nested-PCR (targeting vacA and cagA), SYBR green quantitative PCR (targeting 16S rDNA) tests and compared them with indirect fluorescent-monoclonal antibody (IFA) method for determination of the prevalence of Helicobacter pylori in 118 saliva samples from asymptomatic individuals in Khon Kaen, Thailand. Detection limit of both PCR-based assays was one cell. Prevalence of H. pylori in saliva samples was 55% based on the criterion of positivity of IFA test and one of the PCR-based methods or positivity of both PCR assays. Forty-nine percent of H. pylori detected carried cagA, encoding a cytotoxin associated with severe clinical outcomes. These results imply that the mouth may be an important reservoir for H. pylori, with nearly 50% of the virulent type that could possibly lead to gastroduodenal disease.