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.

Synergistic Effect of Forbesione From Garcinia hanburyi in Combination with 5-Fluorouracil on Cholangiocarcinoma

Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective; thus innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. This study aimed to investigate the synergistic effects of forbesione combined with 5-fluorouracil (5-FU) in hamster cholangiocarcinoma (Ham-1) cells both in vitro and in vivo. The anti-tumor effects of 5-FU combined with forbesione in vitro were determined using the Sulforhodamine B (SRB) assay and the effects in vivo were assessed in transplanted Ham-1 allograph models. Using ethidium bromide/acridine orange (EB/AO) staining, the morphological changes of apoptotic cells was investigated. The expressions of apoptosis-related molecules after combined treatment with forbesione and 5-FU were determined using real-time RT-PCR and western blot analysis. Forbesione or 5-FU alone inhibited proliferation of Ham-1 cells in a dose-dependent manner and their combination showed a synergistic proliferation inhibitory effect in vitro. In vivo studies, forbesione in combination with 5-FU exhibited greater inhibition of the tumor in the hamster model compared with treatment using either drug alone. Forbesione combined with 5-FU exerted stronger apoptotic induction in Ham-1 cells than did single drug treatment. The combination of drugs strongly suppressed the expression of B-cell lymphoma 2 (Bcl-2) and procaspase-3 while enhancing the expression of p53, Bcl-2-associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1), caspase-9 and caspase-3, compared with single drug treatments. These results explained the decreased expression of cytokeratin 19 (CK19) positive cells and proliferation cell nuclear antigen (PCNA) positive cells in Ham-1 cell tumor tissues of the treated hamsters. There was no apparent systemic toxicity observed in the treated animals compared with the control groups. Forbesione combined with 5-FU strongly induced apoptosis in Ham-1 cells. The growth inhibitory effect of combined treatment using these two drugs was much greater than treatment with either drug alone, both in vitro and in vivo.

Burkholderia pseudomallei Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses.

Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.

SXT ELEMENT, CLASS 1 INTEGRON AND MULTIDRUG-RESISTANCE GENES OF VIBRIO CHOLERAE ISOLATED FROM CLINICAL AND ENVIRONMENTAL SOURCES IN NORTHEAST THAILAND.

Emergence of multiple drug resistance in Vibrio cholerae has been increasing around the world including Northeast Thailand. In this study, 92 isolates of V. cholerae (50 O1 and 42 non-O1/non-O139 isolates) from clinical and environmental sources in Northeast Thailand were randomly selected and investigated for the presence of SXT element, class 1 integron and antimicrobial resistance genes. Genotypic-phenotypic concordance of antimicrobial resistance was also determined. Using PCR-based assays, 79% of V. cholerae isolates were positive for SXT element, whereas only 1% was positive for class 1 integron. SXT element harbored antimicrobial resistance genes, dfrA1 or dfr18, floR, strB, sul2, and tetA. Overall phenotypic-genotypic concordance of antimicrobial resistance was 78%, with highest and lowest value being for trimethoprim (83%) and chloramphenicol (70%), respectively. Ninety-two percent of V. cholerae O1 strains isolated from clinical sources harbored both dfrA1 (O1-specific trimethoprim resistance gene) and dfr18 (non-O1-specific trimethoprim resistance gene), whereas only 5% of V. cholerae non-O1/non-O139 strains harbored both genes. All V. cholerae O1 isolated from environmental source harbored dfr18 but 48% of V. cholerae non-O1/non-O139 harbored dfrA1. This study indicates that SXT element was the main contributor to the circulation of multiple-drug resistance determinants in V. cholerae strains in Northeast Thailand and that genetic exchange of SXT element can occur in both V. cholerae O1 and non-O1/non-O139 strains from clinical and environmental sources.

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.

Characterization of a novel two-component system response regulator involved in biofilm formation and a low-iron response of Burkholderia pseudomallei.

Two-component systems (TCSs) regulate an adaptive response to environmental conditions, leading to changes in bacterial cellular processes. In this study, we identified a novel TCS response regulator gene, designated as bfmR (biofilm formation-associated regulator) that regulates biofilm formation by Burkholderia pseudomallei (Bp). An insertion mutant of the Bp bfmR gene resulted in a significant decrease in expression of fimbriae chaperone-usher assembly genes (BPSL2O28 and BPSL22 7), leading to suppression of assembly of fimbriae on the cell surface and reduced biofilm formation. The defective phenotypes of the mutant strain were restored by introducing a complementing plasmid having an intact bfmR gene. Using RT-PCR analyses, we found that bfmR gene expression was upregulated under low-iron growth conditions. In addition, the bfmR mutant strain showed retarded growth in low-iron medium and in phagocytic cells compared to the wild-type strain. These results indicate that bfmR is a novel positive regulator for controlling assembly of fimbriae and biofilm formation, and is upregulated under low-iron conditions.

Epidemiology and identification of potential fungal pathogens causing invasive fungal infections in a tertiary care hospital in northeast Thailand.

Invasive fungal infections (IFIs) are life threatening and associated with a high mortality rate. Here, we describe the distribution of pathogens, host risk factors, and significance of fungi isolated from patients with IFIs. The study included 861 fungal isolates recovered between 2006 and 2011 from 802 patients at Srinagarind Hospital, Thailand. Based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group 2008 criteria, 28.5% (245/861 isolates) of the fungal isolates were considered to be causative agents of IFIs. The most common fungus was Candida albicans (46%, 396/861 isolates). However, the most common yeast causing IFIs was Cryptococcus neoformans (34.7%, 85/245 isolates), while the most common mould was Penicillium marneffei (10.6%, 26/245 isolates). Cryptococcosis was significantly associated with human immunodeficiency virus infections (P < 0.001). Trend analysis revealed that there was no significant increase in IFI cases (P = 0.34) from 2006 to 2011 or from 2007 to 2011 (P = 0.05), but there was a trend toward significant increases in candidiasis (P = 0.04). The fungal isolates were categorized according to the positive predictive value of their recovery in cultures as being true (>95%), moderate (5%-95%), and rare (<5%) pathogens. This classification system could facilitate the prediction of the likelihood of diseases caused by the isolated fungi.

Comparison of stress adaptation and survival rate between Burkholderia pseudomallei with mutant and wild type bfmR.

Burkholderia pseudomallei (Bp) is highly adaptable to a wide range of environmental changes for survival and pathogenesis. However, the underlying mechanisms of such adaptability are still unclear. Two-component system (TCS) is a common signal transduction used by bacteria in response to environmental changes. A gene designated as bfmR (locus tag of BPSL2024) has been proposed to encode a response regulator, a member of the TCS, and was studied by mutagenesis and comparison of its phenotypic changes compared with those of the wild type. The growth rates of the mutant Bp at temperatures of 37 degrees-39 degrees C and pH 5-8 were significantly lower than the wild type strain (p < 0.05), especially at 39 degrees C (p = 0.01) and pH 7 (p = 0.01). The survival rate of the mice infected with the mutant strain is not significantly different from mice infected with wild type strain. The defective phenotypes were recovered in the complemented strain. These results indicated that bfmR is involved in adaptation of Bp to thermal- and pH-induced stress conditions.

Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle.

A tetraplex PCR method was developed for simultaneous detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle samples in comparison with conventional culture method. Specific primers targeting ompW of V. cholerae, tl of V. parahaemolyticus, hsp60 of V. vulnificus and sodB of V. mimicus were employed in the same PCR. Detection limit of the tetraplex PCR assay was 104 cfu/ml (400 cfu/PCR reaction) for pure cultures of all four species of Vibrio. In Vibrio spiked cockle samples, the limit of detection after 6 hours enrichment in alkaline peptone water was 1 cfu/10 g of cockle tissue for three Vibrio spp, except for V. mimicus that was 102 cfu/10 g of cockle tissue. When the tetraplex PCR and culture methods were applied to 100 cockle samples, V. parahaemolyticus, V. vulnificus, V. cholerae and V. mimicus were detected in 100, 98, 80 and 9% of the samples by tetraplex PCR and in 76, 42, 0 and 0% by the culture method, respectively. This developed tetraplex PCR method should be suitable for simultaneous and rapid detection of Vibrio species in food samples and for food safety assessment.

Seroreactivity to specific antigens of Helicobacter pylori infection is associated with an increased risk of the dyspeptic gastrointestinal diseases.

OBJECTIVES: The correlation between seroreactivity to Helicobacter pylori-specific antigens and clinical outcomes in gastrointestinal disease remains unresolved. We investigated the anti-H. pylori antibody profile in northeast Thai dyspeptic patients with gastrointestinal disease in order to identify any H. pylori antigens that may be associated with an increased risk of gastrointestinal disease. PATIENTS AND METHODS: Eighty-nine H. pylori-infected dyspeptic patients (44 non-ulcer, 23 peptic ulcer, 22 gastric cancer) were included in the study. Patients were considered to have H. pylori infection when at least one invasive method (i.e., culture, rapid urease test, and histology on biopsy specimens) and serological tests including a commercial ELISA (Pyloriset EIA-GIII) and a commercial immunoblot (Helicoblot 2.1; Genelabs Diagnostics), were positive. In addition, the sera of 20 H. pylori-infected blood donors and 10 H. pylori-non-infected blood donors were also randomly collected and analyzed for H. pylori infection by ELISA and Helicoblot 2.1. RESULTS: Immunoreactive protein bands at 116-kDa, 89-kDa, 37-kDa, 35-kDa, 30-kDa, 19.5-kDa, and the current infection marker for H. pylori-infected patients had average frequencies of 97.8%, 77.5%, 36.0%, 25.8%, 79.8%, 58.4%, and 69.7%, respectively. The immunoreactive patterns obtained from the H. pylori-infected patients and H. pylori-infected blood donors were similar. The antibodies to VacA and CagA antigens were not significantly different among the H. pylori-infected gastroduodenal patient groups. The simultaneous presence of antibody to 19.5-kDa antigen and absence of antibody to 35-kDa antigen was associated with an increased risk of gastric cancer (p<0.05). The immunoreactive band to 35-kDa antigen was found at significantly higher levels in peptic ulcer patients, and the 37-kDa antigen was found at significantly higher levels in non-ulcer patients (both p<0.05). Significantly low levels of antibodies to 23-kDa and 85-kDa antigens were found associated with peptic ulcer (p<0.05). CONCLUSIONS: We confirm that the universal presence of CagA and VacA in H. pylori-infected patients in Thailand is independent of the gastroduodenal disease. The presence or absence of antibodies to H. pylori-specific antigens may be useful as indirect markers in the screening of H. pylori-infected patients, and may have specific protection roles in H. pylori-related gastroduodenal diseases.

Application of duplex-PCR in rapid and reliable detection of toxigenic Vibrio cholerae in water samples in Thailand.

Toxigenic Vibrio cholerae, the cause of cholera, is a native flora of the aquatic environment which is transmitted through drinking water and still remains the leading cause of morbidity and mortality in many developing countries including Thailand. The culture method (CM), which is routinely used for assessing water quality, has not proven as efficient as molecular methods because the notorious pathogen survives in water mostly in a non-culturable state. We employed duplex-polymerase chain reaction (duplex-PCR) for detection of tcpA and ctxA genes in toxigenic V. cholerae, and compared PCR detection with CM in various waters of Khon Kaen Municipality, Thailand. We also evaluated the effect of different pre-PCR conditions on the results of ctxA and tcpA detection including: 1) water filtered and enriched in alkaline peptone water (APW) for 3 h before PCR, 2) water filtered without enrichment before PCR, and 3) use of only enrichment in APW for 6 h before PCR. Of the 96 water samples (taken from waste-water, potable and waste-water from patients' houses, and from rivers) tested, 48 (50%) were positive for ctxA and tcpA by duplex-PCR, whereas only 29 (30%) were positive for V. cholerae by CM. Of the 29 V. cholerae isolated by CM, 2 (7%) were toxigenic V. cholerae belonging to serovar O1, while the rests were non-O1/ non-O139. Results revealed, therefore, that ctxA and tcpA-targeted duplex PCR is more sensitive than CM for detection of toxigenic V. cholerae from water samples because CM detected much less toxigenic V. cholerae than the non-toxigenic V. cholerae. Template DNA as low as 100 fg or 23 cells of V. cholerae in the water sample was detected in duplex PCR. Pre-PCR filtration followed by enrichment for 3 h significantly increase in the efficiency of duplex-PCR detection of toxigenic V. cholerae.

Enzyme-linked immunosorbent assay for serodiagnosis of Helicobacter pylori in dyspeptic patients and volunteer blood donors.

Helicobacter pylori, an important etiological agent in the development of gastritis, peptic ulcer and gastric carcinoma, can be detected by the enzyme-linked immunosorbent assay (ELISA). Our objectives were: (1) to evaluate the efficacy of a commercial ELISA kit (Pyloriset EIA-G III) in sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for diagnosis of H. pylori infection in Thai dyspeptic patients in Khon Kaen Thailand; and (2) to examine the seroprevalence of H. pylori among blood donors at Srinagarind Hospital's Blood Bank, Khon Kaen University, by the commercial ELISA. Gastric biopsies obtained from 137 dyspeptic patients were diagnosed by culture, rapid urease test (RUT) and histology. Serum samples from the same dyspeptic patients and 100 healthy blood donors were assayed using the commercial ELISA. H. pylori infection in dyspeptic patients was considered positive when the culture or both RUT and histology were positive. Using a cut-off value at a titer of 20 U/ml (as recommended by the manufacturer), we found the commercial ELISA kit had a sensitivity of 93.3%, specificity of 75.3%, PPV of 74.7%, NPV of 93.5% and accuracy of 83.2%. The overall H. pylori seroprevalence in the healthy blood donors was 57%. Of the 100 healthy blood donors, 39 (60.9%) of the males and 18 (50.0%) of the females were seropositive.