Modeling liver fluke transmission in northeast Thailand: Impacts of development, hydrology, and control.

Human infection with the Southeast Asian liver fluke Opisthorchis viverrini and liver fluke-associated cholangiocarcinoma cause significant disease burden in Southeast Asia. While there has been considerable work to understand liver fluke pathology and to reduce infection prevalence, there remains a limited understanding of the environmental determinants of parasite transmission dynamics to inform treatment and control programs. A particular setting where targeted control efforts have taken place is the Lawa Lake complex in northeast Thailand. Here, we describe the recent history of host infections, as well as the hydrologic characteristics of this floodplain ecosystem that influence the extent of snail habitat and fish mobility and the transport of human waste and parasite cercariae. Using mathematical modeling, we outline a framework for reconstructing environmental transmission of O. viverrini over the course of the Lawa Project control program from its inception in 2008 until 2016, using locally acquired but fragmentary longitudinal infection data for both humans and environmental hosts. The role of water flow in facilitating movement between snail, fish, human, and reservoir hosts is a particular focus with respect to its relevant scales and its impact on success of interventions. In this setting, we argue that an understanding of the key environmental drivers of disease transmission processes is central to the effectiveness of any environmental intervention.

Antimicrobial resistance and genetic diversity of the SXT element in Vibrio cholerae from clinical and environmental water samples in northeastern Thailand.

Multidrug resistance in V. cholerae has been increasing around the world including northeastern Thailand. The aquatic environment is a reservoir of V. cholerae and might be an important source of resistant strains. The aims of this study were to investigate the phylogenetic relationships of intSXT gene sequences from 31 clinical and 14 environmental V. cholerae O1 and non-O1/non-O139 isolates and 11 sequences amplified directly from environmental water samples. We also amplified class 1 integrons, the SXT elements (targeting the intSXT gene) and antimicrobial resistance genes directly from water samples. Phylogenetic analysis displayed two major distinct clusters (clusters 1 and 2). Most V. cholerae O1 (19/20, 95%) and non-O1/non-O139 isolates (8/11, 72.7%) from clinical sources, and all sequences obtained directly from water samples, belonged to cluster 1. Cluster 2 mostly comprised environmental non-O1/non-O139 isolates (10/12, 83.3%). We successfully amplified the SXT elements directly from17.5% of water samples. Associated resistance genes were also amplified as follows: sul2 (41.3% of water samples), dfrA1 (60%), dfr18 (33.8%), strB (70%) and tetA (2.5%). Class 1 integrons were not found in water samples, indicating that the SXT element was the major contributor of multidrug resistance determinants in this region. The SXT element and antimicrobial resistance genes could be transferred from clinical V. cholerae O1 to environmental V. cholerae non-O1/non-O139 was demonstrated by conjugation experiment. These findings indicate that there may have been cross dissemination and horizontal gene transfer (HGT) of the SXT element harbored by V. cholerae O1 and non-O1/non-O139 strains isolated from clinical and environmental water sources. Environmental water might be an important source of antimicrobial resistance genes in V. cholerae in this region. Direct detection of antimicrobial resistance genes in water samples can be used for monitoring the spread of such genes in the ecosystem.

New locality record for Haplorchoides mehrai and possible interactions with Opisthorchis viverrini metacercariae in cyprinid fishes in Northeast Thailand.

Metacercariae of Opisthorchis viverrini, a carcinogenic liver fluke, and Haplorchoides sp., a trematode maturing in catfish, are commonly found in cyprinid fish, the second intermediate hosts of both flukes. However, the specific identity of Haplorchoides sp. in Thailand and a precise assessment of the effects of co-infections with O. viverrini have never been clarified. Therefore, we aimed to identify the species of Haplorchoides and to investigate possible interactions of the two trematode species in cyprinid fishes. Based on the morphology and morphometry of the cercaria, metacercaria, and adult stages, the Haplorchoides species found was identified as Haplorchoides mehrai Pande and Shukla 1976. Thailand is formally recorded as a new locality for H. mehrai, where naturally infected hosts include the snail Melanoides tuberculata (first intermediate host), the cyprinid fishes Hampala dispar, Cyclocheilichthys apogon, Puntius leiacanthus, Labiobarbus burmanicus, and Cirrhina jullieni (second intermediate hosts), and a catfish, Mystus nemurus (definitive host). The co-infection rates of O. viverrini and H. mehrai were significantly associated with fish species and fish body region (P < 0.001), with an overall significantly higher average intensity of H. mehrai (126.26 metacercariae/fish) than that of O. viverrini (18.02 metacercariae/fish). Further work is required to demonstrate the extent and mechanisms of possible interactions between these trematode species in the fish host. These data may provide a better understanding of O. viverrini transmission dynamics, and help design integrated control interventions.

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.

DOUBLE-STEP MULTIPLEX REAL TIME PCR WITH MELTING CURVE ANALYSIS FOR DETECTION AND DIFFERENTIATION OF MYCOBACTERIA IN SPUTUM.

Mycobacterium tuberculosis (M. tb) is a causative agent of tuberculosis, a worldwide public health problem. In recent years, the incidence of human mycobacterial infection due to species other than M. tb has increased. However, the lack of specific, rapid, and inexpensive methods for identification of mycobacterial species remains a pressing problem. A diagnostic test was developed for mycobacterial strain differentiation utilizing a double-step multiplex real time PCR together with melting curve analysis for identifying and distinguishing among M. tb, M. bovis BCG, other members of M. tb. complex, M. avium, and non-tuberculosis mycobacteria. The assay was tested using 167 clinical sputum samples in comparison with acid-fast staining and culturing. Using only the first step (step A) the assay achieved sensitivity and specificity of 81% and 95%, respectively. The detection limit was equivalent to 50 genome copies.

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.

Fecal bacterial contamination in natural water reservoirs as an indicator of seasonal infection by Opisthorchis viverrini in snail intermediate hosts.

Opisthorchis viverrini, a carcinogenic liver fluke, requires Bithynia snails as the first intermediate host, which release cercariae after ingesting fluke eggs from contaminated water. Fecal bacterial contamination and O. viverrini-infected Bithynia snails were investigated in samples collected from natural water reservoirs in Ban Phai, Chonnabot and Muang Districts (Ban Lerngpeuy) in Khon Kaen Province, northeast Thailand, where there is a high incidence of cholangiocarcinoma. Water was sampled and examined six times (February, April, June, August, October and December 2006). The most probable number (MPN) index and coliform counts were utilized to evaluate fecal contamination; the cercarial shedding method was conducted for detecting infected snails. The data revealed that all water samples had a high MPN index number, and fecal coliform levels above the WHO standard. This indicated that water in these reservoirs was contaminated with feces or manure constituents. Water sampling from Ban Lerngpeuy showed full-scale bacterial contamination (>1609 MPN index) throughout the year. This finding was correlated with the highest prevalence of O. viverrini-infected snails, which were found nearly all year round in this area. Slightly lower fecal contamination levels were detected in water samples from Chonnabot and Ban Phai, with high MPN index numbers and coliform counts from April to October. This corresponded with the higher recovery of infected snails in June and August, but with relatively lower prevalence than those found in Ban Lerngpeuy. Among the sampling sites, the people in Ban Lerngpeuy live nearer to the reservoir than do those in Ban Phai and Chonnabot. These results indicate that fecal bacterial contamination in natural water reservoirs is an important indicator of seasonal transmission of O. viverrini eggs to snail intermediate hosts. Sanitation improvement is essential and future investigations on the sources of contamination are needed.

Effect of light intensity on Opisthorchis viverrini cercarial shedding levels from Bithynia snails--a preliminary study.

Opisthorchis viverrini requires Bithynia snails as the first intermediate host and cyprinid fish as the second intermediate host. Very low natural infection rates have been reported in Bithynia snails, but very high rates have been found in cyprinid fish in the same endemic region. This study investigated the effect of light intensity, the most important stimulus, on the quantity of O. viverrini cercariae shed from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos snails. Snails were evaluated for cercariae output every hour after exposure to various light intensities for a total period of 7h. The same infected snail was tested under different intensities of light: in the dark, and at 1000, 3000 and 5000 lx. The data showed that under exposure to 1000 and 3000 lx of light, the average percentage and number of cercariae released were higher than that exposed to 5000 lx during the first 2h of the experiment. In contrast, under higher illumination (5000 lx) a longer time (6h) was required to stimulate the peak emergence of cercariae. Darkness was not able to induce O. viverrini cercariae emergence. Among the three intensities of light, exposure at 1000 lx induced the highest average number of released cercariae per snail and the highest percentage of cercarial emergence within the first 2h (125, 54.86%), followed by exposure at 3000 lx (69, 25.58%) and 5000 lx (12, 7.78%). The results suggest that the light intensity of 1000 lx for 2h would be optimal for O. viverrini cercarial shedding from naturally infected B. (D.) siamensis goniomphalos snails.

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample.

Preliminary report of SCCmec-types and antimicrobial susceptibilities of methicillin-resistant Staphylococcus aureus isolates from a university hospital in Thailand.

Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide. It is a major cause of hospital-acquired infections in most hospitals for nearly half century. The present study was conducted to examine the antimicrobial susceptibilities and staphylococcal cassette chromosome mec (SCCmec)-type for MRSA isolates from 237 patients treated at Srinagarind Hospital between September 2002 and August 2003. Antimicrobial susceptibility testing for all isolates was performed using an agar dilution method and SCCmec-types of 81 representatives from 237 isolates were determined using multiplex PCR. The minimum inhibitory concentration (MIC) ranges for the MRSA isolates were as follows: cefazolin 8 to > or =64; erythromycin < or = 0.5 to > or =64; gentamicin < or = 0.5 to > or =64; imipenem < or = 0.5 to >16; ofloxacin < or = 0.5 to > or =64; oxacillin 16 to > or =64; tetracycline 2 to > or =64 and vancomycin < or = 0.5 to 2 microg/ml. All MRSA isolates were susceptible to vancomycin, but only 0.4% to 8.9% was susceptible to the remaining antimicrobial agents. Of the 81 isolates tested, 2 types of SCCmec were found (76 with type III and 2 with type II) and no mecA gene was detected in 3 isolates. Sixty-seven of the 78 isolates carried the mercury-resistant operon. The multilocus sequence type in isolates with type III SCCmec was ST239 and in isolates with type II SCCmec was ST5.

CMY-2, CMY-8b, and DHA-1 plasmid-mediated AmpC ß-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae from a university hospital, Thailand.

Between February 2005 and January 2006 in Srinagarind Hospital, Thailand, 44 from 1730 isolates (2.5%) of Escherichia coli and 8 from 982 isolates (0.8%) of Klebsiella pneumoniae were found to produce plasmid-mediated AmpC ß-lactamases (pAmpCs) as detected by a cefoxitin-Hodge test followed by a multiplex polymerase chain reaction (PCR) technique. Fifteen of the 52 pAmpC-producing isolates also produced extended-spectrum ß-lactamases. The ampC genes found in both organisms were bla(CMY-2) (46 isolates), bla(CMY-8b) (4 isolates), and bla(DHA-1) (2 isolates). These genes were present on plasmids. Twenty-five of the 46 CMY-2-producing isolates could transfer cefoxitin resistance to E. coli UB1637 by conjugation. More than 90% of the pAmpC-producing isolates were resistant to cefoxitin, but 80% to 90% of them were susceptible or intermediately susceptible to ceftazidime or cefotaxime. Enterobacterial repetitive intergenic consensus PCR analysis revealed that most isolates were of different strains, indicating the ease of transmission of these resistance determinants. This is the first report of CMY-2, CMY-8b, and DHA-1 ß-lactamases in Thai isolates.

Antimicrobial susceptibility of Helicobacter pylori isolated from gastric biopsies in dyspeptic patients.

The objective of this study was to evaluate the prevalence of antimicrobial resistance in Helicobacter pylori isolated from the antrum and corpus of dyspeptic patients in Khon Kaen, Thailand, and to compare the antimicrobial susceptibility patterns of H. pylori isolated from the antrum and corpus in individual patients. Antimicrobial susceptibility was determined by disk diffusion, studying susceptibility to metronidazole, clarithromycin, amoxicillin, erythromycin, ciprofloxacin, and tetracycline. The H. pylori resistant rate to at least one of the six antimicrobial agents tested was 37%. The resistance rates were 30.2% for metronidazole, 9.2% for ciprofloxacin, 5% for clarithromycin, 2.4% for amoxicillin, and 1.7% for erythromycin and tetracycline. Single, double, and more than double antimicrobial resistances were found in 27.7, 6.7 and 2.5%, respectively. Antimicrobial susceptibility testing revealed 11 antibiotypes. The most common antimicrobial susceptibility pattern found was sensitivity to 6 antimicrobial agents (63%). H. pylori antimicrobial resistance in specimens isolated from the antrum and corpus were nearly equivalent, 37.3% (22/59) and 36.7% (22/60), respectively. Most of the H. pylori specimens isolated from the antrum and corpus in individual patients were identical (87.7%).

CTX-M extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae in a Thai university hospital.

This study presents updates on molecular epidemiology of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Enterobacteriaceae from Srinagarind Hospital, Khon Kaen University, Thailand. All isolates were screened for the presence of ESBL genes, bla(TEM), bla(SHV), bla(VEB) and bla(CTX-M), using PCR followed by nucleotide sequence determination. The results revealed that beta-lactamase genes among 48 isolates collected between 1998 and 1999 were bla(SHV) (79%), bla(CTX-M-9) (52%), bla(TEM-1) (48%) and bla(VEB) (33%), whereas those found in 52 isolates collected in 2003 were bla(TEM-1) (79%), bla(CTX-M-15) (44%), bla(SHV) (36%), bla(VEB) (36%), bla(CTX -M-14) (11%) and bla(CTX-M-9) (10%). In addition, 45 isolates carried at least two different ESBL genes. Using PCR, part of insertion sequence ISEcpl was found in the upstream regions of bla(CTX-M-14) and bla(CTX-M-15). ERIC-PCR analysis revealed that most ESBL-producing isolates were of different strains. This is the first report of CTX-M-9, CTX-M-14 and CTX-M-15 beta-lactamase genes in Enterobacteriaceae in Thailand.

Detection of heterogeneous, intermediate-vancomycin-resistant Staphylococcus aureus (hVISA) using low-concentration vancomycin disks.

Heterogeneous, intermediate-vancomycin-resistant Staphylococcus aureus (hVISA) represents a threat of an incurable infection since the first report in 1997. The method used to detect hVISA isolates is a population analysis profile (PAP); however, it is impractical for routine laboratory analysis. We therefore tested a simple, reliable and inexpensive method for the detection of hVISA. Eighteen isolates of hVISA and 22 of vancomycin-sensitive S. aureus (VSSA) were included. The organisms were tested by the disk diffusion method, using 15-microg vancomycin disks on four different media: Mueller-Hinton agar (MHA), MHA plus 2% NaCI (MHAS), Brain Heart Infusion agar (BHA), and BHA plus 2% NaCl (BHAS). In addition, two different inoculum sizes, bacterial suspensions adjusted to 0.5 and 2.0 McFarland, were tested. The inhibition zone was read independently by three medical technologists after incubation at 37 degrees C for 24 and 48 hours. The use of MHAS with an inoculum size of 2.0 McFarland and 48-hour incubation period yielded the highest sensitivity (94.4%), specificity (81.8%), positive predictive value (80.9%), and negative predictive value (94.7%). The disk diffusion test with 15-microg vancomycin disk is simple and may be used as a screening method for the detection of hVISA.