Manas Chongsa-nguan

Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3 : K6 and O4 : K68) in Thailand and India

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

Toxic Marine Puffer Fish in Thailand Seas and Tetrodotoxin They Contained

A total of 155 puffers caught from two of Thailand’s seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.

Staphylococcus aureus Clinical Isolates: Antibiotic Susceptibility, Molecular Characteristics, and Ability to Form Biofilm

Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd), PFGE types, accessory gene regulator (agr) groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1–7 (56 isolates) were methicillin resistant (MRSA) and 8–10 (36 isolates) were methicillin sensitive (MSSA). One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq), and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70%) but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.

Specific monoclonal antibodies to Opisthorchis viverrini

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.

Immunogenicity of liposome-associated and refined antigen oral cholera vaccines in Thai volunteers

A mixture of Vibrio cholerae antigens made up of crude fimbrial extract, lipopolysaccharide and procholeragenoid was administered orally to Thai volunteers either as free antigen or associated with liposomes. All vaccines and controls were administered in three doses given at 14 day intervals. Nine volunteers received liposome-associated vaccine and seven received free vaccine. Liposomes without antigens were given to eight volunteers and seven volunteers received 5% NaHCO3 solution alone. Both vaccines had 100% immunogenicity as determined by serum vibriocidal antibody responses. Liposomes were shown by indirect ELISA to localize the immune response against lipopolysaccharide and fimbriae to the intestinal mucosa. Vaccines given liposome-associated antigens had a higher rate of antigen-specific antibody response than did individuals who had received free antigens. The vaccines induced intestinal antibodies of IgM and/or IgA isotypes, but not IgG antibody.

Monoclonal Antibodies to LipL32 Protect Against Heterologous Leptospira spp. Challenge

A non-culture-based leptospirosis vaccine that cross-protects against infection caused by heterologous Leptospira spp. should replace the currently available products, which are qualitatively and quantitatively inadequate. With that in mind, two murine hybridomas secreting monoclonal antibodies (MAb) binding only to homogenates of pathogenic Leptospira spp., and not of the saprophytic L. biflexa, serogroup Patoc, serovar Patoc, were produced. The MAbs of both clones neutralized Leptospira-mediated human red blood cell lysis in vitro and rescued hamsters from lethal infection with heterologous Leptospira spp. The orthologous Leptospira spp. protein carrying the MAb epitope(s) was identified by two-dimensional gel electrophoresis (2DE)-based proteomics and database search. The epitopes of the MAbs were located on the major outer membrane protein LipL32 of the pathogenic Leptospira spp. The MAbs in their humanized version are potential leptospirosis immunotherapeutics. They are also suitable as detection reagents in antigen-based assays for the rapid diagnosis of leptospirosis. Recombinant LipL32 is a good candidate for a broad spectrum, non-culture-based leptospirosis vaccine.

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.

Rapid detection of V. cholerae 01

Abstract Monoclonal antibodies directed against group specific antigen A of Vibio cholerae 01 were obtained through hybridoma technology which involved fusions of Sp 2/0 myeloma cells with splenocytes of Balb/c mice immunized with whole cell lysates of V. cholerae 01. Specificity and crossreactivity of the monoclonal antibodies were determined against whole cell lysates prepared from 38 strains of V. cholerae 01, six strains of V. cholerae non-01, five strains of other vibrios, 45 strains of enterobacteria and Entamoeba histolytica by indirect and dot-blot enzyme-linked immunosorbent assay (ELISA). The monoclonal antibodies (MAb) reacted specifically to the whole cell lysates of the V. cholerae serogroup 01 and did not react to the antigens prepared from other organisms. The MAb were used in a dot-blot ELISA for detecting V. cholerae 01 antigen in 335 seafood specimens and in stools and rectal swabs of patients with acute watery diarrhoea. The dotblot ELISA performed on rectal swab specimens enriched in alkaline peptone water gave 96.0% sensitivity, 99.3% specificity, 94.7% positive predictive value, 99.5% negative predictive value and 98.9% efficacy, respectively, when compared to the conventional culture method. The two methods showed excellent agreement beyond chance, on the basis of a k coefficient value of 94.7%. No V. cholerae 01 was isolated from the 335 seafood samples and the dot-blot ELISA for V. cholerae 01 antigen was also negative, consistent with a high specificity of the assay. The dot-blot ELISA is easy to perform, relatively inexpensive, highly sensitive and specific. It permits multisample analysis at a single time, requires no special equipment and does not pose any disposal problem (compared with the culture method). Most of all, diagnosis of cholera cases could be made accurately within 1–3 h (the dot-blot ELISA takes 1 h, while the culture method takes 2 days). The method is recommended for rapid detection of V. cholerae 01 in contaminated foods, in environmental samples and in stools of diarrhoeic patients.

Immunogenicity of two f0ormulations of oral cholera vaccines in Thai volunteers

A formulation of oral vaccine consisting of Vibrio cholerae lipopolysaccharides (LPS), cell-bound haemagglutinin (CHA) and procholeragenoid (P), namely vaccine A, was compared with another formulation, vaccine B, prepared from killed whole vibrios plus procholeragenoid on their immunogenicity and reactogenicity in Thai male volunteers. Volunteers were randomly allocated into three groups. The first two groups received orally three doses of vaccines A and B, respectively at 14-day intervals. Volunteers in group 3 were controls and received orally 100 ml 5% (w/v) NaHCO3 also at 14-day intervals. Serum samples were collected from all volunteers before each immunization. Intestinal lavage was performed 3 to 7 days before the first dose of vaccine or placebo and 7, 21 and 45 days after the last dose. Serum vibriocidal antibodies were determined and class-specific, antigen-specific antibodies of all serum and lavage samples were assessed by indirect enzyme-linked immunosorbent assay (ELISA) using purified LPS, CHA and cholera toxin (CT) as antigens. Diarrhoea occurred in 10 and 40% of the vaccinees ingesting the vaccines A and B, respectively. The immunogenicity of the vaccine B in terms of seroconversion for vibriocidal antibodies and anti-LPS was higher than the vaccine A. Both vaccines had equal immunogenicity concerning serum anti-CT, while the vaccine A was slightly better than the vaccine B on serum anti-CHA response. The immunogenicity of the two vaccines in evoking intestinal responses was different from the systemic one.(ABSTRACT TRUNCATED AT 250 WORDS)