Wanpen Chaicumpa

Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from poultry in Thailand

We have recently developed a cytolethal distending toxin (cdt) gene‐based species‐specific multiplex PCR assay for identifying Campylobacter jejuni, C. coli and C. fetus. In the present study, the applicability of this assay was evaluated with 34 Campylobacter‐like organisms isolated from poultry in Thailand for species identification and was compared with other assays including API Campy, 16S rRNA gene sequence, and hippuricase (hipO) gene detection. Of the 34 strains analyzed, 20, 10 and 1 were identified as C. jejuni, C. coli, and Arcobacter cryaerophilus, respectively, and 3 could not be identified by API Campy. However, 16S rRNA gene analysis, showed that all 34 strains are C. jejuni/coli. To discriminate between these 2 species, the hipO gene, which is specifically present in C. jejuni, was examined by PCR and was detected in 20 strains, which were identified as C. jejuni by API Campy but not in the remaining 14 strains. Collective results indicated that 20 strains were C. jejuni whereas the 14 strains were C. coli. When the cdt gene‐based multiplex PCR was employed, however, 19, 20 and 19 strains were identified as C. jejuni while 13, 14 and 13 were identified as C. coli by the cdtA, cdtB and cdtC gene‐based multiplex PCR, respectively. Pulsed‐field gel electrophoresis revealed that C. jejuni and C. coli strains analyzed are genetically diverse. Taken together, these data suggest that the cdt gene‐based multiplex PCR, particularly cdtB gene‐based multiplex PCR, is a simple, rapid and reliable method for identifying the species of Campylobacter strains.

Molecular characteristics of Shigella spp. isolated from patients with diarrhoea in a new industrialized area of Thailand

In this study, we used plasmid profile analysis, XbaI macrorestriction with pulsed-field gel electrophoresis (PFGE), and PCR of the ipaH gene, to study the molecular characteristics of 183 Shigella spp. isolated during May 2000 to April 2003 from rectal swabs of patients with watery and/or bloody diarrhoea in a new industrialized area of Thailand. Among the 183 isolates, 167 were S. sonnei and 16 were S. flexneri. For plasmid profile analysis, the 183 isolates revealed 16 different plasmid patterns, designated patterns A to P. The sizes of the plasmid bands were: 6, 5.5, 5, 4.5, 4, 3.25, 2.75, 2.5, 2, 1.75, 1.5 and/or 1.25 kb. The frequency of each plasmid band was 4.5 kb (165 isolates), 3.25 kb (161 isolates), 5.5 kb (129 isolates), 1.75 kb (121 isolates), 1.5 kb (35 isolates), 5 kb (21 isolates), 2 kb (16 isolates), 2.75 kb (12 isolates), 1.25 kb (9 isolates), and 6 kb (8 isolates). PFGE analysis revealed 45 different XbaI macrorestricted DNA banding patterns which could be grouped into 11 groups. All the isolates gave PCR amplicons of the ipaH gene. Plasmid profile analysis and PFGE are powerful tools for differentiation of the Shigella spp. This study provides important data on the molecular characteristics of Shigella isolates in Thailand, which could be useful as an epidemiological baseline for identifying relationships with strains that may emerge in the future.

Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity.

A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins.

Human single chain-transbodies that bound to domain-I of non-structural protein 5A (NS5A) of hepatitis C virus

A safe and broadly effective direct acting anti-hepatitis C virus (HCV) agent that can withstand the viral mutation is needed. In this study, human single chain antibody variable fragments (HuscFvs) to conserved non-structural protein-5A (NS5A) of HCV were produced by phage display technology. Recombinant NS5A was used as bait for fishing-out the protein bound-phages from the HuscFv-phage display library. NS5A-bound HuscFvs produced by five phage transfected-E. coli clones were linked molecularly to nonaarginine (R9) for making them cell penetrable (become transbodies). The human monoclonal transbodies inhibited HCV replication in the HCVcc infected human hepatic cells and also rescued the cellular antiviral immune response from the viral suppression. Computerized simulation verified by immunoassays indicated that the transbodies used several residues in their multiple complementarity determining regions (CDRs) to form contact interface with many residues of the NS5A domain-I which is important for HCV replication complex formation and RNA binding as well as for interacting with several host proteins for viral immune evasion and regulation of cellular physiology. The human monoclonal transbodies have high potential for testing further as a new ramification of direct acting anti-HCV agent, either alone or in combination with their cognates that target other HCV proteins.