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A simple PCR-based typing method was developed to differentiate between strains of Burkholderia pseudomallei. Two pairs of primers, based on sequences from two specific DNA probes, were used to amplify the bacterial DNA by multiplex PCR. We evaluated the PCR method for epidemiological typing of B. pseudomallei and compared this with restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods. In RFLP, the DNA of B. pseudomallei was digested with HindIII and the pKKU-S23L was used as a probe while 5' GTTTCGCTCC 3' primer was used in RAPD. DNA was obtained from 37 B. pseudomallei environmental and clinical isolates from humans or animals. These isolates were also classified by their ability to assimilate L-arabinose. A total of 21 type patterns were identified by multiplex PCR. Among human and animal isolates, multiplex PCR revealed ten types, all of which were arabinose negative (Ara-), whereas six of the 11 types of environmental isolates were Ara-. There are two environmental patterns that also were found in clinical isolates. The RFLP technique showed 12 different types in the 37 isolates, and three of these contained both Ara+ and Ara- isolates. The RAPD technique revealed 33 different types in the 37 isolates. Multiplex PCR, therefore, is the genetic marker that best correlates with the ability of the organism to assimilate L-arabinose. Moreover, two types (M4, M15) correlated with disseminated septicemic melioidosis in the northeast Thailand. If a greater number of isolates are tested, the multiplex PCR technique may prove to be useful for rapid epidemiological typing of B. pseudomallei.

a Laboratory of Immunology, Chulabhorn Research Institute, Bangkok 10210, Thailand b Department of Microbiology, Faculty of Science, Mahidol Uni6ersity, Rama VI Road, Bangkok 10400, Thailand c Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand d Department of Microbiology, Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen, Thailand e National Institute of Health, Department of Medical Ser6ices, Ministry of Public Health, Nonthaburi, Thailand f Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni6ersity, Bangkok, Thailand g Department of Internal Medicine, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand

A highly sensitive, specific, rapid and simple method to detect Burkholderia pseudomallei in blood samples was developed. Two 22-base oligonucleotide primers, based on sequences from a specific DNA probe, were used for amplification of bacterial DNA by the polymerase chain reaction (PCR). Amplification with these primers yielded a 178-base pair product in 100 clinical isolates of B. pseudomallei. As little as 0.5 fg of B. pseudomallei DNA was detectable by this method. Experiments involving inoculation of the organism into uninfected blood samples showed that the method could be used to detect as few as 1 bacterial cell ml-1 of whole blood. Non-specific amplification of other bacterial DNAs from 18 samples of bacteria was not observed. Blood samples from seven patients proven to have melioidosis by haemoculture were positive using these primers. The total time required for sample processing, amplification and visualization was approximately 3.5 h. The high sensitivity, rapidity and simplicity of this method should make it valuable for diagnosis, monitoring of drug treatment and for epidemiological studies of the melioidosis.

Pseudomonas pseudomallei is a causative agent of melioidosis. The disease manifestations range from fulminant sepsis to asymptomatic seroconversion. In septicemic cases, a mortality rate of 80-90% is reported. Rapid and specific diagnosis has become important to the clinical microbiology laboratory. We have developed a P. pseudomallei -specific DNA probe. The cloned fragment, herein designated pKKU-S23L, contained 1·5 kb of P. pseudomallei chromosomal DNA. A radioactively labelled pKKU-S23L insert could detect 1·5 ng of its genomic DNA or 40 000 P. pseudomallei cells. The probe was highly specific for P. pseudomallei DNA and did not cross-hybridize with DNAs prepared from other related bacteria. Using pKKU-S23L as a probe in total cellular DNA digestions and Southern blot hybridization, we were able to classify 60 P. pseudomallei clinical isolates obtained from individual melioidosis patients into eight categories. Therefore, this probe has a potential not only for use in development of specific detection of bacterial DNA in clinical specimens but also for application in epidemiological studies of P. pseudomallei .

The diffusion and activity of ceftazidime (CAZ), imipenem (IPM) and trimethoprim/sulfamethoxazole (TMP/SMX) against Burkholderia pseudomallei biofilms were comparatively tested using the high biofilm-producing strain B. pseudomallei 377 and the biofilm-defective mutant B. pseudomallei M6. Biofilms were generated by inoculation of bacteria on polycarbonate membranes placed on the surface of tryptic soy agar plates. The results showed that diffusion of TMP/SMX through B. pseudomallei biofilms was similar for both strains. However, diffusion of CAZ and IPM was significantly faster through strain M6 biofilm in comparison with strain 377 biofilm. The viabilities of strain 377 biofilm were significantly higher than those observed with strain M6 for all antibiotics challenged at 4 h, suggesting that the biofilm-forming capacity may be involved in antibiotic susceptibilities in B. pseudomallei. These results re-emphasise the importance of biofilm for antibiotic resistance in B. pseudomallei.

Melioidosis is a serious community-acquired infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei. A prospective cohort study identified 2,243 patients admitted to Sappasithiprasong Hospital in northeast Thailand with culture-confirmed melioidosis between 1997 and 2006. These data were used to calculate an average incidence rate for the province of 12.7 cases of melioidosis per 100,000 people per year. Incidence increased incrementally from 8.0 (95% confidence interval [CI] = 7.2-10.0) in 2000 to 21.3 (95% CI = 19.2-23.6) in 2006 (P < 0.001; chi(2) test for trend). Male sex, age >/= 45 years, and either known or undiagnosed diabetes were independent risk factors for melioidosis. The average mortality rate from melioidosis over the study period was 42.6%. The minimum estimated population mortality rate from melioidosis in 2006 was 8.63 per 100,000 people (95% CI = 7.33-10.11), the third most common cause of death from infectious diseases in northeast Thailand after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and tuberculosis.

Leptospira interrogans, the causative agent of leptospirosis, is an important zoonotic bacterium. The mechanisms and roles of cytokine induction in both humans and animals remain unclear. Therefore, the IFN-gamma, IL-6, IL-10 and IL-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) in human THP-1 and mouse RAW 264.7 monocyte cell lines following stimulation with heat-killed Leptospira interrogans serogroup Pomona serovar Pomona, L. biflexa, E. coli or Salmonella group B. The production of IL-6 and IL-12 were higher and rose more rapidly in the RAW 264.7 cells with all bacteria. The IL-10 was not detected in the RAW 264.7 cells when induced by leptospires. The IFN-gamma level in human peripheral blood mononuclear cells (PBMCs) induced by leptospires was also significantly lower than with other bacteria. When IL-10 and IL-12 mRNA expressions were detected in hamster's spleen, their patterns were similar to what was observed in THP-1 in that IL-12 was only slightly increased while IL-10 expression was high. Moreover, the IFN-gamma expression could not be detected in hamsters. The more potent cytokine responses in the RAW 264.7 cells may indirectly reflect the disease outcome in mice which render them to be a good reservoir of leptospirosis. Whether these cytokines have contributed to immunoprotection during the L. interrogans infection remains to be further investigated.

SUMMARY Opisthorchis viverrini infection is associated with human hepatobiliary disease and cholangiocarcinoma, but the role of the immune response in the pathogenesis of infection is unclear. Here ultrasonography was used to examine the biliary tracts of residents from an endemic community. Delayed-type hypersensitivity responses to unrelated antigens, and fluke-specific IgG and IgA levels in serum of this group were also examined. Relationships between immunological parameters, intensity of infection and radiologically measured variables are reported. Immune responsiveness to unrelated antigens did not vary with intensity of parasite infection or disease status. Of all the variables, IgG levels were most markedly elevated in disease cases compared with normal subjects and were closely associated with gall bladder size and dysfunction. This is consistent with the hypothesis that an immunopathologic mechanism is involved in opisthorchiasis and suggests that antibody levels may be useful in screening populations for fluke-associated hepatobiliary disease.

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.

In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny.

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei , undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

The formation of granulation tissue is an important factor promoting recurrence after surgical treatment of laryngotracheal stenosis. Bacterial infection was claimed to be the cause.The present study aimed to identify the bacteriology of granulation tissue in laryngotracheal stenosis patients.Data was collected prospectively. Granulation tissue found in the site of laryngotracheal stenosis was removed and sent to the microbiologic study to identify the organisms.Twenty-four specimens from 17 patients were included in the present study. Coagulase-positive Staphylococcus (45.8%) was the most common gram-positive organism and Pseudomonas aeruginosa as well as Enterobacter species (16.7%) were the most common gram-negative bacteria. Ciprofloxacin may be the oral antibiotic that should be recommended.Coagulase-positive Staphylococcus, Pseudomonas aeruginosa as well as Enterobacter species were the common organisms identified from the granulation tissue in recurrent laryngotracheal stenosis. Oral antibiotics, such as ciprofloxacin, may have benefit in reducing the formation of this granulation tissue.

Heterogeneous patterns were obtained for lipopolysaccharide (LPS) from 1,327 Burkholderia pseudomallei isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and immunoblot analysis. Two LPS serotypes (A and B) possessing different ladder profiles and a rough LPS without ladder appearances were identified. All three LPS types were antigenically distinct by immunoblotting. The predominant type A (97%) produced the lowest amount of biofilm. The two less common types (smooth type B and rough type) were found more in clinical than environmental isolates and more in Australian isolates than Thai isolates. These isolates were more often associated with relapse than with primary infection.

Heterogeneous patterns were obtained for lipopolysaccharide (LPS) from 1,327 Burkholderia pseudomallei isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and immunoblot analysis. Two LPS serotypes (A and B) possessing different ladder profiles and a rough LPS without ladder appearances were identified. All three LPS types were antigenically distinct by immunoblotting. The predominant type A (97%) produced the lowest amount of biofilm. The two less common types (smooth type B and rough type) were found more in clinical than environmental isolates and more in Australian isolates than Thai isolates. These isolates were more often associated with relapse than with primary infection.

Burkholderia pseudomallei is the causative agent of melioidosis but currently the pathogenesis of the disease is still poorly understood. One of the virulent factors of gram-negative bacteria is the ability to produce biofilm to evade host defense. As B. pseudomallei has also been reported to develop the biofilm [1], in the present study, we therefore, quantified the biofilm formation in 50 strains of B. pseudomallei and compared with 50 strains of its avirulent counterpart Burkholderia thailandensis using a modified microtiter-plate test. The results showed that the quantity of biofilm produced by B. pseudomallei was statistically higher (P< 0.01) than that of B. thailandensis (means and SEs of the corrected OD630 were 2.17+/-0.29 and 0.59+/-0.05, respectively). Transmission electron micrographs of the B. pseudomallei strain with high biofilm formation exhibited microcolonies of bacterial cells surrounded by dense extracellular slime matrix comparing with only trace quantity in the low biofilm-producing strain or the biofilm mutants generated by Tn5-OT182 mutagenesis. However, no correlation could be observed between the biofilm formation and virulence, judging from the LD50 values in BALB/c mice. The data obtained with these naturally occurring Burkholderia species and the biofilm mutants are incompatible with the possibility that the biofilm plays a role in the pathogenesis of B. pseudomallei infection.

Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhance resistance against infection in a number of animal models, little is known about the CpG-induced protection against acute fatal sepsis such as that associated with the highly virulent bacterium Burkholderia pseudomallei . We previously demonstrated in an in vitro study that immunostimulatory CpG ODN 1826 enhances phagocytosis of B. pseudomallei and induces nitric oxide synthase and nitric oxide production by mouse macrophages. In the present study, CpG ODN 1826 given intramuscularly to BALB/c mice 2 to 10 days prior to B. pseudomallei challenge conferred better than 90% protection. CpG ODN 1826 given 2 days before the bacterial challenge rapidly enhanced the innate immunity of these animals, judging from the elevated serum levels of interleukin-12 (IL-12)p70 and gamma interferon (IFN-γ) over the baseline values. No bacteremia was detected on day 2 in 85 to 90% of the CpG-treated animals, whereas more than 80% of the untreated animals exhibited heavy bacterial loads. Although marked elevation of IFN-γ was found consistently in the infected animals 2 days after the bacterial challenge, it was ameliorated by the CpG ODN 1826 pretreatment ( P = 0.0002). Taken together, the kinetics of bacteremia and cytokine profiles presented are compatible with the possibility that protection by CpG ODN 1826 against acute fatal septicemic melioidosis in this animal model is associated with a reduction of bacterial load and interference with the potential detrimental effect of the robust production of proinflammatory cytokines associated with B. pseudomallei multiplication.

Opisthorchiasis is the major public health problems in Laos PDR and Thailand. The disease becomes chronic and persists for many years, leading to hepatobiliary disease and cholangiocarcinoma. Less severe manifestations include cholangitis, chronic cholecystitis and cholelithiasis. A significant degree of humoral and cell mediated immune responses to the parasite can be detected both in patients and animal models. The patients IgG levels appear to correlate with gall bladder size and dysfunction and correlated significantly with opisthorchis egg count and decrease after treatment. However, the possible significance of these immune responses to protective immunity is presently unknown. The development of immunodiagnostic method for Opisthorchis viverrini detection has been attempted. The components with molecular weight >116, 89, 78 and 20 kDa appear to be specifically associated with the somatic extract of adult fluke. The 89 kDa protein is the most prominent component found in the in vitro culture fluid of adult worms and the metacercarial extract that can be a candidate with significant immunodiagnostic potential. Highly specific and sensitive monoclonal antibodies for O. viverrini antigens were prepared to detect parasite antigens in stool and antibody in serum. Information regarding the molecular approaches of O. viverrini is very limited. The genome of O. viverrini has neither CpG nor A methylated as found in other parasites. The total length O. viverrini ribosomal DNA is approximately 13 kb. and the presence of a highly repeated DNA specific for the parasite was demonstrated. A O. viverrini specific DNA probe was constructed and PCR based detection with high specificity for amplification of the repeated sequences is performed to detect the presence of eggs’ DNA in stool samples in comparison with classical methods.

B10 mice were immunized with an Opisthorchis viverrini somatic extract and then their responses were analyzed. The antigenic fractions of the extract were separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membranes and solubilized for use in lymphocyte culture. Antibody specificity was also visualized by immunoblotting using immunized mouse sera. The Mr of the main immunogenic fractions for T cells ranged from 28 to 46 kDa, whereas those recognized by antibodies were 45, 52, 56, 59, 65, 69, 75 and 81 kDa. The results indicate a striking difference in the antigenie recognition pattern of T and B cells which may be important for selecting antigen molecules for immunological studies of this trematode infection in man.

SUMMARYThree techniques for estimating the intensity of Opisthorchis viverrini infection in individuals from a Northeast That community are compared. Egg counts were determined using a quantitative formalin/ethyl acetate technique, worm burdens were estimated by expulsion chemotherapy and antibody levels were measured by ELISA. Log-transformed worm and egg counts were closely correlated (r = 0·80), suggesting that both measurements provide good assessments of relative intensity of infection. However, no Opisthorchis worms were recovered from 34 people with high egg counts; probably due to problems with the expulsion technique in some individuals. Examination of egg production per fluke indicated that each fluke contributed an average of 180 eggs per gram (epg) of faeces and fecundity was negatively associated with total worm burden. Serum IgG levels correlated significantly with Opisthorchis egg count (r = 0·61) at two independent assessments. Although significant associations were observed between antibody levels and echinostome infection, analysis suggested that these reflected independent associations between these two variables and Opisthorchis infection and age. We conclude that all three measurements are useful for epidemiological studies.

The humoral immune response in patients with opisthorchiasis was investigated using an enzyme-linked immunosorbent assay. IgG antibody reactive with Opisthorchis viverrini antigens was present in the serum of all patients. The infection also stimulated specific IgA and IgE antibody responses in most patients and, in practically all patients, there was a marked increase of total IgE. There was a moderate but significant correlation between serum IgG antibody level and severity of infection as judged from the quantity of eggs in the stool of the patients. There was also a significant elevation of antibody in the bile and serum of O. viverrini-infected patients who also had biliary obstruction. Analysis of paired samples from individual patients showed that while IgG was the predominant class of antibody in the serum of all patients, IgA was present at approximately the same level as IgG or higher in the bile of many patients. In addition to IgA and IgG antibodies, IgE antibody was also detectable in 50% of the bile samples. The high level of IgA antibody in the bile together with its presence in association with the secretory component suggested a selective transport and/or local production of IgA antibody by the hepatobiliary system of these patients.

Leptospirosis, caused by Leptospira spp., is a zoonotic disease found worldwide. Killed whole cell leptospiral vaccines have been used as effective vaccines to elicit specific antibodies for protection. However, the involvement of cytokine responses after vaccination is not well characterized. Hamsters were immunized with killed L. interrogans serovar Autumnalis before challenge to study cytokine mRNA expression levels (interferon [IFN]-gamma, tumor necrosis factor [TNF]-alpha, interleukin [IL]-10, and IL-4). Vaccinated groups showed 92-100% survival rates, whereas control hamsters died within 6-10 days. However, live organisms were detected in vaccinated groups, and mild to moderate pathology was observed early in infection. IFN-gamma and TNF-alpha mRNA expression levels correlated with the severity of infection and lung pathology, whereas IL-4 and IL-10 expression levels were significantly higher in vaccinated groups. In summary, commonly used vaccines changed the cytokine profiles and protected hamsters from death but failed to stimulate sterile immunity and were unable to prevent the occurrence of pathology.

For diagnosis of melioidosis, we compared polymerase chain reaction (PCR)-based DNA detection and three serologic methods with the culture method currently used as gold standard. The diagnostic values of the serologic methods were evaluated in 130 patients. All these patients resided in an endemic area. An enzyme-linked immunosorbent assay (ELISA) gave slightly higher specificity (86.2%) than a dot immunoassay (DOT) (85.3%), but was superior to an indirect hemagglutination assay (IHA) (79.8%). The sensitivities of the DOT (85.7%) and ELISA (71.4%) were considerably higher than that of IHA (61.9%). However, the PCR was the most sensitive (95.2%) and specific (91.7%). Nevertheless, DOT and ELISA are more practical for local hospitals. With the high negative predictive value of both the ELISA (94.0%) and DOT (96.9%) in a high prevalence area, clearly these methods can rule out most of the non-melioidosis patients.

Opisthorchis viverrini infection is an endemic disease that causes a serious public health problem in southeast Asia, especially in northeast Thailand, We have developed a PCR method using a pair of primers named OV-6F/OV-6R for detecting O. viverrini eggs in stool samples and compared it with Stoll's egg-count method. The primers were designed based on the pOV-A6 specific DNA probe sequence which gave a 330 base pair product. The PCR method can detect a single egg in artificially inoculated faeces or as little as 2 × 10 -17 ng of O. viverrini genomic DNA. The method gave 100% sensitivity in all hamster groups except in animals exposed to the lowest intensity of infection (1 metacercaria/hamster). In the first month of infection, the PCR method was more sensitive than using the egg-count method in all infected groups especially in the light infections. 'l'he PCR method was also successfully used in monitoring a therapeutic study. Since the PCR method showed no cross-reaction with Heterophyid flukes, it can be useful for specific identification of O. viverrini eggs in stool samples without the risk of false positives. It also has great potential for application in clinical epidemiological studies.