Induction of regulatory T cells by Opisthorchis viverrini.

Opisthorchis viverrini causes public health problems in South-East Asia. Recently, TGF-ß and IL-10 have been reported to increase in O. viverrini-infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4+ T cells in infected hamsters were investigated. It was demonstrated that IL-4+ CD4+ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL-10+ CD4+ T cells were also discovered at a significant level while Treg (T regulatory)-like TGF- ß+ CD4+ T cells were in MLNs of infected hamsters. Moreover, the CD4+ CD25+ Foxp3+ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T-cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF-ß+ CD4+ while TCC35, TCC41 and TCC108 were IL-4+ CD4+ and TCC23 was IFN-γ+ CD4+ . This TGF-ß+ CD4+ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF-ß-mediated mechanisms. This study indicated that O. viverrini-infected hamsters could induce TGF-ß+ CD4+ Treg-like cells. The CSAg-specific Tregs secreted high TGF-ß, and limited immune cell proliferation.

Use of a low-dose steroid as an adjunct in the treatment, in mice, of severe sepsis caused by Burkholderia pseudomallei.

Human melioidosis caused by Burkholderia pseudomallei is a severe septic disease that is associated with high mortality, even under appropriate antibiotic treatment. The therapeutic effects of low-dose hydrocortisone plus ceftazidime, and of ceftazidime alone, have recently been investigated in the treatment of acute, severe sepsis caused by B. pseudomallei, both in normal BALB/c mice and in BALB/c mice with streptozotocin-induced diabetes. The mice were infected and then treated intravenously, from day 1 or day 2 post-infection, with saline (as a control, given twice daily for 10 days), low-dose hydrocortisone (given in twice-daily doses of 5 mg/kg, for 5 days) plus ceftazidime (given in twice-daily doses of 1200 mg/kg, for 10 days), or the same doses of ceftazidime alone. Although the infected, untreated mice all died within 14 days, almost all of the treated animals were still alive at the end of the follow-up, 30 days post-infection. The addition of the steroid appeared to have no benefit, with bacterial loads and plasma concentrations of tumour necrosis factor, aspartate aminotransferase, alanine aminotransferase and creatinine decreasing similarly in all the treated groups. The infected diabetic mice given hydrocortisone-ceftazidime from day 1 (but not those given just ceftazidime from day 1) showed an increase in their blood glucose concentrations. When infected mice were treated with the low-dose steroid and lower doses of the antibiotic (in twice-daily doses of 120-600 mg/kg), the steroid not only offered no apparent benefit but seemed to reduce survival. It therefore appears that low-dose hydrocortisone, as an adjunct to antibiotic treatment, does not provide benefit in the treatment of murine melioidosis and may have negative effects on human cases of the disease who have diabetes mellitus.

TLR4 genetic variation is associated with inflammatory responses in Gram-positive sepsis.

OBJECTIVES: To identify important pathogen recognition receptor (PRR) pathways regulating innate immune responses and outcome in Staphylococcus aureus sepsis. METHODS: We analysed whether candidate PRR pathway genetic variants were associated with killed S. aureus-induced cytokine responses ex vivo and performed follow-up in vitro studies. We tested the association of our top-ranked variant with cytokine responses and clinical outcomes in a prospective multicentre cohort of patients with staphylococcal sepsis. RESULTS: An intronic TLR4 polymorphism and expression quantitative trait locus, rs1927907, was highly associated with cytokine release induced by stimulation of blood from healthy Thai subjects with S. aureus ex vivo. S. aureus did not induce TLR4-dependent NF-κB activation in transfected HEK293 cells. In monocytes, tumor necrosis factor (TNF)-α release induced by S. aureus was not blunted by a TLR4/MD-2 neutralizing antibody, but in a monocyte cell line, TNF-α was reduced by knockdown of TLR4. In Thai patients with staphylococcal sepsis, rs1927907 was associated with higher interleukin (IL)-6 and IL-8 levels as well as with respiratory failure. S. aureus-induced responses in blood were most highly correlated with responses to Gram-negative stimulants whole blood. CONCLUSIONS: A genetic variant in TLR4 is associated with cytokine responses to S. aureus ex vivo and plasma cytokine levels and respiratory failure in staphylococcal sepsis. While S. aureus does not express lipopolysaccharide or activate TLR4 directly, the innate immune response to S. aureus does appear to be modulated by TLR4 and shares significant commonality with that induced by Gram-negative pathogens and lipopolysaccharide.

Evaluation of PCR based coprodiagnosis of human opisthorchiasis.

In this study, a recently developed PCR test for the detection of Opisthorchis viverrini in human faecal samples was evaluated using two parasitological methods as references. During a survey of foodborne trematodes (FBT) in the Vientiane Province, Lao PDR, 85 samples were collected and evaluated for FBT eggs by the Kato Katz (KK) technique, the formalin ethyl acetate concentration technique (FECT) and a PCR analysis for the distinction between O. viverrini and other FBT. The two parasitological methods did not differ in the ability of detecting FBT eggs, and a single KK reading was characterized by a sensitivity of 85% when compared to two FECT readings. The PCR tested positive only in cases where eggs had been demonstrated by parasitological examination. However, the PCR tested negative in some samples with very high egg counts. Demonstrating a PCR sensitivity of approximately 50% in samples with faecal egg counts>1000, the previously reported PCR sensitivity based on in vitro studies was not supported. It is believed that technical problems rather than diagnostic reference related issues were responsible for the relatively low PCR performance. Further studies should aim at optimizing DNA extraction and amplification, and future PCR evaluation should include specificity control such as the scanning electron microscopy of eggs in test samples or the expulsion of adult trematodes from PCR tested individuals.

Isolation of fetal nucleated red blood cell from maternal blood using immunomagnetic beads for prenatal diagnosis.

The immunomagnetic beads method for isolation of fetal nucleated red blood cells (FNRBCs) from peripheral blood of 78 pregnant women for prenatal diagnosis was developed. The study subjects were classified into 8-10 and 11-14 weeks of gestation (n = 39 each). Peripheral blood cells were divided into two for the FNRBCs isolation using two protocols, one with anti-CD45 depletion followed by anti-CD71 and anti-GPA monoclonal antibodies and another without CD45 depletion. The use of CD45 depletion gave a slightly higher number of sorted cells but not significantly different (p > 0.05). The percentage of CD71+ and GPA+ cells obtained from 8-10 weeks and 11-14 weeks of gestation was not different (p > 0.05). The sensitivity in determining the sorted FNRBCs for male fetal sex by PCR using 8-10 and 11-14 weeks of gestation was generally 50 and 69%, respectively. The method so developed is simple and cost effective and may thus be applied for prenatal diagnosis.

Drug susceptibility and biofilm formation of Burkholderia pseudomallei in nutrient-limited condition.

Burkholderia pseudomallei is the causative agent of melioidosis, which can form biofilms and microcolonies in vivo and in vitro. One of the hallmark characteristics of the biofilm-forming bacteria is that they can be up to 1,000 times more resistant to antibiotics than their free-living counterpart. Bacteria also become highly tolerant to antibiotics when nutrients are limited. One of the most important causes of starvation induced tolerance in vivo is biofilm growth. However, the effect of nutritional stress on biofilm formation and drug tolerance of B. pseudomallei has never been reported. Therefore, this study aims to determine the effect of nutrient-limited and enriched conditions on drug susceptibility of B. pseudomallei in both planktonic and biofilm forms in vitro using broth microdilution method and Calgary biofilm device, respectively. The biofilm formation of B. pseudomallei in nutrient-limited and enriched conditions was also evaluated by a modified microtiter-plate test. Six isolates of ceftazidime (CAZ)-susceptible and four isolates of CAZ-resistant B. pseudomallei were used. The results showed that the minimum bactericidal concentrations of CAZ against B. pseudomallei in nutrient-limited condition were higher than those in enriched condition. The drug susceptibilities of B. pseudomallei biofilm in both enriched and nutrient-limited conditions were more tolerant than those of planktonic cells. Moreover, the quantification of biofilm formation by B. pseudomallei in nutrient-limited condition was significantly higher than that in enriched condition. These data indicate that nutrient-limited condition could induce biofilm formation and drug tolerance of B. pseudomallei.

Molecular analysis of T and B cell repertoires in mice immunized with Opisthorchis viverrini antigens.

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 antigenic recognition pattern of T and B cells which may be important for selecting antigen molecules for immunological studies of this trematode infection in man.

Characterization of humoral immune response in the serum and bile of patients with opisthorchiasis and its application in immunodiagnosis.

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.

Comparison of the polymerase chain reaction and serologic tests for diagnosis of septicemic melioidosis.

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.

Short report: evaluation of a monoclonal antibody-based latex agglutination test for rapid diagnosis of septicemic melioidosis.

A monoclonal antibody (MAb)-based latex agglutination (MAb-LA) test was developed to rapidly identify Burkholderia pseudomallei in hemoculture of patients with septicemic melioidosis. The method was evaluated in a clinical situation on 396 hemocultures positive for bacterial growth, of which 75 cultures were positive for B. pseudomallei by conventional biochemical tests. The sensitivity and specificity of the MAb-LA test were 95% and 100%, respectively. The positive and negative predictive values were 100% and 99%. The method is highly reliable and suitable for rapid diagnosis of septicemic melioidosis, reducing the time normally required from a minimum of 3-4 days by conventional methods to less than 30 hr. Most of these 30 hr are involved in growing up enough bacteria to perform the MAb-LA test, which itself takes only 1 min.

Immunology and molecular biology of Opisthorchis viverrini infection.

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.

Use of multiplex PCR patterns as genetic markers for Burkholderia pseudomallei.

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.

Ribotyping of Burkholderia pseudomallei from clinical and soil isolates in Thailand.

Burkholderia pseudomallei is a soil saprophyte that causes melioidosis in humans and animals. Restriction fragment length polymorphism of the ribosomal DNA regions (ribotyping) were analyzed in 577 isolates comprising 371 clinical and 206 soil isolates collected throughout Thailand in 1997. A total of 77 distinct ribotype patterns consisting of 2-9 bands with sizes ranging from 2.8 to >23 kb were found. Twelve major ribotypes were identified of which types 3, 8 and 23 were commonly found (278/577, 48.2%) in both clinical (217/371, 58.5%) and environmental isolates (61/206, 29.6%). Three unique environmental types were found whereas a unique clinical type was not observed. Even though ribotypes show high heterogeneity in the rDNA region, the unique environmental patterns were clearly different from the clinical patterns as clearly seen by UPGMA dendrogram. Moreover, the three major types (types 3, 8 and 23) were discovered in nearly half of B. pseudomallei isolates. Subtyping of these major ribotypes in correlation with clinical profiles may help researchers to identify the virulence factor of the organism.

Detection of Paragonimus heterotremus in experimentally infected cat feces by antigen capture-ELISA and by DNA hybridization.

An antigen capture enzyme-linked immunosorbent assay (antigen capture-ELISA) and DNA hybridization technique were developed and evaluated for their application in the detection of Paragonimus heterotremus infection in experimentally infected cats. An IgG fraction prepared from serum of a rabbit immunized with P. heterotremus excretory-secretory (ES) products was used as the capture antibody. An IgG1 monoclonal antibody specific to the 22- and 31.5-kDa ES products of P. heterotremus was used as the antigen probe. As little as 0.24 ng of the ES products could be detected by this technique. A specific P. heterotremus DNA probe derived from the P. heterotremus genomic DNA library containing 1,500 base pairs was used in a dot-blot hybridization assay for the detection of parasite DNA. The radioactively labeled probe could detect DNA released from as few as 2 P. heterotremus eggs. Both ELISA and DNA hybridization were found to have 100% specificity, with sensitivities of 73.7% and 100%, respectively.

Immunization of hamsters against Opisthorchis viverrini infection.

Attempts were made to induce acquired immunity against Opisthorchis viverrini infection in hamsters by immunizing them with aqueous somatic extract and metabolic products of adult worms, crude adult worm homogenates and metacercarial somatic extracts via either the intraperitoneal or combined intraperitoneal and oral routes. These procedures failed to stimulate significant protective response in animals that had never been exposed to O. viverrini. However, the protective response reached a significant level (30% worm reduction) in hamsters that had been infected with a small member of flukes prior to immunization with aqueous somatic extract of adult worms. Although these findings indicate that it may be possible to reduce reinfection in people in the endemic area by immunization, it appears that a better method currently available for the control of O. viverrini infection is health education aimed at changing food habits and improving sanitation and personal hygiene.

Analysis of Opisthorchis viverrini antigens: physicochemical characterization and antigen localization.

Antigens of Opisthorchis viverrini were identified and characterized by enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis following radioimmuno-precipitation, and indirect immunofluorescence. The immunoreactive protein with a relative molecular weight (Mr) of 89 kD was the predominating component of the parasite metabolic products. An immunofluorescence study showed it to be associated with parasite eggs, linings of the reproductive system and secretions therein. Protein of the surface tegument had Mr of greater than 116, 108, 64, 38, 34, 20 and 16-17 kD. The 16-17 kD surface molecule was the predominant protein, representing approximately 50% of the total protein in crude aqueous extracts of adult worms. However, it was poorly immunogenic when compared with the 89 kD molecule. Together with data presented previously, it appears that in addition to the 89 kD protein, several tegumental molecules are also specific for O. viverrini and have immuno-diagnostic potential.

Effect of praziquantel treatment on antibody levels and lymphoproliferative responses in patients with opisthorchiasis.

The purpose of this study was to explore alternative method(s) to monitor the efficacy of anthelmintic treatment of patients with opisthorchiasis. Therefore, in our initial attempt, we studied the changes in antibody levels and lymphoproliferative responses in O. viverrini infected patients before and 2 months after successful praziquantel treatment. The results showed that although a substantial reduction of the antibody levels occurred after such a treatment, it did not occur in all patients. In those showing reduction, the final level were still above 2 standard deviations of the normal mean value. The reduction was more profound for IgG antibody. With regard to the IgA antibody isotype, the reduction was not as marked. In contrast, IgE antibody levels in most patients not only failed to decline, but instead, showed a tendency to be elevated after praziquantel treatment. Unlike the antibody levels, there was no alteration in the lymphoproliferative response to PHA stimulation and therefore this parameter is not useful for our intended objective. It was suggested that studies of a more specific O. viverrini component may be more reliable than the current method of parasitological examination of eggs in the feces of suspected individuals.

Use of culture-filtrated antigen in an ELISA and a dot immunoassay for the diagnosis of melioidosis.

An enzyme-linked immunosorbent assay (ELISA) and a dot immunoassay with culture-filtrated antigen were developed for detection of Burkholderia pseudomallei specific antibodies in melioidosis patients. Sixty-eight sera of bacteriologically confirmed melioidosis patients, 45 sera of other bacterial infected patients and 80 sera of healthy blood donors from endemic area were investigated. The samples were subjected to those assays im comparison with indirect hemagglutination (IHA). The sensitivity, specificity, positive and negative predictive values in this dot immunoassay were 94.1%, 99.2%, 98.5% and 96.9%, respectively, with cut-off dilution at 1:4,000, whereas those in ELISA were 92.6%, 96.8%, 94.0% and 96.0%, respectively, with cut-off value of OD = 0.47 at 490 nm. Meanwhile, those in IHA were 64.7%, 93.6%, 84.6%, 83.0% respectively, with a cut-off value of > or = 1:80. The results in this study demonstrated that the dot immunoassay was more reliable and rapid than ELISA as the serological test for diagnosis of melioidosis.

Characterization of Pseudomonas pseudomallei antigens by SDS-polyacrylamide gel electrophoresis and western blot.

Immunological characterization of various Pseudomonas pseudomallei preparations was carried out by SDS-PAGE and Western blot using sera from infected humans and from patients with other bacterial infections. Somatic (SOM) and partially purified cell extracts (PCE) gave more complex SDS-PAGE patterns: M(r) ranged from 86 to 12.7 and 48 to 10 kDa, respectively. The culture-filtrated antigens (CF) from 3 different kinds of synthetic media consisted of fairly simple profiles with common bands M(r) of 40, 26 and 16 kDa. PCE and CF reacted specifically with infected human sera; SOM did not. The components with M(r) of 40 kDa in CF reacted consistently with all infected sera but failed to react with sera infected with Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Staphylococcus aureus, Streptococcus spp., Pseudomonas aeruginosa and P. stutzeri. This peptide was demonstrated to be a major component in CF thus suggesting its potential for development of immunodiagnostic methods for melioidosis.

Comparison between the antimicrobial susceptibility of Burkholderia pseudomallei to trimethoprim-sulfamethoxazole by standard disk diffusion method and by minimal inhibitory concentration determination.

Melioidosis, an infection caused by Burkholderia pseudomallei, usually occurs in immunocompromised patients and requires prolonged antibiotic therapy. Previously, oral trimethoprim-sulfamethoxazole (TM/SM), an inexpensive and effective drug has been used as a maintenance therapy. The susceptibility of B. pseudomallei to TM/SM by the standard disk diffusion method is very low. However, some patients who were treated with TM/SM as a maintenance therapy despite the in vitro resistance showed good clinical responses. There were no data comparing the susceptibility of B. pseudomallei by the standard disk diffusion method with other quantitative susceptibility tests. The objective of this study was to determine the agreement between the antimicrobial susceptibility of B. pseudomallei to TM/SM by standard disk diffusion and minimal inhibitory concentration determination (MIC). We performed the susceptibility test of 144 strains of B. pseudomallei to TM/SM by both the standard disk diffusion and microbroth dilution MIC. The sensitivity results were 53.5 per cent and 84.0 per cent respectively. The agreement between the 2 tests was very poor (Kappa = 0.14; 95% CI = -0.01 to 0.29). The false resistant rate by the standard disk diffusion test was 67.9 per cent. Further in vitro susceptibility and clinical study are needed to define the interpretive criteria that correlate with clinical response.