A Use of 56-kDa Recombinant Protein of Orientia tsutsugamushi Karp Serotype in Serodiagnosis of Scrub Typhus by Enzyme-Linked Immunosorbent Assay in Thais.

Scrub typhus is a mite-borne disease caused by a Gram-negative obligately intracellular bacillus, Orientia tsutsugamushi. The disease is endemic in the Asia-Australia-Pacific region, including Thailand. Scrub typhus generally manifests as acute undifferentiated febrile fever along with myalgia, rash, and lymphadenopathy. An eschar can be a valuable diagnostic clue, but this skin lesion may be missed in some patients. The disease symptoms resemble those of other febrile illnesses such as leptospirosis, typhoid, murine typhus, malaria, and dengue fever, making a laboratory diagnosis necessary for the definitive diagnosis. In this study, we expressed a recombinant protein derived from 56-kDa type-specific antigen of O. tsutsugamushi Karp serotype and tested its ability to detect and differentiate scrub typhus infection. IgM and IgG antibodies were determined in sera from scrub typhus (n = 92) and other febrile illness patients (murine typhus (n = 25), melioidosis (n = 36), leptospirosis (n = 42), and dengue (n = 35)) from Thailand. Sensitivities of 87.0% and 59.8% with a specified assay cut-off were obtained for IgM and IgG indirect ELISAs, respectively, with a specificity of 100% in both tests. The sensitivity was increased to 95.7% when a combination of IgM and IgG ELISAs results was considered. Our study suggested a potential of the 56-kDa recombinant protein for further development and evaluation for use in scrub typhus serodiagnosis.

Evaluation of a genus-specific rGroEL1-524 IgM-ELISA and commercial ELISA kits during the course of leptospirosis in Thailand.

In the present study, we developed a genus-specific rGroEL1-524 IgM-ELISA assay for use in screening diagnosis of suspected leptospirosis among acute undifferentiated febrile illness patients during acute fever. The diagnostic accuracies of the rGroEL1-524 IgM-ELISA, commercial Panbio IgM-ELISA, and Virion-Serion Classic IgG-ELISA were evaluated using 133 Thai leptospirosis sera and 210 controls. Sensitivities were 91.7%, 59.6%, and 17.7% for acute infection, and the specificities were 92.6%, 90.2%, and 88.3% for the non-leptospirosis control, respectively. The rGroEL1-524 IgM-ELISA had high sensitivity, at 92.3% and 91.7%, among culture-positive and MAT-negative cases at 1-3 days post-onset of symptoms (DPO1-3), respectively. Impaired specificity on scrub typhus was found, possibly from antibody cross-reaction to ortholog GroEL. Commercial Panbio IgM-ELISA had sensitivities at DPO1-3 of 30.8% and 41.7% for culture-positive and MAT-negative cases whereas Virion-Serion IgG-ELISA showed sensitivities of 5.9% and 13.3%, respectively. The rGroEL1-524 IgM-ELISA could be useful as a screening test for early diagnosis. The performance of the commercial ELISA suggests the applicability of IgM-ELISA for diagnosis, while IgG-ELISA is useful for seroprevalence surveys. However, confirmation by reference tests is recommended.

Antimicrobial Peptide Modifications against Clinically Isolated Antibiotic-Resistant Salmonella.

Antimicrobial peptides are promising molecules to address the global antibiotic resistance problem, however, optimization to achieve favorable potency and safety is required. Here, a peptide-template modification approach was employed to design physicochemical variants based on net charge, hydrophobicity, enantiomer, and terminal group. All variants of the scorpion venom peptide BmKn-2 with amphipathic α-helical cationic structure exhibited an increased antibacterial potency when evaluated against multidrug-resistant Salmonella isolates at a MIC range of 4-8 µM. They revealed antibiofilm activity in a dose-dependent manner. Sheep red blood cells were used to evaluate hemolytic and cell selectivity properties. Peptide Kn2-5R-NH2, dKn2-5R-NH2, and 2F-Kn2-5R-NH2 (variants with +6 charges carrying amidated C-terminus) showed stronger antibacterial activity than Kn2-5R (a variant with +5 charges bearing free-carboxyl group at C-terminus). Peptide dKn2-5R-NH2 (d-enantiomer) exhibited slightly weaker antibacterial activity with much less hemolytic activity (higher hemolytic concentration 50) than Kn2-5R-NH2 (l-enantiomer). Furthermore, peptide Kn2-5R with the least hydrophobicity had the lowest hemolytic activity and showed the highest specificity to Salmonella (the highest selectivity index). This study also explained the relationship of peptide physicochemical properties and bioactivities that would fulfill and accelerate progress in peptide antibiotic research and development.

Genetic association study of interferon lambda 3, CD27, and human leukocyte antigen-DPB1 with dengue severity in Thailand.

BACKGROUND: Dengue patients develop different disease severity ranging from mild (dengue fever [DF]) to severe forms (dengue hemorrhagic fever [DHF] and the fatal dengue shock syndrome [DSS]). Host genetics are considered to be one factor responsible for the severity of dengue outcomes. To identify genes associated with dengue severity that have not been studied yet, we performed genetic association analyses of interferon lambda 3 (IFNL3), CD27, and human leukocyte antigen-DPB1 (HLA-DPB1) genes in Thai dengue patients. METHODS: A case-control association study was performed in 877 children (age ≤ 15 years) with dengue infection (DF, n = 386; DHF, n = 416; DSS, n = 75). A candidate single nucleotide polymorphism of each of IFNL3, CD27, and HLA-DPB1 was selected to be analyzed. Genotyping was performed by TaqMan real-time PCR assay, and the association with dengue severity was examined. RESULTS: The rs9277534 variant of HLA-DPB1 was weakly associated with DHF. The genotype GG and G allele conferred protection against DHF (p = 0.04, odds ratio 0.74 for GG genotype, p = 0.03, odds ratio 0.79 for G allele). The association became borderline significant after adjusting for confounders (p = 0.05, odds ratio 0.82). No association was detected for IFNL3 or CD27. CONCLUSIONS: The present study demonstrated the weak association of the rs9277534 variant of HLA-DPB1 with protection against DHF. This variant is in the 3' untranslated region and affects HLA-DPB1 surface protein expression. Our finding suggests that HLA-DPB1 may be involved in DHF pathogenesis.

Interferon lambda 1 is associated with dengue severity in Thailand.

OBJECTIVES: Patients with dengue exhibit a range of symptoms from an acute febrile illness (dengue fever, DF), to dengue hemorrhagic fever (DHF), and to the most severe outcome, dengue shock syndrome (DSS). This study was performed to determine the host genetic factors responsible for dengue severity. Two single nucleotide polymorphisms (SNPs) of the interferon lambda 1 (IFNL1) gene (rs30461 and rs7247086) were analyzed for their association with dengue severity in a Thai population. METHODS: This was a case-control association study involving 877 patients under the age of 15 years (DF, n = 386; DHF, n = 416; DSS, n = 75). Genotyping was performed by TaqMan real-time PCR assay. RESULTS: It was found that the rs7247086 variant of IFNL1 was associated with DHF, but not DSS. Genotypes CT and TT and the T allele were protective against DHF (p = 0.03, odds ratio 0.62 for CT, odds ratio 0.13 for TT; and p = 0.01, odds ratio 0.54 for the T allele). The other SNP tested was not associated with DHF or DSS. CONCLUSIONS: The rs7247086 variant of IFNL1 (the T allele) was found to be protective against DHF, suggesting that IFNL1 may play a role in the pathogenesis of DHF.

Immunodominance of LipL3293-272 peptides revealed by leptospirosis sera and therapeutic monoclonal antibodies.

BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.

ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand.

BACKGROUND: Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. METHODS: To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 µg/ml, and anti-human IgG diluted at 1:4000. RESULTS: The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11). CONCLUSIONS: Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis.

MULTIPLE-LOCUS VARIABLE-NUMBER TANDEM REPEAT ANALYSIS OF BRUCELLA ISOLATES FROM THAILAND.

Brucellosis-induced abortion can result in significant economic loss to farm animals. Brucellosis can be transmitted to humans during slaughter of infected animals or via consumption of contaminated food products. Strain identification of Brucella isolates can reveal the route of transmission. Brucella strains were isolated from vaginal swabs of farm animal, cow milk and from human blood cultures. Multiplex PCR was used to identify Brucella species, and owing to high DNA homology among Brucella isolates, multiple-locus variable-number tandem repeat analysis (MLVA) based on the number of tandem repeats at 16 different genomic loci was used for strain identification. Multiplex PCR categorized the isolates into B. abortus (n = 7), B. melitensis (n = 37), B. suis (n = 3), and 5 of unknown Brucella spp. MLVA-16 clustering analysis differentiated the strains into various genotypes, with Brucella isolates from the same geographic region being closely related, and revealed that the Thai isolates were phylogenetically distinct from those in other countries, including within the Southeast Asian region. Thus, MLVA-16 typing has utility in epidemiological studies.

Third-stage Gnathostoma spinigerum larva excretory secretory antigens modulate function of Fc gamma receptor I-mediated monocytes in peripheral blood mononuclear cell culture.

BACKGROUND: Third (infective)-stage Gnathostoma spinigerum larvae (L3) mainly cause human gnathostomiasis. G. spinigerum L3 migrate throughout the subcutaneous tissues, vital organs, and central nervous system and can cause various pathogenesis including sudden death. Interestingly, G. spinigerum L3 can survive and evade host cellular immunity for months or years. The effects of G. spinigerum excretory-secretory (ES) products involved in larval migration and immune-evasive strategies are unknown. Monocytes are innate immune cells that act as phagocytic and antigen-presenting cells and also play roles against helminthic infections via a complex interplay between other immune cells. Fc gamma receptor I (FcγRI) is a high-affinity receptor that is particularly expressed on monocytes, macrophages, and dendritic cells. The cross-linking of FcγRI and antigen-antibody complex initiates signal transduction cascades in phagocytosis, cytokine production, and antibody-dependent cell-mediated cytotoxicity (ADCC). This study investigated whether ES antigen (ESA) from G. spinigerum L3 affects monocyte functions. RESULTS: Cultures of normal peripheral blood mononuclear cells (PBMC) separated from healthy buffy coats were used as a human immune cell model. ESA was prepared from G. spinigerum L3 culture. Using Real-Time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the effect of ESA to down-regulate FcγRI mRNA expression in monocytes during 90 min of observation was not well delineated. Flow cytometry analysis revealed a significant phenotypic-decreased FcγRI expression on the monocyte surface at 12 hours (h) of cultivation with the ESA (p = 0.033). Significantly reduced monocyte-mediated phagocytosis capacity was consistently observed after 12 h of ESA pretreatment (p = 0.001). CONCLUSIONS: Our results suggest that G. spinigerum ESA modulates monocyte function via depletion of FcγRI expression. This study provides preliminary information for future in-depth studies to elucidate mechanisms of the immune-evasive strategy of G. spinigerum larvae.

Cloning and expression of a 16-kDa recombinant protein from Angiostrongylus cantonensis for use in immunoblot diagnosis of human angiostrongyliasis.

Angistrongylus cantonensis is a zoonotic nematode parasite and causative agent of human angiostrongyliasis, which clinically presents as eosinophilic meningitis or meningoencephalitis. Diagnosis of the disease is problematic since parasitologic findings are infrequent, and infection determinations must be based on the clinical symptoms and serological tests with limited specificities and sensitivities. The aim of the present study was to identify and generate a novel recombinant protein from A. cantonensis and evaluate its efficacy in the diagnosis of human angiostrongyliasis when incorporated into a Western blot serodiagnostic system. A cDNA protein expression library from adult A. cantonensis was constructed, followed by immunoscreening with serum from confirmed infected patients to identify and isolate immunoreactive clones. One clone, designated fAC40, possessed a partial sequence encoding a LisH protein domain with a predicted molecular weight of 16 kDa and containing four predicted antigenic peptides. By incorporating recombinant fAC40 in Western immunoblot tests using a serum panel consisting of confirmed and clinically diagnosed cases of human angiostrongyliasis and other helminthic infections, fAC40 exhibited a sensitivity and specificity of 91.8 and 100 %, respectively, and a positive and negative predictive value of 100 and 97.19 %, respectively, in the diagnosis of angiostrongyliasis. Importantly, it was not reactive with antibodies from serum of patients infected with Gnathostoma spinigerum and Cysticercus cellulosae, infections that clinically present neurological symptoms similar to angiostrongyliasis. These data demonstrate that the 16-kDa recombinant protein from A. cantonensis possesses high potential as a candidate antigen for a more sensitive and specific serodiagnosis of human angiostrongyliasis.

The Immunoreactive Exo-1,3-ß-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.

Molecular genotyping of human papillomavirus l1 gene in low-risk and high-risk populations in Bangkok.

BACKGROUND: Human papillomavirus (HPV) infections in Thailand are a public health concern, but information on HPV infection in sex workers and men who have sex with men (MSM) is limited. The aim of this study was to measure the prevalence and genotype distribution of HPV among low- and high-risk, HIV-negative populations. METHODS: A total of 300 participants were categorized as general women, female sex workers, MSM, and MSM sex workers. Human papillomavirus infections were identified by the Papanicolaou test and nested polymerase chain reaction. A phylogenetic analysis of partial HPV L1 genes was performed. RESULTS: Abnormal cytology was found in 5% of general women, 10% of female sex workers, 24% of MSM, and 28% of MSM sex workers. Human papillomavirus was detected in 9% of general women, 13% of female sex workers, and 30% in both MSM and the MSM sex workers. The prevalence of HPV high-risk genotypes was significantly higher in female sex workers and MSM, whereas low-risk genotypes and genital warts were significantly higher in MSM sex workers. Significantly more patients with genital warts and cervical intraepithelial neoplasia I/anal intraepithelial neoplasia I harbored low-risk genotypes, whereas those with cervical intraepithelial neoplasia II/anal intraepithelial neoplasia II harbored high-risk genotypes. CONCLUSIONS: High- and low-risk HPV genotypes persist in high-risk groups in Bangkok. Some genotypes infecting at-risk populations are not vaccine preventable. These findings may help to elucidate the prevalence of HPV infections in Thailand and serve as the basis for additional investigations into risk factors for these populations.

ELISA for brucellosis detection based on three Brucella recombinant proteins.

Control of brucellosis among farm animals, wildlife and humans require reliable diagnosis. Rose Bengal serological test (RBT) is based on lipopolysaccharide antigen of Brucella, which may cross react with other gram-negative bacteria and produce false positive result. Immunoreactive proteins, such as outer-membrane protein BP26, ribosome recycling factor protein CP24 and Brucella lumazine synthase (BLS), previously reported to be recognized by infected sheep sera, were selected for production of recombinant proteins for use in an ELISA in order to investigate immune response among goats and cows, in comparison with commercial RBT. Cut-off value for ELISA was based on the immune response of in vitro fertilized goats and cows. Goats positive for Brucella culture or by RBT were ELISA positive for either IgG or IgM against at least one recombinant protein. For animals with negative RBT, animals with positive ELISA could be detected, and 61.6% possessed ELISA values as high as in infected animals. Thus, this ELISA procedure is proposed as an alternative to RBT for screening of brucellosis in farm animals.

Cloning and application of recombinant dengue virus prM-M protein for serodiagnosis of dengue virus infection.

We studied the use of the precursor to the M structural protein (prM) found only on the surface of mature dengue virus as a target protein to detect dengue virus infection. Recombinant D2-16681 prM-M protein was constructed and tested for immunogenicity with dengue and Japanese encephalitis patient sera by Western blot analysis and indirect ELISA. The sensitivity and specificity of indirect ELISA were 48.1 and 85.5%, respectively, and Western blot assay were 23.1 and 98.7%, respectively, for detection of dengue virus. Although the sensitivity of the indirect ELISA is low, the indirect ELISA using recombinant D2-16681 prM-M proteins as antigen may be used for early detection of dengue virus infection.

Genetic speciation of environmental Legionella isolates in Thailand.

Legionella-like organisms were isolated during 2003-2007 from various water resources by culturing on selective media of Wadowsky-Yee-Okuda agar. The 256 isolates were identified as belonging to the Legionella genus based on detection of 108 bp PCR product of the 5S rRNA gene, while the inclusion as Legionella pneumophila were confirmed by PCR detection of a specific mip gene region of 168 bp. The 50 isolates, identified as non-pneumophila, were then subjected to DNA tree analysis, based on mip gene of ~650 bp and rnpB genes product ranged from 304 to 354 bp. Phylogenetic tree was constructed to predict their species in relative to the available database. The isolates of which their speciation, based on those two genes were inconclusive, were then investigated for the almost full-length of 16S rRNA sequences. The isolates were assigned as 16 known Legionella species, and proposed seven novel species based on their unique 16S rRNA sequence.

Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand.

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.

Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein.

The outer membrane protein LipL21, LipL32, LipL41 and Loa22 of Leptospira interrogans serovar Copenhageni were previously revealed by immunoproteomic analysis, using sera from acute phase infection in a guinea pig. The full-length DNA of each protein was then cloned from the same serovar and expressed in pRSET vector. The obtained molecular weight (MW) of recombinant proteins rLipL21, rLipL32 and rLoa22 were slightly higher than the MW predicted from nucleotide sequences of each inserted gene, while only the N-terminal half of rLipL41 was obtained. Mice antiserum raised against each purified recombinant protein could react with the whole cell lysate of leptospiral serovars, implying that leptospiral native proteins shared a common epitope with recombinant protein. Serodiagnosis using recombinant protein antigen based on indirect ELISA procedure was developed in this study. The optimization of the ELISA components lead to determination of optical density (OD) from a single serum-dilution of 1:1000 in the leptospirosis patients group and normal healthy control group. The cut off OD values for both IgG and IgM class were investigated, and based on this fixed dilution only the IgG class could be used for differential diagnosis of patients and normal individuals. Compared with the MAT assay, ELISA assay utilizing both rLipL32 and rLoa22 as antigen, gave high accuracy and could thus be useful as a confirmative serology test.

Roles of partially purified antigens from Gnathostoma spinigerum larvae on antibody production by human B cell culture.

A 24 kDa protein from advanced third stage Gnathostoma spinigerum larvae (GsAL3) is used for gnathostomiasis serodiagnosis. This study investigated whether partially purified protein antigen (Ag) from GsAL3 (Gnath Ag), prepared by simple gel filtration chromatography, could be used for serodiagnosis. Using DNA microarray analysis, significant gene expression related to immunoreactivity was examined in peripheral blood mononuclear cells (PBMC) cocultured with Gnath Ag. Antigenicity was then determined by its capacity to induce antibody production among purified naive B cells stimulated with Gnath Ag and anti-CD40. Seven and 14 days post-exposure, immunoglobulin levels (Igs) in culture supernatants were determined by enzyme-linked immunosorbent assay. The Gnath Ag stimulated PBMC had a significant increase in gene expression related to an innate immune response and decreased cell mediated immunity, but the expression of gene related antibody production was not markedly increased. The Gnath Ag stimulated naive B cells or lipopolysaccharide primed B cells to produce low levels of specific antibody. Our findings support the assertion that partially purified Gnath Ag possess low antigenicity for Ig induction. Further studies are needed to improve G. spinigerum larva Ag for serodiagnosis.

Application of recombinant SMR-domain containing protein of angiostrongylus cantonensis in immunoblot diagnosis of human angiostrongyliasis.

The aim of this study was to find novel proteins expressed from an Angiostrongylus cantonensis adult female worm cDNA library for serodiagnosis of angiostrongyliasis. An immuno-dominant clone, fAC22, was identified by immunoscreening with pooled positive sera from proven angiostrongyliasis patients. The clone contained an open reading frame of 2,136 bp encoding a 80.5 kDa protein with a predicted isoelectric point of 5.8. The deduced amino acid sequence (712 amino acids) contained the conserved domain of Small mutS related (Smr) superfamily protein, with similarity with the Smr domain protein of Brugia malayi. The fusion His-tagged 81 kDa recombinant protein expressed as inclusion body in Escherichia coli was solubilized and purified by Ni-affinity chromatography for use in immunoblot analysis. Its sensitivity, specificity, positive and negative predictive values in immunodiagnostic test was 93.5, 91.5, 79.0 and 97.5%, respectively. Although some cross-reactivity of the antigen was observed among gnathostomiasis, bancroftian filariasis, ascariasis, echinococcosis, paragonimiasis and opisthorchiasis, sera from 14 other infections were all negative. These data indicate its possible application in immunodiagnosis of clinically suspected angiostrongyliasis. Key words: Angiostrongylus cantonensis,eosinophilic meningitis, recombinant fusion protein, immunodiagnosis

Iso-electricfocusing of Bithynia snail antigens for IgG- and IgG(1-4)-ELISA detection of human opisthorchiasis.

Diagnosis of opisthorchiasis is confirmed by the presence of characteristic eggs and worms. However, misdiagnosis may occur in light infections, and also due to the morphological similarity of opisthorchid eggs to other species. A finding of specific immune mediators can help confirm infection. This study used indirect ELISA to detect total IgG and IgG(1-4) with selected antigens of Bithynia siamensis goniomphalos extract, which were derived by liquid-phase iso-electricfocusing (IFE). Antigens (Iso-AgF) from 20 IEF fractionated fractions were selected based on a high ELISA-OD ratio between pooled-positive and pooled-negative sera. Iso-AgF 7, 7, 6, 2, and 10 resulted in high OD-ratio to total IgG, IgG1, 2, 3, and 4, respectively. A full-scale ELISA was conducted with sera from 50 opisthorchiasis cases, 196 from other parasitic-disease cases, and 35 healthy controls. Iso-AgF7 to IgG1 showed the best result, with sensitivity, specificity, positive and negative predictive value of 100, 96, 86, and 100%, respectively, at a cut-off 0.221. Low cross-reactivity to IgG1 was found in one case each of gnathostomiasis, trichinellosis, toxocariasis, angiostrongyliasis, bancroftian filariasis, enterobiasis, neurocysticercosis, and taeniasis. Thus, Iso-AgF7 to IgG1 was a good candidate antigen to be developed for detection of antibodies against Opisthorchis viverrini.