Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

Background & objectives: El Tor Vibrio cholerae O1 carrying ctxBC trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. Methods: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpAC, tcpAE, hlyAC and hlyAE were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. Results: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. Interpretation & conclusions: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxBC).

Affinity purified oval antigen for diagnosis of Opisthorchiasis viverrini.

Monoclonal antibodies (MAb) were raised against an oval antigen of the liver fluke Opisthorchis viverrini which is the causative agent of a parasitosis, i.e. opisthorchiasis in Thailand. The antibodies were used in an affinity column to purify the O. viverrini oval antigen from a crude extract of adult parasites by chromatography. The oval antigen was then used in a membrane (dot) ELISA for detecting antibodies in serum samples of parasitologically confirmed Opisthorchis viverrini infected individuals (adult parasites were found in stools after praziquantel treatment and salt purgation), as well as of individuals infected with other parasites and parasite-free controls. The MAb-based dot-ELISA using the affinity purified O. viverrini oval antigen revealed 100% sensitivity, specificity and accuracy for detecting O. viverrini infection. The test is simple, rapid and highly reproducible. Several samples can be tested at the same time without the requirement for special equipment or much increase in testing time; thus it is suitable for mass screening for O. viverrini exposure, especially in new endemic areas. Furthermore using serum specimens could increase patient and community compliance compared to the conventional parasitological survey which uses stool samples for the detection of O. viverrini ova, without treatment and subsequent salt purgation, this conventional method shows a low sensitivity and is also unpleasant to both the sample donors and the laboratory technicians which has historically shown a further negative impact on the final outcome.

Validation of salmonellosis and shigellosis diagnostic test kits at a provincial hospital in Thailand.

Rapid Diagnosis of salmonellosis and shigellosis was performed using six different diagnostic test kits which recently have been made available commercially. They were Salmo-Dot, Typhi-Dot, Shigel Dot A, B, C, and D test kits for detection of Salmonella spp., group D salmonellae, and groups A, B, C, and D Shigella spp., respectively. The principle of all test kits is a membrane (dot) ELISA using specific monoclonal antibodies to the respective pathogens as the detection reagents. The present study was designed to validate the accuracy of the test kits, at a laboratory in a provincial hospital in Thailand, in comparison with the conventional bacterial culture method alone or with the combined results of the culture and the Western blot analysis (WB) for detecting the respective bacterial lipopolysacchharides (LPS) in specimens. Five hundred rectal swab samples of patients with diarrhea who seeked treatment at the hospital, were evaluated. The diagnostic accuracy of the Salmo-Dot was 91.0% when compared with the conventional bacterial culture method alone but was 100.0% in comparison with the combined results of the culture and the WB. The Typhi-Dot and the Shigel-Dot A, B, C, and D showed 100%, 99.2%, 95.0%, 94.0% and 96.4%, respectively when compared with the culture alone and all were 100% in comparison with the combination of the results of the bacterial culture and the WB. The Shigel-Dot A revealed antigen of type 1 Shigella dysenteriae in several specimens in which the bacteria could not be recovered by the culture method. This difference is important as type 1 Shigella dysenteriae have high epidemic potential and often cause severe morbidity. Unawareness of their presence by the conventional culture may have great impact on disease surveillance for public health. The pathogen detection using the six diagnostic test kits is sensitive, specific, rapid, and relatively simple and less expensive. Several specimens can be tested at the same time without much increase in turn around time. Moreover, these kits produce no contaminated waste as compared with the bacterial culture method. The test kits should be used for rapid screening of specimens of patients with diarrhea especially in areas where culture facilities are inadequate.

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

Detection of Salmonella contamination in food samples by dot-ELISA, DNA amplification and bacterial culture.

A dot-blot enzyme-linked immunosorbent assay (dot-ELISA) employing a genus Salmonella specific monoclonal antibody (MAb) was used for detection of the bacteria in food samples in comparison with the conventional culture method and the DNA amplification. Among the 200 chicken and pork samples (100 each) tested, 9% and 33%, 7% and 20% and 7 and 23% were positive for salmonellae by the dot-ELISA, the culture method and the DNA amplification, respectively. Statistical analyses revealed that the sensitivity, specificity, efficacy, and positive and negative predictive values of the detection of Salmonella in the food samples by dot-ELISA compared with the culture method were 93.33%, 91.76%, 92%, 66.66% and 98.73%, respectively. Comparison of the DNA amplification and the culture method revealed the sensitivity, specificity, efficacy, and positive and negative predictive values of 100%, 91.58%, 92%, 65.21% and 100%, respectively. The dot-ELISA and the DNA amplification results were in a better agreement when the two assays were compared. The sensitivity, specificity, efficacy, positive and negative predictive values of the dot-ELISA compared to the DNA amplification were 91.3%, 100%, 98%, 100% and 97.5%, respectively. From this study, the dot-ELISA is rapid, simple, sensitive, specific at low cost with limited amount of infectious waste to be disposed and offers another advantage in that it detects only the smooth LPS of Salmonella which implies the possible presence of the virulent organisms.

Diagnosis of enteric fever caused by Salmonella spp. in Vietnam by a monoclonal antibody-based dot-blot ELISA.

Enteric fever caused by Salmonella spp. is prevalent in Vietnam. None of the currently available diagnostic methods meets the ideal criteria on rapidity, simplicity, sensitivity, specificity, cost-effectiveness and practicality for developing areas. In this study, a recently developed monoclonal antibody-based dot-blot ELISA was used in comparison with the hemoculture method and the classical Widal test for diagnosis of salmonellosis in 171 Vietnamese patients presenting with clinical features of enteric fever. Urine samples of 50 healthy counterparts were used as negative controls. Salmonella spp. were isolated from 77 of 171 patients (45%) while 98 and 111 patients were positive by dot-blot ELISA and Widal test, respectively. The diagnostic sensitivity, specificity, accuracy, positive predictive value and negative predictive value of the ELISA performed on three serial urine samples collected at 2 hour intervals of the 171 patients were 92.2%, 71.3%, 80.7%, 72.4% and 91.8%, respectively when compared with the culture method. The Widal test performed on acute and convalescence serum samples showed 87.0%, 46.8%, 68.4%, 60.4% and 83.3% diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values, respectively when compared with the bacterial culture method. Kappa coefficience revealed very good agreement beyond chance between the MAb-based ELISA and the culture method. The ELISA was not reactive when tested on urine samples of 50 healthy individuals which indicates 100% specificity. The Salmonella antigenuria of the patients as detected by ELISA lasted 10.3+/-3.9 days after initiating antibiotic treatment. The MAb-based dot-blot ELISA is easy to perform. It is rapid, sensitive, specific, inexpensive, and non-invasive and does not require equipment, thus is suitable for developing areas. It can detect acute/recent infection and can be used for evaluation of the efficacy of the treatment.

A dot-ELISA test using monoclonal antibody-purified antigens for the diagnosis of paragonimiasis caused by Paragonimus heterotremus.

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody-affinity chromatography was developed for detecting antibodies to Paragonimus heterotremus in four groups of subjects. They consisted of 30 patients with P. heterotremus infection, 93 patients with other parasitic infections, 18 patients with pulmonary tuberculosis and 30 normal, healthy controls. Sensitivity, specificity, as well as positive and negative predictive values of the test were 100, 97, 88, and 100%, respectively.

Parasites elicited cross-reacting antibodies to Opisthorchis viverrini.

Two batches of crude antigens extracted from adult Opisthorchis viverrini worms were compared. One was derived from adult worms harvested from the livers of laboratory infected hamsters and another was obtained from worms sedimented from the faeces of opisthorchiasis patients following treatment with Praziquantel. SDS-PAGE and Coomassie brilliant blue staining revealed that the two preparations had similar protein components of which the predominant ones were the 17-18 kDa doublet. The antigens were used in an indirect ELISA for the detection of antibodies against O. viverrini in the sera of four groups of patients, ie. patients with opisthorchiasis (group 1), patients with mixed infections of O. viverrini and other parasites (group 2), patients with other parasitic infections (group 3), and normal-heathy, parasite-free individuals (group 4). The sensitivity of the test was high (91-92%), regardless of the batch of the antigen used. However, its specificity was relatively low (70-80%). Cross-reaction was observed with patients infected with Paragonimus heterotremus, Schistosoma spp.; Taenia spp.; Trichinella spiralis; Strongyloides stercoralis; hookworms; Plasmodium spp.; hookworms and Plasmodium spp.; S. stercoralis, Blastocystis hominis and yeast; and hookworms, Ascaris lumbricoides, Trichuris trichiura and P. falciparum. Western blot analysis revealed that sera of patients infected with these heterologous organisms contained antibodies reactive to O. viverrini antigenic components ranging from Mr 15.5 to 144.

Cow milk protein allergy during the first year of life: a 12 year experience at the children's hospital, Bangkok.

CMA should be suspected for patients aged less than one year who had persistent diarrhea and/or hematemesis with no enteric pathogen found. Confirmed diagnosis could be made by Goldman challenge test. Patients with confirmed CMA should be treated by changing the cow milk feeding to soy milk feeding. However, in our study, 17% of CMA patients were also allergic to soy protein. Thus the soy milk was replaced by the elemental formula for successful treatment of this group of patients. Beside persistent diarrhea, hematemesis, anemia and hypoalbuminemia were other possible findings among patients with CMA with or without soy protein allergy.

Monoclonal antibody-based dot-blot ELISA for the detection of Salmonella in foods.

Monoclonal antibody (MAb) produced to polysaccharides in the LPS molecule of salmonellae was used in a dot-blot ELISA for detecting Salmonella in 873 food samples, ie 100 fresh chicken, 261 frozen chicken, 78 pork, 84 beef, 100 hen eggs, 100 duck eggs, 50 sea-mussels, 50 shrimps and 50 squids in comparison with the conventional culture method. Salmonella culture from foods involved the following steps: pre-enrichment, enrichment in selective medium, isolation on selective and indicator media, followed by biochemical and serological identification of appropriate colonies, respectively. The whole culture procedure took 5 days. Food samples from the selective enrichment medium were also subjected to the MAb-based dot-blot ELISA. The whole procedure of dot-blot ELISA took less than 2 hours. Among 873 food samples, salmonellae could be recovered from 7.4% of the samples by the bacterial isolation method (16% of fresh chicken, 8.8% of frozen chicken, 24.4% of pork, 3.6% of beef and 2% each of hen eggs and duck eggs, respectively). Salmonella derby were predominant among pork samples while S.paratyphi B biovar java predominated in chicken. The MAb-based dot-blot ELISA were positive in 19.5% of the food samples, i.e. 30% of fresh chicken, 27.6% of frozen chicken, 34.6% of pork, 21.4% of beef, 20% of shrimp, 16% of sea-mussels, 2% of hen eggs and 4% of duck eggs. The sensitivity and specificity of the MAb-based dot-blot ELISA compared to the bacterial culture method were 81.5% and 85%, respectively. The discrepancy of the data between the culture method and the dot-blot ELISA might be due to the fact that the culture method could detect only living cells at numbers that gave at least one isolated colony on the selective/differential plate while the dot-blot ELISA detects any form of Salmonella antigen. The monoclonal antibody-based dot-blot ELISA offers several advantages over the conventional bacterial culture method when it is used for screening of Salmonella contamination in foods, especially export foods. These include rapidity, cost-effectiveness and simplicity (the dot-blot ELISA does not need highly trained personnel or equipment, in contrast to the culture method). The test can be performed in field conditions and the result can be read visually. It also offers multisample analysis at one time which renders more samples of food for screening possible, thus false negative results are fewer which, in turn, assures the quality of the export food in a cost-saving, short time frame.

A monoclonal antibody-based dot-blot ELISA diagnostic kit for the detection of Vibrio cholerae 01 in stools of diarrheic patients and household contacts.

A "cholera diagnostic kit" was developed for sensitive, specific, rapid, and inexpensive detection of Vibrio cholerae 01. The monoclonal antibody specific to antigen A of Vibrio cholerae 01 was used as an antigen detection reagent and the principle of dot-blot ELISA was adopted. The kits were used in seven Regional Medical Sciences Centres, Ministry of Public Health, located at various regions of Thailand where diarrhea occurs frequently. Diagnostic efficiency of the kits in the detection of Vibrio cholerae 01 from rectal swabs of the diarrheic patients and their household contacts was evaluated in comparison with the conventional culture method. The two methods were found to have excellent degree of agreement (kappa values > 95%). The dot-blot ELISA has several advantages over the culture methods, ie rapid (dot-blot ELISA takes 1-2 hours while the culture method takes at least two days) and inexpensive. It requires no sophisticated equipment. The procedure is not complicated thus it is easy to train personnel. The diagnostic kits are recommended for use in the detection of severe diarrhea caused by V. cholerae 01 not only in hospitals and health centres where adequate treatment of the patients is required as a life-saving measure but also for early recognition of cholera cases and their contacts so that other action, ie prevention and control of outbreaks and surveillance can be promptly implemented.

Immunodiagnosis of trichinellosis: efficacy of somatic antigen in early detection of human trichinellosis.

Crude antigens prepared from the infective stage larvae of Trichinella spiralis were used for antibody detection by indirect ELISA and Western blotting in serum samples taken from trichinellosis patients and from normal, parasite-free controls. The serum specimens were collected from acute ill, symptomatic patients on the first day of treatment (Day 0), and then two months (M2) and 4 months (M4) later. The sensitivities of the indirect ELISA and Western blotting on Day 0 were 81% and 92%, respectively. Both tests were 100% sensitive for M2 and M4 serum samples. Every serum sample from the parasite-free controls tested negative by both immunological assays, indicating 100% specificity. Crude somatic antigens can therefore be used for the early detection of human trichinellosis (acute trichinellosis).

Partially purified antigens of Paragonimus heterotremus for serodiagnosis of human paragonimiasis.

The preparative crude extract of Paragonimus heterotremus was fractionated by isoelectric focusing. Fractions at pH 5 which contained a specific antigen with a relative molecular weight of 31.5 kDa were pooled and used in an indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis for diagnosis of human paragonimiasis. The sensitivity and specificity of ELISA were found to be 100% and 99% respectively. The band of 31.5 kDa antigenic component was found to give consistent reaction with paragonimiasis sera. The sensitivity, specificity and predictive value (positive and negative) of immunoblot analysis for the 31.5 kDa band were all 100%.

Localization of Vibrio cholerae O1 in the intestinal tissue.

Colonization of V. cholerae O1 in vivo is known to be a non-invasive type which the vibrios are confined only to the intestinal tissues. The pathway by which the vibrio antigens reach the lymphoid cells and subsequently give rise to the immune responses is not entirely clear. Thus, experiments were performed in experimental rats by inoculating live V. cholerae O1 into the ligated ileal loops. The fate of the vibrios in the intestinal tissues was then studied by transmission electron microscopy at different times after the inoculation. It was concluded that live V. cholerae O1 were initially taken up by the M cells which overlay Peyer's patches and which subsequently delivered the intact vibrios to phagocytic cells in the Peyer's patches. These phagocytic cells processed (digested) the vibrios while the lymphocytes and plasma cells infiltrated around them. During the late period of infection (12-15 hours after inoculation of the vibrios), vibrios were also found passing through the loose intercellular spaces between the absorptive epithelial cells into the underlying intestinal tissues.

Comparison of the protective efficacy on mekongi schistosomiasis in mice induced by antigens derived from cercariae, schistosomulae and adult worms.

Protective efficacy of the extracts of cercariae, schistosomulae and adult worms of S. mekongi was studied in mice receiving immunizations with these extracts emulsified with Freund's complete adjuvant initially and incomplete adjuvant subsequently, and compared with mice receiving physiological saline with or without adjuvants as controls. After challenge with cercariae, the animals were sacrificed and the larvae or adult worms harvested by lung recovery and perfusion techniques on day 5 and weeks 6-8, respectively. Worm reduction rates were significantly higher in mice receiving extracts of schistosomula (59%) and adult worms (51%) than in those receiving the cercarial extracts (31%). Similar findings were obtained with the perfusion technique showing worm reduction rates of 57%, 53% and 30% in mice receiving extracts of schistosomulae, adult worms and cercariae, respectively. ELISA antibody titers were correspondingly increased in mice receiving extracts of schistosomulae and adult worms, but not in those receiving cercariae. This apparent association may be inadequate to suggest that the increase in ELISA titer be used as an indicator for resistance in mekongi schistosomiasis.

Immunological studies on some bacterial and parasitic diseases in Thailand.

Over the past two decades a wide range of immunological studies have been carried out on organisms responsible for causing serious bacterial and parasitic diseases in Thailand. Particular emphasis has been placed on cholera, where investigations have been directed towards vaccine development and rapid diagnosis and on diagnosis of typhoid and enterotoxigenic Escherichia coli. In the area of parasitic diseases emphasis has been directed especially to immunodiagnosis of helminthic diseases, including gnathostomiasis, paragonimiasis, opisthorchiasis, strongyloidiasis and trichinellosis. These studies are reviewed in this report.

Enzyme-linked immunosorbent assay for diagnosis of human strongyloidiasis.

Crude antigen (CA) was prepared from Strongyloides stercoralis filariform larvae obtained from in vitro culture of the human feces containing rhabditiform larvae. The lyophilized filariform larvae were ground and ultrasonicated in distilled water then the soluble antigenic preparation was delipidized. The protein content of the crude soluble antigen was 20% of the original dried larvae. The CA was passed through a gel filtration chromatography column and yielded three different protein fractions namely F1, F2 and F3. CA and its fractions were used in the indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to S. stercoralis in serum samples of 5 groups of individuals. These were patients with parasitologically confirmed strongyloidiasis (group 1), patients with mixed S. stercoralis and other parasitic infections (group 2), non-strongyloidiasis patients with other worm infestation(s) (group 3), normal parasite-free Thais (group 4) and normal parasite-free Swedes (group 5). It was found that F2 was the best antigen in the ELISA. The sensitivity, specificity and positive and negative predictive values of the test using F2 as the antigen were 95.0%, 96.4%, 95.0% and 96.4%, respectively.

Immunogenicity of liposome-associated oral cholera vaccine prepared from combined Vibrio cholerae antigens.

Liposomes were prepared from bovine brain sphingomyelin and cholesterol. They were reinforced by incorporation of osmium tetroxide to prevent their immediate degradation inside the host. Combined Vibrio cholerae antigens (lipopolysaccharide, crude cell-bound hemagglutinin and procholeragenoid) were orally administered to experimental rats either as free or liposome-associated. A total of 70 experimental rats was utilized in experiments comparing the immune responses of rats to liposome-associated vaccine, free vaccine, liposomes, or placebo, and to vaccines where the lipid or antigen levels were reduced. Immediately after feeding with sodium bicarbonate to lower the gastric acidity, they were fed either cholera vaccines or placebo. Results from serum ELISA revealed that the liposomes localized the immune response to the intestinal mucosa. They displayed an adjuvant property in terms of evoking a higher immune response to V. cholerae antigens, as measured by the appearance of specific antibody-producing cells in the intestinal mucosa, than when the antigens were fed alone. The adjuvanticity was found to be lipid dose dependent. Liposomes prepared with high lipid content enhanced immunogenicity of the admixture antigens to a greater degree.