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.

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.

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.