Comparative identification of protein profiles and major allergens of saliva, salivary gland and whole body extracts of mosquito species in Thailand.

Allergic reactions to mosquito bites, such as generalized urticaria or severe local reactions are common problems worldwide. The diverse sources of allergen prepared from different mosquito body parts usage are a major obstacle to obtaining safe and effective tests and immunotherapy for mosquito bite allergy. Thus, the reactions are often not recognized and allergen immunotherapy is seldom used for severe reaction to mosquito bites. In a search for appropriate allergen sources, the protein profiles of saliva, salivary glands and whole body extracts were comparatively analyzed from 4 common mosquito species of Thailand and/or South East Asia; viz. Culex quinquefasciatus, Aedes aegypti, Aedes albopictus and a zoophilic strain, Anopheles minimus. The major allergens in the extracts which elicited specific IgE responses in the pooled sera of subjects allergic to mosquito bites were identified. It was concluded that mosquito saliva was the best source of allergens. Additionally, both species-specific and species-shared allergens of the 4 mosquito species were identified. The major saliva allergens having MWs of 36, 32 and 22 kDa were identified. The identificstion of major allergens should facilitate the production of specific recombinant allergens and contribute to improvement in the diagnosis and specific immunotherapy of Thai mosquito bite allergy patients.

Determination of fecal occult blood in primary schoolchildren infected with Trichuris trichiura.

A correlation of Trichuris trichiura infection and fecal occult blood detection was conducted in 146 primary schoolchildren in Narathiwat Province, Thailand. The Kato-Katz thick smear method was used for determining egg counts and stated as eggs per gram of feces (epg). The number of T. trichiura eggs was categorized as class I (1-499 epg), class 11 (500-4,999 epg), and class III (> 5,000 epg), according to the relation between infection intensity and reduced hemoglobin concentration. Each fecal sample was processed to detect occult blood using a guaiac-based test (Hema-Screen, USA) and an immunochromatographic-based test (HEXAGON OBTI test, Germany). There were 50 schoolchildren without parasitic infection in the control group. Of 96 cases with T. trichiura infection, 85 and 11 children were classified in the class I and class II groups, respectively, but no subjects were in the class III group. Positive occult blood detection results in the control, class I, and class II groups using the guaiac and the immunochemical tests were 0, 3.5, and 9.1% (p=0.19), and 0, 2.4, and 36.4%, (p<0.0001) respectively. This study suggests that T. trichiura infection with an intensity of 500 epg or greater may be associated with intestinal bleeding.

Impact of a filariasis control program on intestinal helminthic infections; a pilot study in Narathiwat Province, Thailand.

This study was conducted in 9 villages located in endemic areas for brugian filariasis in Narathiwat Province, Thailand. Parasitological and anthropometric examinations were cross-sectionally performed to assess the prevalence of intestinal parasitic infections of 539 villagers. Paired stool samples were collected before and after mass treatment for the filariasis control program in 150 participants in order to study the impact of the filariasis control program on intestinal helminthiasis. The results found that 50.3% of the villagers were infected with one or more types of intestinal parasites. Double and triple infections were found in 10.9% and 1.6% of infected individuals respectively. The prevalence of intestinal parasitic infections peaked in the 1-10 year old age-group, which are pre-school and young school-age children. A significant reduction of intestinal helminthic infections in the post-treatment stool sample was observed in the 150 participants who were examined six months after mass treatment. Integrating an intestinal helminthic control program alongside the existing filariasis control program would be an appropriate and cost-effective strategy in the control of intestinal helminths. However, reinfection of parasites was observed.

An antigen detection assay for diagnosing filariasis.

In this study we examined the diagnostic potential of monoclonal antibodies (MAb) reactive to antigens of adult Brugia malayi, their microfilariae and antigen of Dirofilaria immitis. The MAb of clone 17E10, which were of IgM isotype, reacted to the inner cuticles and internal content of both male and female worms and also to the sheath and internal content of microfilariae in utero. However, these MAb did not react to the sheath of blood circulating microfilariae. The MAb 17E10 produced a smear pattern between 37 to > 200 kDa in the Western blot analysis against a SDS-PAGE separated extract of B. malayi. The epitopes were non-protein in nature as indicated by their resistance to proteinase-K treatment. The MAb 17E10 were applied in a sandwich ELISA to detect filarial antigen in the buffy coat and plasma of patients. We tested patients with different clinical manifestations of brugian filariasis, i.e. microfilaremia (M), lymphangitis (L) and elephantiasis (E), as well as non-symptomatic inhabitants of a filariasis endemic area (NE), and compared them to samples from non-symptomatic inhabitants of disease non-endemic areas (NNE). It was found that 22 of 31 (70.9%) of M, 7 of 13 (53.8%) of L, 2 of 14 (14.2%) of E, 10 of 100 (10.0%) of NE and none (0%) of the NNE were positive for antigenaemia. The assay was also positive in 14 of 15 (93.3%) blood samples from B. malayi microfilaremic cats and in 7 of 7 (100%) blood samples of Dirofilaria immitis microfilaremic dogs. The so-developed test has a high potential for routine diagnosis of active filariasis, for epidemiological studies in both humans and reservoir animals and for monitoring treatment efficacy.

A comparative study of the major allergenic components in house dust extracts between siriraj and commercially prepared extracts.

The quantity and concentration of major allergens in mite allergenic extracts are crucial for skin testing, which is the recommended standard method for the diagnosis of house dust mite allergic disease. The purposes of this study were 1) To compare the constituents of major mite allergens in 3 types of mite extracts, i.e., extracts from mite reared in-house (Dermatophagoides pteronyssinus and Dermatophagoides farinae), extracts from commercial mite products and commercial ready-made mite extracts. These in-house extracts were prepared either with or without the preservative, glycerine. The concentrations of the major constituents of group 1 and 2 allergens of the extracts were determined by a two-site monoclonal based ELISA. 2 ) Biological assays were also carried out to determine the relative potency of the extracts in 120 allergic patients by skin prick test. It was found that the mean concentrations of Der p1, Der f1 and group 2 mite allergens in extracts from mites reared in-house were 102, 195 and 94 ตg/ml, respectively, compared to 169, 238 and 91 ตg/ml, respectively in commercial mite extracts. The commercial with product had the lowest concentrations of mite allergens (1 ตg/ml). Comparison of mite extracts with and without glycerine preservative showed no significant difference in concentrations of major allergens. Reduction of allergens concentration from 10,000 to 1,000 PNU/ml also reduced the concentration of mite allergen proportionately. Siriraj mite extracts were stable for at least 1 year at 4 oC without any significant change in composition or concentration. In conclusion, mite reared in-house can be used as raw material for preparation of mite allergenic vaccine.