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.     

Reduced levels of transforming growth factor-beta1, interleukin-12 and increased migration inhibitory factor are associated with severe malaria

In the present study, we investigated plasma levels of interleukin (IL)-12 and transforming growth factor (TGF-beta1) in malaria patients as these two cytokines regulate the balance between pro- and anti-inflammatory cytokines. We compared plasma IL-12 and TGF-beta1 levels in groups of malaria patients categorized as uncomplicated, severe, cerebral and placental malaria. Both TGF-beta1 and IL-12 levels were significantly reduced in peripheral plasma of adults with severe and cerebral malaria as well as in plasma of Tanzanian children with cerebral malaria (P<0.05). Similar results were observed with both placental and peripheral plasma of pregnant women who were infected with Plasmodium falciparum. IL-18, a cytokine known to be critical for the induction of IFN-gamma along with IL-1, was produced more in uncomplicated adult patients than in aparasitimic healthy controls (P<0.05). However, IL-18 response rate declined as the symptoms of the disease became more severe suggesting that the IL-18 response may be impaired with increased malaria severity. Together, the results of the three cytokines support the notion that imbalance between pro- and anti-inflammatory cytokines may contribute to the development of severe malaria infection. With malaria infection during pregnancy, we demonstrated that macrophage migration inhibitory factor (MIF) levels in infected placental plasma were significantly higher than those in the paired peripheral plasma (P<0.05). MIF, therefore, may play an important role in the local immune response to placental P. falciparum infection.     

Preferences for treatment of diarrhoea and dysentery in Kaengkhoi district, Saraburi province, Thailand

To estimate the proportion of cases missed in a passive surveillance study of diarrhoea and dysentery at health centres and hospitals in Kaengkhoi district, Saraburi province, Thailand, a community-based cluster survey of treatment-seeking behaviours was conducted during 21-23 June 2002. Interviews were conducted at 224 households among a study population of 78,744. The respondents reported where they sought care for diarrhoea and dysentery in children aged less than five years and adults aged over 15 years. Health centres or hospitals were the first treatment choice for 78% of children with dysentery (95% confidence interval [CI] 63-94%), 64% of children with diarrhoea (95% CI 54-74%), 61% of adults with dysentery (95% CI 40-82%), and 35% of adults with diarrhoea (95% CI 17-54%). A high degree of heterogeneity in responses resulted in a relatively large design effect (D=3.9) and poor intra-cluster correlation (rho=0.3). The community survey suggests that passive surveillance estimates of disease incidence will need to be interpreted with caution, since this method will miss nearly a quarter of dysentery cases in children and nearly two-thirds of diarrhoea cases in adults.     

CpG DNA, liposome and refined antigen oral cholera vaccine

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.     

Risk factors for tuberculosis infection among household contacts in Bangkok, Thailand

A cross-sectional study was conducted to determine the prevalence of tuberculosis infection and risk factors for tuberculosis infection among household contacts aged less than 15 years in Bangkok, Thailand, between August 2002 and September 2003. During the study period, 342 index cases with sputum smear positive pulmonary tuberculosis patients were recruited into the study and their 500 household contacts aged under 15 years were identified. The prevalence of tuberculosis infection among household contacts was found to be 47.80% (95%CI = 43.41-52.19). In multivariate analysis, a generalized estimating equation (GEE) was used to determine the risk factors for tuberculosis infection among household contacts. The results indicated that the risk of tuberculosis infection was significantly associated with close contact (adjusted OR = 3.31, 95%CI = 1.46-7.45), exposure to female index case (adjusted OR = 2.75, 95%CI = 1.25-6.08), exposure to mother with tuberculosis (adjusted OR = 3.82, 95%CI = 1.44-10.14), exposure to father with tuberculosis (adjusted OR = 2.55, 95%CI = 1.19-5.46), exposure to index case with cavitation on chest radiograph (adjusted OR = 4.43, 95%CI = 2.43-8.05), exposure to index case with 3+ sputum smear grade (adjusted OR = 3.85, 95%CI = 1.92-7.70), and living in crowded household (adjusted OR = 2.63, 95%CI = 1.18-5.85). The distribution of tuberculosis infection and risk factors among contact cases are significant for health care staff in strengthening and implementing tuberculosis control programs in Thailand.     

Effect of norethisterone enanthate on endometrial bleeding and changes in coagulation function, ovarian hormones and endometrial histology in Thai women

Endometrial bleeding and alteration in blood coagulation, fibrinolysis, ovarian function and endometrial morphology were studied in twelve normally menstruating women who received the injectable contraceptive norethisterone enanthate (NET-EN) 200mg at 60-day intervals. Levels of clotting factor VII declined significantly during episodes of irregular bleeding compared to those during both normal pretreatment menstruation and the bleeding-free period during treatment. Antithrombin III and fibrinolytic activity, expressed by euglobulin lysis time, showed no marked change. The average level of progesterone during the bleeding-free period was slightly but significantly lower than that during the bleeding period. There were no significant alterations in the mean levels of estradiol and the ratio of estradiol to progesterone. The endometrial biopsies showed considerable individual variation and seem to be independent of the effects of NET-EN on ovarian function.     

A Study Failing to Determine Significant Benefits from Assisted Hatching: Patients Selected for Advanced Age, Zonal Thickness of Embryos, and Previous Failed Attempts

Purpose: Pregnancy and implantation rates after mechanical assisted hatching (AH) in patients aged 38 years, with embryos 15 m in zonal thickness and two or more failed attempts, were assessed at two infertility centers using fresh and frozen embryo transfer (FET) cycles. Methods: AH was performed on 3-day-old embryos. Spare embryos cryopreserved at the two-pronucleus stage were subjected to AH after 2 days of culture and transferred to artificially prepared uteri. Results: In fresh cycles, no significant differences in pregnancy rates (clinical and ongoing) and implantation rates were observed between the AH and the controls for all three selected patient groups (Centers 1 and 2). In FET cycles, AH tended to give poor results for 38 year olds (clinical pregnancy rates of 0 and 5.0% with AH vs 13.3 and 16.7% for controls at Centers 1 and 2, respectively). With AH, embryos with thick zonae implanted to the same extent as those in the control group and achieved pregnancies for patients with multiple failures (four to six attempts for some) in both fresh and FET cycles. Conclusions: AH failed to show significant benefits in all three patient groups. A larger study group may confirm the effects of AH on frozen/thawed embryos and outcomes for multiple failure cases.     

Periplaneta americana arginine kinase as a major cockroach allergen among Thai patients with major cockroach allergies

Periplaneta americana is the predominant cockroach (CR) species and a major source of indoor allergens in Thailand. Nevertheless, data on the nature and molecular characteristics of its allergenic components are rare. We conducted this study to identify and characterize the P. americana allergenic protein. A random heptapeptide phage display library and monoclonal antibody (MAb) specific to a the P. americana component previously shown to be an allergenic molecule were used to identify the MAb-bound mimotope and its phylogenic distribution. Two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, peptide mass fingerprinting, and BLAST search were used to identify the P. americana protein containing the MAb-specific epitope. We studied the allergenicity of the native protein using sera of CR-allergic Thai patients in immunoassays. The mimotope peptide that bound to the MAb specific to P. americana was LTPCRNK. The peptide has an 83-100% identity with proteins of Anopheles gambiae, notch homolog scalloped wings of Lucilia cuprina, delta protein of Apis mellifera; neu5Ac synthase and tyrosine phosphatase of Drosophila melanogaster, and a putative protein of Drosophila pseudoobscura. This finding implies that the mimotope-containing molecule of P. americana is a pan-insect protein. The MAb-bound protein of P. americana was shown to be arginine kinase that reacted to IgE in the sera of all of the CR-allergic Thai patients by immunoblotting, implying its high allergenicity. In conclusion, our results revealed that P. americana arginine kinase is a pan-insect protein and a major CR allergen for CR-allergic Thai patients.