Population genetic data for ten miniSTR loci in the Sri Lankan population.

Allele frequencies and forensically important parameters of ten autosomal miniSTR loci, D1S1677, D2S1776, D10S1248, D11S4463, D12SATA, D14S1434, D17S974, D18S853, D20S482, and D22S1045, were obtained for 278 unrelated adults from the Sri Lankan population. The combined power of discrimination and probability of exclusion was found to be 0.999999999621539 and 0.9979620, respectively. No significant deviations from Hardy-Weinberg equilibrium were observed except for D20S482 which conformed to HW expectations only after the application of a Bonferroni correction. The study suggests the potential use of these miniSTRs as a supplement or as a stand-alone STR marker system for the analysis of highly degraded evidence in Sri Lanka.

Heavy metal mediated immunomodulation of the Indian green frog, Euphlyctis hexadactylus (Anura:Ranidae) in urban wetlands.

Impacts of heavy metal toxicity on the immune system of the Indian green frog, Euphlyctis hexadactylus, in Bellanwila Attidiya, an urban wetland polluted with high levels of heavy metals, compared to the reference site in Bolgoda, in Sri Lanka was investigated. Significantly higher accumulation of selected heavy metals, copper (Cu), zinc (Zn), lead (Pb), and cadmium (Cd) were detected by AAS in frog liver and gastrocnemius muscle, in the polluted site than in the reference site. Non-functional immunotoxicity tests; total WBC, splenocyte and bone marrow cell counts, spleen weight/body weight ratio, neutrophil/lymphocyte ratio and basal immunoglobulin levels, and phagocytic capacity of peritoneal macrophages (immune functional test) were carried out using standard methodology. Test parameters recorded significantly lower values for frogs of the polluted site compared with their reference site counterparts, indicative of lowered immune response of frogs in the former site. In vitro phagocytic assay based on NBT dye reduction, measured the stimulation index (SI) of E. hexadactylus blood leukocytes, splenocytes and peritoneal macrophages, where SIs of frogs in the polluted site were significantly lower. Also, in vitro exposure of frog phagocytes to Cu, Zn, Pb and Cd at 10(-2)-10(-10)M, showed immunomodulation i.e. low concentrations stimulated phagocytosis while increased concentrations showed a trend towards immunosuppression. IC50 values indicated Cd>Zn>Cu>Pb as the decreasing order of the potential of phagocytosis inhibition. In conclusion, this study clearly demonstrated immunomodulation of E. hexadactylus, stimulated by heavy metals. In-vitro studies evidently suggested the use of phagocytosis as a biomarker in Ecoimmunotoxicology to detect aquatic heavy metal pollution.

Cost-effective diagnosis of male oxidative stress using the nitroblue tetrazolium test: useful application for the developing world.

Seminal oxidative stress plays an important role in male factor infertility (MFI), worldwide. A study was thus undertaken for the first time to establish seminal reactive oxygen species (ROS) as a clinical marker of MFI in a cohort of Sri Lankan males. The nitro blue tetrazolium (NBT) assay for ROS estimation and modified Endtz test for detecting leucocytes were carried out on semen samples (N = 102) of subfertile males. Age-matched individuals (N = 30) with proven past paternity served as controls. Significantly higher ROS production was evident in individuals with asthenozoospermia and unexplained infertility (Mann-Whitney U-test, P = 0.000), than in the fertile and the other subfertile groups tested. Receiver operating characteristic plot analysis established cut-off points of 40.57 and 42.02 µg formazan/10(7) spermatozoa for ROS to distinguish fertile males from asthenozoospermics (71.4% sensitivity: 70% specificity; AUC = 0.82), and from unexplained infertile males (74.1 % sensitivity: 73.3% specificity; AUC = 0.85) respectively. As ROS appear to be a potential marker of male infertility, it is imperative to validate this test as a simple, cost-effective hence a widely accessible diagnostic tool to be included in MFI investigations in the developing world.

Ocular and subcutaneous dirofilariasis in a Sri Lankan infant: an environmental hazard caused by dogs and mosquitoes.

An 11-month-old infant presented with a subcutaneous nodule in the right cheek which was found by ultrasonography to be a worm. Following treatment with di-ethylcarbamazine, a worm emerged from the left upper eyelid which was confirmed to be Dirofilaria repens. Dirofilariasis usually manifests as a single lesion and is rare in infants.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

Genetic complexity of Plasmodium vivax parasites in individual human infections analyzed with monoclonal antibodies against variant epitopes on a single parasite protein.

Monoclonal antibodies against variant epitopes of a highly polymorphic protein (PV200) in schizonts of Plasmodium vivax have been used to analyze the variety of genetically distinct populations of parasites present in the peripheral blood of individual P. vivax infections in Sri Lanka. In 9 out of 10 isolates of freshly drawn P. vivax infected blood from different individuals, parasites of only 1 PV200 serotype was found within each individual infection, even though parasites were serotypically distinct between individuals. In 1 isolate parasite population, 3 distinct PV200 serotypes were identified. Thus, most P. vivax infections appeared to consist of a single genetically homogeneous population of parasites within the detection limits of the technique. The prevalence of P. vivax infections in an area of malaria transmission in southern Sri Lanka and the densities of oocysts in mosquitoes fed on P. vivax infected individuals indicated that parasite populations would be transmitted many times before encountering parasites of other origins, and that individual populations would tend to reduce to genetic homogeneity during transmission. These expectations are consistent with the high proportion of genetically homogeneous P. vivax isolates observed.

Plasmodium vivax: cloning and expression of a major blood-stage surface antigen.

Plasmodium vivax is a highly prevalent malaria pathogen of man; the following report is the first to describe the cloning and expression of a major asexual erythrocytic stage antigen of this species. The screening of a genomic DNA expression library with a monoclonal antibody directed against a 200-kDa surface component (Pv200) of the more mature schizonts of P. vivax led to the selection of a recombinant bacterial clone which produced a fusion protein. Mouse and rabbit immune sera raised against the purified fusion protein recognized the 200-kDa parasite antigen on Western blots and reacted with the surface of segmenters by immunofluorescence. Sequencing of the 1.9-kb P. vivax DNA insert coding for this fusion protein revealed a 45-47% homology at the nucleotide level with the P. falciparum gene of a parasite surface antigen, Pf195, which has been shown to be a promising candidate for a malaria vaccine in primates and in man.

Monoclonal and polyclonal antibodies both block and enhance transmission of human Plasmodium vivax malaria.

Antibodies against gametes of the malarial parasite inhibit the development of the parasite in the mosquito and curtail the transmission of malaria. We now report that a monoclonal antibody against gametes of the human malaria pathogen Plasmodium vivax and antibodies induced during natural infections of P. vivax in humans which suppress infectivity of the parasites to the vector at high concentrations can, at lower concentrations, have the opposite effect and enhance the level of malaria infection in the mosquitoes. Infectivity enhancing effects of up to 12-fold were demonstrated when a transmission blocking monoclonal antibody and immune human sera were diluted, in some undiluted immune human sera, and in the sera of vivax malaria patients during convalescence after drug cure.

Immunoelectron microscopy of Schüffner's dots in Plasmodium vivax-infected human erythrocytes.

Plasmodium vivax induces morphologic alterations in infected host erythrocytes that are visible by light microscopy in Romanovsky-stained blood smears as multiple brick-red dots. These morphologic changes, referred to as Schüffner's dots, are important in the identification of this species of malarial parasite and have been associated by electron microscopy with caveolavesicle complexes along the erythrocyte plasmalemma. We have produced a monoclonal antibody (MAb A 20) that identifies an antigen in Plasmodium vivax-infected erythrocytes that is associated with the caveola-vesicle complexes of the parasitized host cell. This monoclonal antibody reacts with air-dried P vivax-infected erythrocytes to produce a pattern by the indirect immunofluorescence test (IFT) that is evocative of Schüffner's dots. Immunoelectron microscopy of P vivax-infected human erythrocytes using MAb A 20 confirmed the location of this antigen within vesicles of caveola-vesicle complexes. On Western blots MAb A 20 recognized four polypeptides of 54, 64, 72, and 86 kd. MAb A 20 reacted by IFT with 90% of Sri Lankan isolates and with a Brazilian P vivax isolate, which indicates that the epitope identified by this monoclonal is conserved.

Demonstration of antigenic polymorphism in Plasmodium vivax malaria with a panel of 30 monoclonal antibodies.

A panel of 30 monoclonal antibodies was established against asexual erythrocytic stages of Plasmodium vivax and used to investigate the antigenic composition of the parasite. At least 38 different antigenic polypeptides of P. vivax were characterized by the Western blot technique. The possible location of these antigens, as well as their stage and species specificity, was determined on the basis of the staining patterns produced by these antibodies on air-dried parasites in the indirect immunofluorescence test. Immunofluorescence performed with 30 different monoclonal antibodies on 50 different isolates of P. vivax obtained from patients showed that a high level of antigenic polymorphism prevailed in P. vivax. Only six monoclonal antibodies reacted with epitopes that were represented in more than 80% of parasite isolates, and therefore, appeared to be relatively conserved among different isolates. The other 24 monoclonal antibodies reacted with only 20 to 70% of parasite isolates.