Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6M urea for the first wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n=16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p<0.05) over the first 24 weeks. In Week 4, all animals had avidity values <35% and by Week 8, 8/16 animals had avidity values of >35%. These results suggest that an avidity value of <35% indicates a recent primary infection while a value of >35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody influence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep.
Toxoplasmosis, being one of the TORCH's infections in pregnant women, is caused by Toxoplasma gondii, an obligate intracellular protozoan parasite. This parasitic infection in pregnancy congenitally causes severe outcomes to their fetus and newborn. This study aimed to determine the seroprevalence and stages of Toxoplasma infection in pregnant women and its associated risks exposures.
Plasmodium knowlesi is now known as the fifth Plasmodium species that can cause human malaria. The Plasmodium merozoite surface protein (MSP) has been reported to be potential target for vaccination and diagnosis of malaria. MSP-1(33) has been shown to be immunogenic and its T cell epitopes could mediate cellular immune protection. However, limited studies have focused on P. knowlesi MSP-133. In this study, an approximately 28-kDa recombinant P. knowlesi MSP-1(33) (pkMSP-1(33)) was expressed by using an Escherichia coli system. The purified pkMSP-1(33) reacted with serum samples of patients infected with P. knowlesi (31 of 31, 100%) and non-P. knowlesi malaria (27 of 28, 96.43%) by Western blotting. The pkMSP-1(33) also reacted with P. knowlesi (25 of 31, 80.65%) and non-P. knowlesi malaria sera (20 of 28, 71.43%) in an enzyme-linked immunosorbent assay (ELISA). Most of the non-malarial infection (49 of 52 in by Western blotting and 46 of 52 in the ELISA) and healthy donor serum samples (65 of 65 by Western blotting and ELISA) did not react with recombinant pkMSP-1(33).
Toxoplasmosis is an infection caused by the parasite Toxoplasma gondii. Chronically-infected individuals with a compromised immune system are at risk for reactivation of the disease. In-vivo induced antigen technology (IVIAT) is a promising method for the identification of antigens expressed in-vivo. The aim of the present study was to apply IVIAT to identify antigens which are expressed in-vivo during T. gondii infection using sera from individuals with chronic toxoplasmosis. Forty serum samples were pooled, pre-adsorped against three different preparations of antigens, from each in-vitro grown T. gondii and Escherichia coli XLBlue MRF', and then used to screen a T. gondii cDNA expression library. Sequencing of DNA inserts from positive clones showed eight open reading frames with high homology to T. gondii genes. Expression analysis using quantitative real-time PCR showed that SAG1-related sequence 3 (SRS3) and two hypothetical genes were up-regulated in-vivo relative to their expression levels in-vitro. These three proteins also showed high sensitivity and specificity when tested with individual serum samples. Five other proteins namely M16 domain peptidase, microneme protein, elongation factor 1-alpha, pre-mRNA-splicing factor and small nuclear ribonucleoprotein F had lower RNA expression in-vivo as compared to in-vitro. SRS3 and the two hypothetical proteins warrant further investigation into their roles in the pathogenesis of toxoplasmosis.
Toxoplasma gondii is an important human pathogen with a worldwide distribution. It is primarily of medical importance for pregnant women and immunocompromised patients. Primary infection of the former is often associated with fetal infection, which can lead to abortion or severe neonatal malformation. Immunocompromised patients are at risk of contracting the severe form of the disease that may be fatal. Thus, detection of T. gondii infection with high sensitivity and specificity is crucial in the management of the disease. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to toxoplasma antigens in the patient's serum sample. Most of the commercially available tests use T. gondii native antigens and display wide variations in test accuracy. Recombinant antigens have great potential as diagnostic reagents for use in assays to detect toxoplasmosis. Thus in this review, we address recent advances in the use of Toxoplasma recombinant proteins for serodiagnosis of toxoplasmosis.
Indirect fluorescent antibody (IFA) tests and enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies to Plasmodium falciparum in an indigenous population in an area of Malaysia with high malaria prevalence. The results of three surveys were analyzed to examine the relation of these serologic measures with age, parasite rate, and spleen size. For children 0-4 years old, increasing spleen size was associated with an increasing likelihood of malaria parasitemia, while for 5-9 year olds the two variables were unrelated. Parasite rate declined with age and ELISA titre increased with age in all surveys; IFA titre was consistently high and did not vary with age. Neither antibody measure was significantly correlated with either the presence or the actual density of parasitemia. These antibody measures are most useful as adjuncts to the more traditional techniques of malaria assessment.
Most studies on human immunity to malaria have focused on the roles of immunoglobulin G (IgG), whereas the roles of IgM remain undefined. Analyzing multiple human cohorts to assess the dynamics of malaria-specific IgM during experimentally induced and naturally acquired malaria, we identified IgM activity against blood-stage parasites. We found that merozoite-specific IgM appears rapidly in Plasmodium falciparum infection and is prominent during malaria in children and adults with lifetime exposure, together with IgG. Unexpectedly, IgM persisted for extended periods of time; we found no difference in decay of merozoite-specific IgM over time compared to that of IgG. IgM blocked merozoite invasion of red blood cells in a complement-dependent manner. IgM was also associated with significantly reduced risk of clinical malaria in a longitudinal cohort of children. These findings suggest that merozoite-specific IgM is an important functional and long-lived antibody response targeting blood-stage malaria parasites that contributes to malaria immunity.
Cattle in Peninsular Malaysia were examined for evidence of infection with Babesia ovata, B. bigemina and B. bovis by an enzyme-linked immunosorbent assay for detection of antibody to the three Babesia species. All of the test samples when assayed with B. ovata antigen, resulted in low value indicating low probability of cattle infected with B. bigemina, 74.4% were positive for B. bovis and 72.6% were positive for both Babesia species. In addition, a serological survey with regard to age difference was carried out on a milk production farm. High reactivity antibody to B. bigemina and B. bovis was detected in calves less than 1 month of the age. The reactivity decreased in calves 1-3 months of the age. Then, the reactivity increased for both Babesia species in 6 months old calves. These results suggested that cattle infected with B. bigemina and B. bovis were widespread throughout Peninsular Malaysia and that both parasites might exist as an enzootical parasite.
The seroprevalence of toxoplasmosis among 505 of human immunodeficiency virus (HIV)/AIDS patients was 226 (44.8%; 95% CI 42.64-51.76): 27 (47.4%) and 199 (44.4%) showed Toxoplasma seropositivity with and without toxoplasmic encephalitis (TE), respectively (P <0.05). The majority of these patients were in the 25-34 age group (44 versus 39%), male (86 versus 76%), and Chinese (49 versus 53%), though no statistical significance was found between the two. Significant differences between these two groups were noted, however, in terms of marital status, occupation, and present address. The heterosexual exhibited the most frequent behavior at risk for HIV infection, and accounted for 51 and 59% of patients with and without TE, respectively. Only 17/260 (6.5%) and 1/137 (0.7%) of them later acquired TE after receiving primary chemoprophylaxis (cotrimoxazole) and antiretroviral therapy including HAART (P <0.05). Fifty-seven (11.3%) out of those 505 patients were diagnosed with AIDS-related TE. The most common clinical manifestation was headache (56%). The computed tomography scan findings showed most lesions to be multiple (96.4%), hypodense (66.7%), and in the parietal region (39.3%). Twenty-seven (47.4%) patients had chronic (latent) Toxoplasma infection as evidenced by seropositivity for anti-Toxoplasma (IgG) antibody. At the time of diagnosis, the range of CD4 cell count was from 0-239 with a median of 25 cells/cumm. We also found that a CD4 count of less than 100 cells/cumm was significantly associated with development of TE (P <0.05). Clinical outcomes showed that among those who survived, 21 (36.8%), 16 (28.1%), and 2 (3.5%) of patients had completed treatment, transferred out, and were lost to follow up, respectively. Unfortunately, 18 (31.6%) of the cases were officially pronounced dead. Overall, 7 (12.3%) patients were detected as recurrent TE in this study.
An accurate diagnosis for toxoplasmosis is crucial for pregnant women as this infection may lead to severe sequelae in the fetus. The value of IgG avidity assay as a tool to determine acute and chronic toxoplasmosis during pregnancy was evaluated in Universiti Kebangsaan Malaysia Medical Centre (UKMMC). In this study, 281 serum samples from 281 pregnant women in various trimesters were collected. These samples were assayed using specific anti-Toxoplasma IgM and IgG antibodies, followed by IgG avidity test. The overall seroprevalence of toxoplasmosis in pregnant women was 35.2% (33.5% for anti-Toxoplasma IgG and 1.8% for both anti-Toxoplasma IgG and IgM antibodies). Of 5 (1.8%) serum samples positive for IgM ELISA, 4 had high-avidity antibodies, suggesting past infection and one sample with borderline avidity index. Two samples with low avidity were from IgM negative serum samples. The IgG avidity assay exhibited an excellent specificity of 97.6% and a negative predictive value (NPV) of 95.6%. The study also demonstrated no significant correlation between avidity indexes of the sera with IgG (r=0.12, p=0.24) and IgM (r=-0.00, p=0.98), suggesting the complementary needs of the two tests for a better diagnosis outcome. These findings highlight the usefulness of IgG avidity assay in excluding a recently acquired toxoplasmosis infection in IgM-positive serum sample.
Entamoeba histolytica causes amoebic diarrhoea, colitis and liver abscess (ALA). Diagnosis of ALA is difficult, as most patients do not have simultaneous intestinal amoebic infection. At Hospital Universiti Sains Malaysia (HUSM), diagnosis of ALA relies on a combination of clinical findings, ultrasound examination of the liver and serodiagnosis using a commercial kit. In this study, two in-house indirect ELISAs were developed and evaluated. One of the in-house assays utilises E. histolytica crude soluble antigen (CSA) to detect serum IgG specific to the parasite whereas the other uses E. histolytica ether extract antigen (EEA). Preparation of CSA requires a sonicator to lyse the amoeba whereas EEA was prepared by chemically solubilizing the trophozoites. Based on the cut-off value of mean optical density + 3SD, CSA-ELISA showed 100% (24/24) sensitivity and 93.33% (210/225) specificity; while EEA-ELISA showed 91.67% (22/24) sensitivity and 95.11% (214/225) specificity. In conclusion, both the in-house indirect ELISAs were found to be efficacious for diagnosis of ALA; and the EEA is easier to prepare than the commonly used CSA.
Schizophrenia is a pervasive neuropsychiatric disease of unknown cause. Previous studies have reported that toxoplasmosis may be a possible cause of schizophrenia. To ascertain possible relationship between Toxoplasma gondii and schizophrenia, a cross sectional study, employing an enzyme-linked immunosorbent assay (ELISA) was performed to study the seroprevalence of anti-T. gondii IgG antibody in schizophrenic patients. Furthermore, demographic data analysis from schizophrenic patients were analysed to associate toxoplasmosis with schizophrenia. A total of 288 serum samples from schizophrenic patients (n=144) and psychiatrically healthy volunteers (n=144) were recruited in this study. Interestingly, a significant result in the serointensity rate of anti-T. gondii IgG antibody (> 60 IU/mL) in schizophrenic patients (61.1%) was demonstrated as compared to psychiatrically healthy volunteers (40.8%) (X² = 4.236, p < 0.050). However, there was no significant difference between the seropositivity rate of anti-T. gondii IgG antibody between the two groups. Analysis from demographic data revealed that the seropositivity rate of anti-T. gondii IgG antibody in schizophrenic patients was significantly associated with age group of more than 40 years old (p=0.007) and between ethnic (p=0.046). Nevertheless, no significant association between seropositivity rate of anti-T. gondii IgG antibody with gender (p=0.897), duration of illness (p=0.344) and family history of schizophrenia (p=0.282) in these patients. Thus, this finding is essential as a preliminary data in Malaysia to establish the association between T. gondii and schizophrenia.
In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.
We report a case of unusual cutaneous toxoplasmosis manifestation in a HIV-positive patient. He presented with hard and painful nodular lesions on the arms, hands and chest. Serology tests for anti-Toxoplasma antibody were negative. However, histopathologic examination of the lesion revealed foci of macrophages containing crescent-shaped organisms resembling the zoites of the protozoan parasite Toxoplasma gondii. Ultrastructure examination under electron microscopy and PCR confirmed the organism as T. gondii.
Toxoplasma gondii is a protozoan parasite that is capable of causing a zoonotic disease, known as toxoplasmosis. Vertical transmission of T. gondii from the mother to the fetus, during pregnancy may cause severe complications to the developing fetus. This current study aimed to determine the seroprevalence and investigate the associated risk factors of Toxoplasma infection in pregnant women (n=219) visiting the antenatal clinic at UMMC. While the elevated level of anti-Toxoplasma IgG and IgM antibodies indicates the presence of infection, it fails to differentiate between a past and a recent infection. Thus, the study also demonstrates the usefulness of IgG avidity in validating the timing of infection. The serum samples were tested for the presence of anti-Toxoplasma IgG and IgM antibodies by ELISA test, and the seropositive samples for both anti-Toxoplasma IgG and IgM antibodies were further evaluated by IgG avidity. The results showed that the overall prevalence of T. gondii seropositivity was 34.7%. Of these, 30.6% (67/219) were positive for anti-Toxoplasma IgG antibody only, 2.3% (5/219) were positive for anti-Toxoplasma IgM only, and the remaining 1.8% (4/219) was positive for both anti-Toxoplasma IgG and IgM antibodies. All of the pregnant women who were positive for both anti-Toxoplasma IgG and IgM antibody were found to have past infection when evaluated by IgG avidity. In this study, Malay ethnicity and the number of existing previous children were significantly associated with T. gondii seropositivity (p<0.05). Based on these findings, information and education on the transmission and prevention of congenital toxoplasmosis are very crucial as a public health effort towards a healthier society.
The IgG avidity assay is an important tool in the management of suspected toxoplasmosis in pregnant women. This study aimed to produce new Toxoplasma gondii recombinant proteins and to assess their usefulness in an IgG avidity assay. Toxoplasma positive and negative serum samples were used, the former were categorized into low (LGA) and high (HGA) IgG avidity samples. Immunoblots were performed on 30 T. gondii cDNA clones to determine the reactivity and IgG avidity to the expressed proteins. Two of the clones were found to have diagnostic potential and were analyzed further; AG12b encoded T. gondii apical complex lysine methyltransferase (AKMT) protein and AG18 encoded T. gondii forkhead-associated (FHA) domain-containing protein. The His-tagged recombinant proteins, rAG12b and rAG18, were expressed and tested with LGA and HGA samples using an IgG avidity western blot and ELISA. With the IgG avidity western blot, rAG12b identified 86.4% of LGA and 90.9% of HGA samples, whereas rAG18 identified 81.8% of both LGA and HGA samples. With the IgG avidity ELISA, rAG12b identified 86.4% of both LGA and HGA samples, whereas rAG18 identified 77.3% of LGA and 86.4% of HGA serum samples. This study showed that the recombinant antigens were able to differentiate low avidity and high avidity serum samples, suggesting that they are potential candidates for use in the Toxoplasma IgG avidity assay.
Toxoplasma gondii primary infection in pregnancy is associated with poor obstetric outcomes. This study aimed to determine the seroprevalence of Toxoplasma infection in pregnant migrant and refugee women from Myanmar attending antenatal care in Thailand. A random selection of 199 residual blood samples from first antenatal screen in 2014-2015 was tested for Toxoplasma IgG and IgM antibodies. Seroprevalence of Toxoplasma infection was 31.7% (95% confidence interval = 25.6-38.4). Avidity testing in the three positive IgM cases indicated all were past infections. Multiparity (≥ 3 children) was significantly associated with higher Toxoplasma seropositivity rates. Seroprevalence of T. gondii infection in this pregnant population is similar to the only other report from Myanmar, where multiparity was also identified as a significant association. Toxoplasma infection is important in pregnant women. Nevertheless, in this marginalized population, this infection may be given less priority, due to resource constraints in providing the most basic components of safe motherhood programs.
Crude soluble antigen (CSA) produced from Entamoeba histolytica trophozoite is conventionally used for serodiagnosis of invasive amoebiasis. However, high background seropositivities by CSA-assay in endemic areas complicate the interpretation of positive result in clinical settings. Instead, incorporating a second assay which indicates active or recent infection into the routine amoebic serology could possibly complement the limitations of CSA-assay. Hence, the present study aimed to evaluate the diagnostic efficacies of indirect ELISAs using CSA and excretory-secretory antigen (ESA) for serodiagnosis of amoebic liver abscess (ALA). Reference standard for diagnosis of ALA at Hospital Universiti Sains Malaysia is based on clinical presentation, radiological imaging and positive indirect haemagglutination assay (titer ≥256). Five groups of human serum samples collected from the hospital included Group I - ALA diagnosed by the reference standard and pus aspirate analysis using real-time PCR (n=10), Group II - ALA diagnosed by the reference standard only (n=41), Group III - healthy control (n=45), Group IV - other diseases control (n=51) and Group V - other infectious diseases control (n=31). For serodiagnosis of ALA serum samples (Group I and II), CSA-ELISA showed sensitivities of 100% for both groups, while ESA-ELISA showed sensitivities of 100% and 88%, respectively. For serodiagnosis of non-ALA serum samples (Group III, IV and V), CSA-ELISA showed specificities of 91%, 75% and 100%, respectively; while ESA-ELISA showed specificities of 96%, 98% and 100%, respectively. Indirect ELISAs using CSA and ESA have shown distinct strength for serodiagnosis of ALA, in terms of sensitivity and specificity, respectively. In conclusion, parallel analysis by both assays improved the overall efficacies of amoebic serology as compared to either single assay.
Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.