Mice were chronically infected with cysts of ME49 strain of Toxoplasma gondii. At different periods post-infection, their spleens were removed and single cell suspensions were made. Lymphocyte transformation experiments were performed on the lymphocyte suspensions using three different kinds of antigens of ME49 strain of T. gondii, namely soluble, excretory/secretory and cystic forms. The results showed that the pattern of lymphocyte responsiveness was dependent on the kind of antigen employed for induction of the blastogenesis. Using soluble and cystic forms of the antigen, different periods of lymphocyte suppression and lymphocyte proliferation were demonstrated. However, with the use of excretory/secretory antigen, no significant suppression of lymphocyte stimulation was noted throughout the course of infection. Thus excretory/secretory antigen may be the best form of antigen for stimulation of the cell-mediated immune response and hence it appears to be a good candidate for vaccine in toxoplasmosis.
C57BL/6 mice were orally infected with different doses of cysts of ME49 strain of Toxoplasma gondii to produce groups of acutely and chronically infected mice. Sera were obtained at different periods post-infection. SDS-PAGE was ran with excretory/secretory antigens of ME49 and RH strains of T. gondii, followed by Western blot analyses using the above sera and anti- IgA, IgM, IgG as conjugates. The SDS-PAGE profiles of the two antigens were similar. However the antigenic bands showed variations in all blots, most evidently in IgA blots of chronic sera. IgG blots showed greatest similarities in reactive bands. In IgM blots, more common bands were shown in chronic sera than in acute sera. Variations and similarities in prominence of some bands and time of their appearance were also noted, especially in IgM and IgG blots of chronic sera. Thus antigenic variations and similarities are present in excretory/secretory products of different strains of T. gondii.
Toxoplasmosis can cause serious disease in immunocompromised patients and to congenitally infected foetuses. Appropriate laboratory investigations in potential cases of acute Toxoplasma infection are important. Excretory secretory antigen (ESA) is immunogenic during both human and experimental infections, therefore is considered as a good candidate for investigation into new infection markers. In this study, ESA was prepared from in vitro cultures of Toxoplasma gondii to identify T. gondii ESA antigenic component(s) that is/are most reactive with serum samples from probable acute cases of toxoplasmosis. Serum samples were obtained from several categories of individuals with the following Toxoplasma serology: Group I: IgM+ IgG+ (low IgG avidity) or IgM+ IgG- from sera of patients who had clinical query of toxoplasmosis (n=35). Group II: IgM- IgG+ (high IgG avidity) from chronically infected individuals (n=30). Group III: normal/healthy individuals with anti-Toxoplasma IgMIgG- (n=20). Group IV: individuals with other infections who had anti-Toxoplasma IgM- IgG- (n=10). The ESA was subjected to SDS-PAGE, followed by Western blot analysis using the above sera and probed with peroxidase conjugated anti-human IgM and IgA antibodies. The blots were then developed using chemiluminescence substrate. The selected antigenic band was excised from the gel after two dimensional electrophoresis and sent for mass spectrometry analysis using MALDI TOF-TOF. The most promising antigenic band was a 10 kDa protein which showed sensitivity of 80% in both IgM and IgA blots, and specificity of 96.7% with sera from other infections and healthy controls. The two best identifications for the 10 kDa band were ubiquitin (ribosomal protein CEP52 fusion protein) and polyubiquitin.
Microscopic detection of active phase of lymphatic filariasis is indicated by the presence of microfilaria in whole blood. This method is not sensitive and requires relatively large amount of blood sample. PCR allows very sensitive detection of the parasite DNA using a smaller amount of blood; and the use of dried blood spots facilitates sample transportation. Nevertheless, limited studies have been reported on PCR using dried blood spot for detection of Brugia malayi. In this study, we investigated the effects of concentrating whole blood genomic DNA sample and the amplification methods [conventional PCR (C-PCR) and real-time PCR] on the detection of B. malayi DNA from dried blood spots from a very low endemic area in Malaysia. Both C-PCR and real-time PCR detected 2 out of 18 (11%) samples as positive from non-concentrated genomic DNA preparations. After the DNA samples were pooled and concentrated, both C-PCR and realtime PCR detected B. malayi DNA amplifications in 7 out of 18 (39%) samples. However one sample which showed faint band in C-PCR was detected as highly positive in real-time PCR. In conclusion, both C-PCR and real-time PCR using dried blood spots from a low endemic area demonstrated equal sensitivity for detection of B. malayi DNA.
Strongyloidiasis is an infection caused by the intestinal nematode Strongyloides stercoralis. Infected healthy individuals are usually asymptomatic, however it is potentially fatal in immunocompromised hosts due to its capacity to cause an overwhelming hyperinfection. Strongyloidiasis could be missed during routine screening because of low and intermittent larval output in stool and variable manifestations of the symptoms. We present two cases of strongyloidiasis occurring in children with solid organ malignancies suspected to have the infection based on their clinical conditions and treatment history for cancer. Both patients were diagnosed by molecular and serological tests and were successfully treated. Thus, strongyloidiasis in patients undergoing intensive treatment for malignancies should be suspected, properly investigated and treated accordingly.
Transmission of soil-transmitted helminthes infection is by faecal oral route, and is influenced by food preference. Kelantanese love to consume ulam which are raw vegetables and herbs. Some of the herbs grow on grounds with high humidity and are abundant near drainage areas, these are also places with higher likelihood of harbouring viable parasite ova. The aim of this study was to determine the prevalence of soiltransmitted helminthes in vegetables, herbs and fruits found in our local setting. The results by microscopy showed that there was no helminthes ovum or protozoan parasite in the samples. However, Strongyloides stercoralis rhabdatiform larvae were identified in water samples used to wash pegaga, kesum and water spinach, and the number of larvae observed were 152, 9 and 16 respectively. Analysis by real-time PCR confirmed the microscopic observation of this helminth. This study highlighted that vegetables and herbs are likely sources of Strongyloides stercoralis infection in Kota Bharu, Kelantan. Thus vegetable sellers as well as the food handlers are the two important groups who are at high risk of acquiring the infection.
In vitro culture of Toxoplasma gondii can provide tachyzoites which are active, viable and with desirable purity. Thus the aim of this study was to optimize the cell culture method for T. gondii propagation to obtain a consistent source of parasites with maximum yield and viability, but minimum host cell contamination for use in production of excretory-secretory antigen. Tachyzoites with seed counts of 1x10(6), 1x10(7) and 1x10(8) harvested from infected mice were added to VERO cells of different degrees of confluence, namely 50%, 85% and 100%, and examined periodically using an inverted microscope. When the maximum release of the tachyzoites was observed from the host cells, the culture supernatant was removed and the tachyzoites harvested. Using a Neubauer chamber, the percentages of viable tachyzoites and host cell contamination were determined using trypan blue stain. Parameters that gave the best yield and purity of viable tachyzoites were found to be as follows: VERO cells at 85% confluence in DMEM medium and inoculum comprising 1x10(7) tachyzoites. After about 3 days post infection, the tachyzoites multiplied 78x, with a yield of ~7.8x10(8) per flask, 99% viability and 3% host cell contamination. This study has successfully optimized the method of propagation of T. gondii tachyzoites in VERO cells which produce parasites with high yield, purity and viability.
We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies.
Currently, the laboratory diagnosis of toxocariasis, caused by Toxocara canis or T. cati, mainly relies on serological tests. Unfortunately, however, the specificities of most of the commercial tests that are available for the serodiagnosis of this disease are not very high and this may cause problems, especially in tropical countries where co-infections with other helminths are common. In an effort to develop a serological assay with improved specificity for the detection of Toxocara infection, an IgG(4)-ELISA based on a recombinant version (rTES-30USM) of the 30-kDa Toxocara excretory-secretory antigen (TES-30) has recently been developed. To produce the antigen, the TES-30 gene was cloned via assembly PCR, subcloned into a His-tagged prokaryotic expression vector, and purified by affinity chromatography using Ni(2+)-nitrilotriacetic-acid (Ni-NTA) resin. The performance of the ELISA based on the recombinant antigen was then compared with that of commercial kit, based on an IgG-ELISA, for the serodiagnosis of toxocariasis (Toxocara IgG-ELISA; Cypress Diagnostics, Langdorp, Belgium). Both assays were used to test 338 serum samples, including 26 samples from probable cases of toxocariasis. Assuming that all the probable cases were true cases, the assay based on rTES-30USM demonstrated a sensitivity of 92.3% (24/26) and a specificity of 89.6% (103/115) whereas the commercial kit exhibited a sensitivity of 100% (26/26) but a specificity of only 55.7% (64/115). The high sensitivity and specificity exhibited by the new IgG(4)-ELISA should make the assay a good choice for use in tropical countries and any other area where potentially cross-reactive helminthic infections are common.
We conducted a field study of a rapid test (Brugia Rapid) for detection of Brugia malayi infection to validate its sensitivity and specificity under operational conditions. Seven districts in the state of Sarawak, Malaysia, which are endemic for brugian filariasis, were used to determine the test sensitivity. Determination of specificity was performed in another state in Malaysia (Bachok, Kelantan) which is non-endemic for filariasis but endemic for soil-transmitted helminths. In Sarawak both the rapid test and thick blood smear preparation were performed in the field. The rapid test was interpreted on site, whereas blood smears were taken to the district health centres for staining and microscopic examination. Sensitivity of Brugia Rapid dipstick as compared with microscopy of thick blood smears was 87% (20/23; 95% CI: 66.4-97.2) whereas the specificity was 100% (512/512). The lower sensitivity of the test in the field than in laboratory evaluations (> or =95%), was probably due to the small number of microfilaraemic individuals, in addition to difficulties in performing the test in remote villages by field personnel. The overall prevalence of brugian filariasis as determined by the dipstick is 9.4% (95% CI: 8.2-0.5) while that determined by microscopy is 0.90% (95% CI: 0.5-1.3) thus the dipstick detected about 10 times more cases than microscopy. Equal percentages of adults and children were found to be positive by the dipstick whereas microscopy showed that the number of infected children was seven times less than infected adults. The rapid dipstick test was useful as a diagnostic tool for mapping and certification phases of the lymphatic filariasis elimination programme in B. malayi-endemic areas.
Strongyloides stercoralis infection can persist in the host for several decades, and patients with cancer and other clinical conditions who are exposed to immunosuppressive therapy are at risk of developing hyperinfection.
Toxocariasis is a zoonotic helminthic infection of humans caused by the dog roundworm (Toxocara canis) or cat roundworm (Toxocara cati). There are two main human syndromes: visceral larva migrans (VLM), which are characterized by symptoms associated with major organs and ocular larva migrans (OLM), in which pathological effects on the host are restricted to the eye and the optic nerve. The present study evaluated the seroprevalence of toxocariasis among the Orang Asli with an IgG4-ELISA using recombinant antigens (rTES-26, rTES-30 and rTES-120) and an IgG-ELISA commercial kit (Cypress Diagnostic, Belgium). A total of 188 serum samples were analyzed using IgG4-ELISA recombinant antigens while 83 were tested using IgG-ELISA. Overall, 9 out of 188 (4.8%) samples were positive with the former assay: rTES-26 (2.7%) and rTES-30 (2.1%); and 63 out of 83 (75.9%) were positive with the IgG-ELISA. In general, the seroprevalence of toxocariasis among males (9.5%) was higher compared to females (1%). Children below 12 years (6.3%) have higher seroprevalence rate compared to adults (1.2%). Out of 59 IgG positive samples, 56 (94.9%) were also positive with soil-transmitted helminth (STH) infections which may indicate high false positivity. None of the IgG4- ELISA positive samples were positive with STH infections. Of 9 positive samples with IgG4-ELISA, 7 were also positive with IgG-ELISA giving the probability of true cases. The present finding indicated that exposure to Toxocara infection is not unusual among Malaysian aborigines, and it affects both sexes and all age groups. As a prevention strategy, more effective public health programmes to promote better understanding on the consequences of toxocariasis among the Orang Asli communities are deemed necessary.
Sera from fifty subjects with different presentations of Brugian filariasis and from common soil-transmitted helminth infections were tested for specific anti-filarial IgG and its subclasses. Anti-filarial IgG, IgG1 and IgG3 showed cross-reactivities with soil-transmitted helminthic infections and no significant differences in optical densities among the various groups of filarial patients. In comparison with other groups of subjects, IgG4-ELISA of sera from microfilaraemic patients and some previously microfilaraemic patients showed a significant increase in optical density readings, while IgG2-ELISA showed elevated optical density readings in sera of patients with chronic elephantiasis. Therefore IgG2-ELISA is potentially useful in the diagnosis of brugian chronic elephantiasis while IgG4-ELISA may be beneficial for follow-up diagnosis of treated microfilaraemic patients.
A total of 1,134 finger-pricked blood samples were collected from residents of Setiu, Terengganu. A drop of blood was used to make thick blood smear and about four drops were used for obtaining serum. The smears were stained and examined by the State Vector Control Unit in Kuala Terengganu, while the serum samples were tested for specific IgG4 antibodies to a novel recombinant antigen using Brugia-Elisa. Prevalence of filariasis in these areas were found to be 0.26% (3/1,134) using thick blood smear examination and 2.47% (28/1,134) using Brugia-Elisa, thus demonstrating the greater sensitivity of the latter test. In addtion, Brugia-Elisa showed a high level of specificity (97.8%, 1,106/1,131) when compared to thick blood smear examination.
Brugian filariasis infects 13 million people in Asia. The routine prevalence survey method using night thick blood smear is not sensitive enough to reflect the actual infection prevalence. In 1997-2001, only three microfilaraemic cases (of 5601 individuals screened; 0.05%) were reported in Pasir Mas, a district in Kelantan (Malaysia), which shares a border with Thailand. We therefore investigated the infection prevalence in this district by employing a sensitive and specific serological assay (Brugia-Elisa). This test is based on detection of specific IgG4 antibody against a Brugia malayi recombinant antigen. A total of 5138 children, aged 7-12 years, from 16 primary schools, were tested. Eighteen pupils in eight schools, located in five subdistricts, tested positive, giving an overall prevalence rate of 0.35%. Infection in these children is significant as they represent more recent cases. These subdistricts should be included in the national filariasis elimination programme.
We have recently reported that a dipstick colloidal dye immunoassay (DIA) that detect parasite antigens in human serum is sensitive and specific for the diagnosis of active infection of lymphatic filariasis. Rabbit polyclonal antibodies (RbBmCAg) labelled with a commercial dye, palanil navy blue was used to detect filarial antigenemia among Indonesian and Bangladeshi immigrant workers (N= 630) at oil palm estates at Hulu Trengganu District, Peninsular Malaysia. Microfilaremia with Brugia malayi were detected in 51 (8.10 %) individuals, of which 42 (6.67 %) were among the Indonesians and 9 (1.98 %) among the Bangladeshis. Microfilaremia with Wuchereria bancrofti were detected in 33 (5.24 %) individuals of which 15 (2.38 %) were among the Indonesians and 18 (2.86 %) among the Bangladeshis workers. The DIA detected 96 (15.24 %) antigenemic cases which comprise of all the microfilaremic cases and 15 (2.38 %) amicrofilaremic cases. The amicrofilaremic cases with filarial antigenemia consisted of 9 (1. 43 %) Indonesians and 6 (0.95%) Bangladeshis. We have used 6 ul of the RbBmCAg and diluted (1:10) patients' sera per dipstick which make the DIA reagent conservative. The DIA is a rapid test and can be read in approximate 2 hours.. Additionally, coloured dots developed in the DIA can be qualitatively assessed visually for intensity. The DIA does not require sophisticated equipment or radioactivity, and therefore suitable for field application.
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.
Amoebic serodiagnosis at Hospital Universiti Sains Malaysia (HUSM), Kelantan employs an indirect haemagglutination assay (IHA) which detects anti-Entamoeba histolytica antibodies in patients' serum samples. In an amoebiasis endemic area such as Kelantan, interpretation of a positive IHA result can be problematic due to the high background antibody levels. The TechLab E. histolytica II ELISA is a commercial kit for detection of specific Gal/GalNAc lectin antigen in stool samples, and has been reported to be able to detect the antigen in serum samples from patients with amoebic liver abscess (ALA). Thus in this study we investigated the usefulness of TechLab E. histolytica II ELISA for diagnosis of ALA by comparing it with IHA. This is a cross sectional study involving 58 suspected ALA patients who were admitted to the surgical ward, HUSM, Kelantan. The diagnosis of ALA was established based on clinical symptoms and signs, ultrasound and/or CT scan results. The serum specimens obtained from the patients were tested with IHA (Dade Behring Diagnostics, Marburg, Germany) and TechLab E. histolytica II ELISA (Techlab, Blacksburg, Virginia, USA) according to the manufacturers' instructions. Of the 58 patients, 72.4% (42) were positive by IHA and only 8.6% (5) were positive by the TechLab E. histolytica II ELISA. Agreement between the IHA and ELISA was poor (kappa value 0.019, p=0.691). There was also no correlation between ELISA results and IHA antibody titers. The TechLab E. histolytica II ELISA was not sensitive in detecting amoebic antigen in samples from ALA patients. In addition the results of the test did not correlate with the IHA anti-E. histolytica antibody titres. Therefore, the TechLab E. histolytica II ELISA was found not to be useful for serological diagnosis of ALA at HUSM.
Western blot analyses were performed on 444 serum specimens: 40 sera from microfilaraemic individuals, 10 sera from elephantiasis patients, 24 treated individuals, 50 sera from residents of endemic areas without anti-filarial IgG4 antibodies (endemic normals), 20 sera from amicrofilaraemic individuals with high anti-filarial IgG4 antibodies, 200 sera from healthy city-dwellers (non-endemic samples), and 100 sera from soil-transmitted helminth-infected individuals. Phast electrophoresis system was used to electrophorese Brugia malayi soluble adult worm antigen on 10-15% SDS-PAGE gradient gels followed by electrophoretic transfer onto PVDF membranes. Membrane strips were then successively incubated with blocking solution, human sera, and monoclonal anti-human IgG4 antibody-HRP, with adequate washings done in between each incubation step. Luminol chemiluminescence detection was then used to develop the blots. An antigenic band with the MW of approximately 37 kDa was found to be consistently present in the Western blots of all microfilaraemic sera, all amicrofilaraemic sera with high titres of anti-filarial IgG4 antibodies, some treated patients, and some elephantiasis patients. The antigen did not occur in immunoblots of individuals with other helminthic infections, normal endemic individuals, and city dwellers. Therefore the B. malayi antigen of with the MW of approximately 37 kDa demonstrated specific reactions with sera of B. malayi-infected individuals and thus may be useful for diagnostic application.