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.
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.
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.
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.
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.
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.
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.
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.
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.
This study investigated the prevalence of parasitic infections among aborigine children at Post Brooke, Kelantan. Eighty-four formalin-fixed specimens and 78 PVA-fixed specimens were obtained. 79.8% and 35.9% of the samples were positive for helminth ova and protozoa respectively. The parasites detected (single plus mixed infections) were A. lumbricoides (50/84, 59.5%), T. trichiura (35/84, 41.7%), hookworm (5/84, 6.0%), S stercoralis (1/84, 1.2%), G. intestinalis (18/78, 23.1%), E. histolytica (7/78, 9.0%) and E. coli (7/78, 9.0%). Two hundred thick blood film examinations detected only one case of Plasmodium falciparum infection. A high prevalence of intestinal parasitic infections among the children at Post Brooke was demonstrated in this study; thus there is an urgent need to improve the hygiene, education and living standards of this population.
An IgG4 ELISA based on a novel recombinant antigen was evaluated for detection of Brugia malayi infection, using 2487 sera from various institutions: 2031 samples from Universiti Sains Malaysia, 276 blinded sera from 2 other institutions in Malaysia, 140 blinded sera from India and 40 blinded sera from Thailand. These sera were from various groups of individuals, i.e., microfilaraemics, chronic patients, endemic normals, non-endemic normals and individuals with other parasitic and bacterial infections. Based on a cut-off optical density reading of 0.300, the IgG4 ELISA demonstrated specificity rates of 95.6-100%, sensitivity rates of 96-100%, positive predictive values of 75-100% and negative predictive values of 98.9-100%. These evaluation studies demonstrated the high specificity and sensitivity of this test for the detection of active B. malayi infection. Thus, the IgG4 ELISA would be very useful as a tool in diagnosis and in elimination programmes for brugian filariasis.
A polymerase chain reaction assay based on the enzyme-linked immunosorbent assay (PCR-ELISA) has been developed to detect Brugia malayi infection in an area of low endemicity in Malaysia. Blood samples from 239 subjects were tested: 192 amicrofilaraemic individuals, 14 microfilaraemic persons and 3 chronic elephantiasis cases from endemic areas and 30 city-dwellers (non-endemic controls). PCR products were examined by ELISA and Southern hybridization. In the PCR-ELISA, digoxigenin-labelled PCR products were hybridized to a biotin-labelled probe. This was followed by incubation in streptavidin-coated microtitre wells and detection using anti-digoxigenin-peroxidase and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)]. All microfilaraemic samples were positive by PCR-ELISA and Southern hybridization and all samples from non-endemic subjects and chronic elephantiasis patients were negative. The PCR-ELISA detected 12 times as many B. malayi infections as did thick blood film examination. Nineteen of the 194 samples from the endemic area gave positive results by both PCR-ELISA and Southern hybridization, and an additional 5 samples were positive by PCR-ELISA only. The PCR-ELISA was specific and sensitive, detected more infections, and was more reproducible than Southern hybridization.