Laboratory diagnosis of toxocariasis is still a challenge especially in developing endemic countries with polyparasitism. In this study, three Toxocara canis recombinant antigens, rTES-26, rTES-30, and rTES-120, were expressed and used to prepare lateral flow immunoglobulin G4 (IgG4) dipsticks. The concordance of the results of the rapid test (comprising three dipsticks) with a commercial IgG-enzyme-linked immunosorbent assay (ELISA) (Cypress Diagnostics, Belgium) was compared against the concordance of two other commercial IgG-ELISA kits (Bordier, Switzerland and NovaTec, Germany) with the Cypress kit. Using Toxocara-positive samples, the concordance of the dipstick dotted with rTES-26, rTES-30, and rTES-120 was 41.4% (12/29), 51.7% (15/29), and 72.4% (21/29), respectively. When positivity with any dipstick was considered as an overall positive rapid test result, the concordance with the Cypress kit was 93% (27/29). Meanwhile, when compared with the results of the Cypress kit, the concordance of IgG-ELISA from NovaTec and Bordier was 100% (29/29) and 89.7% (26/29), respectively. Specific IgG4 has been recognized as a marker of active infection for several helminthic diseases; therefore, the two non-concordant results of the rapid test when compared with the NovaTec IgG-ELISA kit may be from samples of people with non-active infection. All the three dipsticks showed 100% (50/50) concordance with the Cypress kit when tested with serum from individuals who were healthy and with other infections. In conclusion, the lateral flow rapid test is potentially a good, fast, and easy test for toxocariasis. Next, further validation studies and development of a test with the three antigens in one dipstick will be performed.
SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
An enzyme-linked immunosorbent assay using excretory-secretory antigens of the second stage larvae maintained in vitro was used to determine the seroprevalence of Toxocara antibodies in Orang Asli (aborigines) of Peninsular Malaysia. The mean + 3 SD optical density of 30 healthy subjects was used as the cut-off point. Overall prevalence was found to be 31.9%. No significant relationship was found between positive rates with sex and age groups, though children between 0 to 9 years recorded the highest positive rates. Eosinophil counts were found to be closely related to the proportion of positivity to toxocaral infection and mean optical densities. There was some degree of cross-reaction with Trichuris trichuria positive sera.
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 dot enzyme immunoassay for determination of antibodies to Japanese encephalitis virus was designed for use as a field technique for the surveillance of Japanese encephalitis virus activity among domestic pigs. The test was compared with the neutralization test and the hemagglutination inhibition test and found to be more sensitive than the hemagglutination inhibition test and comparable to the neutralization test in sensitivity but more simple to perform than either the neutralization or the hemagglutination inhibition tests. An IgM capture ELISA for the determination of JEV specific porcine IgM was also utilized to determine current infection rates in pigs. The tests which do not involve the determination of specific IgM are better used for testing sentinel animals for providing clues as to the rate of transmission of JEV among pigs. IgM tests determining acute infection are less likely to be useful unless animals are tested very frequently or if a great number of animals are tested at any one time.
Current diagnosis of melioidosis is based on bacterial culture and/or serology which is becoming increasingly useful. An IgM-ELISA using heat-killed whole cells of Pseudomonas pseudomallei was developed and compared with an indirect haemagglutination technique (IHAT) and an indirect immunofluorescent technique(IFAT). The IgM-ELISA using a P:N ratio of > or = 2 had a sensitivity of 91% and a specificity of 96%. All 3 assays were further used in a seroepidemiological survey amongst different groups of patients and healthy individuals. It was found that the IFAT performed better than the IHAT, detecting antibodies to P. pseudomallei in 6% of diabetics, 5% of pyrexics, 8% of pregnant women and 3% of farmers. For the same groups the IgM-ELISA detected antibodies in 1% of pyrexics, 8% of pregnant women and a further 14% of farmers. The IgM-ELISA was found to be sensitive and useful for the serological diagnosis of acute melioidosis.
The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.
An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.
A comparative seroprevalence study on bovine trypanosomiasis and anaplasmosis was conducted. Sera of adult cattle and buffaloes of different breeds from farms from five different states in Malaysia were collected and tested for the presence of Trypanosoma evansi antibodies by CATT and Anaplasma marginale antibodies by c-ELISA. Of the 116 samples, 14.7% tested positive for bovine trypanosomiasis and 77.6% for bovine anaplasmosis.
IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.
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.
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.
The purpose of this study was to retrospectively detect Aspergillus antigens in sera obtained from immunocompromised host using an in-house modified double antibody sandwich ELISA.
Brugia malayi infection is endemic in several Asian countries. Filaria-specific IgG4 antibody detection based on BmR1 recombinant antigen has been shown to be sensitive and specific for the diagnosis of brugian filariasis. Two formats of the test has been reported ie indirect ELISA (BE) and rapid dipstick test (BR). Since different test formats use different amounts of sample and reagents which may affect its sensitivity and specificity, this study was performed to compare these two test formats in the detection of B. malayi. A total of 264 blinded serum samples from India and Malaysia were employed. Group 1 comprised 164 samples from actively infected individuals and group 2 comprised 100 samples from filaria non-endemic areas. Sensitivity was 96.3% (158/164) and 90.8% (149/164) for rapid test and ELISA respectively; chi-square p=0.00. Both test formats demonstrated 100% specificity. Therefore the rapid test format was equally specific but more sensitive than the ELISA format. The ELISA format would be able to demonstrate decline in IgG4 titer post-treatment while the rapid test would be very useful for screening and diagnosis in the field.
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.
In recent years, environmental concerns over ultra-trace levels of steroid estrogens concentrations in water samples have increased because of their adverse effects on human and animal life. Special attention to the analytical techniques used to quantify steroid estrogens in water samples is therefore increasingly important. The objective of this review was to present an overview of both instrumental and non-instrumental analytical techniques available for the determination of steroid estrogens in water samples, evidencing their respective potential advantages and limitations using the Need, Approach, Benefit, and Competition (NABC) approach. The analytical techniques highlighted in this review were instrumental and non-instrumental analytical techniques namely gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), enzyme-linked immuno sorbent assay (ELISA), radio immuno assay (RIA), yeast estrogen screen (YES) assay, and human breast cancer cell line proliferation (E-screen) assay. The complexity of water samples and their low estrogenic concentrations necessitates the use of highly sensitive instrumental analytical techniques (GC-MS and LC-MS) and non-instrumental analytical techniques (ELISA, RIA, YES assay and E-screen assay) to quantify steroid estrogens. Both instrumental and non-instrumental analytical techniques have their own advantages and limitations. However, the non-instrumental ELISA analytical techniques, thanks to its lower detection limit and simplicity, its rapidity and cost-effectiveness, currently appears to be the most reliable for determining steroid estrogens in water samples.
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.
Hendra virus (HeV) and Nipah virus (NiV) are the causative agents of emerging transboundary animal disease in pigs and horses. They also cause fatal disease in humans. NiV has a case fatality rate of 40 - 100%. In the initial NiV outbreak in Malaysia in 1999, about 1.1 million pigs had to be culled. The economic impact was estimated to be approximately US$450 million. Worldwide, HeV has caused more than 60 deaths in horses with 7 human cases and 4 deaths. Since the initial outbreak, HeV spillovers from Pteropus bats to horses and humans continue. This article presents a brief review on the currently available diagnostic methods for henipavirus infections, including advances achieved since the initial outbreak, and a gap analysis of areas needing improvement.
Accurate and timely diagnosis of dengue virus is important for early detection of dengue virus infection. In this study, the usefulness of the dengue NS1 antigen test was evaluated as a routine, rapid diagnostic test for dengue virus infection. A total of 208 sera from patients suspected of having dengue virus infection were collected and tested for dengue antibody, dengue genome and dengue NS1 antigen. Dengue antibody test, dengue PCR test and dengue antigen test were able to detect dengue virus infection from Days 1 to 8 in 72.8, 52.8 and 44.0% of samples, respectively. Of the 208 sera tested, 69.2% (144/208) of the acute sera were positive for dengue virus infection based on IgM antibody, IgG antibody, NS1 antigen and PCR tests. Thirty-two point two percent of the samples (67/208) were found positive for dengue NS1 antigen, 38.5% (80/208) were PCR positive, 40.9% (85/208) were IgM positive and 36.1% (75/208) were IgG positive for dengue virus. The results reveal the detection rate of dengue virus infection was similar for PCR and dengue antibody (65.9%) and for NS1 antigen and dengue antibody (62.0%) combinations. Therefore, the dengue NS1 antigen test can be used to complement the current antibody test used in peripheral laboratories. Thus, the combination of the NS1 antigen and antibody tests could increase the diagnostic efficiency for early diagnosis of dengue infection.