The concentration of plasma sialic acid was estimated using the modified chemical method and the more sensitive enzymatic method in 20 subjects with impaired glucose tolerance and 20 control subjects. The mean sialic acid concentration values of the control subjects and subjects with impaired glucose tolerance using the enzymatic method were 1.747 +/- 0.047 and 2.583 +/- 0.070 mmole/l and 1.753 +/- 0.067 and 2.591 +/- 1.02 mmole/l for the chemical method. The intra-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 1.963% and 1.583%, respectively, for the enzymatic assay and 2.728% and 2.431%, respectively, for the chemical assay. The inter-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 2.686% and 2.723% for the enzymatic assay, and 3.819% and 3.95% for the chemical assay. Since the values do not differ significantly, the chemical assay is a cost effective method that can be used in large epidemiological studies.
Three MAbs 1C4.2D8, 1C4.2C4 and 1C4.1F5 were produced using sonicated adult worm antigens of Angiostrongylus malaysiensis and they were found to be secreters of IgG1. The MAbs 1C4.2C4 and 1C4.2D8 were found to react with antigens of A. malaysiensis and cross-react with the closely related A. cantonensis but not with other helminths. A total of 108 human sera collected from Orang Asli (aborigenes) from Grik, in the State of Perak were tested for A. malaysiensis infection using the MAb-ELISA. MAb 1C4.1F5 and 25 (23%) were positive. Twenty of these positive samples were tested with the MAb 1C4.2D8 and none was found to be positive.
A dot-blot ELISA was compared with a previously performed sandwich ELISA for the detection of Parastrongylus cantonensis antigens in sera from patients. Using the same monoclonal antibody and the same sera, 6 of 10 sera (60%) from parastronglyiasis patients were positive in dot-blot ELISA, whereas with sandwich ELISA, 5 of the same patient sera (50%) were positive. The specificity in both assays was 100% using 50 sera from patients with other parasitic diseases; of these, 10 each were from patients with cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 sera from normal health Thais and Malaysians. The sensitivity of the assays was, however, slightly better with dot-blot ELISA and because it is simple, quick and cost-effective, it may be a test of choice for specific diagnosis of human parastrongyliasis.
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.
A specific monoclonal antibody (AW-3C2) as revealed by ELISA was produced against the adult worm antigens of Parastrongylus cantonensis and used in a sandwich ELISA for the detection of circulating antigens in the sera of parastrongyliasis patients and those with other parasitic diseases. A total of 60 sera was used in this study. Of these, 10 each were from patients with parastrongyliasis, cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 serum samples from normal healthy Thais and Malaysians. The mean +/- optical density (OD) values for the normal Thai and Malaysian groups were 0.126 +/- 0.028 and 0.124 +/- 0.029, respectively. The mean OD values of the parastrongyliasis patient group differed significantly from that of the normal groups as well as those of other parasitic infections. Using a cut-off point of OD +/- 3SD of the control groups as indicating a positive reading, the specificity of the assay with this monoclonal antibody was 100% while the sensitivity was 50%.
Two monoclonal antibodies (MAbs), one produced against Plasmodium falciparum (PF-IG8) and the other against P. cynomolgi (PC-IE12) schizont antigens were used in a sandwich ELISA for the detection of circulating plasmodial antigens in sera of patients infected with either P. falciparum, P. vivax or P. malariae. The mean +/- SD optical density (OD) values for the normal control group using PF-108 and PC-1E12 were 0.351 +/- 0.036 and 0.205 +/- 0.044, respectively. Mean OD values for the three infected groups were found to be significantly higher than those of the normal control group for both MAbs. However, ELISA values for individual serum specimens did not correlate with the level of parasitemia in the infected blood. Using a cut-off point of mean OD +/- 3 SD of the normal control group as indicating a positive reading, the specificity of this assay with both MAbs was 100%. The sensitivity of the assay using PF-1G8 was 95% while that obtained with PC-1E12 was 98%.
The highly sensitive AFRIMS format IgM capture ELISA for the diagnosis of dengue virus infections requires the use of mouse brain derived hemagglutinins and consequently also the use of 20% acetone extracted normal human serum to eliminate high background. These reagents are not always easily available and we have thus compared the AFRIMS format with another published format which uses cell culture derived antigens (culture fluid, CF, format) in order to determine if it is reasonable to use cell culture derived antigens in situations where hemagglutinins and normal human serum are difficult to obtain. The study shows that using AFRIMS results as the reference point, the CF format described here has a sensitivity of 90% and a specificity of 96%.
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.
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.
House crows (Corvus splendens) in Selangor, Malaysia were examined for the presence of Campylobacter species, Salmonella species, Mycoplasma gallisepticum and Mycoplasma synoviae by serology, culture and pcr. For the detection of Campylobacter and Salmonella species swabs were taken either from the intestine or cloaca. For the detection of mycoplasmas, swabs were taken either from the choanal cleft or trachea for culture and pcr and serum samples were tested by the rapid serum agglutination (rsa) and monoclonal antibody-blocking elisa (mbelisa) for antibodies to M gallisepticum and M synoviae. For campylobacter, 25.3 per cent of the crows were positive by culture, and the species identified were Campylobacter jejuni and Campylobacter coli. No Salmonella species were isolated. Four of 24 swabs were positive for M gallisepticum dna but none gave positive results for M synoviae dna. No M gallisepticum or M synoviae antibodies were detected by rsa but 60 per cent of the sera gave positive reactions for M gallisepticum and 13 per cent gave positive reactions for M synoviae by mbelisa.
Dengue infection is a major public health problem affecting millions of people living in tropical countries. With no suitable vaccines and specific antiviral drugs, treatment for dengue is usually symptomatic and supportive. Early diagnosis and recognition of severe disease is therefore crucial for better management of the patient. Two-dimension electrophoresis was used to identify disease-associated proteins that can be used for diagnosis and as drug targets for treatment. Two markers, identified by mass spectrometry analysis as alpha1-antitrypsin and NS1 proteins were found to be upregulated in dengue fever (DF; n=10) and dengue haemorrhagic fever (DHF; n=10) patients compared with healthy individuals (n=8). Both alpha1-antitrypsin and NS1 proteins were overexpressed two-fold in DHF patients compared with DF patients. Our study suggests that alpha1-antitrypsin and NS1 protein could be used as biomarkers as early indicators of DHF risk among patients with suspected dengue infection.
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.
Identification of pregnant women infected with Chlamydia trachomatis is essential to allow early antibiotic treatment in order to prevent adverse pregnancy outcomes. In this study, two nucleic acid amplification tests (NAAT) namely nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA) were evaluated in terms of sensitivity and specificity for the detection of C. trachomatis DNA in pregnant women with preterm complications. A cross-sectional study was carried out in two public hospitals in Southern Selangor, Malaysia. Endocervical swabs obtained were subjected to DNA amplification using nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA). A total of 83 endocervical swabs obtained from pregnant women of less than 37 weeks gestation and presented with preterm complications were subjected to chlamydial DNA detection using both assays. The study shows that Amplicor CT/NG assay is more effective in the detection of C. trachomatis DNA from endocervical swabs compared to Biosewoom nested PCR kit. Agreement between the two assays were poor (kappa=0.094) with nested PCR showing a low sensitivity of 10.81% and a 97.83% specificity when compared to Amplicor CT/NG. The results obtained indicated that BioSewoom nested PCR was less sensitive than Amplicor CT/ NG for detecting C. trachomatis in endocervical specimens and that another more reliable test is required for confirmatory result.
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.
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.
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.
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 detection of leptospires in patient blood in the first week of the disease using PCR provides an early diagnostic tool. PCR using two sets of primers (G1/G2 and B64-I/B64-II) tested with samples seeded with 23 leptospiral strains from pathogenic and non-pathogenic strains was able to amplify leptospiral DNA from pathogenic strains only. Of the 39 antibody negative samples collected from patients suspected for leptospirosis, only 1 sample (2.6%) was PCR positive. Using LSSP-PCR, the G2 primers allowed the characterization of Leptopira species to 10 different genetic signatures which may have epidemiological value in determining species involved in outbreaks. Leptospiral outer membrane proteins from three strains were purified and reacted against patients sera and gave rise to different profiles for pathogenic and non-pathogenic strains. Lymphocytes of mice injected with OMPs proliferated and released IFN(-3) when stimulated in vitro using Leptospira OMP as antigens. This suggests that an immune response could be established using leptospiral OMPs as a putative vaccine. OMPs were also used in a Dot-ELISA to detect antibodies against Leptospira pathogens in humans.