A supersensitive ELISA was developed for measurement of thyroid-stimulating hormone (TSH) concentrations in serum using in-house rabbit polyclonal antisera and a commercial monoclonal antibody. The assay was optimised and validated by recovery, linearity and cross-reactivity experiments and further compared to other available assays and EQAS samples. Good precision was obtained with a working assay range of 0.2 to 100 mIU/L with < 10% coefficient of variation (CV) for both intra and interassay. The assay is highly sensitive and specific with a minimum detectable limit of 0.07 mIU/L and negligible cross-reactivities against LH, FSH, HCG and other pituitary peptides. Good correlations were obtained when compared to Abbott hTSH EIA (r = 0.993; p < 0.001; n = 85) and NETRIA IRMA (r + 0.995; p < 0.001; n = 76). The normal reference range established was 0.4 to 4.0 mIU/L (n = 76). TSH levels in serum of thyrotoxic patients (n = 83) were significantly lower (0.07 to 0.20 mIU/L, p < 0.0001) and completely distinct from normal values thereby obviating the requirement of a TRH-stimulation test. Stability studies showed that coated wells can be stored at 4 degrees C for at least 2 months. This highly sensitive in-house hTSH ELISA which is cheap, stable and readily available is useful for diagnosis and management of patients with various thyroid disorders.
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 study to determine the sensitivity and specificity of the Directogen Group A Streptococcal Test was carried out in a primary care clinic in University Hospital, Kuala Lumpur. The study also looked at the prevalence of streptococcal pharyngitis in the clinic. We found that the rapid test has a sensitivity of 90.9% and a specificity of 90.7%. Viral pharyngitis is the commonest form (63%) of pharyngitis followed by Group A Beta haemolytic streptococcal pharyngitis (14.2%). The presence of exudates, tonsillar enlargement and the presence of tender cervical lymph nodes have a significantly higher association with streptococcal pharyngitis.
Study site: Primary care clinic, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia
Classifications of adult low energy nutritional status based on the Quetelet or body mass index (weight/height2) have often assumed independence of age and sex. This paper reports findings of a study of 447 men and 564 women aged over 18 years and belonging to the Iban tribe of central Sarawak, East Malaysia. BMI and fat-free mass fell markedly in both sexes, and fat mass in women but not men, after about 40 years of age. In men over age 40, and women aged 18-40, BMI was sensitive to reported morbidity. For subjects aged over 40 years, BMI was related to morbidity independently of age effects in men, and to age alone in women. These findings suggest that the functional significance of low BMI differs between the sexes and with age.
Data are presented for 2382 children investigated for fever in a Malaysian hospital between 1984 and 1987 when Widal tests and blood cultures were a routine part of every fever screen. There were 145 children who were culture positive (TYP-CP) for Salmonella typhi, while 166 were culture negative but were diagnosed as having typhoid (TYP-CN). Analyses of the sensitivity and specificity of combinations of initial Widal titres in predicting a positive S. typhi culture in a febrile child (culture positive vs the rest) showed the best model to be an O- and/or H-titre of > or = 1 in 40 (sensitivity 89%; specificity 89%). While the negative predictive value of the model was high (99.2%) the positive predictive value remained below 50% even for very high titres of O and H (> 1 in 640), at which point the specificity was 98.5%, supporting the clinical view that a high proportion of the TYP-CN patients really were typhoid but were missed by culture. The TYP-CN patients showed a very similar clinical and age profile to TYP-CP patients. The length of history of fever did not affect the initial Widal titre in culture positive cases. The Widal test in children remains a sensitive and specific 'fever screen' for typhoid although it will not identify all cases. In children, lower cut-off points for O- and H-titres should be used than are generally recommended.
An indirect ELISA was used to detect antibodies against outer membrane protein preparations (OMPs) from Salmonella typhi. Sera from patients with a definitive diagnosis of typhoid fever (TF) gave a mean absorbance reading, at 414 nm, of 1.52 +/- 0.23 as compared to 0.30 +/- 0.11 for sera from healthy individuals. This gave a positive to negative ratio of absorbance readings of approximately 5.1. Suspected TF patients (no isolation of S. typhi), with positive and negative Widal titers had mean absorbance readings of 1.282 +/00.46 and 0.25 +/- 0.19, respectively. Sera from patients with leptospirosis, rickettsial typhus, dengue fever, and other infections gave mean absorbances of 0.20 +/- 0.08, 0.24 +/- 0.08, 0.27 +/- 0.08, and 0.31 +/- 0.16, respectively. The sensitivity, specificity, positive and negative predictive values were 100%, 94%, 80% and 100%, respectively. The antibody response detected in the definitive TF cases was predominantly IgG in nature and no cross-reactivity was seen with OMP preparations extracted from E. coli. Variable reactivity was noted with OMP preparations obtained from other Salmonella spp. Three major OMPs are presented in the antigen preparation and strong binding of positive sera was detected to all three bands.
Our experience with 1,094 consecutive fine needle aspirations (FNAs) of the breast in 918 cases is presented. Correlation between the FNA cytology and subsequent histology of the lesions was available in 211 cases. FNA had a sensitivity rate of 87.3%, a specificity rate of 99.3% and a positive predictive value of 98.2%. A false negative diagnosis rate of 5.1% occurred. The overall diagnostic accuracy rate was 95.7%.
A simple, non-isotopic in-house enzyme-linked immunoabsorbant assay (ELISA) for human growth hormone (GH) was developed. The assay involved using in-house polyclonal anti-GH adsorbed onto 96-well microtitre plates, commercially prepared mouse monoclonal anti-GH, and goat anti-mouse IgG horseradish peroxidase detection system. Results of recovery and parallelism studies ranged from 95%-106% and 98%-101% respectively, of the expected values. The detection limit of the assay was 0.008 mIU/well or the equivalent to 0.4 mIU/L of undiluted serum. Intra- and interassay coefficients of variations were 4.8%-7.9% and 6.5%-8.7% respectively. Serum GH levels measured in this assay correlated well with those measured in established in-house radioimmunoassays (r = 0.985, p < 0.001) and immunoradiometric assay from NETRIA (r = 0.984, p < 0.001).
An indirect enzyme linked immunosorbent assay (ELISA) was developed for the diagnosis of allergy to a house dust mite, Dermatophagoides farinae. The efficacy of the ELISA was then evaluated against a prick test using a commercial allergen. Eighty five suspected allergic rhinitis patients from the Otorhinolaryngology Department, Kuala Lumpur General Hospital, were tested with the ELISA and prick test. Prick test and ELISA results were positive in 84.7% and 80.0% of the patients respectively. The ELISA was found to have 87.5% sensitivity, 61.5% specificity, 92.6% positive predictive value, 47.1% negative predictive value, 7.4% false positive and 52.9% false negative. There was total agreement between the prick test and ELISA for prick test grades of 3+ and 4+. It is concluded that the ELISA is a useful assay for detection of individuals who are highly sensitive to D. farinae.
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.
The changes in serum prealbumin (transthyretin) and serum albumin in acute and chronic liver diseases were investigated. Albumin has long been used as a useful indicator of liver function but serum prealbumin has recently been noted for its clinical significance in acute liver diseases. Serum prealbumin concentrations and liver function tests (albumin, bilirubin, alanine aminotransferase) were determined on blood obtained from normal donors (n = 148) and from patients suffering from liver diseases (n = 78) such as acute viral hepatitis, chronic active hepatitis, cirrhosis and hepatoma. The mean serum prealbumin concentration in normal subjects was 29.6 +/- 4.82 mg/dl while the mean serum prealbumin concentration in patients with liver disease was greatly reduced (acute viral hepatitis = 15.3 +/- 7.4mg/dl; chronic active hepatitis = 10.2 +/- 6.6mg/dl; cirrhosis = 9.9 +/- 6.4mg/dl and hepatoma = 10.7 +/- 4.2). Albumin concentrations dropped slightly (13% compared to control) in acute viral hepatitis but dropped markedly (28% compared to control) in chronic liver diseases. The study suggests that serum prealbumin concentration might be a more sensitive indicator than albumin in assessing liver dysfunction in acute liver diseases.
In Peninsular Malaysia, only Wuchereria bancrofti and Brugia malayi are reported to cause human filariasis. Brugia pahangi infects many of the same animal hosts as the zoonotically transmitted subperiodic B. malayi. There is a well-recognized need for improved diagnostic techniques for lymphatic filariasis. Parasite antigen detection is a promising new approach, and it will probably prove to be more sensitive and specific than clinical, microscopic and antibody-based serological methods. We recently generated monoclonal antibodies (MAb XC3) from in vitro culture products of adult B. pahangi (B.p. IVP). Filarial antigenemia was quantitated in various hosts including the sera from 6 Malaysian Aborigines with acute lymphatic filariasis. In hosts infected with brugian filariasis and dirofilariasis, antigenemia was scored ranging from 90 ng/ml to 960 ng/ml. None of the control animal and human sera had antigenemia above 90 ng/ml. In addition, MAb XC3 and B.p. IVP were applied in several seroepidemiological surveys among household cats in Kuala Selangor in order to correlate information gathered for future studies of possible cases of human infection. Out of the 81 cats surveyed, 10 (12.35%) and 5 (6.17%) were parasitologically positive for B. pahangi and B. malayi, respectively. However, 21 (25.92%) were antigenemia positive when serologically investigated with MAb XC3. Antifilarial antibodies to B.p. IVP by direct ELISA showed very high cross-reactivity with non-filarial gut worm infections. 16 (19.75%) cats had reciprocal titers ranging from 320 to 2,560. Only 1 (1.23%) cat from this group was antigenemic.
The VP4 genetic groups of 151 field strains of human rotaviruses obtained from infants and young children with diarrhea from four locations in Malaysia were analyzed. The strains were adapted to growth in tissue culture and studied further by molecular hybridization of northern blotted RNA to PCR-generated cDNA probes representing amino acids 84-180 of the KU strain VP4, 83-181 of the DS-1 strain VP4, and 83-180 of either the 1076 or K8 strain VP4, representing VP4 genetic groups 1-4 (P1A, P1B, P2, and P3), respectively. The majority (79% of the field strains hybridized with the KU VP4 genetic group 1 probe and were associated with G1, G3, G4, untypable, or mixed G serotypes. VP4 genetic group 1 (P1A) strains were the most common in all locations in Malaysia between 1978-1988. Three strains which exhibited G3 and subgroup I specificity hybridized with the K8 VP4 genetic group 4 probe. These three VP4 genetic group 4 (P3) strains were detected in two different years and locations, extending the initial detection of this VP4 genetic group (the K8 strain) in Japan to a larger geographical area of Asia.
The BamHI Z EBV replication activator (ZEBRA) protein is involved in the switch from latency to productive cycle of Epstein-Barr virus. A recombinant ZEBRA protein was synthesized and assessed in enzyme-linked immunosorbent assay (ELISA) for serum IgG response in nasopharyngeal carcinoma (NPC) patients. In 100 NPC serum samples that were positive for IgA to the EBV viral capsid antigen (VCA), 75% had IgG anti-ZEBRA antibodies. In contrast, only 3/83 (3.6%) serum samples from healthy donors and 2/50 (4%) from other cancers were positive for IgG to ZEBRA. Interestingly, in a selected group of 100 NPC sera negative for IgA to VCA, 25% contained IgG anti-ZEBRA antibodies. This suggests that the ELISA for IgG anti-ZEBRA may also identify earlier cases of NPC not detected by the conventional immunofluorescence test for IgA to VCA.
This is a study of 13 Malaysian patients with clinically definite Multiple sclerosis (MS) subjected to a hot bath test with VEPs, BAEPs, median nerve SSEPs before and after heating. Five patients (38%) developed neurological changes with the rise in body temperature. There was an average of 0.46 new sign per patient. Four patients had motor disturbances attributed mainly to aggravation of spinal cord dysfunction. Two patients had additional visual deterioration, one patient has associated VEP change. This study shows that though Uhthoff's phenomenon has not been noted in the reports of Asian MS patients, when subjected to rigorous testing, Asian MS patients also show sensitivity to body temperature change. However, the percentage of positivity of the hot bath test is much lower than that reported for Caucasians. Thus this study shows further differences between Asian and Caucasian MS patients.
The aim of this study was to optimize the conditions for the passive adsorption of polyclonal antibody onto plain surface polystyrene latex particles and its performance in a slide latex agglutination test for rotavirus antigen detection. Cleaning of latex particles by washing through repetitive centrifuging, decanting and resuspending in distilled water was adequate in removing surfactants from the particles' surfaces to enable coating. A study of antibody concentration, incubation temperature and buffer pH revealed that optimum coating was achieved with a 3-fold excess of antibody to the calculated total particle surface capacity for the antibody in a glycine-saline buffer of pH 9.2 at 40 degrees C for 4 hours. The ionic strength and pH of the latex suspending buffer and the sample buffer were critical factors determining the sensitivity of the test and the appearance of non-specific agglutination. Ultrasonication, addition of glycerol and Tween 20, either individually or in combination, were able to suppress non-specific agglutination in some batches of latex reagents. Polyethylene glycol 6000 enhanced the quality of agglutination as well as reduced the time of its appearance, especially in reagents that produced poor agglutination.
Dengue viruses pose a considerable global public health problem with an estimated 100 million cases of illness every year. This illustrates the need for rapid and reliable diagnostic methods for proper patient management and disease control. Currently, laboratory diagnosis depends on serology or virus isolation, with both methods having certain drawbacks. Alternatively, reverse transcription and polymerase chain reaction (RT-PCR) offers the potential for the rapid, highly sensitive and specific detection of dengue viruses. Since we occasionally encounter the problem of insufficient amounts of patient serum for the direct detection of dengue viruses, a method was developed for the extraction of viral RNA after biological amplification in mosquito larvae. Using this method, 15 of 19 clinical samples tested were correctly identified using RT-PCR.
Two commercial serological kits, Pylori-set (Orion Diagnostica, Finland) and Helico-G (Cambridge Biomedical Ltd, UK), and an in-house ELISA were evaluated with sera from 24 Helicobacter pylori-positive and 146 H. pylori-negative dyspeptic patients. Sensitivity, specificity, positive and negative predictive values of Pylori-set were lower than that of Helico-G and in-house ELISA. Helico-G was more sensitive (91.7%) than in-house ELISA (83.3%) and both had comparable negative predictive values of 98.3% and 97.3%, respectively. However, specificity (97.9%) and positive predictive value (86.9%) of an in-house ELISA were much higher than specificity (80.1%) and positive predictive value (43.1%) of Helico-G. Kappa index of agreement between the three serological tests (Pylori-set, Helico-G or in-house ELISA) and the presence of H. pylori in antral biopsies was very low (k = 0.13; z = 1.9; p > 0.05), moderate (k = 0.49; z = 7.1; p < 0.0001), or substantial (k = 0.82; z = 10.8; p < 0.0001), respectively. Overall, statistical evaluations demonstrated that both commercial kits were not as reliable as the in-house ELISA for serodiagnosing H. pylori infection.