Typhoid fever remains a common problem in Malaysia, but for its diagnosis both blood culture and the Widal test have drawbacks. A dot enzyme immunoassay (EIA) has been developed which detects IgM and IgG antibodies to a specific 50 kDa outer membrane protein on Salmonella typhi. This study was performed among outpatients attending the university hospital in Kelantan, a state on the east coast of Peninsular Malaysia where typhoid is endemic. The dot EIA was done on 149 outpatients of all ages in whom typhoid was suspected. Of these, 60 were not analysable due to insufficient data. The other 89 were retrospectively classed as typhoid (total = 21), or not typhoid (total = 68). The criteria for diagnosis of typhoid was either, blood culture was positive, or with blood culture negative, temperature was at least 38 degrees C and Widal O and/or H titer greater than or equal to 1/160. We then compared the diagnosis with the EIA result. For the result where either IgM or IgG was positive, sensitivity was 90%, specificity 91% and negative predictive value 97%. For IgM positive, specificity was 100%. But the specificity of IgG positive alone was reduced by six false positives, which were probably due to persistence of IgG after acute infection. Other cases were found where IgG positive alone appeared in the first week of typhoid fever, probably due to rapid response in a second or subsequent infection. We also found that IgM-producing patients were significantly younger than those showing IgG alone positive.
Study site: Community Medicine clinic, Accident & emergency department, Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia
Surface plasmon resonance (SPR) is a medical diagnosis technique with high sensitivity and specificity. In this research, a new method based on SPR is proposed for rapid, 10-minute detection of the anti-dengue virus in human serum samples. This novel technique, known as rapid immunoglobulin M (IgM)-based dengue diagnostic test, can be utilized quickly and easily at the point of care. Four dengue virus serotypes were used as ligands on a biochip. According to the results, a serum volume of only 1 μl from a dengue patient (as a minimized volume) is required to indicate SPR angle variation to determine the ratio of each dengue serotype in samples with 83-93% sensitivity and 100% specificity.
Brucellosis is a zoonotic disease which can be transmitted by direct or indirect contact with infected animal or their products. It is an important public health problem but little is known on brucellosis in the Malaysian population. The aim of this study was to determine the presence of Brucella antibodies using commercial Brucella IgG and IgM ELISA kits (Vircell, SL, Barcelona Spain). A total of 184 sera from suspected patients were received from 16 hospitals in Malaysia over the years 2004 to 2009. Only 10 serum samples (5.4%) were positive for Brucella antibodies in which 5 showed the presence of both IgM and IgG. Most of the positive patients were occupationally involved with animals. This study suggests the seroprevalance of brucellosis among individuals who have contact with infected animals in Malaysia is low.
BACKGROUND: Dengue has been recognized as a potential hazard to tourists. A prospective, controlled study in the outpatient clinic of a German infectious disease clinic was conducted to assess the prevalence of dengue virus infection among international travelers.
METHODS: Serum samples from 130 patients with signs or recent history clinically compatible with dengue (fever, headache, muscle and joint pain, or rash), 95 matched controls with diarrhea, and 26 patients who never visited a country endemic for dengue were investigated.
RESULTS: Nine (6.9%) of the 130 patients with compatible symptoms and 1 (1%) of the 95 controls with diarrhea developed rising antibody titers against dengue virus. Of these 10 patients with probable dengue infection, 6 had been to Thailand, 2 to Malaysia, and 1 each to Indonesia and Brazil.
CONCLUSIONS: Infection with dengue virus appears to be a realistic threat to travelers to Southeast Asia. Symptoms commonly associated with dengue, such as fever, myalgia, arthralgia, and vomiting, can be helpful for diagnosis when present, but the absence of typical symptoms does not exclude infection.
The object of this study was to determine the effect of prepartum supplementation of vitamin E with or without injective vitamin E and selenium (Se) on productive and reproductive performances and immune function in dairy cows. Sixty multiparous Holstein dairy cows were divided randomly into three groups at the end of gestation. Cows in each group received one of three treatments: (1) a single intramuscular (im) injection of vit. E + selenium 3 weeks prepartum; (2) daily supplementation of oral vit. E given from 3 weeks prepartum to parturition; (3) injective vit. E + Se with daily supplementation of oral vit. E. Blood samples were collected from cows at calving and from calves at 0 and 7 days of age. Concentration of IgG in serum of cows and calves as well as in colostrum was determined. No significant differences among treatments occurred in the concentrations of IgG, animal, and calf production and reproduction performance. Due to the lack of significant difference between injection and oral supplementation, it is recommended to replace the injection with oral supplementation.
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.
The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5(+)/6(+)) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients.
Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.
Cystic echinococcosis (CE) caused by infection with Echinococcus granulosus is of major concern for humans in many parts of the world. Antigen B was prepared from E. granulosus hydatid fluid, and Western blots confirmed eight batches showing a band corresponding to the 8-/12-kDa subunit with positive serum and no low-molecular mass band (< 15 kDa) with negative serum. The batches were pooled and used to prepare lateral flow immunoglobulin G4 (IgG4) and IgG dipsticks. Diagnostic sensitivity was determined using serum samples from 21 hydatidosis patients, and diagnostic specificity was established using sera from 17 individuals infected with other parasites and 15 healthy people. IgG4 dipstick had a diagnostic sensitivity of 95% (20 of 21) and a specificity of 100% (32 of 32). The IgG dipstick had a sensitivity of 100% (21 of 21) and a specificity of 87.5% (28 of 32). Thus, both IgG and IgG4 dipsticks had high sensitivities, but IgG4 had greater specificity for the diagnosis of human CE.
Toxoplasma gondii is a protozoan parasite that can infect all mammals, serving as intermediate hosts. The cause of congenital toxoplasmosis is transplacental transmission of the parasite to the foetus, resulting in wide range of manifestations from mild chorioretinitis to miscarriage. Its frequency can be reduced by early screening of pregnant women which is based mainly on tests for anti-Toxoplasma antibodies. We collected serum samples of 594 pregnant women (subjects) after taking their consent over a period of two years (2016-2018) and analyzed them for anti-Toxoplasma IgG by ELISA. The positive samples were then analyzed for IgG avidity test which could differentiate between recent and past infections. The seroprevalence was also correlated with the age of the subjects and their contact with cats. 162 subjects were found positive out of which only three showed a recent infection. After following up until delivery, one of them delivered a baby who had jaundice and was diagnosed with anti-Toxoplasma IgM at birth. The foetus of the second subject died in-utero, while the third woman delivered a normal baby after being given spiramycin when diagnosed with toxoplasmosis in the first trimester. It was found that most of the positive subjects had frequent contact with cats. Invasion of the parasite during third trimester resulted in death in-utero and jaundice. Most common cause of pregnancy wastage during our study was spontaneous abortions while pregnancy loss due to congenital anomalies was rare.
Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii that is prevalent in humans and animals. This study was aimed to determine the seroprevalence of T. gondii infection among hemato-oncology patients and its association with sociodemographic and behavioural characteristics. This cross-sectional study was conducted at the Hospital Universiti Sains Malaysia (USM) involving 56 blood samples from hemato-oncology patients. Anti-T. gondii IgG and IgM antibodies and IgG avidity were determined using enzyme-linked immunosorbent assays (ELISA). The association of T. gondii exposure, sociodemographic, and behavioural characteristics were assessed by a questionnaire and face-to-face interviews. Twenty-eight (50%) patients were seropositive for T. gondii antibodies, where 27 (48.21%) patients were IgG+/IgM- and one patient (1.79%) was IgG+/IgM+ with high avidity index, indicating infection of more than 20 weeks. A univariate analysis showed that age, gender, ethnicity, marital status, educational level, employment status, stem cell transplant, blood transfusion, close contact with cats, water supply, and consumption of undercooked meat were not significantly associated with Toxoplasma seropositivity (p < 0.05). Our study has demonstrated, for the first time, the serological evidence of T. gondii exposure among hemato-oncology patients in Hospital USM. Our findings indicated that latent toxoplasmosis was relatively prevalence among our patients. Therefore, serological screening tests should be considered for immunocompromised patients as well as the implementation of health education programmes to encourage a healthy lifestyle and the consumption of healthy food among them.
Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) are mosquito-borne flavivirus of medical importance in tropical countries such as Malaysia. However, much remains unknown regarding their prevalence among the underserved indigenous people (Orang Asli) living in communities in the forest fringe areas of Peninsular Malaysia. Information on the prevalence of diseases is necessary to elevate the effectiveness of disease control and preventive measures. This study aimed to determine the seroprevalence of the three major flaviviruses among the Orang Asli and investigate the association between demographic factors and seropositivities. Sampling activities were conducted in the Orang Asli villages to obtain serum samples and demographic data from consenting volunteers. The presence of DENV, JEV, and ZIKV immunoglobulin G (IgG) antibodies in the sera were examined using commercial enzyme-linked immunosorbent assay kits. A focus reduction neutralization assay was performed to measure virus-specific neutralizing antibodies. A total of 872 serum samples were obtained from the Orang Asli volunteers. Serological assay results revealed that DENV IgG, JEV IgG, and ZIKV IgG seropositivities among the Orang Asli were at 4.9%, 48.4%, and 13.2%, respectively. Neutralizing antibodies (FRNT50 ≥ 1:40) against JEV and ZIKV were found in 86.7% and 100.0%, respectively, out of the samples tested. Positive serology to all three viruses corresponded significantly to the age of the volunteers with increasing seropositivity in older volunteers. Findings from the study suggest that Orang Asli are at significant risk of contracting JEV and ZIKV infections despite the lack of active transmission of the viruses in the country.
A commercially available enzyme-linked immunosorbent assay (ELISA) (PanBio Dengue Screening ELISA) that utilized both immunoglobulin M (IgM) and IgG capture in the same microtiter well for the diagnosis of dengue infection was evaluated. Sensitivity in primary and secondary dengue was 95%, while specificity was 94%.
BACKGROUND: Chlamydia pneumoniae, a bacterium that causes respiratory infections, is probably under-diagnosed. There is also interest in its possible role in the aetiology of coronary heart disease. This is the first population-based seroprevalence survey of C. pneumoniae infection in Singapore.
METHODS: A random sample of 1,068 people aged 18-69 years was selected from the participants of the Singapore National Health Survey conducted in 1998. Sera and data on certain clinical measurements and conditions had been collected. IgG antibodies for C. pneumoniae were detected using an indirect microimmunofluorescence test and positivity graded. Seropositivity was defined as IgG titre >/=1:16.
RESULTS: There were no statistically significant differences in the prevalence rates of seropositivity to C. pneumoniae for age group 18-69 years among the three ethnic groups, i.e. Chinese (males 76.7%, females 68.3%), Malays (males 75.4%, females 59.1%), and Asian Indians (males 74.6%, females 59.4%). The seropositivity rate for people aged 18-69 years in Singapore was 75.0% for males and 65.5% for females (difference of 9.5%, P < 0.001). In both genders combined, seropositivity increased from 46.5% in the age group 18-29 to reach a plateau of 78.9% in the age group 40-49, which remained stable to 60-69 years. There was no association of seropositivity with smoking, diabetes mellitus, hypertension or body mass index after adjustment for age and gender.
CONCLUSION: The high prevalence rates in our study population and the higher rate in males compared to females are consistent with studies from other parts of the world. No significant difference in prevalence rates was observed among Chinese, Malays and Indians. The pattern of rising and levelling off of seropositivity with age suggests that C. pneumoniae infection occurs early in life, and in older ages the high level of seropositivity is probably maintained by re-infections or chronic infections. Chlamydia pneumoniae infection was not found to be associated with the cardiovascular risk factors examined.
There is still substantial uncertainty concerning the association between Chlamydia pneumoniae and ischemic heart disease. This may partly be explained by the adjustment for potential confounders in different population studies. This is the first study in Singapore to look at the association of C. pneumoniae seropositivity with ischemic heart disease in a multivariate analysis. A random sample of 714 persons aged between 35 and 69 years was selected from the participants of the Singapore National Health Survey conducted in 1998. Data on clinical measurements and conditions were collected using biochemical tests and interviewer-based questionnaires. Ischemic heart disease was defined by the Rose questionnaire and included history suggestive of angina and/or myocardial infarction. Immunoglobulin G antibodies for C. pneumoniae were detected using an indirect microimmunofluorescence test, and seropositivity was defined as IgG titers > or = 1:16. There were no statistically significant differences in the prevalence rates of seropositivity to C. pneumoniae among the three ethnic groups, that is, Chinese (80.4%), Malays (74.0%), and Asian Indians (73.2%). There was no association between seropositivity and ischemic heart disease after adjustment for age alone (OR 1.00, 95% CI 0.54-1.83) or for age, sex, and other risk factors of atherosclerosis (OR 0.99, 95% CI 0.53-1.84). C. pneumoniae Immunoglobulin G seropositivity was not associated with an increased risk of ischemic heart disease as defined by the Rose angina questionnaire in Singapore.
Toxoplasma gondii is an important human pathogen with a worldwide distribution. It is primarily of medical importance for pregnant women and immunocompromised patients. Primary infection of the former is often associated with fetal infection, which can lead to abortion or severe neonatal malformation. Immunocompromised patients are at risk of contracting the severe form of the disease that may be fatal. Thus, detection of T. gondii infection with high sensitivity and specificity is crucial in the management of the disease. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to toxoplasma antigens in the patient's serum sample. Most of the commercially available tests use T. gondii native antigens and display wide variations in test accuracy. Recombinant antigens have great potential as diagnostic reagents for use in assays to detect toxoplasmosis. Thus in this review, we address recent advances in the use of Toxoplasma recombinant proteins for serodiagnosis of toxoplasmosis.
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.
INTRODUCTION: The aim of this report is to establish an accurate diagnosis of acute dengue virus infection early, in order to provide timely information for the management of patients and early public health control of dengue outbreak.
METHODS: 224 serum samples from patients with a clinical diagnosis of acute dengue infection, which were subsequently confirmed by laboratory tests, were used to evaluate the performance of a commercially-available dengue NS1 antigen-capture ELISA kit.
RESULTS: The dengue NS1 antigen-capture ELISA gave an overall sensitivity rate of 93.3 percent (209/224). The sensitivity rate was significantly higher in acute primary dengue (97.4 percent) than in acute secondary dengue (68.8 percent). In comparison, the virus isolation gave an overall positive isolation rate of 64.7 percent, with a positive rate of 70.8 percent and 28.1 percent, for acute primary dengue and acute secondary dengue, respectively. Molecular detection of dengue RNA by RT-PCR gave an overall positive detection rate of 63.4 percent, with a positive rate of 62.5 percent and 68.8 percent, for acute primary dengue and acute secondary dengue, respectively. Of the 224 acute serum samples from patients with laboratory-confirmed acute dengue infection, dengue IgM was detected in 88 specimens, comprising 68 acute primary dengue specimens and 20 acute secondary dengue specimens. NS1 antigen-capture ELISA kit gave an overall sensitivity rate of 88.6 percent in the presence of anti-dengue IgM and 96.3 percent in the absence of anti-dengue IgM.
CONCLUSION: Of the 224 acute serum samples, the sample ages of 166 acute serum samples are known. The positive detection rate of dengue NS1 antigen-capture ELISA, on the whole, was higher than the other three established diagnostic test methods for laboratory diagnosis of acute dengue infection.
Rapid diagnosis of dengue infection is essential to patient management and disease control. The development of a rapid (5 min) immunochromatographic test and a 2 h commercial capture enzyme linked immunosorbent assay (ELISA) for anti-dengue IgM and IgG antibodies may lead to more rapid and accurate testing in peripheral health settings and diagnostic laboratories.
In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P