A sensitive and specific RT-nested PCR coupled with an ELISA detection system for detecting Newcastle disease virus is described. Two nested pairs of primer which were highly specific to all the three different pathotypes of NDV were designed from the consensus fusion gene sequence. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. Based on agarose electrophoresis detection, the RT-nested PCR was about 100 times more sensitive compared to that of a non-nested RT-PCR. To facilitate the detection of the PCR product, an ELISA detection method was then developed to detect the amplified PCR products and it was shown to be ten times more sensitive than gel electrophoresis. The efficacy of the nested PCR-ELISA was also compared with the conventional NDV detection method (HA test) and non-nested RT-PCR by testing against a total of 35 tissue specimens collected from ND-symptomatic chickens. The RT-nested PCR ELISA found NDV positive in 21 (60%) tissue specimens, while only eight (22.9%) and two (5.7%) out of 35 tissue specimens were tested NDV positive by both the non-nested RT-PCR and conventional HA test, respectively. Due to its high sensitivity for the detection of NDV from tissue specimens, this PCR-ELISA based diagnostic test may be useful for screening large number of samples.
The performance of a commercial immunochromatography test for rapid detection of dengue NS1 antigen present in serum or plasma of patients was evaluated against a commercial dengue NS1 antigen-capture ELISA. The rapid immunochromatography test gave an overall sensitivity of 90.4% with a specificity of 99.5%. The sensitivity was highest for serum samples from which virus was isolated (96.3%) and lowest for those from which virus was not isolated and RT-PCR was negative (76.4%). The sensitivity was significantly higher for serum samples from patients with acute primary dengue (92.3%) than those from patients with acute secondary dengue (79.1%). The positive predictive value and negative predictive value of this commercial immunochromatography test were 99.6% and 87.9% respectively.
The Crimean-Congo haemorrhagic fever virus (CCHFV), which is transmitted by the ticks of Hyalomma spp. in general and H. marginatumin particular, can cause severe disease in humans, with mortality rates of 3-30%. Other than from the bites of infected ticks, CCHFV can also be transmitted through contact with patients with the acute phase of infection or contact with blood or tissues from viraemic livestock. Outbreaks of human cases of haemorrhagic manifestations have been documented since 1945 and described in parts of Africa, Asia, Eastern Europe and the Middle East and most recently India in 2011. In addition, serological evidence of the disease has been reported in some countries where no human cases were reported. As regional neighbours China and India have been affected by this virus, this study was conducted to determine the seroprevalence of CCHFV among Orang Asli population of Malaysia as the most at risk people who residing in the deep forests.
The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.
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.
Adiponectin, an adipocyte-derived hormone has been implicated in the control of blood glucose and chronic inflammation in type 2 diabetes. However, limited studies have evaluated dietary factors on plasma adiponectin levels, especially among type 2 diabetic patients in Malaysia. The aim of this study was to investigate the influence of dietary glycemic index on plasma adiponectin concentrations in patients with type 2 diabetes. A cross-sectional study was conducted in 305 type 2 diabetic patients aged 19-75 years from the Penang General Hospital, Malaysia. Socio-demographic information was collected using a standard questionnaire while dietary details were determined by using a pre-validated semi-quantitative food frequency questionnaire. Anthropometry measurement included weight, height, BMI and waist circumference. Plasma adiponectin concentrations were measured using a commercial ELISA kit. Data were analyzed using multiple linear regression. After multivariate adjustment, dietary glycemic index was inversely associated with plasma adiponectin concentrations (β =-0.272, 95% CI -0.262, - 0.094; p<0.001). It was found that in individuals who consumed 1 unit of foods containing high dietary glycemic index that plasma adiponectin level reduced by 0.3 μg/mL. Thirty two percent (31.9%) of the variation in adiponectin concentrations was explained by age, sex, race, smoking status, BMI, waist circumference, HDL-C, triglycerides, magnesium, fiber and dietary glycemic index according to the multiple linear regression model (R2=0.319). These results support the hypothesis that dietary glycemic index influences plasma adiponectin concentrations in patients with type 2 diabetes. Controlled clinical trials are required to confirm our findings and to elucidate the underlying mechanism.
Plasmodium knowlesi is the fifth species identified to cause malaria in humans and is often misdiagnosed as Plasmodium malariae due to morphological similarities. The development of an inexpensive, serological detection method utilizing antibodies specific to P. knowlesi would be a valuable tool for diagnosis. However, the identification of specific antigens for these parasites remains a major challenge for generating such assays. In this study, surface protein containing an altered thrombospondin repeat domain (SPATR) was selected as a potentially specific antigen from P. knowlesi. Its multistage expression by sporozoites, asexual erythrocytic forms and gametocytes, along with its possible role in liver cell invasion, suggests that SPATR could be used as a biomarker for diagnosis of P. knowlesi.
Amoebic liver abscess (ALA) is the most frequent clinical presentation of extra-intestinal amoebiasis. The diagnosis of ALA is typically based on the developing clinical symptoms, characteristic changes on radiological imaging and serology. Numerous serological tests have been introduced for the diagnosis of ALA, either detecting circulating amoebic antigens or antibodies. However those tests show some pitfalls in their efficacy and/or the preparation of the tests are costly and tedious. The commercial IHA kit that used crude antigen was reported to be useful in diagnosis of ALA, however high antibody background in endemic areas may cause problems in its interpretation. Thus, discovery of well-defined antigen(s) is urgently needed to improve the weaknesses of current serodiagnostic tests.
Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus that causes regular outbreaks in East Asia with mortality rate exceeding 75%. Major cellular targets of NiV infection are endothelial cells and neurons. To better understand virus-host interaction, we analyzed the transcriptome profile of NiV infection in primary human umbilical vein endothelial cells. We further assessed some of the obtained results by in vitro and in vivo methods in a hamster model and in brain samples from NiV-infected patients. We found that NiV infection strongly induces genes involved in interferon response in endothelial cells. Among the top ten upregulated genes, we identified the chemokine CXCL10 (interferon-induced protein 10, IP-10), an important chemoattractant involved in the generation of inflammatory immune response and neurotoxicity. In NiV-infected hamsters, which develop pathology similar to what is seen in humans, expression of CXCL10 mRNA was induced in different organs with kinetics that followed NiV replication. Finally, we showed intense staining for CXCL10 in the brain of patients who succumbed to lethal NiV infection during the outbreak in Malaysia, confirming induction of this chemokine in fatal human infections. This study sheds new light on NiV pathogenesis, indicating the role of CXCL10 during the course of infection and suggests that this chemokine may serve as a potential new marker for lethal NiV encephalitis.
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.
Following a series of H5N1 cases in chickens and birds in a few states in Malaysia, there was much interest in the influenza A viruses subtypes that circulate among the local pig populations. Pigs may act as a mixing vessel for avian and mammal influenza viruses, resulting in new reassorted viruses. This study investigated the presence of antibodies against influenza H1N1 and H3N2 viruses in pigs from Peninsular Malaysia using Herdcheck Swine Influenza H1N1 and H3N2 Antibody Test Kits. At the same time, the presence of influenza virus was examined from the nasal swabs of seropositive pigs by virus isolation and real time RT-PCR. The list of pig farms was obtained from the headquarters of the Department of Veterinary Services, Malaysia, and pig herds were selected randomly from six of 11 states in Peninsular Malaysia. A total of 727 serum and nasal swab samples were collected from 4- to 6-month-old pigs between May and August 2005. By ELISA, the seroprevalences of swine influenza H1N1 and H3N2 among pigs were 12.2% and 12.1% respectively. Seropositivity for either of the virus subtypes was detected in less than half of the 41 sampled farms (41.4%). Combination of both subtypes was detected in 4% of all pigs and in 22% of sampled farms. However, no virus or viral nucleic acid was detected from nasal samples. This study identified that the seropositivity of pigs to H1N1 and H3N2 based on ELISA was significantly associated with factors such as size of farm, importation or purchase of pigs, proximity of farm to other pig farms and the presence of mammalian pets within the farm.
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.
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.
Vitamin E is divided into two subgroups; tocopherols and tocotrienols. Both have protective roles in biological systems. The present study was conducted to compare the effect of short-term supplementation at 200 mg/d of either alpha-tocopherol or a tocotrienol-rich fraction (TRF) from palm oil on immune modulation and plasma vitamin E levels in normal healthy Asian volunteers. In a randomised, double-blind placebo-controlled trial conducted, fifty-three healthy volunteers aged 20-50 years were recruited based on the study's inclusion and exclusion criteria. They were randomly assigned into three groups, i.e. two experimental groups that received daily supplementation at 200 mg of either alpha-tocopherol or the TRF, and the control group that received a placebo. Blood was drawn on days 0, 28 and 56 for several laboratory analyses. Differences in the production of IL-4 or interferon-gamma by concanavalin A-stimulated lymphocytes isolated from these volunteers were not significant (P>0.05). There were no significant differences observed in immune parameters between the healthy volunteers who received daily supplementation with either alpha-tocopherol or the TRF. As these observations were made in the absence of any immunogenic challenge, we feel it would be of benefit to study if there would be any differences observed when an immunogenic challenge such as vaccination were introduced.
Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.
Tick-borne encephalitis virus (TBEV) and Crimean-Congo haemorrhagic fever virus (CCHFV) are important tick-borne viruses. Despite their wide geographical distribution and ease of acquisition, the prevalence of both viruses in Malaysia is still unknown. This study was conducted to determine the seroprevalence for TBEV and CCHFV among Malaysian farm workers as a high-risk group within the population.
In this study, recombinant proteins that encompassed the AD I-AD III regions of 56 kDa immunodominant gene of 2 Orientia tsutsugamushi (OT) serotypes; Gilliam and TA763 were expressed in Escherichia coli. Both recombinant proteins exhibited serologic cross-reactivity with the rabbit antisera against various OT serotypes, as evaluated by enzyme-linked immunosorbent assay (ELISA), but not against other rickettsial species, including Rickettsia typhi, R. prowazekii and TT118 SFG rickettsiae. The feasibility of using the recombinant proteins as a diagnostic reagent was further evaluated by ELISA using sera from blood donors and scrub typhus patients. The results suggested a higher affinity of the antihuman IgM than IgG with both recombinant proteins. The IgM ELISA findings were agreeable with the results of indirect immunoperoxidase (IIP) assay especially with sera of high antibody (1:1600). However, more than one antigen are probably needed for development of an effective assay for serodiagnosis of scrub typhus in endemic areas.
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.
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%.
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.