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.
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.
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.
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.
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.
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.
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.