Dengue is a major threat to public health globally. While point-of-care diagnosis of acute/recent dengue is available to reduce its mortality, a lack of rapid and accurate testing for the detection of previous dengue remains a hurdle in expanding dengue seroepidemiological surveys to inform its prevention, especially vaccination, to reduce dengue morbidity. This study evaluated ViroTrack Dengue Serostate, a biosensors-based semi-quantitative anti-dengue IgG (immunoglobulin G) immuno-magnetic agglutination assay for the diagnosis of previous and recent dengue in a single test. Blood samples were obtained from 484 healthy participants recruited randomly from two communities in Petaling district, Selangor, Malaysia. The reference tests were Panbio Dengue IgG indirect and capture enzyme-linked immunosorbent assays, in-house hemagglutination inhibition assay, and focus reduction neutralization test. Dengue Serostate had a sensitivity and specificity of 91.1% (95%CI 87.8-93.8) and 91.1% (95%CI 83.8-95.8) for the diagnosis of previous dengue, and 90.2% (95%CI 76.9-97.3) and 93.2% (95%CI 90.5-95.4) for the diagnosis of recent dengue, respectively. Its positive predictive value of 97.5% (95%CI 95.3-98.8) would prevent most dengue-naïve individuals from being vaccinated. ViroTrack Dengue Serostate's good point-of-care diagnostic accuracy can ease the conduct of dengue serosurveys to inform dengue vaccination strategy and facilitate pre-vaccination screening to ensure safety.
Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.
Previous studies have shown that lipid peroxidative processes may play a role in disease pathogenesis in lupus-prone MRL/lpr mice. Studies were thus performed to determine if an immune response against malondialdehyde (MDA), a highly reactive byproduct of lipid peroxidation, was present in these mice. By using MDA-modified mouse serum albumin (MSA) as antigens in ELISA, we found that these mice produce high levels of MDA-specific antibodies in the complement-fixing IgG2a and IgG2b subclasses. Anti-MDA antibodies were also found in MRL/+ mice but in significantly lower levels. The specificity of these antibodies was verified by inhibition ELISA. MDA may contribute to disease pathogenesis in these mice by altering the immunogenicity of self molecules, eliciting an immune response and forming immune complexes that may deposit in tissues.
A simple adherence test to detect IgM antibodies in patients with typhoid is described. The test utilises the IgM-"capture" approach, in which the test serum is applied to microtitration plate wells previously coated with anti-human IgM, followed by application of a stained Salmonella typhi antigen suspension which shows adherence in positive cases. By this test, 58 (95%) of 61 sera from confirmed cases of typhoid possessed IgM antibodies to the H or O or both antigens of S. typhi. In patients for whom a diagnosis of typhoid was based only on a significant Widal-test titre, 31 (41%) of 76 sera had IgM antibodies to the H or O or both antigens of S. typhi. Some cross-reactivity of the IgM antibodies was detected, especially with the O antigens of S. paratyphi A and B. A total of 82 sera from non-typhoidal fevers (leptospirosis, typhus, dengue fever) showed no reactivity in this test. In normal sera there was no detectable IgM to the O antigen of S. typhi and only a small number (3.9%) had low levels of IgM to the H antigen. The significance and potential importance of this simple, sensitive, specific and economical test is discussed.
The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups. The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups. However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7. The IgA response was intermediate with GMTIs ranging from 119.2 to 170. The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections. As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts. None of the sera from the control groups had any significant titres of antibodies.
Malaria causes high global mortality and morbidity annually. Plasmodium knowlesi has been recognised as the fifth human Plasmodium sp. and its infection is widely distributed in Southeast Asia. Merozoite surface protein-119 (MSP-119) appears as a potential candidate for malaria blood stage vaccine as it could induce protective immunity. In this study, codon optimized P. knowlesi MSP-119 (pkMSP-119) was expressed and purified in yeast Pichia pastoris expression system. The purified recombinant protein was further evaluated using Western blot assay using knowlesi malaria, non-knowlesi human malaria, non-malarial parasitic infections and healthy serum samples (n = 50). The sensitivity of purified pkMSP-119 towards detection of knowlesi infection was as 28.6% (2/7). pkMSP-119 did not react with all non-malarial parasitic infections and healthy donor sera, yet reacted with some non-knowlesi human malaria sera, therefore lead to a specificity of 86.0% (37/43).
Toxoplasmosis can cause serious disease in immunocompromised patients and to congenitally infected foetuses. Appropriate laboratory investigations in potential cases of acute Toxoplasma infection are important. Excretory secretory antigen (ESA) is immunogenic during both human and experimental infections, therefore is considered as a good candidate for investigation into new infection markers. In this study, ESA was prepared from in vitro cultures of Toxoplasma gondii to identify T. gondii ESA antigenic component(s) that is/are most reactive with serum samples from probable acute cases of toxoplasmosis. Serum samples were obtained from several categories of individuals with the following Toxoplasma serology: Group I: IgM+ IgG+ (low IgG avidity) or IgM+ IgG- from sera of patients who had clinical query of toxoplasmosis (n=35). Group II: IgM- IgG+ (high IgG avidity) from chronically infected individuals (n=30). Group III: normal/healthy individuals with anti-Toxoplasma IgMIgG- (n=20). Group IV: individuals with other infections who had anti-Toxoplasma IgM- IgG- (n=10). The ESA was subjected to SDS-PAGE, followed by Western blot analysis using the above sera and probed with peroxidase conjugated anti-human IgM and IgA antibodies. The blots were then developed using chemiluminescence substrate. The selected antigenic band was excised from the gel after two dimensional electrophoresis and sent for mass spectrometry analysis using MALDI TOF-TOF. The most promising antigenic band was a 10 kDa protein which showed sensitivity of 80% in both IgM and IgA blots, and specificity of 96.7% with sera from other infections and healthy controls. The two best identifications for the 10 kDa band were ubiquitin (ribosomal protein CEP52 fusion protein) and polyubiquitin.
This study probes into the prospect of cross-reactivity of HCMV with RCMV which has not been acknowledged to date. We describe the uncovering of a protein with an estimated size of between 61-68 kDa from local RCMV strains which reacted with HCMV positive sera. Our findings are a first disclosure of a plausible immunological cross-reactivity between RCMV with its human counterpart which grounds substantial interest implying existence of conserved determinants between rat and human CMV polypeptides. The cross-reactive protein most likely represents an enveloped glycoprotein, though the precise identification and its degree of similarity needs to be evidently defined and further elucidated in forthcoming experiments.
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.
The Indirect Fluorescent Antibody Test (IFAT) and thin blood smears were conducted to establish the prevalence of Ehrlichia canis infection in dogs presented for treatment by pet owners at five private and one government veterinary clinic. Results showed that 15% of the dogs were positive for the parasite via IFAT, but none using blood smears. However, infected dogs did not show severe clinical symptoms of canine monocytic ehrlichiosis (CME).
Antibodies to the protozoan parasite, Toxoplasma gondii were assayed in sera of 200 goats, 100 pigs, 126 cattle from various states of Malaysia, and 135 dogs and 55 cats around Ipoh region using an indirect fluorescence antibody test (IFAT, cut-off titer 1:200); antibodies were found in 35.5% of goats, 14.5% cats, 9.6% dogs, 7.9% local cattle and 4% yellow cattle but not in pigs. Results indicate that infection is most prevalent in goats.
Flourescent antibody test (FAT) was applied to determine the cross-reactivities of monoclonal (mAb), polyclonal (pAb) antibodies to Neospora, Toxoplasma and Cryptosporidium and antisera from cattle naturally infected with Neospora canium against antigens from a number of sources. Both mAb and pAb to Neospora reacted strongly (FAT titre up to 2560) with the homologous antigens and demonstrated weak titre (80) or no reaction with both Toxoplasma and Cryptosporidium antigens. Also mAb and pAb to Toxoplasma gondii reacted at titres of 80 - 640 with homologous antigens and at titres of 10-40 with N. caninum. No cross-reactions with either mAb or pAb antibodies to N. caninum and T. gondii were observed with Cryptosporidium parvum. The same results were observed with C. parvum mAb when tested with both N. caninum and T. gondii antigens. Sera from cattle naturally infected with N. caninum had titres ranging from 80- 640 with N. caninum antigens, and 10- 40 with T. gondii and C. parvum antigens. At low dilutions, the complete surfaces of Neospora and Toxoplasma parasites were fluorescent, while in higher dilutions only dotted fluorescence appeared on the apical complex. These results indicated the presence of cross-reactivity between Neospora and Toxoplasma but not with Cryptosporidium. Accordingly the recommended cut-off antibody titre for diagnosis of neosporosis is 80.
A truncated form of surface antigen 2 (SAG2) of the protozoan parasite Toxoplasma gondii was cloned and expressed in the methylotrophic yeast Pichia pastoris. This recombinant antigen, designated as recSAG2-N, contained only the N-terminal half of the native SAG2. The recSAG2-N was secreted by the Pichia pastoris into the culture supernatant, and it was harvested by using the trichloroacetic acid precipitation method. Specificity of recSAG2-N was evaluated in western blot assays. Fifty human serum samples, including 32 from confirmed cases of toxoplasmosis, were tested. Results from the assays showed that recSAG2-N reacted with sera from the toxoplasmosis cases only. In vivo experiments showed that serum from mice which received recSAG2-N reacted with the native SAG2 of T. gondii.
The objective of this study was to elucidate the association of various risk factors with dengue cases reported in Lundu district, Sarawak, by analyzing the interaction between environmental, entomological, socio-demographic factors. Besides conventional entomological, serological and house surveys, this study also used GIS technology to generate geographic and environmental data on Aedes albopictus and dengue transmission. Seven villages were chosen based on the high number of dengue cases reported. A total of 551 households were surveyed. An overall description of the socio-demographic background and basic facilities was presented together with entomological and geographical profiles. For serological and ovitrap studies, systematic random sampling was used. Serological tests indicated that 23.7% of the 215 samples had a history of dengue, either recent or previous infections. Two samples (0.9%) were confirmed by IgM ELISA and 49 samples (22.8%) had IgG responses. A total of 32,838 Aedes albopictus eggs were collected in 56 days of trapping. Cluster sampling was also done to determine whether any of the risk factors (entomological or geographical) were influenced by geographical location. These clusters were defined as border villages with East Kalimantan and roadside villages along Lundu/Biawas trunk road. The data collected were analyzed using SPSS version 10.01. Descriptive analysis using frequency, means, and median were used. To determine the association between variables and dengue cases reported, and to describe the differences between the two clusters of villages, two-sample t-test, and Pearson's Chi-Square were used. Accurate maps were produced with overlay and density function, which facilitates the map visualization and report generating phases. This study also highlights the use of differential Global Positioning System in mapping sites of 1m accuracy. Analysis of the data revealed there are significant differences in clusters of villages attributable to container density, house density, distance of the house from the main road, and number of Ae. albopictus eggs from ovitraps set indoor, outdoor and in dumping sites (Person's Chi-Square = 6.111, df = 1, p < 0.01). Further analysis using t-test showed that house density, container density, indoor mosquitoes egg count, outdoor mosquitoes egg count, and dumping sites mosquitoes egg count were higher at the roadside villages compared to border villages. A number of potential risk factors including those generated from GIS were investigated. None of the factors investigated in this study were associated with the dengue cases reported.
This research is focussed to quantify IGF1 by electroanalytical analysis on InterDigitated electrode surface and characterized by the microscopic observations. For the detection, antibody and aptamer were used to analyze the level of IGF1. The sandwich pattern (aptamer-IGF1-antibody) was designed on the chemically modified IDE surface and reached the limit of detection to 10 fM with 100 folds enhancement in the sensitivity. Different control experiments (absence of IGF1, binding with IGF2 and with non-complementary aptamer) were failed to show the current changes, discriminated the specific detection. A good detection strategy is to complement the currently following imaging systems for AAA.
Evidence of influenza A virus (IAV) infection in dogs, a major companion animal of humans, suggests the possibility that they may constitute a new source for transmission of novel influenza viruses to humans. The potential public health risk posed by this possibility of interspecies spread of IAV between dogs and humans necessitated surveillance for the virus in dogs and their human contacts. Sera from 239 asymptomatic pet and hunting dogs in Oyo state, Nigeria were screened for anti-IAV nucleoprotein antibodies using competitive enzyme-linked immunosorbent assay (ELISA) while haemagglutination inhibiting (HI) antibodies in the positive sera were detected using influenza virus H3 and H5 subtypespecific antigens. Suspensions prepared from 239 and 39 nasal swabs from dogs and human contacts, respectively were tested for presence of the highly conserved IAV matrix gene by reverse transcriptase-polymerase chain reaction (RT-PCR). Only 4 (1.7%) of the 239 sera tested were positive by the ELISA. The HI test confirmed the presence of H3 influenza virus subtype-specific antibodies in one (25.0%) of the 4 ELISA-positive sera with a titre of 1:128 while none was positive for H5 subtype-specific antibodies. All the nasal swabs assayed by RT-PCR were negative for IAV nucleic acid. The detection of IAV antibodies in pet and hunting dogs in this study, although at a low rate, suggests that these dogs could play a crucial role in the zoonotic transmission of influenza viruses especially considering the close interaction between them and their human contacts. Continuous surveillance for IAV among dog populations in Oyo State (and Nigeria) is therefore advocated to facilitate early detection of infection or emergence of novel influenza virus strains that could be potentially harmful to humans and or animals.
Leptospirosis is a worldwide zoonotic disease caused by spirochetes of the genus Leptospira. The clinical manifestation of leptospirosis is non-specific and frequently misdiagnosed as other illnesses. The aim of this study was to compare the diagnostic accuracies of two commercial tests for early diagnosis of Leptospira species: the IgM latex agglutination test (IgM LAT) and the IgM enzyme-linked immunosorbent assay (IgM ELISA). A total of 140 serum samples were obtained from patients suspected of leptospirosis at the Universiti Kebangsaan Malaysia Medical Centre (UKMMC). These serum samples were tested for the presence of Leptospira sp. using IgM LAT, IgM ELISA and MAT. From Table 1, IgM LAT showed 21% (n = 29) positive, 18% (n = 25) inconclusive and 61% (n = 86) negative, while IgM ELISA showed 6% (n = 8) positive, 6% (n = 8) inconclusive, 88% (n = 124) negative and MAT showed 11% (n = 16) positive, 47% (n = 65) inconclusive, 42% (n = 59) negative. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IgM LAT were 68.8%, 57.6%, 30.6% and 87.2% respectively, while for IgM ELISA they were 37.5%, 89.8%, 50% and 84.1%, respectively as compared to MAT (Table 2). The results showed that IgM LAT had higher sensitivity but lower specificity compared to IgM ELISA. In conclusion, IgM LAT can be useful as an early screening test for early diagnosis of Leptospira sp., while IgM ELISA is a suitable method for reducing false negative detection of Leptospira sp. As both tests show moderate percentages (~65%) in accuracy, an additional test is required for better detection of Leptospira sp.
A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development.
Nipah virus (NiV) is a paramyxovirus that causes severe disease in humans and animals. There are two distinct strains of NiV, Malaysia (NiVM) and Bangladesh (NiVB). Differences in transmission patterns and mortality rates suggest that NiVB may be more pathogenic than NiVM. To investigate pathogenic differences between strains, 4 African green monkeys (AGM) were exposed to NiVM and 4 AGMs were exposed to NiVB. While NiVB was uniformly lethal, only 50% of NiVM-infected animals succumbed to infection. Histopathology of lungs and spleens from NiVB-infected AGMs was significantly more severe than NiVM-infected animals. Importantly, a second study utilizing 11 AGMs showed that the therapeutic window for human monoclonal antibody m102.4, previously shown to rescue AGMs from NiVM infection, was much shorter in NiVB-infected AGMs. Together, these data show that NiVB is more pathogenic in AGMs under identical experimental conditions and suggests that postexposure treatments may need to be NiV strain specific for optimal efficacy.
Matched MeSH terms: Antibodies, Monoclonal/therapeutic use
In 1973, 2610 sera were collected from adults living in 22 localities in four states in Peninsular Malaysia and tested by IFAT for Plasmodium falciparum antibodies. A larger number of thin films were examined. The attack phase of the Malaria Eradication Programme (MEP) in these areas was started between 1968 and 1973. The results showed that the highest prevalence rates and geometrical mean reciprocal titres (CMRT) were among adults from Kelantan where the antibody prevalence varied greatly among the adults and there was active transmission in at least three areas. The values were lowest for Kedah. The P. falciparum antibody prevalence rates were higher than the parasite rates as revealed in single thin film examinations but a number of the positive sera were reactive only at low titres. The low concentration probably indicated the residual antibody from cured cases or past infections and cross reactions to P. vivax and P. malariae infections. The strong reactions probably indicated current P. falciparum transmission as shown by positive thin films. The present study showed that the antibody profile of adults, as shown by IFAT, is of considerable value in assessing the malaria situation in a given area and that it would be useful as a malariometric tool in epidemiological studies to evaluate the progress of malaria eradication/control programmes.