Antigen Detection Enzyme-linked Immunosorbent Assay Research Articles

Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena

BackgroundAntigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA.MethodsWe report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland–Altman (BA) analysis and Lin’s concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA.ResultsThe TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin’s CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin’s CCC: 0.24) but increased on days 28 and 90 PI (Lins’ CCC: 0.88 and 0.98, P < 0.001).Conclusions/significanceThe TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.Graphical

Epidemiology of Rotavirus Infection in Dairy Calves in Central Ethiopia

Rotavirus is a major pathogen responsible for diarrheal disease in calves and neonates resulting in loss of productivity and economic losses in dairy farms in developed and developing countries. In Ethiopia, the diarrheal disease caused by Rotavirus is not so far studied adequately. Across-sectional study was conducted from October 2019 to May 2020 to estimate the prevalence, assess associated factors, and isolate Rotavirus in calves below two months of age in the central part of Ethiopia, namely Holeta, Sululta, Bishoftu, and Sebeta. A total of 404 fecal samples, comprising 213 diarrheic and 191 non-diarrheic cases, were collected from calves of less than two months of age by simple random sampling technique. Rotavirus infection was detected using antigen detection Enzyme-linked immunosorbent assay technique. All samples positive for ELISA were propagated in Madin Darby bovine kidney cells. Data was analyzed using STATA version 13. Fisher’s exact test and logistic regression analysis were used to determine the association between prevalence of rotavirus and potential risk factors. An overall rotavirus prevalence of 1.24% (95%CI 0.40 – 2.86%) was found. The prevalence of rotavirus in diarrheic calves (1.41%) was not significantly different from the prevalence of rotavirus in non-diarrheic calves (1.04%) (p&gt;0.05). Similarly, the origin, breed, sex, age, management, and type of farm had no significant association with the prevalence of rotavirus infection of calves (p&gt;0.05). After 3 subsequent passages, progressive cytopathic effect i.e. Cell swelling and obscure cell boundaries, detachment and floatation of cells, cytoplasmic vacuolationas, increased cell granularity and cytoplasmic stranding were observed in all samples. The presence of rotavirus in dairy calves might entail morbidity and mortality in calves. Moreover, rotavirus infection might also lead to zoonotic transmission. Therefore, further detailed molecular epidemiological studies are warranted. Good hygienic and husbandry practices are essential to limit the spread of infection.

Enhanced detection and serotyping of foot-and-mouth disease virus serotype O, A, and Asia1 using a novel multiplex real-time RT-PCR.

Rapid and accurate detection and serotyping of foot-and-mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription-polymerase chain reaction (RT-PCR)/sequencing (VP1 RT-PCR/sequencing) and antigen detection enzyme-linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real-time RT-PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real-time RT-PCR using these primers and probes demonstrated serotype-specific detection with enhanced sensitivity compared to VP1 RT-PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real-time RT-PCR and previous VP1 RT-PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real-time RT-PCR and VP1 RT-PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real-time RT-PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real-time RT-PCR and VP1 RT-PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real-time RT-PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.

Combining a Universal Capture Ligand and Pan-Serotype Monoclonal Antibody to Develop a Pan-Serotype Lateral Flow Strip Test for Foot-and-Mouth Disease Virus Detection.

Foot-and-mouth disease virus (FMDV) causes FMD, a highly contagious disease of cloven-hoofed animals including cattle, goats, pigs and sheep. Rapid detection of FMDV is critical to limit the devastating economic losses due to FMD. Current laboratory methods for FMDV detection such as virus isolation, real-time reverse transcription PCR and antigen detection enzyme-linked immunosorbent assay (AgELISA) are labor-intensive, requiring trained personnel and specialized equipment. We present the development and validation of a pan-serotype lateral flow strip test (LFST) that uses recombinant bovine integrin αvβ6 as a universal capture ligand and a pan-serotype monoclonal antibody (mAb) to detect FMDV. The LFST detected all seven FMDV serotypes, where the diagnostic sensitivity was comparable to the AgELISA, and the diagnostic specificity was 100% without cross-reactivity to other viruses causing vesicular disease in livestock. This rapid test will be useful for on-site FMDV detection, as well as in laboratories in endemic countries where laboratory resources are limited.

Molecular and Serological Epidemiology of Foot-and-Mouth Disease Virus in North Region of Cameroon

The serological prevalence of foot and mouth disease virus (FMDV) among the cattle population in the North region of Cameroon was determined using ELISA (Enzyme Linked Immunosorbent Assays) serological tests for structural as well as non-structural proteins. In these cattle, FMDV RNA was identified, amplified, sequenced and the sequences were used to construct a phylogenetic tree. A sedentary cattle population randomly selected from six veterinary centres in the North region was sampled twice, six months apart. High prevalence of FMDV antibody was recorded in the first (402/466 (85.84%)) and second (358/411 (86.90%)) sampling periods. There was no significant difference (P > 0.05) in prevalence of FMDV antibody between the two sampling periods. Goudali and Peulh breeds of cattle and animals of three to five years old were the most infected with FMDV and mostly in the months of May and August. A seroprevalence of 100% (n = 14) of FMDV against serotypes A and O was observed in sera from convalescent animals in the study area. FMDV antigen detection ELISA showed a prevalence of 18/37 (48.65%) for serotypes SAT1 (8.1%), SAT2 (35.1%), A (10.8%) and O (2.7%) among the clinically infected animals. There was no significant difference (P > 0.05) in prevalence of FMDV RNA between the sampling periods. A prevalence of FMDV RNA (17.5% (n = 120) and 16.7% (n = 240)) was observed among the sedentary animals that were sampled four to five months apart. FMDV RNA prevalence of 28/37 (75.6%) among clinically infected animals was also observed, thus confirming all the 12 outbreaks investigated. Sequence analysis of VP1 coding gene of the SAT2 serotype showed that it was homologous to the Libyan isolates (that caused epidemics in northern Africa in 2012) and also clustered with the serotypes isolated from both Nigeria and Sudan in 2007.

Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer–antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Molecular and serological typing of foot-and-mouth disease virus serotypes currently circulating in Egypt

Foot-and-mouth disease (FMD) is an extremely contagious viral disease affects all cloven- hoofed animals. The present study aimed to investigate the epidemiological situation of FMD in Egypt during 2017 and 2018, based on antigenic and genetic characterizations of FMD virus (FMDV). Thirty oral epithelia were collected from vaccinated animals (14 native cattle and 16 water buffaloes) showed clinical signs of FMD in four Egyptian governorates having outbreaks. In all collected samples the antigen detection was performed using enzyme-linked immunosorbent assay (ELISA), while the genetic characterization was done by using conventional reverse transcription polymerase chain reaction (RT-PCR), Sequencing and phylogenetic analysis were constructed for genetic characterization. The obtained results of FMDV antigen detection ELISA indicated that 50% of the examined samples were positive for FMDV and serotyped as serotype O 40%, serotype SAT2 33% and serotype A 27% respectively. RT-PCR confirmed the results of FMDV antigen detection by ELISA. Six amplicons were sequenced and phylogenetically analyzed for viral protein 1 (VP1) of FMD. Results demonstrated that genotype O was related to East Africa-3 (EA-3) topotype with 12.7% difference from vaccine strain O-IRN-8-2005-Pan-Asia-2. Furthermore, genotype A clustered into Asia topotype with 6% difference from vaccine strain A-IRN-1-2005. Meanwhile genotype SAT2 in 2018 was related to VII topotype but it was in close relation with strains isolated from Libya in 2012 with 94.3% amino acid identity that differ from the previously circulated SAT2 since 2012 and recorded recently in Egypt. The presented results confirmed the circulation of a new topotype of serotype SAT2.

Isolation and Characterization of Coronavirus and Rotavirus Associated with Calves in Central Part of Oromia, Ethiopia.

Background Coronavirus and rotavirus are most commonly associated etiologies for calves' diarrhoea, resulting in loss of productivity and economy of farmers. However, various facets of diarrheal disease caused by coronavirus and rotavirus in calves in Ethiopia are inadequately understood. A cross-sectional study was conducted with the aim of isolation and molecular characterization of coronavirus and rotavirus from calves in the central part of Oromia (Bishoftu, Sebata, Holeta, and Addis Ababa), Ethiopia, from November 2018 to May 2019. The four study areas were purposively selected and faecal samples were collected by simple random sampling for diagnosis of coronavirus and rotavirus infection by using the antigen detection enzyme-linked immunosorbent assay (Ag-ELISA) kit. In addition, this study was carried out to have insight in prevalence and associated risk factors of coronavirus and rotavirus infection in calves. Result During the study, 83 diarrheic and 162 nondiarrheic faecal samples collected from calves less than 4 weeks of age were screened for coronavirus and rotavirus. Of the 83 diarrheic samples, 1 sample (1.2%) was positive for coronavirus antigen and 6 samples (7.2%) were found to be positive for rotavirus antigen by Ag-ELISA. All the nondiarrheic samples were negative for both coronavirus and rotavirus Ag. The overall prevalence of coronavirus and rotavirus infection in calves was estimated at 0.4% (1/245) and 2.45% (6/245), respectively. All samples (7) of ELISA test positive of both coronavirus and rotavirus were propagated in Madin-Darby bovine kidney (MDBK) cells. After 3 subsequent passages, progressive cytopathic effect (CPE), i.e., rounding, detachment, and the destruction of monolayer cell of five samples (1 sample of coronavirus and 4 samples of rotavirus) (71.4%) were observed. At the molecular stage, reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to determine the presence of coronavirus and rotavirus nucleic acid by using specific primers. The 5 samples that were coronavirus and rotavirus antigen positive by ELISA and develop CPE on cell culture were also positive on RT-PCR technique. The prevalence of infection peaked at 1st and 2nd weeks of age in male calves. Conclusion Diarrheal disease caused by coronavirus and rotavirus has a great health problem in calves that interrupts production benefits with reduced weight gain and increased mortality and its potential for zoonotic spread. So, the present findings show coronavirus and rotavirus infection in calves in Ethiopia that needs to be addressed by practising early colostrum feeding in newborn calves, using vaccine, or improving livestock management.

Performance of Ag-ELISA in the diagnosis of Taenia solium cysticercosis in naturally infected pigs in Tanzania

BackgroundTaenia solium is a zoonotic parasite responsible for neurocysticercosis—a major cause of late-onset acquired epilepsy in humans. Lack of affordable, specific and sensitive diagnostic tools hampers control of the parasite. This study assessed the performance of an antigen detection enzyme-linked immunosorbent assay (Ag-ELISA) in the diagnosis of viable T. solium cysticercosis in naturally infected slaughter-age pigs in an endemic area in Tanzania.MethodsA total of 350 pigs were bled before they were slaughtered and their carcases examined. Serum was analyzed for circulating antigens by using a monoclonal antibody-based B158/B60 Ag-ELISA. Each carcase was examined for the presence of Taenia hydatigena cysticerci and half carcase musculature together with the whole brain, head muscles, tongue, heart and diaphragm were sliced with fine cuts (< 0.5 cm) to reveal and enumerate T. solium cysticerci. Half carcase dissection can detect at least 84% of infected pigs. Prevalence and their 95% confidence intervals (CI) were calculated in Stata 12. Sensitivity, specificity, predictive values and likelihood ratios were determined.ResultsTwenty–nine pigs (8.3%, 95% CI: 5.6–11.7%) had viable T. solium cysticerci while 11 pigs had T. hydatigena cysticerci (3.1%, 95% CI: 1.6–5.5%). No co-infection was observed. Sixty-eight pigs (19.4%, 95% CI: 15.4–20%) tested positive on Ag-ELISA; of these, 24 had T. solium cysticerci and 7 had T. hydatigena cysticerci. Sensitivity and specificity were determined to be 82.7% and 86.3%, respectively. Positive and negative predictive values were 35.2% and 98.2%, respectively. Likelihood ratios for positive and negative Ag-ELISA test results were 6.0 and 0.2, respectively. There was a significant positive correlation between the titre of circulating antigens and intensity of T. solium cysticerci (r(348) = 0.63, P < 0.001).ConclusionsThe Ag-ELISA test characteristics reported in this study indicate that the test is more reliable in ruling out T. solium cysticercosis in pigs, than in confirming it. Hence, a negative result will almost certainly indicate that a pig has no infection, but a positive result should always be interpreted with caution. Estimates of T. solium prevalence based on Ag-ELISA results should, therefore, be adjusted for test performance characteristics and occurrence of T. hydatigena.

Development and validation of a simplified serotyping ELISA based on monoclonal antibodies for the diagnosis of foot-and-mouth disease virus serotypes O, A, C and Asia 1.

This study describes the development and validation of a simplified enzyme-linked immunosorbent assay (ELISA) for the detection and discrimination of foot-and-mouth disease virus (FMDV) serotypes O, A, C and Asia 1. The multiplex ELISA was designed using selected, type-specific monoclonal antibodies (MAbs) coated onto ELISA plates as catching antibodies and a unique pan-FMDV MAb (1F10) as detector conjugate. Capture MAbs with the broadest intratypic reactivity were selected for each of the four FMDV serotypes by screening large panels of candidate MAbs with a wide spectrum of representative FMDV isolates. An additional pan-FMDV ELISA using 1F10 MAb for both capture and detection was used to complement the specific typing ELISAs to detect virus isolates, which might escape binding to the selected serotype-specific MAbs. This multiplex ELISA was prepared in a stabilized format, with immunoplates pre-coated with six MAbs and positive antigen controls already trapped by the relevant MAb, with the view to make available a diagnostic kit. Diagnostic performance of the MAbs-multiplex ELISA was analysed using 299 FMDV-positive epithelial suspensions representative of the antigenic and genomic variability within each serotype. Overall, the results provided evidence that the diagnostic performance of this assay platform is improved compared to the more complex polyclonal-based antigen detection ELISA; combining data from different serotypes and referring to the gold standard tests (i.e. virus isolation and/or RT-PCR), the MAbs-multiplex ELISA showed a sensitivity of 79% for the serotype-specific ELISA, compared to 72% for the polyclonal ELISA. The absence of reactivity of a minority of FMDV strains using the MAbs-multiplex ELISA can largely be attributed to deteriorated or low antigen concentration in the sample. This multiplex ELISA is simple, rapid and stable. FMDV antigenic diversity was adequately covered by the selected MAbs. Therefore, it can be used to replace existing polyclonal ELISAs for FMDV detection and serotyping.

Molecular Characterisation and Clinical Correlates of Rotavirus in Children and Adults in a Tertiary Care Centre, Chennai, South India

Aims: This study was undertaken to determine the rate of detection of rotavirus causing diarrhoea among children and adults, identify the common genotypes circulating and determine clinical correlates. Settings and Design: This is a cross-sectional study in a tertiary care centre. Materials and Methods: Stool samples were collected from adults and children, transported on ice, aliquoted and stored at − 80°C. Rotavirus antigen detection enzyme-linked immunosorbent assay was performed on all samples. Representative samples were typed by conventional hemi-nested VP7 and VP4 reverse transcription-polymerase chain reaction. Statistical Analysis Used: Test of proportion, Student’s t-test and Chi-square test were used for statistical analysis. Results: A total of 444 stool samples were collected and tested over 14 months. Among these, 116 were paediatric with a rate of positivity of 36.21% and 328 were adults with rate of positivity of 20.73%. Among children under 5 years (n = 90), the rate of positivity was 41.11%. Vesikari scale was used for clinical assessment. The mean ± standard deviation Vesikari score in rotavirus-infected children and rotavirus-uninfected children was 11.2 ± 3.2 and 8.9 ± 3.6, respectively, and the difference was statistically significant. Nineteen samples were genotyped in children < 5 years, 94.7% were of G1P[8] and 5.3% were of G9P[4] genotype. Genotyping of 14 adult samples, G1P[8](85.7%) was found as the predominant genotype, two samples (14.3%) were partially typed (G9PUT and G12PUT). Conclusions: The rate of positivity of rotavirus in children under 5 years was 41.11%. G1P[8] is the most common strain circulating across all age groups.

Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.

Molecular Evaluation of Conventional Microscopic Method Versus Fecal Antigen Capture Enzyme-Linked Immunosorbent Assay and Rapid Immunochromatographic Assay for Diagnosis of Cryptosporidium Infection

Background: Cryptosporidiumhasemergedasanimportantcauseofdiarrheal illness worldwide, especially among young children and patients with immune deficiencies. Aim: The present study aimed to investigate the frequencyofCryptosporidium infection among suspected versus nonsuspected patients and to evaluate the performances of conventional microscopic assay, Cryptosporidium antigen detection enzyme-linked immunosorbent assay (CAD-ELISA), Crypto-Antigen Rapid Test (CA-RT), for diagnosis of Cryptosporidium infection using Cryptosporidium-specific polymerase chain reaction (CS-PCR) as criterion standard test. Methods: A total of 181 diarrheic stool samples including 105 from suspected patients and 76 from nonsuspected ones were collected. Results: The results revealed higher rate of infection among suspected patients (25% among diarrheic children, 24.2% among elderly/immune suppressed patients) as compared with nonsuspected patients (6.6% among immune-competent apparently healthy adults). Furthermore, conventional microscopic assay exhibited higher sensitivity (83.3%) as compared with CAD-ELISA (75%) and CA-RT (45.8%). On the other hand, higher specificity was shown for CA-RT (99.4%) and CAD-ELISA (98.7%) as compared with conventional assay (92.9%). Meanwhile, CA-RT and CAD-ELISA showed higher positive predictive values (91.7%, 90%) and lower negative predictive values (92.3%, 96.9%) than conventional assay (64.5%, 97.3%). Conclusions: No single test is perfect for diagnosis of cryptosporidiosis, anditisworthmentioningthatasignificantpercentageofCryptosporidium infections would have been missed if any of these assays had been the sole method of diagnosis. It is suggested to include an antigen detection immunoassay, preferably ELISA, in addition to microscopy for better diagnosis of Cryptosporidium infection.

Sensitivity and Specificity of an In-house Sandwich ELISA Kit for Newcastle Disease Virus Antigen Detection

Of all serological tests enzyme-linked immunosorbent assay (ELISA) is still considered the gold standard for the detection of antigens and antibodies of either macro or micro-organisms worldwide. The ELISA kits for serum antibody detection against many viruses and other micro-organisms of both man and animals are available in the market. Whereas, antigen detection ELISA kits for Newcastle disease virus (NDV) is not yet available in Bangladesh. The Present study was designed for the development of an economically feasible In-house Sandwich ELISA and to test its sensitivity and specificity for the detection of NDV antigens from clinically suspected field samples. 96- well flat bottom polystyrene plates coated with hyperimmune polyclonal serum against NDV raised in rabbits was used to capture NDV antigens. The anti-rabbit IgG and DAB with 30% H2O2 were used as conjugate and substrate respectively for standardization of the test method. The plate coated with serum diluted 10-3 was found suitable for capturing maximum antigen of NDV by the In-house Sandwich ELISA. The cut-off value of the present ELISA test was calculated as 0.855 and was able to capture the viral antigen present in the 10-4 fold dilution of allantoic fluid (AF) which is equivalent to 1HA unit, indicating the highest degree of sensitivity of the newly developed ELISA. In case of field samples, the newly developed ELISA kit was able to detect 100% viral antigens of NDV present in the feces, 95.50% of the brain tissue and oro-nasal swab and 94.12% of colon swab samples of either naturally and experimentally infected birds in this study. The ND virus specific polyclonal antibody used in the kit bind only with ND virus without any cross reactive antigens of other viruses of chicken like Avian influenza virus (AIV) and Infectious bursal disease viruses (IBDV). Therefore, findings of the present study clearly indicates that the newly developed In-house Sandwich ELISA kit can be used for rapid confirmatory diagnosis of Newcastle disease (ND) with minimum cost, using any kind of field samples from either sick or dead birds. DOI: http://dx.doi.org/10.3329/mh.v1i2.14093 Microbes and Health, 2012 1(2): 65-71

Neurocysticercosis: HP10 Antigen Detection Is Useful for the Follow-up of the Severe Patients

BackgroundThe most severe clinical form of neurocysticercosis (NC) occurs when cysticerci are located in the subarachnoid space at the base of the brain (SaB). The diagnosis, monitoring and treatment of NC-SaB, constitutes a severe clinical challenge. Herein we evaluate the potential of the HP10 antigen detection enzyme-linked immunosorbent assay (HP10 Ag-ELISA) in the long term follow-up of NC-SaB cases. Assay performance was compared with that of Magnetic Resonance Imaging (MRI). In addition, the robustness of the HP10 Ag-ELISA was evaluated independently at two different institutions.Methodology/Principal FindingsA double-blind prospective cohort trial was conducted involving 38 NC-SaB cases and a total of 108 paired serum and cerebrospinal fluid (CSF) samples taken at intervals of 4 to 8 months for up to 43 months. At each medical visit, results of sera and CSF HP10 Ag-ELISA and MRI obtained at last visit were compared and their accuracy was evaluated retrospectively, considering radiological evolution between appointments. In the long-term follow-up study, HP10 Ag-ELISA had a better agreement than MRI with retrospective radiological evaluation. High reproducibility of HP10 Ag-ELISA between laboratories was also demonstrated.ConclusionsResults reported in this study establish for the first time the usefulness of the comparatively low cost HP10 Ag-ELISA for long term follow-up of NC-SaB patients.

Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis

Introduction:Cystic echinococcosis (CE) is a zoonotic disease of humans with variable clinical manifestations. Imaging and immunological methods are currently the mainstay of diagnosis of this disease. Although the immunological tests for detection of anti-echinococcal antibodies have several disadvantages, they are widely being used. Antigen is far more superior than antibody detection test as they can provide a specific parasitic diagnosis.Materials and Methods:A sandwich enzyme linked immunosorbent assay (ELISA) was designed using antibodies to 24 kDa urinary hydatid antigen for the detection of hydatid antigens in urine, serum and cyst fluid specimens. The performance of this novel test was compared with that of other hydatid antibody detection ELISA and enzyme immune transfer blot (EITB) using radiological and surgical confirmation as the gold standard.Results:The antigen detection ELISA showed 100% sensitivity and specificity when tested with cyst fluid. On testing urine and serum, the antigen detection ELISA was found to be more specific than antibody detection ELISA. EITB was found to be the most sensitive and specific test.Conclusions:ELISA using polyclonal antibodies against 24 kDa urinary hydatid protein was moderately sensitive to detect hydatid antigen in serum and urine. Hence polyclonal antibodies to 24 kDa urinary hydatid antigen can be used as an alternative source of antibody to detect hydatid antigen in serum, urine and cyst fluid. In the present study, EITB was found to be highly specific test for detection of hydatid antibodiesin serum. 24 kDa protein was found to be specific and of diagnostic value in CE.

Evaluation of Ziehl-Neelsen staining, auramine phenol staining, antigen detection enzyme linked immunosorbent assay and polymerase chain reaction, for the diagnosis of intestinal cryptosporidiosis

Background and Objectives:Cryptosporidiosis is a very important opportunistic infection and is responsible for significant morbidity and mortality in HIV/AIDS patients. The objective of this study is to evaluate Ziehl-Neelsen staining, auramine phenol staining, antigen detection enzyme linked immunosorbent assay and polymerase chain reaction, for the diagnosis of intestinal cryptosporidiosis.Materials and Methods:The study was designed to determine the efficacy of modified Ziehl-Neelsen (ZN), Auramine-Phenol (AP) staining, antigen detection enzyme linked immunosorbent assay (ELISA) and nested polymerase chain reaction (PCR) for detection of cryptosporidia in 671 HIV-seropositive patients, 353 HIV-seronegative patients including 198 children with diarrhea and 50 apparently healthy adults.Results:Cryptosporidium was detected in 26 (3.9%), 37 (5.5%), 32 (4.8%) and 40 (6%) HIV-seropositive and 8 (2.3%), 10 (2.9%), 9 (2.6%) and 9 (2.6%) HIV-seronegative patients by ZN staining, AP staining, antigen detection ELISA and PCR, respectively. None of the healthy controls were infected with Cryptosporidium. Based on criteria of ‘true positive’ samples, i.e. positive by any two of the four techniques out of ZN, AP, antigen detection ELISA and PCR, sensitivity of ZN and ELISA was 79.06% and 95.35% respectively. AP and PCR were found to be 100% sensitive. Specificity of ZN and ELISA was 100% while specificity of AP and PCR was 99.59% and 99.39% respectively.Conclusions:Auramine phenol staining is a rapid, sensitive and specific technique for diagnosis of intestinal cryptosporidiosis.

First detection of pike fry-like rhabdovirus in barbel and spring viraemia of carp virus in sturgeon and pike in aquaculture in the Czech Republic

Rapid antigen detection enzyme-linked immunosorbent assay (ELISA) testing of cell cultures with organ homogenate from fish, collected from farms with a predominance of common carp or in natural aquaculture in the Czech Republic between 1995 and 2008, identified piscine vesiculovirus in 27 of 178 samples. Using reverse transcription semi-nested PCR, targeting a 550 nucleotide region of the glycoprotein (G) gene, piscine vesiculovirus was confirmed in 23 of the 27 organ samples diagnosed by ELISA as infected. PCR products were amplified and sequenced from 18 isolates from common carp Cyprinus carpio (family Cyprinidae), 2 isolates from northern pike Esox lucius (family Esocidae), and 1 isolate each from Siberian sturgeon Acipenser baerii (family Acipenseridae), common barbel Barbus barbus (family Cyprinidae), and koi carp Cyprinus carpio koi (family Cyprinidae). The sequences (based on 401 nucleotides) clustered into 2 genogroups. The majority of isolates (n = 22), including those from sturgeon and pike, grouped with the spring viraemia of carp virus (SVCV) Genogroup I and Subgroup Id. The 22 isolates could be further subdivided into 2 groups: Id1 (n = 20) and Id2 (n = 2). A marker (a non-conservative nucleotide substitution) for the Id1 SVCV group was identified. It was specifically found in all sequences of Id1 isolates when testing SVCV originating from different countries. The remaining isolate from barbel, was classified in the pike fry-like rhabdovirus Genogroup IV. This is the first confirmation of natural SVCV infection in sturgeon and pike, and pike fry-like rhabdovirus infection in barbel. In the case of the pike fry-like rhabdovirus, this is also its first identification in the Czech Republic. According to the presence/absence of evident clinical signs of rhabdoviral disease in the 3 infected hosts, only the sturgeon seemed to be susceptible to the monitored rhabdovirus.

Performance Evaluation of Five Detection Tests for Avian Influenza Antigen with Various Avian Samples

In this paper, we report on the evaluation of five influenza antigen detection tests by avian influenza H5N 1 virus-positive swab samples to estimate their diagnostic sensitivity. The tests included two chromatographic immunoassays, an H5 avian influenza-specific antigen detection enzyme-linked immunosorbent assay (ELISA), an influenza A antigen detection ELISA, and an H5 rapid immunoblot assay. The results showed that the overall sensitivities of these tests ranged from 36.3% to 51.4% (95% confidence interval ranging from 31.0% to 57.0%), which were comparable to Directigen Flu A antigen detection tests but substantially lower than genome detection methods. Diagnostic sensitivity performance is a function of the concentration of antigens in samples and the analytical sensitivity of the individual test. The test sensitivities were significantly higher for sick and dead birds by cloacal, tracheal, or tissue swabs than for fecal swabs from apparently healthy birds, and these tests would not be suitable for surveillance testing of clinically healthy birds. Furthermore, the sensitivity for testing tracheal and cloacal swabs from waterfowl and wild birds was not as good as for chickens. This was most likely to be associated with variation in virus titers between specimens from different bird species. However, the tests showed good sensitivities for testing brain swabs from clinically affected waterfowl species. The results indicate that these antigen detection tests could be used for preliminary investigations of H5N 1 outbreaks as a low-cost, simple flock test in sick and dead birds for the rapid detection of H5N1 infection. However, the relatively low sensitivity of the tests as individual bird tests means that they should be used on optimal clinical specimens from diseased birds, testing birds on a flock basis, or testing samples as close to the onset of disease as possible before viral titers diminish. They should be followed up by confirmatory tests, such as reverse transcription polymerase chain reaction or viral culture, wherever possible but could assist in facilitating rapid investigations and control interventions.

Detection of Ebola Virus in Oral Fluid Specimens during Outbreaks of Ebola Virus Hemorrhagic Fever in the Republic of Congo

Patients who have refused to provide blood samples has meant that there have been significant delays in confirming outbreaks of Ebola virus hemorrhagic fever (EVHF). During the 2 EVHF outbreaks in the Republic of Congo in 2003, we assessed the use of oral fluid specimens versus serum samples for laboratory confirmation of cases of EVHF. Serum and oral fluid specimens were obtained from 24 patients with suspected Ebola and 10 healthy control subjects. Specimens were analyzed for immunoglobulin G antibodies by enzyme-linked immunosorbent assay (ELISA) and for Ebola virus by antigen detection ELISA and reverse-transcriptase polymerase chain reaction (RT-PCR). Oral fluid specimens were collected with a commercially available collection device. We failed to detect antibodies against Ebola in the oral fluid specimens obtained from patients whose serum samples were seropositive. All patients with positive serum RT-PCR results also had positive results for their oral fluid specimens. This study demonstrates the usefulness of oral fluid samples for the investigation of Ebola outbreaks, but further development in antibodies and antigen detection in oral fluid specimens is needed before these samples are used for filovirus surveillance activities in Africa.