Positive Enzyme-linked Immunosorbent Assay Results Research Articles

Prevalence of Different Courses of Feline Leukaemia Virus Infection in Four European Countries.

Prevalence of progressive feline leukaemia virus (FeLV) infection is known to still be high in cats in Europe, especially in Southern Europe, but the prevalence of other outcomes of FeLV infection has not been determined in most countries. The present study aimed to investigate the prevalence of progressive, regressive, abortive, and focal infection in four European countries, two with a high (Italy, Portugal) and two with a low expected prevalence (Germany, France). Blood samples of 934 cats (Italy: 269; Portugal: 240; France: 107; Germany: 318) were evaluated for the p27 antigen, as well as anti-whole virus, anti-SU, and anti-p15E antibodies by enzyme-linked immunosorbent assay (ELISA) in serum and for proviral DNA by quantitative polymerase chain reaction (qPCR) in whole blood. Positive p27 antigen ELISA results were confirmed by reverse transcriptase-qPCR (RT-qPCR) detecting viral RNA in saliva swabs and/or blood. The outcome of FeLV infection was categorised as progressive (antigen-positive, provirus-positive), regressive (antigen-negative, provirus-positive), abortive (antigen- and provirus-negative, antibody-positive), and focal (antigen-positive, provirus-negative) infection. Overall FeLV prevalence was 21.2% in Italy, 20.4% in Portugal, 9.5% in Germany, and 9.3% in France. Prevalence of progressive, regressive, abortive, and focal infection in Italy was 7.8%, 4.5%, 6.3%, and 2.6%; in Portugal 3.8%, 8.3%, 6.7%, and 1.7%; in Germany 1.9%, 1.3%, 3.5%, and 2.8%; in France 1.9%, 3.7%, 2.8%, and 0.9%, respectively. In conclusion, overall FeLV prevalence is still very high, especially in Southern European countries. Therefore, testing, separation of infected cats, and vaccination are still important measures to reduce the risk of FeLV infection.

Associations between Johne’s disease and fertility in UK dairy herds

The objective of this observational study was to quantify associations between Mycobacterium avium subspecies paratuberculosis (MAP) antibody status and a variety of fertility outcomes, in UK dairy cattle. Longitudinal milk recording, fertility and MAP antibody enzyme-linked immunosorbent assay (ELISA) milk test data were collated retrospectively from 121,762 lactations in 78 herds. Datasets were structured into appropriate units to suit outcomes and enable temporal association between current and future MAP status, and fertility measures. Current MAP status was categorised according to most recent status within 180 days, with time-related future MAP status assigned based on MAP antibody ELISA milk test data for each cow. Multilevel multivariable logistic regression models were used to evaluate associations between MAP status and 21-day pregnancy and submission rate and conception risk. Posterior predictions and cross-validation techniques were used to assess model fit and check model building assumptions. A negative association was found between risk of insemination (Odds Ratio [OR], 0.78; 95% Credible Interval [CI], 0.66–0.92) and conception occurring (OR, 0.65; CI, 0.5–0.84) and transition from negative to non-negative MAP test status in the next 30–90 days. A positive association was observed between risk of insemination (OR, 1.34; CI, 1.16–1.52) and conception occurring (OR, 1.26; CI, 1.11–1.43) and transition from negative to non-negative MAP test status in the next 90–180 days. Current positive MAP test status was negatively and positively associated with insemination (OR, 0.59; CI, 0.49–0.70) and conception risk (OR, 1.12; CI, 0.96–1.30), respectively. Herd managers will have had access to test results, declaring cows with past recent or multiple positive MAP antibody ELISA results not to be bred, negatively influencing insemination risk. Overall, these results demonstrate the temporal association between a positive MAP antibody ELISA result and dairy cow fertility outcomes, with particular variability prior to a positive MAP antibody ELISA result.

Evaluation of glutamic acid decarboxylase (GAD) 65 antibody detection methods for neurological and diabetic investigation in an Australian diagnostic laboratory

The role of anti-glutamic acid decarboxylase (GAD) 65 autoantibodies in autoimmune neurological conditions is evolving, but testing recommendations remain unchanged in Australia with GAD enzyme-linked immunosorbent assay (ELISA) and immunoblot as the only two Therapeutic Goods Administration approved testing methods available in Australia. Common practice is for use of ELISA in diagnosis of type 1 diabetes mellitus (T1DM) and use of immunoblot for diagnosis of GAD65-associated neurological disease. We observed a cohort of patients with negative immunoblot results and positive ELISA in the context of GAD-associated neurological disease without T1DM. In the absence of robust consensus guidelines on preferred testing modalities, we sought to determine if ELISA could have a superior role in the diagnosis of GAD-associated neurological disease when compared to immunoblot in this paper. We tested for anti-GAD65 autoantibodies on 55 patient samples, 40 samples requested for neurological disease and 15 type 1 diabetes samples with detectable anti-GAD65, using two testing platforms: Euroimmun anti-GAD enzyme-linked immunosorbent assay (ELISA) and. Euroimmun EuroLine immunoblot for paraneoplastic neurologic syndromes. These results were correlated against the clinical scenario. Positive ELISA results had a sensitivity of 100% and specificity of 91% for GAD65-related neurological disease. Immunoblot showed sensitivity of 43% and specificity of 76% for GAD65-related neurological disease. ELISA proved more sensitive and specific for GAD65-related neurological disease compared to immunoblot, raising questions about the role of this testing modality in neurological disease. We propose that ELISA should be used as a sole diagnostic method for all GAD65 antibody-related neurological disease over immunoblot. The presence of anti-GAD65 antibody on immunoblot is of doubtful clinical significance.

Combining Immunoassays to Identify Zika Virus Infection in Dengue-Endemic Areas.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged as a global health threat. The rise in ZIKV infections has driven an increased incidence of neonates born with microcephaly or other neurological malformations. Therefore, screening for ZIKV infection can considerably impact pregnant women, especially during the first trimester. The majority of ZIKV infections are mild or asymptomatic, and clinical diagnosis is inaccurate. Moreover, given the high level of cross-reactivity among flaviviruses, serological approaches to distinguish ZIKV from dengue virus (DENV) infections are complicated. We used the combination of DENV and ZIKV nonstructural protein 1 (NS1) IgG enzyme-linked immunosorbent assay (ELISA) and ZIKV NS1 blockade-of-binding (BOB) ELISA to test the convalescent sera of non-flavivirus, primary DENV, secondary DENV, and ZIKV infections. Our findings indicate that primary testing using a ZIKV NS1 IgG ELISA, the test of choice for large-scale ZIKV serosurvey studies, provided relatively high sensitivity. Moreover, the confirmation of positive ELISA results using the ZIKV NS1 BOB ELISA increased average specificity to 94.59% across serum samples. The combined use of two simple ELISAs for ZIKV serosurveys and the monitoring of ZIKV infection during pregnancy can elucidate the epidemiology, pathogenesis, and complications of ZIKV in DENV-endemic areas.

Aquaporin-4 Autoantibody Detection by ELISA: A Retrospective Characterization of a Commonly Used Assay.

Objective Aquaporin-4 (AQP4) serum autoantibodies are detected by a variety of methods. The highest sensitivity is achieved with cell-based assays, but the enzyme-linked immunosorbent assay (ELISA) is still commonly utilized by clinicians worldwide. Methods We performed a retrospective review to identify all patients at the University of Utah who had AQP4 ELISA testing at ARUP Laboratories from 2010 to 2017. We then reviewed their diagnostic evaluation and final diagnosis based on the ELISA titer result. Results A total of 750 tests for the AQP4 ELISA were analyzed, and 47 unique patients with positive titers were identified. Less than half of these patients (49%) met the clinical criteria for neuromyelitis optica spectrum disorder (NMOSD). In cases of low positive titers (3.0–7.9 U/mL, n = 19), the most common final diagnosis was multiple sclerosis (52.6%). In the moderate positive cohort (8.0–79.9 U/mL, n = 14), only a little more than half the cohort (64.3%) had NMOSD. In cases with high positives (80–160 U/mL, n = 14), 100% of patients met clinical criteria for NMOSD. Conclusions Our data illustrates diagnostic uncertainty associated with the AQP4 ELISA, an assay that is still commonly ordered by clinicians despite the availability of more sensitive and specific tests to detect AQP4 autoantibodies in patients suspected of having NMOSD. In particular, low positive titer AQP4 ELISA results are particularly nonspecific for the diagnosis of NMOSD. The importance of accessibility to both sensitive and specific AQP4 testing cannot be overemphasized in clinical practice.

Evaluation of the ELISA and immune blotting value for the diagnosis of echinococcosis

Background: The main role in the diagnosis of human echinococcosis belongs to instrumental methods. Enzyme-linked immunosorbent assay (ELISA) for determinftion the specific echinococcosis IgG is important in predicting the development of relapses and evaluating the effectiveness of treatment. This method has significant deficiency – false-positive results due to cross-reactions in patients with other parasitic or allergic diseases. Purpose: To evaluate the value of ELISA and immunoblotting for diagnosis of echinococcosis and monitoring the effectiveness of treatment. Methods & Materials: We analyzed 1500 serum samples of patients with echinococcosis and persons suffered from another parasitic or allergic diseases. The study was carried out by ELISA for detection of specific Echinococcus granulosus IgG. Also 66 serum samples containing specific IgG were studied by immunoblotting. Results: ROC analysis showed that the sensitivity of ELISA in determination of specific Echinococcus granulosus IgG was 92.23%, specificity 88.87%, AUC is 0.91. The predictive value of a positive result was 56.89%, negative – 98.63%. Immunoblotting results of 66 samples containing IgG showed: in 6 patients with enterobiosis a positive result was due to non-specific P39 protein (39 kDa). In 3 patients who underwent surgical treatment due to alveococcosis P16/18 (16–18 kDa) proteins were detected, specific for all another echinococcosis except echinococcus granulosus. Specific P7 protein (7 kDa) and non-specific P39 were detected before surgery in 15 patients with isolated uncomplicated echinococcosis. Six months after surgery and antigelminthic treatment we observed 5-fold decrease in level of IgG and only non-specific P39 in 6 patients, in 9 cases it was happen only after 3 years. In 18 patients with multiple recurrent echinococcosis highly specific P7, non-specific P39 and P16/18 (16–18 kDa) proteins were detected one month after repeated surgical treatment. One year after the completion of 6-month course of anti-relapse therapy a decrease of level the specific immunogobulins was noted, and positive results in ELISA was due to non-specific P16/18. Conclusion: The use of serological methods in the diagnosis of echinococcosis and monitoring the effectiveness of surgical and anti-relapse treatment is justified. The method of immunoblotting allows to differentiate echinococcosis and detect false positive ELISA results with other parasitic invasions.

Anti-HIV-1 antibodies based confirmatory results in Wuhan, China, 2012-2018.

The number, intensity and order of emergence of HIV-1 specific antibodies in serum or plasma were associated with the stage of HIV-1 infection. In this study, we retrospectively analyzed the HIV-1 confirmatory results tested by western blot (WB) or recombination immunoblot assay (RIBA) in Wuhan, 2012–2018, to access the profiles of HIV-1 specific antibodies. A total of 14432 HIV-suspected serum or plasma samples collected from local hospitals and other HIV screening laboratories were further screened by two 4th generation enzyme-linked immunosorbent assay (ELISA) kits in our laboratory, of which 11068 specimens (76.69%) had at least one positive ELISA result and thereby were finally confirmed with WB or RIBA. RIBA had identified 652 (81.09%) positive and 13 (1.62%) indeterminate cases from July 1, 2014 to January 7, 2015, while WB had identified 8358 (81.43%) positive and 643 (6.26%) indeterminate cases in the other times during 2012–2018. The indeterminate rate of WB was significant higher than that of RIBA (p<0.001). Although the number of HIV-1 infected subjects increased significantly from 2012 (n = 911) to 2018 (n = 1578), the positive rate of HIV-1 antibodies decreased markedly from 70.08% in 2012 to 58.79% in 2018 (p<0.001). The most commonly observed antibody profile was gp160+gp120+p66+(p55+)p51+gp41+p31+p24+p17+ (4131, 49.43%) for WB-MP and gp160+gp120+gp41+p31+p24+p17+ (382, 58.59%) for RIBA-WANTAI, and the absence of reactivity to three possible serologic markers for recent HIV-1 infection, p31, p66, and p51, increased significantly from 2012 to 2018, with the overall rate of 17.03%, 9.40%, and 15.15%, respectively. The suspected acute HIV-1 infection was also observed to be increased in recent years, with an overall rate of 1.00%. Our results indicated the detection rate had decreased for HIV-1 infection, but increased for suspected recent and acute HIV-1 infection during 2012–2018, reflecting the efforts of intervention among high risk population.

Effectiveness of personal protective equipment in preventing severe acute respiratory syndrome coronavirus 2 infection among healthcare workers

IntroductionInformation on the effectiveness of personal protective equipment (PPE) for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among healthcare workers (HCWs), especially among HCWs with frequent contact with patients with SARS-CoV-2, is limited. MethodsWe conducted a prospective cohort study on 49 HCWs who worked in close contact with patients with SARS-CoV-2 infection. HCWs had blood samples taken every 2 weeks to test for SARS-CoV-2 antibodies using two different types of assay. ResultsForty-nine participants (31 nurses, 15 doctors, 3 other workers) were enrolled. In total, 112 blood samples are obtained from participants. The median work days in 2 weeks was 9 (interquartile range (IQR): 5–10) days. In a single work day, 30 of the 49 participants (61.5%) had contact with patients with suspected or conformed SARS-CoV-2 at least 8 times, and approximately 60% of participants had more than 10 min of contact with a single patient. The median self-reported compliance to PPE was 90% (IQR: 80–100%). Seven participants tested positive for SARS-CoV-2 antibody using enzyme-linked immunosorbent assay (ELISA); however, none were seropositive for SARS-CoV-2 neutralizing antibody, so the positive ELISA results were assumed to be false-positive. ConclusionsThe study provides evidence that appropriate PPE is sufficient to prevent infection amongHCWs. It is necessary to establish a system that provides a stable supply of PPE for HCWs to perform their duties.

Molecular Detection of Group A Rotavirus in under Five Years Old Children with Acute Diarrhoea Admitted to Yangon Children’s Hospital

Rotaviruses are regarded as the most common cause of viral gastroenteritis and are responsible for considerable morbidity and mortality among children especially under five years of age worldwide. In developing countries like Myanmar, where diarrhoea is in the priority childhood disease, rotavirus surveillance and detection of rotavirus genotypes are utmost important. A hospital-based, cross-sectional descriptive study was conducted at Yangon Children‟s Hospital among under five children admitted for acute diarrhoea from January to October 2016. This study includes detection of Group A rotavirus antigen by commercial enzyme-linked immunosorbent assay (ELISA) and genotyping by multiplex RT-PCR. From a total of 488 collected samples, rotavirus antigen was detected in 219 samples (45%). Rotavirus diarrhoea was most common among the age of 6-11 months (38.8%) followed by 12-23 months (37.9%). The results showed that boys were more commonly affected than girls. Detection of rotavirus positivity was peak in February (57.6 %). Out of 219 stool samples with positive ELISA result, 40 stool samples with high optical density value were proceeded for further determination of G and P genotypes. Regarding distribution of G genotypes, the most common G genotype was G9 which comprised 45%, and that of P genotype was P[8] which comprised 92.5%. Regarding combination of G and P genotypes, the most frequent combination is G9P[8], and it constituted 42.5%. Untypable genotypes were seen in 30% of G and 2.5% of P typing. As rotavirus infection can be prevented by vaccine, WHO recommended that rotavirus vaccination should be included in national immunization program especially in countries where prevalence of rotavirus is high. The distribution of G and P genotypes is important in consideration of appropriate vaccine in pre-vaccination and evaluation of effectiveness of vaccine in post-vaccination period. Therefore, the information on currently circulating genotypes of rotavirus in this study will serve as valuable data for vaccination programme.

First dengue virus seroprevalence study on Madeira Island after the 2012 outbreak indicates unreported dengue circulation

BackgroundIn 2012, the first dengue virus outbreak was reported on the Portuguese island of Madeira with 1080 confirmed cases. Dengue virus of serotype 1 (DENV-1), probably imported from Venezuela, caused this outbreak with autochthonous transmission by invasive Aedes aegypti mosquitoes.ResultsWe investigated the seroprevalence among the population on Madeira Island four years after the outbreak. Study participants (n = 358), representative of the island population regarding their age and gender, were enrolled in 2012 in a cross-sectional study. Dengue antibodies were detected with an in-house enzyme-linked immunosorbent assay (ELISA) using the dimer of domain III (ED3) of the DENV-1 envelope protein as well as commercial Panbio indirect and capture IgG ELISAs. Positive ELISA results were validated with a neutralization test. The overall seroprevalence was found to be 7.8% (28/358) with the in-house ELISA, whereas the commercial DENV indirect ELISA detected IgG antibodies in 8.9% of the individuals (32/358). The results of the foci reduction neutralization test confirmed DENV-1 imported from South America as the causative agent of the 2012 epidemic. Additionally, we found a higher seroprevalence in study participants with an age above 60 years old and probable secondary DENV infected individuals indicating unreported dengue circulation before or after 2012 on Madeira Island.ConclusionsThis study revealed that the number of infections might have been much higher than estimated from only confirmed cases in 2012/2013. These mainly DENV-1 immune individuals are not protected from a secondary DENV infection and the majority of the population of Madeira Island is still naïve for DENV. Surveillance of mosquitoes and arboviruses should be continued on Madeira Island as well as in other European areas where invasive vector mosquitoes are present.

Aflatoxin M1 in cow, sheep, and donkey milk produced in Sicily, Southern Italy.

Samples (n = 485) of raw (n = 394) or heat-treated (n = 91) milk of three different species (cow, n = 170; sheep, n = 133; donkey, n = 84), collected 2013-2016 in Western Sicily (Southern Italy), were analyzed for aflatoxin M1 (AFM1) by enzyme-linked immunosorbent assay (ELISA). Positive ELISA results were further analyzed by HPLC with fluorescence detection. Both methods had a detection limit for AFM1 in milk of 7ngkg-1. ELISA yielded 12.9 and 5% positives in cows and sheep milk, respectively, all samples of donkey milk were negative. Levels of AFM1 were in most cases at 0.007-< 0.05μgkg-1, only two samples (sheep milk) slightly exceeded the European Union maximum level of 0.05μgkg-1. Only 6% of the samples were positive for AFM1 in a concentration range of 0.008-0.15μgkg-1. Only milk samples collected directly from farms were positive. Overall, the levels were much lower than previously reported for Southern Italy cow and sheep milk samples purchased in retail stores. The results of this work indicate a continuous improvement of the feeding techniques on dairy farms of Southern Italy, which is essential to ensure consumers' protection.

HITTING the Diagnosis: Testing for Heparin-Induced Thrombocytopenia in Cancer Patients.

To evaluate the use of a pretest probability score (4Ts score) in cancer patients to guide ordering of laboratory screening tests for heparin-induced thrombocytopenia (HIT). A retrospective chart review was conducted for patients (n = 140) in whom laboratory testing for HIT was requested. 4Ts scores were calculated and correlated with heparin-endogenous platelet factor 4 antibody enzyme-linked immunosorbent assay (ELISA) test results. All patients with a high pretest probability of HIT (4Ts score = 6-7) had positive ELISA results, compared to 26.1% of patients with intermediate (4Ts score = 4-5) and 4.3% of patients with low (4Ts score ≤3) pretest probability. No patients with 4Ts scores of 2 or less had positive ELISA results. HIT can be ruled out in cancer patients (negative predictive value and sensitivity = 100%) with low pretest probability, defined by 4Ts scores of 2 or less, significantly reducing the need for laboratory testing in this patient population.

High prevalence of sensitization to gibberellin‐regulated protein (peamaclein) in fruit allergies with negative immunoglobulin E reactivity to Bet v 1 homologs and profilin: Clinical pattern, causative fruits and cofactor effect of gibberellin‐regulated protein allergy

Gibberellin-regulated protein (GRP) is a new allergen in peach allergy, with an amino acid sequence very well conserved through several botanical species. We investigated the allergenicity of GRP in fruit allergies other than peaches and identified the clinical characteristics of fruit allergy patients with GRP sensitization. One hundred consecutive Japanese patients with fruit allergies were enrolled in the present study. To identify the features of GRP sensitization, we selected patients with negative ImmunoCAP results for Bet v 1 homologs and profilin, which are marker allergens for pollen-food allergy syndrome (PFAS), or lipid transfer protein. These patients underwent specific immunoglobulin E measurements by enzyme-linked immunosorbent assay (ELISA) and skin prick tests (SPT) using purified nPru p 7. Twenty of 100 consecutive patients with fruit allergies had negative ImmunoCAP results for Bet v 1 homologs and profilin. Thirteen (65.0%) of the 20 patients had positive ELISA and/or SPT results using nPru p 7, whereas one of the 20 patients had positive ImmunoCAP results for Pru p 3. In 13 nPru p 7-sensitized patients, the causative foods were peaches (92.3%), apricots (61.5%), oranges (46.2%) and apples (30.8%). Ten patients (76.9%) had multiple causative fruits. Frequent symptoms included facial edema (92.3%) and laryngeal tightness (66.7%). In eight patients (61.5%), exercise or aspirin intake enhanced the allergic reaction onset as cofactors. The prevalence of GRP sensitization was high in Japanese fruit allergy patients except for PFAS patients. In conclusion, GRP-sensitized patients may have allergies to multiple fruits and may show peculiar characteristics such as facial swelling and cofactor dependence.

Nonstrangulating intestinal infarction associated with Strongylus vulgaris in referred Danish equine cases.

Strongylus vulgaris is a pathogenic helminth parasite infecting horses and was once considered to be the primary cause of colic. Migrating larvae cause ischaemia and infarction of intestinal segments. This knowledge is derived from case reports and experimental inoculations of parasite-naïve foals, and it remains unknown to what extent the parasite is associated with different types of colic. To evaluate the role of S. vulgaris as a risk factor for different types of colic in horses. A retrospective case-control study among horses referred with abdominal pain to the University of Copenhagen Large Animal Teaching Hospital during 2009-2011. Each colic case was matched with an equid of the same type (pony, Warmblooded or Coldblooded), age, sex and admitted in the same month and year but for problems unrelated to the gastrointestinal tract. Serum samples were analysed for antibodies to migrating S. vulgaris larvae using a recently developed enzyme-linked immunosorbent assay (ELISA). The following 4 case definitions were used: colic sensu lato, i.e. all horses presenting with colic (n = 274), with further subgroups, i.e. undiagnosed colics (n = 48), strangulating obstructions (n = 76) and nonstrangulating infarctions (n = 20). Strongylus vulgaris antibody levels were similar to control values in colics sensu lato and horses with undiagnosed colic. In contrast, nonstrangulating intestinal infarctions were significantly associated with positive S. vulgaris ELISAs (odds ratio 5.33, 95% confidence interval 1.03-27.76, P = 0.05). Also, horses with nonstrangulating infarctions had a significantly higher occurrence of positive ELISAs than horses with strangulating obstructions (odds ratio 3.79, 95% confidence interval 1.34-10.68, P = 0.01) and the colic sensu lato group (odds ratio 3.09, 95% confidence interval 1.20-8.01, P = 0.02). Nonstrangulating intestinal infarction was strongly associated with S. vulgaris-specific antibodies, whereas the more broadly defined colic categories were not associated with positive ELISA results. Thus, the ELISA holds potential to become a helpful adjunct in diagnosis and management of horses with colic.

Serum Strongylus vulgaris-specific antibody responses to anthelmintic treatment in naturally infected horses.

Strongylus vulgaris is the most pathogenic helminth parasite of horses, causing verminous endarteritis with thromboembolism and infarction. A serum enzyme-linked immunosorbent assay (ELISA) has been validated for detection of antibodies to an antigen produced by migrating larvae of this parasite. The aim was to evaluate ELISA responses to anthelmintic treatment in cohorts of naturally infected horses. Fifteen healthy horses harboring patent S. vulgaris infections were turned out for communal grazing in May 2013 (day 0). On day 55, horses were ranked according to ELISA titers and randomly allocated to the following three groups: no treatment followed by placebo pellets daily; ivermectin on day 60 followed by placebo pellets daily; or ivermectin on day 60 followed by daily pyrantel tartrate. Fecal and serum samples were collected at ∼28-day intervals until study termination on day 231. Increased ELISA values were observed for the first 53 days following ivermectin treatment. Titers were significantly reduced 80 days after ivermectin treatment. Horses receiving daily pyrantel tartrate maintained lower ELISA values from 137 days post ivermectin treatment until trial termination. These results illustrate that a positive ELISA result is indicative of either current or prior exposure to larval S. vulgaris infection within the previous 5 months.

Gluten detection in foods available in the United States – A market survey

Many gluten-free (GF) food choices are now available in supermarkets. However, the unintentional presence of gluten in these foods poses a serious health risk to wheat-allergic and celiac patients. Different GF labelled foods (275) and non-GF labelled foods, without wheat/rye/barley on the ingredient label (186), were analysed for gluten content by two different enzyme linked immunosorbent assay (ELISA) kits. Considering the gluten threshold of 20ppm, GF labelled foods had 98.9% GF labelling compliance with 1.1% (3 out of 275) of foods being mislabelled/misbranded. Among the non-GF labelled foods, 19.4% (36 out of 186) of foods had >20ppm of gluten, as measured by at least one ELISA kit, of which 19 foods had >100ppm of gluten. The presence of oats in non-GF labelled foods was strongly correlated with a positive ELISA result. Gluten was also found in a significant number of foods with gluten/wheat-related advisory warnings.

High False Positive Rate Of An ELISA Screen For The Detection Of Anti-Factor VIII Antibodies In Congenital Hemophilia A

IntroductionAnti-factor VIII antibodies are a serious complication of factor replacement therapy in hemophilia A patients. Neutralizing antibodies could previously only be detected via the functional and gold-standard Bethesda assay with or without the Nijmegen modification. The introduction of an enzyme-linked immunosorbent assay (ELISA) screening test provides a less laborious alternative to the Bethesda assay and also has a high sensitivity. Unlike the Bethesda assay, the ELISA can potentially detect non-neutralizing antibodies, which raise the possibility of false positive screens. As it is important that both clinicians and the coagulation laboratory understand the clinical performance of ELISA screening, this study evaluated the sensitivity and specificity of ELISA and Bethesda assays used in both a laboratory validation study and in subsequent clinical experience. MethodsResults from all active adult and pediatric congenital hemophilia A patients who underwent both Bethesda assay and ELISA in British Columbia, Canada as of July 2013 were included in this study. The sensitivity and specificity were compared to the provincial coagulation laboratory validation study of the GTI Factor 8 Antibody ELISA kit against the classical Bethesda assay conducted in both hemophilia A and normal controls in 2010. Optical density (OD) readings were retrieved for eligible samples and from the laboratory validation study. Results35 samples from 26 different patients were used in the validation study performed in 2010. Of 147 adult and 86 pediatric hemophilia A clinic patients active during the study period, 56 samples from 16 adults and 17 children underwent concurrent Bethesda assay and ELISA screen between November 2010 and June 2013. Since ELISA implementation, 389 ELISA screens and 187 Bethesda assays were performed. ELISA screens resulted in the avoidance of Bethesda assays in 85% (330/389) of samples submitted during that period. Specificities and positive predictive values were lower in the clinical sample due to a larger number of false positives (n=18; 32%) relative to the validation study (n=3; 9%), while sensitivity and negative predictive values remained at 100% (Table 1). Interestingly, 67% (2/3) of false positive ELISA screens in the laboratory setting had Bethesda positive histories; however, in the clinical sample, only 6% (1/18) had a distant history of inhibitors. ODs were available for all validation study samples and for 54 clinical samples. Redefinition of our OD cutoff to improve specificity was prevented by false positive patients with strongly positive ODs.Table 1ELISA pre-clinical and clinical performance summaryData sourceSample sizeSensitivitySpecificityPositive predictive valueNegative predictive valueLaboratory validation study351009057100Clinic561005547100Pediatric331004350100Adult231006840100 ConclusionsThe ELISA screen used in this setting is highly sensitive for anti-factor VIII antibody detection in congenital hemophilia A. However, compared to pre-clinical data, our 2.5 years of clinical experience reveals a high incidence of false positives resulting in a significantly lower specificity. Our approximate cost savings in British Columbia as a result of avoided Bethesda assays due to ELISA screens (n=330) is $90 per test, and may exceed $200 per test in the United States. Additional testing and follow-up on positive ELISA results can be an inconvenience in both time and blood sampling, especially in the pediatric population where inhibitor status is more closely monitored. The cost ramifications of this follow up testing could not be quantified in this study. The potential implications for the detection of non-neutralizing anti-FVIII antibodies in hemophilia A need to be further explored and long-term study and monitoring of these discrepant patients is warranted. Disclosures:No relevant conflicts of interest to declare.

Preliminary evaluation of a quantitative polymerase chain reaction assay for diagnosis of feline immunodeficiency virus infection.

Enzyme-linked immunosorbent assay (ELISA) has high sensitivity and specificity for detection of feline immunodeficiency virus (FIV) antibodies, but does not distinguish between infection and vaccination. Real-time quantitative PCR (qPCR) assays may distinguish infected from vaccinated cats. Performance of a commercial qPCR assay was assessed with blood samples from 29 FIV-infected non-vaccinated, 26 FIV-uninfected vaccinated and 35 FIV-uninfected non-vaccinated cats. FIV infection status of cats was assigned based on a combination of vaccination and medical history, prevention of contact with potentially infected cats and two FIV antibody ELISA results. Test sensitivity and specificity were determined against this gold standard. The qPCR test yielded positive results in samples from 23/29 FIV-infected non-vaccinated, 2/26 FIV-uninfected vaccinated and 0/35 FIV-uninfected non-vaccinated cats. It was concluded that the qPCR test was moderately sensitive and highly specific for the diagnosis of FIV infection, and that it may be suitable for ruling out FIV infection in cats with a positive antibody ELISA result and unknown vaccination history.

False‐positive results in ELISA‐based anti FVIII antibody assay may occur with lupus anticoagulant and phospholipid antibodies

The evaluation of a prolonged aPTT often includes Lupus Anticoagulant, Antiphospholipid Antibodies, and Factor VIII (FVIII) inhibitors. We have noticed that patient samples positive for lupus antibody (LA) are frequently also positive for FVIII IgG antibodies in an enzyme-linked immunosorbent assay (ELISA), indicating the need for follow-up testing with a more labour-intensive functional assay for FVIII inhibition. This study evaluates the potential for a FVIII IgG ELISA to yield false-positive results in patient samples positive for LA or other antiphospholipid antibodies. A total of 289 residual de-identified patient samples positive for LA (n = 143), anti-cardiolipin IgG (n = 84), or beta2-glycoprotein antibody (n = 62) were tested for FVIII IgG using a commercial ELISA. Samples with positive FVIII IgG ELISA results were further tested for FVIII activity using a clot-based FVIII inhibitor assay. The FVIII IgG ELISA yielded positive results in 39 (13%) of the samples tested, including 13/143 (13%) LA-positive, 15/85 (18%) aCL IgG-positive and 6/62 (10%) β2-glycoprotein IgG-positive samples. The clot-based FVIII inhibitor assay yielded negative results in all 39 FVIII IgG-positive specimens tested, indicating discrepancy with the FVIII IgG ELISA results. Patient specimens positive for LA, aCL IgG, or β2-glycoprotein IgG may yield false-positive results for FVIII antibodies. Caution is warranted in interpreting FVIII antibody results in these cases.

Analysis of available diagnostic tests for latex sensitization in an at-risk population

BackgroundLack of a Food and Drug Administration (FDA)–approved skin testing reagent for latex allergy in the United States requires reliance on patient history and serologic assays for diagnosis. ObjectiveTo determine the diagnostic sensitivity, specificity, and predictive values of an FDA-cleared antilatex IgE serology test and an enzyme-linked immunosorbent assay (ELISA) with various sources of latex protein antigens in an at-risk but unselected population of health care workers. MethodsHealth care workers underwent duplicate latex and serologic testing for latex specific IgE with the CAP assay and ELISA from June 1, 1998, through December 31, 2002. Logistic regression with receiver operating characteristic curve analysis determined the values, resulting in 98% and 99% specificity for the CAP assay and ELISA, respectively. ResultsResults of paired skin and serologic tests were available for 792 participants. Forty duplicate skin test results (5%) were positive. For the CAP assay, sensitivity was 35%; specificity, 98%; positive predictive value, 48.3%; and negative predictive value, 96.6%. ELISA demonstrated similar results. Multivariable logistic regression yielding a 98% or 99% specificity for the various ELISAs demonstrated that the adjusted odds of a positive skin test result significantly increased with positive CAP assay and ELISA results using a powdered glove extract. ConclusionsThe performance of the FDA-cleared antilatex IgE serologic test for latex allergy has much lower sensitivity than previously reported. This finding confirms that this serologic test should be used only for patients with a history of latex allergy and not for screening the population with a low prevalence of latex sensitization.