Positive Enzyme-linked Immunoassay Research Articles

Prevalence of Skin-specific Autoantibodies in HIV-infected Patients and Uninfected Controls.

Various autoantibodies are detected more frequently in HIV-infected individuals than in HIV-negative controls; however, limited data exist regarding autoimmune blistering skin diseases. Using enzyme-linked immunoassay (ELISA) and indirect immunofluore-scence, no difference in the frequency and magnitude of autoantibodies against BP180, BP230, desmoglein 1 and 3 was found between 594 HIV-infected patients and 248 uninfected controls in this cross-sectional study (16.0% vs. 11.7%, respectively, for at least one positive ELISA, p = 0.11). Interestingly, reactive syphilis serology in both HIV-infected individuals and uninfected controls was associated with positive anti-BP180 ELISA results (adjusted odds ratio (OR) 2.14, 95% confidence interval (CI) 1.07-4.29, p = 0.03 and OR 4.70, CI 1.3-16.86; p = 0.0180). Our study shows a comparably low prevalence of cutaneous autoantibodies in both HIV-infected patients and uninfected controls lacking signs of autoimmune blistering skin disease. Positive BP180 ELISA in the absence of clinical signs of bullous pemphigoid should prompt further evaluation for syphilis antibodies.

Anti-PF4/Heparin Antibody Levels in Bacteremia, Fungemia and Sepsis

Heparin-induced thrombocytopenia (HIT) is caused by antibodies targeting platelet factor 4 (PF4)/heparin complexes. The immune response leading to HIT remains perplexing with many paradoxes. Unlike other drug induced reactions, anti-PF4/heparin antibody generation does not follow the classic immunologic response. As Greinacher and colleagues have shown, the primary immune response lacks IgM precedence and class switching, and heparin-induced antibodies can induce HIT by day 5 in heparin-naïve patients.Continued exposure to heparin also is puzzling with a weak or declining secondary immune response. Research by Krauel and colleagues suggests that that there is close interplay among infection, PF4 and the immune system. In 2010 they demonstrated that human and murine PF4 bind to Gram positive (S.aureus, S.pneumoniae, L.monocytogenes) and Gram negative (E.coli, N.meningitidis) bacteria in vitro, with bacterial surfaces acting as polyanions. High dose heparin inhibited this binding and anti-PF4/heparin antibodies from patients with HIT reacted with these PF4/bacterial complexes (S. aureus and E. coli). Using a murine model, they went on to show that polymicrobial sepsis in the absence of heparin led to antibody generation. In a separate study, Krauel and colleagues also showed that PF4 binds specifically to the lipid A component of Gram negative bacteria.In this analysis, we report on anti-PF4/heparin antibody levels in groups of patients hospitalized for sepsis, as compared to a control group without sepsis. We examined 200 patients with sepsis, retrospectively identified, from a hospital database of anti-PF4/heparin testing done in medical inpatients with thrombocytopenia but low pretest probability of HIT. This included patients with bacteremia (57), fungemia (7) and sepsis without septicemia (136). For comparison, data from 50 patients without sepsis during the same time period was used. Inclusion criteria for all groups were age 18 years and older and antibody testing within 4 days of admission. Exclusion criteria were diagnosis of HIT or heparin allergy, prior hospitalization or heparin exposure within 90 days of admission, cardiopulmonary bypass or orthopedic surgery within 6 months, hemodialysis, active or past malignancy, antiphospholipid syndrome, autoimmune disease or immunosuppressive therapy.All patients studied were on subcutaneous heparin at prophylactic doses only (i.e. no intravenous use, no therapeutic anticoagulation). UFH use predominated with prevalence of >85% in all groups. Testing was done using a commercially available standardized solid phase enzyme-linked immunoassay (EIA) to detect antibodies (IgG/IgA/IgM) directed against PF4 complexed with polyvinylsulfonate (Genetic Testing Institute, Wisconsin). All assays were performed in the central hospital laboratory according to manufacturer's specifications and measured in optical density (OD) units. The data sets demonstrated continuous unimodal distribution with high OD outliers, indicative of varying immune responses along a continuum. Statistical significance was calculated using independent t-testing with p-value set at 0.05 for significance.Results showed that patients hospitalized with sepsis have higher anti-PF4/heparin antibody levels. Both patients with bacteremia, and sepsis without bacteremia, had significantly higher OD than patients without sepsis (p<0.05). There was no significant difference between Gram negative and Gram positive bacteremia and antibody levels. This suggests that bacterial cell wall components of both classes have similar antigenicity. Interestingly, patients with fungemia had much lower antibody levels compared to bacteremia and sepsis. Despite the small sample size for fungemia, this difference trended strongly towards statistical significance (p=0.05). The threshold for a positive EIA is currently established at OD>0.400, a value based on sensitivity and set by the manufacturer. When the prevalence of a positive EIA was assessed, 16% patients with sepsis and bacteremia tested positive compared to 4% in the control group. In summary, there is an increased prevalence of anti-PF4/heparin antibodies in patients hospitalized with bacterial but not fungal sepsis. These results support the theory that bacterial infection has a role to play in preimmunization leading to anti-PF4/heparin antibody generation. DisclosuresNo relevant conflicts of interest to declare.

Enzyme-linked Immunoassay Index for Anti-NC16a IgG and IgE Auto-antibodies Correlates with Severity and Activity of Bullous Pemphigoid.

Bullous pemphigoid (BP) is characterized by IgG and IgE autoantibodies to the NC16a domain of BP180. This study evaluated the correlation between body surface area (BSA), total serum IgE and enzyme-linked immunoassay (ELISA) for IgG and IgE anti-NC16a in 77 patients with BP in the active stage, and the degree of conversion to negative of the studied parameters in clinical remission. Statistically positive correlations were observed between BSA and examined parameters (correlation index 0.2548, 0.2491, 0.311, respectively). Originally, patients with BP with positive ELISAs for both IgG and IgE autoantibodies presented twice as extensive skin lesions as those with positive IgG and negative IgE ELISAs. Conversion of ELISAs for IgG and IgE to negative in clinical remission occurred in 9.3% and 81% of patients with BP, respectively. This confirmed that ELISA for IgE anti-NC16a is a helpful parameter in the modification of current treatment and the assessment of risk of relapse in BP.

Standardization of an Indirect ELISA for Serological Diagnosis of Aspergilloma

Background: So far several literatures have discussed that GMP as a major cell wall component in Aspergillus species and can also be secreted into the medium as a component of the exoantigen. Moreover, one of the critical points in designing an ELISA is the preparation of the antigen. Objectives: The main aim of this study was the identification of GMP and standardization of an indirect ELISA for the serological diagnosis of Aspergilloma. Materials and Methods: An indirect enzyme linked immunoassay (ELISA) was developed and standardized for the serological diagnosis of Aspergilloma. Results: Among 500 serum samples, 35 had positive and 465had negative ELISA test against Aspergilloma. In comparison, serum samples were tested by immunoblotting test, considered as the standard. In relation to the immunoblotting, the ELISA presented 94.2% sensitivity, 99.5% specificity, 94.2% positive predictive value, 99.5% negative predictive value and 99.2% precision. Conclusions: It was concluded that the ELISA is a suitable method for large scale screening of antibodies against Aspergilloma. The easy techniques have shortened the required time of tests (approximately 1.30 hours for our ELISA, as opposed to 2.30 hours with commercial ELISA kit). This may be a substantial benefit for large numbers of samples that should be tested.

The Pattern of Indeterminate Human Immunodeficiency Virus Test and Follow-Up Evaluation in Pregnant Women

We studied the pattern of indeterminate HIV serological tests among pregnant women with follow-up testing in the postpartum period. Medical records of pregnant women were reviewed over a 2-year period. Of 16,596 pregnant women, 127 (0.8%) had positive HIV enzyme-linked immunoassay (ELISA) result. With Western blot (WB) test, 54 (0.33%) were positive, 43 (0.26%) were negative, and 30 (0.18%) were indeterminate. One of the 30 women (3.3%) with indeterminate WB converted to positive WB during pregnancy. White and black women were more likely to have an unconfirmed positive ELISA (indeterminate or negative WB) than Hispanics ( P = 0.021). The positive WB rate for black women was significantly higher ( P < 0.001) than other racial/ethnic groups. The postpartum follow-up testing of 14 women with indeterminate WB varied between 4 to 20 weeks; 16 did not have any postpartum follow-up test. The common bands in indeterminate WB were P24, P18, and nonviral proteins. The pattern of indeterminate WB result and its follow-up was variable during pregnancy and postpartum period. There is a need for development of national standards of care for indeterminate WB mothers and their infants in the postpartum period. Additional studies are needed to determine the cause of indeterminate tests, reducing their occurrence in the testing process, and the optimum time for testing in the postpartum period.

Prediction of Heparin Induced Thrombocytopenia: Validation of an Experience-Based Computer Model.

Background: Diagnosis of heparin-induced thrombocytopenia (HIT) requires certain clinical features along with the detection of platelet activating antibodies induced by heparin interaction with platelet factor 4 (PF4). Although the PF4 enzyme-linked immunoassay (ELISA) is a highly sensitive laboratory test, it is not specific. Adding excess heparin to the sample can confirm a positive PF4 ELISA result when it decreases antibody binding by 50% or more, potentially improving the specificity of the assay. We sought to determine the clinical value of the PF4 ELISA and confirmatory test by developing a predictive algorithm for HIT.Methods: We retrospectively identified 116 patients with positive anti-PF4/heparin antibodies at Duke University Medical Center in 2005. We collected data on all patients to allow for clinical classification as HIT positive or HIT negative. HIT positive patients had at least a 30% decline in platelet count after 4–14 days of heparin exposure, or within 48 hours if recent (within last 100 days) heparin exposure. Anti-PF4/heparin antibody titers were determined by ELISA using a confirmatory step with excess heparin. A multivariate logistic regression model was fitted to the 2005 data, to estimate the relationship between patient characteristics (including age, gender, race, and clinical service), laboratory findings (including peak PF4 titer, confirmatory positive status), and clinical HIT status (HIT positive versus HIT negative). The model was then validated on an independent sample of 97 patients with positive anti-PF4/heparin antibodies retrospectively identified at Duke University Medical Center from January to July of 2006.Results: We found no significant relationship between age, race, or gender and clinical HIT status. In multivariate analysis, the peak PF4 titer and confirmatory positive status were independent predictors of HIT. Figure 1 depicts median, 25th, and 75th percentiles of the predicted probabilities for the HIT-positive and HIT-negative patients in the 2005 training and 2006 test populations. The predictive accuracy on the 2005 training set (c-index 0.783, Somer's Dyx 0.566) was maintained when the algorithm was applied to the independent 2006 test population (c-index 0.799, Somer's Dyx 0.597). Figure 2 depicts the ROC curves for both patient populations.Conclusions: This is the first study to our knowledge that has demonstrated the clinical utility of the Heparin/PF4 ELISA confirmatory test for the diagnosis of HIT. Based upon the PF4 ELISA and confirmatory assays, a predictive computer algorithm can distinguish patients likely to have HIT from those who are not. Accurate predicted probabilities of HIT will enable clinicians to more rapidly initiate appropriate therapy. [Display omitted] [Display omitted]

Differential diagnoses of suspected Lyme borreliosis or post-Lyme-disease syndrome

The symptoms of Lyme borreliosis are similar to those of a variety of autoimmune musculoskeletal diseases. Persistence of complaints is frequently interpreted as unsuccessful antibiotic treatment of Borrelia-associated infections. However, such refractory cases are rare, and re-evaluation of differential diagnoses helps to avoid the substantial risk of long-term antibiotic therapy. In this study, we analyzed patients who presented to our rheumatology unit with previous suspected or diagnosed Lyme borreliosis. Eighty-six patients from a 3.5-year period were evaluated. The mean age of patients was 49.2 +/- 17.2 years; 60% (n = 52) reported a tick bite and 33% (n = 28) an erythema. Forty-seven percent (n = 39) had positive enzyme-linked immunoassay results and Western blots (Mikrogen, Martinsried, Germany). All but 12 patients had already received antibiotic treatment previously. Nine percent (n = 8) had ongoing or recent Lyme borreliosis. Twenty-nine percent (n = 25) showed clinical symptoms and radiographic changes compatible with degenerative disorders of the cervical and/or lumbar spine. These patients were significantly older when compared to the other patients (59.3 +/- 13.7 years vs 46.1 +/- 17.2 years, p = 0.001). Seventeen percent (n = 16) had arthropathies related to psoriasis or rheumatoid arthritis. Twelve percent (n = 10) were positive for the HLA B27 antigen. Other diseases were less frequent. Six patients (7%) could not be diagnosed conclusively, and four of these patients had negative Borrelia immunoassay results. In conclusion, Borrelia-associated diseases were rare in this study. Differential diagnoses helped to initiate a successful disease-specific therapeutic strategy.

The Incidence of Thrombosis in Patients with Isolated Heparin Induced Thrombocytopenia.

Introduction: Heparin-induced thrombocytopenia type-II (HIT) is a serious prothrombotic disorder caused by heparin exposure. The incidence of thrombosis in patients with isolated HIT, defined as HIT without clinically evident thrombosis at the time of diagnosis, is not well established.Aim: The purpose of this prospective study was to determine the total incidence of thrombotic events after diagnosis of isolated HIT from radiographic evidence of asymptomatic deep venous thrombosis (DVT) plus radiographic confirmation of symptomatic thrombosis.Patients and Methods: We evaluated all patients with a positive enzyme-linked immunoassay (ELISA) for heparin-platelet factor 4 (PF4) antibody (Ab) daily at Massachusetts General Hospital from 10/10/05 to 5/13/06. Inpatients with (1) a positive PF4 Ab test, (2) thrombocytopenia, as defined by a ≥50% drop from baseline platelet count and/or a fall in platelet count to <150×109/L, in association with heparin exposure, (3) no signs or symptoms of thrombosis at time of the positive Ab test, and (4) no other definitive etiology of thrombocytopenia were considered to have isolated HIT and included for study. Patients with a prior diagnosis of HIT, DVT, pulmonary embolism, or peripheral arterial thrombosis were excluded. Within 72 hours of diagnosis and of initiation of a non-heparin anticoagulant, all included patients underwent radiographic examination for asymptomatic DVT in the lower extremities (LE). Objective evidence of thrombotic events other than LE DVT after the diagnosis was also recorded. Daily platelet count, type and timing of all anticoagulants, use of blood products, and PF4 Ab titer were collected to determine if there was an association between these factors and development of thrombosis. Mortality rate during hospitalization was also recorded.Results: Of the 158 patients with a positive heparin-PF4 Ab, 64 patients met criteria for study, 14 of which were lost to follow-up. Among the 50 remaining eligible patients, the total incidence of thrombosis was 20% (12% were found to have an asymptomatic thrombotic event and 8% developed a symptomatic thrombotic event). Development of thrombosis was independently associated with platelet transfusion (p=0.005) and with the degree of platelet count nadir as expressed by platelet count (p=0.038) or by percent decrease from baseline (p=0.031). There was no association between the PF4 Ab titer or the type and timing of non-heparin anticoagulant and development of thrombosis. The overall mortality rate in patients diagnosed with isolated HIT during hospitalization was 22%.Conclusion: The total incidence of thrombotic events in isolated HIT was 20%, with greater than half of the events being asymptomatic thromboses found only by radiographic examination. This high incidence of asymptomatic LE DVT suggests that routine investigation for LE DVT should be performed in this patient population and that patients with isolated HIT should be treated with a non-heparin anticoagulant. Our findings also confirm the current recommendation to avoid platelet transfusions in patients with isolated HIT as we found an increased rate of thrombosis associated with this practice.

HLA-Specific antibodies are associated with high-grade and persistent-recurrent lung allograft acute rejection

Background The impact of HLA-specific antibodies is not well established in the acute rejection of lung allografts. Acute rejection represents the most important risk factor for the development of chronic lung allograft dysfunction. Methods We analyzed the pattern of HLA antibodies before and after transplantation in 54 patients, and correlated our data with the presence and frequency of high-grade and persistent-recurrent acute rejection, during the first 18 post-operative months. The diagnosis of acute rejection was based on histologic International Society for Heart and Lung Transplantation (ISHLT)-published criteria. Results Ten of 54 patients had a positive enzyme-linked immunoassay (ELISA) post-transplantation. In 90% of ELISA-positive patients, the presence of HLA antibodies was associated with persistent-recurrent acute rejections, compared with 34% in the ELISA-negative group ( p < 0.005). There were 28 high-grade acute rejection episodes in the ELISA-positive group, compared with 36 in the ELISA-negative group ( p < 0.0001). The ELISA-positive patients required a greater intensity of immunosuppressive therapy. The patients with ELISA-detected anti-HLA antibodies were at least 3-fold more likely to develop high-grade acute rejection and persistent-recurrent acute rejection, and 7-fold more likely to develop multiple episodes of persistent-recurrent acute rejection, compared with ELISA-negative patients. Conclusions ELISA-based screening for the development of HLA antibodies is a reliable method that can identify lung transplant recipients at increased risk for high-grade and persistent-recurrent acute rejection. Although bronchiolitis obliterans appears as a point of no return in the evolution of lung-transplanted patients, early detection of risk factors for acute rejection could indirectly decrease the incidence of bronchiolitis obliterans. These lung-transplanted patients may benefit from an altered strategy of immunosuppression.

Comparison of antigen-capture ELISA to stool-culture methods for the detection of asymptomatic Entamoeba species infection in Kafer Daoud, Egypt.

We performed a prospective field study in the village of Kafer Daoud in Menofia, Egypt to compare the fecal culture method with enzyme linked immuno assay (ELISA) for detection of 170 kDa lectin antigen in feces for diagnosis of asymptomatic Entamoeba histolytica and Entamoeba dispar infection. All subjects with E. histolytica or E. dispar infection detected by culture also had positive ELISA for amebic antigen in their feces and an additional 57 Entameoba infections missed by culture were detected by ELISA (P < 0.001 compared to culture). The presence of fecal anti-lectin IgA antibodies and serum anti-LC3 (recombinant cysteine-rich lectin protein) IgG antibodies were positive predictors for E. histolytica infection (P < 0.03). Of interest, infection with Trichomonas hominis but not Blastocystis hominis was positively associated with E. histolytica infection (P < 0.05). In conclusion, ELISA for detection of fecal lectin antigen is a more sensitive method than fecal culture for detecting asymptomatic E. histolytica infection.

Ocular Toxocara canis infections: diagnosis by enzyme immunoassay.

An enzyme-linked immunoassay (ELISA) was used to establish the sero-prevalence of Toxocara canis infection in several New Zealand populations including those who, through environment or occupation, had close contact with dogs. The prevalence of positive ELISA values found reflected the degree of dog contact, e.g. positive ELISA's occurred in 2.8% of urban adults, 13.9% of dog breeders and 28.4% of hydatid control officers emphasizing that a history of dog contact needs to be considered when ELISA results are interpreted in a clinical context. Ophthalmoscopy of 102 hydatid officers, 28.4% of whom were seropositive, disclosed no evidence of ocular toxocariasis and suggested that the officers might be immunoprotected. Preliminary immunoblot analysis of the range of toxocara excretory/secretory antigens bound to serum immunoglobulin from patients with presumed visceral larva migrans and ocular toxocariasis, showed the same reactivity profile as a positive reference serum and serum from a seroresponsive hydatid control officer.