Enzyme-linked Immunoassay Tests Research Articles

High prevalence of hepatitis B and HIV among women survivors of sexual violence in South Kivu province, eastern Democratic Republic of Congo.

Limited data are available on the prevalence rates of hepatitis B and acquired immunodeficiency syndrome (AIDS) among women survivors of sexual violence (WSSV) in South Kivu province, in the eastern part of the Democratic Republic of Congo (DRC), where armed conflicts persist. Here, we aimed to assess the prevalence of these two infections in this vulnerable local population. A total of 1002 WSSV, aged from 18 to 70 years old were enrolled from May 2018 to May 2020 at three healthcare facilities (Panzi, Mulamba and Bulenga hospitals), which are called "The One-Stop Centre Care Model" for the management of sexual violence in South Kivu. Blood samples were collected and tested for hepatitis B virus (HBV) and human immunodeficiency virus (HIV) antigens and antibodies using enzyme-linked immunoassay (ELISA) methods. Subsequently, viral load quantification for HBV and HIV were performed using the GeneXpert. Univariate and multivariate logistic regression models were used to assess factors associated with HIV-positive and HBV-positive status. For HBV, overall prevalence was 8.9% (95% CI; 7.2-10.8%), 32.1% (95% CI; 29.3-35.0%), and 14.5% (95% CI; 12.3-16.8%) for HBsAg, anti-HBc and anti-HBs antibodies, respectively. Among the 89 HBsAg-positive patients, 17 (19.1%) were HBeAg-positive. The median age of individuals with a positive HBsAg test was higher than those with a negative test (median: 40 years (IQR 30-52) compared to 36 years (IQR 24-48)). Risk factors for HBV infection were age (≥35 years) (AOR = 1.83 [1.02-3.32]; p = 0.041), having no schooling (AOR = 4.14 [1.35-12.62]; p = 0.012) or only primary school-level (AOR = 4.88 [1.61-14.75]; p = 0.005), and multiple aggressors (AOR = 1.76 [1.09-2.84], p = 0.019). The prevalence of HIV was 4.3% [95% CI: 3.1-5.7%]. HIV/HBV co-infection occurred only in 5 individuals (0.5%). The HBV viral load was detectable (> 1 log10 UI/mL) in 61.8% of HBsAg-positive subjects and 64.8% HIV-positive subjects had a high viral load (≥ 3 log10 copies/mL). This study revealed a high prevalence of HBV and HIV infections among WSSV in South Kivu. The results generated highlight the urgent need for systematic screening of HBV and HIV by integrating fourth-generation ELISA tests in HIV and HBV control programs.

Expression of a single-chain monellin (MNEI) mutant with enhanced stability in transgenic mice milk.

Monellin is a sweet protein that may be used as a safe and healthy sweetener. However, due to its low stability, the application of monellin is currently very limited. Here, we describe a wild-type, a double-sites mutant (E2N/E23A) and a triple-sites mutant (N14A/E23Q/S76Y) of single-chain monellin (MNEI) expressed in transgenic mice milk. Based on enzyme-linked immunoassay (ELISA), Western blot, and sweetness intensity testing, their sweetness and stability were compared. After boiling for 2min at different pH conditions (2.5, 5.1, 6.8, and 8.2), N14A/E23Q/S76Y-MNEI showed significantly higher sweetness and stability than the wild-type and E2N/E23A-MNEI. These results suggest that N14A/E23Q/S76Y-MNEI shows remarkable potential as a sweetener in the future.

Comparative analysis of contemporary anti-double stranded DNA antibody assays for systemic lupus erythematosus.

Elevated double-stranded DNA (dsDNA) antibody levels in blood serum are considered a disease-specific marker in systemic lupus erythematosus (SLE), correlate with disease activity and the incidence of lupus nephritis, and can be detected in up to 86% of all SLE cases. Despite the high clinical relevance, the variety of dsDNA antibody testing methods with heterogenous performance in clinical use remains challenging. This study is the first to prospectively investigate the performance of two of today's most commonly applied anti-dsDNA testing methods head-to-head under real-world conditions, as well as their correlation with other clinical and serological disease parameters in SLE patients. In this prospective study, all SLE patients undergoing treatment at the Department of Rheumatology at the University Hospital Bonn within a 13-months period (n=41) and control patients without connective-tissue disease (n=51) were consecutively enrolled and examined. For all study participants' serum samples both anti-dsDNA-NcX enzyme-linked immunoassay testing EUROIMMUN, Luebeck, Germany) and the fluorescence immunoassay ELiA dsDNA (Thermo Fisher Scientific, Waltham, USA) were performed. In addition, demographic data, further laboratory values and disease activity parameters were recorded. Clinical disease activity was assessed by SLEDAI-2K. Both assays showed high specificity (anti-dsDNA-NcX ELISA: 0.9, ELiA dsDNA: 0.959), but there were notable differences in sensitivity (anti-dsDNA-NcX ELISA: 0.51, ELiA dsDNA: 0.38). Pearsons's correlation yielded a positive correlation between anti-dsDNA concentrations and CRP concentrations for the anti-dsDNA-NcX ELISA (R=0.22; p=0.038) and a mild-to-moderate inverse correlation between concentrations of anti-dsDNA and complement C4 for the ELiA dsDNA test (R=-0.22; p=0.045) when SLE and control patients were considered together. Other than, no significant correlation between anti-dsDNA concentrations and clinical or laboratory findings was found for either test procedure. Both anti-dsDNA antibody assays represent reliable examination methods with high specificity for the diagnosis of SLE that fulfill EULAR/ACR requirements. However, the anti-dsDNA-NcX ELISA showed superior sensitivity and significant correlation with disease activity (as measured by CRP concentrations).

Abstract 11896: Spontaneous Heparin-Induced Thrombocytopenia Presenting as Acute Stent Thromboses Following an Acute St-Segment Elevation Myocardial Infarction

Introduction: Spontaneous heparin-induced thrombocytopenia (HIT) is a rare clinical variant that develops without prior exposure to heparin. We present a 40-year-old man who was diagnosed with spontaneous HIT after an acute ST-segment elevation myocardial infarction complicated by multiple acute stent thromboses and left ventricular (LV) thrombus. Case: A 40-year-old previously healthy man with no known history of heparin exposure presented to our emergency department with an acute ST-segment elevation myocardial infarction. Emergent coronary angiography revealed a severely thrombotic 100% occlusion of his distal left anterior descending artery for which thrombectomy and percutaneous coronary intervention (PCI) were performed with placement of a drug eluting stent. Prior to angiography, the patient received 4000 units of unfractionated heparin and an additional 2000 units intra-arterially during the case. Labs prior to PCI revealed thrombocytopenia and HIT enzyme-linked immunoassay testing was sent. He underwent repeat angiography two additional times during his course for recurrent chest pain which both revealed acute stent thrombosis. HIT testing eventually returned positive for the presence of platelet factor 4 antibodies. A subsequent serotonin release assay returned positive, confirming the diagnosis of HIT. A transthoracic echocardiogram during his hospitalization also revealed an LV thrombus. Discussion: Heparin-induced thrombocytopenia (HIT) is traditionally diagnosed after thrombocytopenia develops 5 to 10 days after exposure to heparin. However, there have been rare reports of a spontaneous form of HIT that can occur without prior exposure to heparin. This case highlights a rare presentation of spontaneous HIT. Although infrequent, cardiologists should be aware of atypical presentations of HIT, since almost all patients will receive heparin prior to or during coronary angiography and coronary interventions. Spontaneous HIT should be considered in all patients who present with unexplained thrombocytopenia with arterial and/or venous thrombi. Failure to recognize this clinical entity could lead to potentially life-threatening thrombotic complications from further administration of heparin.

West Nile virus in the Republic of Serbia-Diagnostic performance of five serological tests in dog and horse sera.

West Nile virus (WNV) is a zoonotic mosquito-borne virus classified as family Flaviviridae and genus Flavivirus. The first WNV outbreak in humans in the Republic of Serbia was recorded in 2012. Equids and dogs can show clinical symptoms after WNV infection and are often used as sentinels. This study aimed to (i) give insight into seropositivity for WNV in clinically healthy dog and horse sera in different regions of Serbia and (ii) compare diagnostic value of 'in-house' and commercially available indirect immunofluorescence (IFA) and enzyme-linked immunoassay (ELISA) tests to 'gold standard' virus neutralization test (VNT). Due to cross-reactivity, sera were tested for Usutu virus and tick-borne encephalitis virus in VNT based on the epidemiological data of field presence. Blood sera of dogs (n=184) and horses (n=232) were collected from 2011 to 2013. The seropositivity was confirmed by VNT in 36.9 % tested dog sera and 34.9% tested horse sera with highest positivity in regions near two big rivers, while in four dog and seven horse sera, positivity resulted from Usutu virus infection. Comparative results of diagnostic tests in dogs ranged from 18.7 % seropositivity by 'in-house' ELISA to 31.9% by commercially available ELISA. In horses, seropositivity ranged from 36.2% by 'in-house' IFA to 32.5% by commercially available IFA and from 26.3% by 'in-house' IgG ELISA to 20.9% by commercially available ELISA. There were no statistically significant differences according to the McNemar test between 'in-house' and commercially available IFA and ELISA test in horse sera, while the same was not true for two ELISAs used in dog sera (χ2 =8.647, p=.003). Established seropositivity in dogs and horses was in accordance with the epidemiological situation and WNV spread in the Republic of Serbia and proven Usutu virus co-circulation. 'In-house' tests remain a valuable tool in early diagnostics of WNV.

Moving ahead in HCV diagnostics: Need for the introduction of HCV core antigen assay in low and middle-income countries

Hepatitis C virus (HCV) infections are associated with significant morbidity and mortality globally. The diagnosis of HCV is primarily based on indirect serological assays such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), and rapid diagnostic tests to detect HCV antibodies. Direct tests detect/quantify components of HCV virions, such as HCV ribonucleic acid (RNA) (nucleic acid test or nucleic acid amplification test [NAT]) and HCV core antigen (HCVcAg). The HCVcAg assay (CIA, Abbott ARCHITECT) is an immune assay used for the quantitative determination of the HCVcAg. This test is simple and fast with the potential to be incorporated into diagnostic guidelines and be used in combination with anti-HCV (CIA) as an effective screening test. HCVcAg can also be used as a potential biomarker for treatment initiation and monitoring patients to assess the treatment response. Apart from this, the scope for implementation of the HCVcAg assay in resource limited setting lies in screening of immune compromised patients where anti-HCV serology is not dependable. However, concerns related to lower sensitivity compared to HCV RNA do exist. Nevertheless, the HCVcAg assay can make a significant difference in the measures taken for the control and eradication of hepatitis C and its complications in India.

Seroprevalence of Rubella Virus-specific Antibodies in Women and the Diagnostic Efficacy of Enzyme-linked Immunoassay and Rapid Immunochromatographic Tests.

IntroductionRubella is an infectious disease caused by the Rubella virus. The disease was previously called German measles and is transmitted through respiratory aerosols. Rubella causes both clinical and subclinical infections in children and young adults. Rubella virus has teratogenic capabilities and may cause severe complications in the fetuses of women who acquire Rubella viral infection during their pregnancy. The present study aims to evaluate the seroprevalence of anti-Rubella virus immunoglobulin (Ig) G and IgM antibodies in both pregnant and non-pregnant women and assess the diagnostic efficacy of enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic tests.MethodsThe study included 240 females in the age range of 16-45 years. The study subjects included both pregnant women and non-pregnant women. After informed consent, 5 milliliters of blood was collected from each participant, and serum was separated and tested for the presence of antibodies (IgG and IgM) against the Rubella virus using both the traditional ELISA (Delta Biologicals, Pvt. Ltd., China) and a rapid ELISA-immunochromatographic test (ICT) (Span Biotech. Ltd., China). The data collected were systematically entered into Microsoft Excel sheets (Microsoft Corporation, Redmond, Washington) and were analyzed using SPSS Statistics for Windows, Version 17.0, 2008 (SPSS Inc., Chicago, Illinois).ResultsThe study revealed an overall seroprevalence of 31.66% for Rubella-specific IgG and IgM antibodies. Out of the 125 pregnant women included in the study, 49 (39.20%) were seropositive for Rubella IgG antibodies, and among the 115 non-pregnant women tested, 24 (20.86%) were positive for Rubella IgG antibodies. Four (5.26%) of the 76 seropositive women revealed IgM antibodies. The sensitivities of both the ELISA (40.61%) and rapid immunochromatographic (39.20%) tests were observed to be low and the specificities of both methods were similar (79.13%).ConclusionThe seroprevalence of Rubella-specific IgG antibodies was observed to be low as compared to the other regions of India. The low seroprevalence may predispose pregnant women to Rubella viral infection and may lead to increased incidences of congenital Rubella syndrome (CRS). Both the ELISA and immunochromatographic tests showed low sensitivity and similar specificities.

Meta-analysis of diagnostic performance of serology tests for COVID-19: impact of assay design and post-symptom-onset intervals

ABSTRACT Serology detection is recognized for its sensitivity in convalescent patients with COVID-19, in comparison with nucleic acid amplification tests (NAATs). This article aimed to evaluate the diagnostic accuracy of serologic methods for COVID-19 based on assay design and post-symptom-onset intervals. Two authors independently searched PubMed, Cochrane library, Ovid, EBSCO for case–control, longitudinal and cohort studies that determined the diagnostic accuracy of serology tests in comparison with NAATs in COVID-19 cases and used QUADAS-2 for quality assessment. Pooled accuracy was analysed using INLA method. A total of 27 studies were included in this meta-analysis, with 4 cohort, 16 case–control and 7 longitudinal studies and 4565 participants. Serology tests had the lowest sensitivity at 0–7 days after symptom onset and the highest at >14 days. TAB had a better sensitivity than IgG or IgM only. Using combined nucleocapsid (N) and spike(S) protein had a better sensitivity compared to N or S protein only. Lateral flow immunoassay (LFIA) had a lower sensitivity than enzyme-linked immunoassay (ELISA) and chemiluminescent immunoassay (CLIA). Serology tests will play an important role in the clinical diagnosis for later stage COVID-19 patients. ELISA tests, detecting TAB or targeting combined N and S proteins had a higher diagnostic sensitivity compared to other methods.

Utility and Pitfalls of Stepwise Laboratory Testing for Heparin Induced Thrombocytopenia: Retrospective Review from an Academic Medical Center

Introduction: Heparin induced thrombocytopenia (HIT) occurs in up to 5% of adults exposed to heparin due to formation of heparin-dependent antibodies to the heparin/platelet factor 4 (PF4) complex. Patients develop thrombocytopenia and are at risk for severe thrombotic complications. Mortality rates can be as high as 20%, but are often much lower with prompt recognition, cessation of heparin, and treatment with alternative anticoagulants. However, these alternative medications are often both labor-intensive drips and expensive, highlighting the need for quick decision making in such challenging cases. The classic workup of HIT involves assessing clinical suspicion (often using the "4T score" on a scale of 0-8), followed by lab testing for detection of the heparin-dependent antibody and then a functional assay to confirm pathogenicity of the antibody. Our institution uses an in-house rapid Particle ImmunoFiltration Assay (PIFA), which is a same-day test and reported as "positive" or "negative." Other testing available includes send-out tests for Enzyme-Linked Immunoassay (ELISA) to IgG, A, and M of the PF4 antibody as well as the C-14 Serotonin Release Assay (SRA) functional assay. The goal of this retrospective review is to analyze the utility of an algorithmic approach to laboratory testing in aiding the rapidity of diagnosis of HIT without missing possible critical cases. Methods: This was a single institutional study at a large urban academic medical center. We reviewed inpatient charts from 2015-2018 of patients who had any lab testing for HIT. As per institutional guidelines, first-line testing is recommended by using the in-house same-day testing PIFA. If positive, a sample is automatically reflexed and sent-out for PF4 ELISA testing and SRA. Furthermore, clinicians are able to directly order ELISA and SRA testing at their medical discretion. Our analysis looked at those patients with at least two different "HIT-related" laboratory tests to best analyze the concordance and discordance rates of the above testing to assess for sensitivity, specificity, and overall accuracy of using a stepwise testing approach. We used a cutoff value of >0.4 optical density (OD) for ELISA testing, and >20% release at low-dose heparin concentration for SRA testing. Results: There were 118 patients who had at least two different HIT-related laboratory tests sent. 91 patients had both PIFA and ELISA testing, with 37/79 (47%) positive concordance rate and 6/12 (50%) negative concordance rate, for a sensitivity of 86% and specificity of 13%. 3 patients with positive PIFA also had positive SRA, and there were 2 patients with negative PIFA with positive SRA testing (see attached Table). When comparing ELISA testing to SRA, 4/41 (10%) had concordant positive testing, while no patient with a negative ELISA test had a positive SRA (28 concordant negative cases). Overall, of 94 SRA tests run, 5 were positive, of which 2/5 had negative PIFA and 0/4 had negative ELISA testing. Conclusions: While PIFA testing had a high sensitivity compared to ELISA, the overall accuracy compared to ELISA was low, while ELISA testing was 100% sensitive in this analysis. Furthermore, there was still a risk of missing cases of HIT using PIFA testing alone. In both cases of positive SRA with a negative PIFA, patients had a high 4T score of >6, consistent with a high clinical suspicion for HIT. We conclude that PIFA testing is not equivalent to ELISA testing, and that use of a laboratory "algorithm only" approach would be inappropriate in the diagnosis of HIT. Our results highlight the importance of using both clinical scoring systems and appropriate lab testing together in the workup and diagnosis of HIT. Disclosures No relevant conflicts of interest to declare.

Possible role of circulating endothelial cells in patients after acute myocardial infarction

Acute myocardial infarction (AMI) occurs as a result of insufficient myocardial perfusion leading to cell necrosis. This is most commonly due to the obstruction of the coronary artery by ruptured atherosclerotic plaque and thrombosis. Damaged ischemic and necrotic myocardial cells release pro-inflammatory substances in tissue and plasma, leading to a systemic inflammatory response. Profound systemic inflammatory response during ischemia/reperfusion injury causes disruption of endothelial glycocalyx and detachment of endothelial cells that express von Willebrant factor (vWF). We hypothesize that circulating vWF+ endothelial cells could act as antigen presenting cells which interact with T and NK cells directly, by cell to cell contact and indirectly by cytokine and chemokine secretion, leading to the immune response towards inflammation. Analyzing the frequency, phenotype and pro-inflammatory substances produced in circulating vWF positive (+) cells in patients with AMI could be beneficial to determine the severity of the pro-inflammatory response, according to the level of endothelial dysfunction in the early period of AMI. To evaluate these hypotheses, we suggest to determine frequency, phenotype, and ability of cytokine/chemokine production in circulating vWF+ endothelial cells by simultaneous surface and intracellular cell staining, and flow cytometry analysis. Secretion of pro-inflammatory cytokines and chemokines, pro-atherogenic substances and the components of glycocalyx might be measured in supernatants of magnetically separated or sorted vWF+ endothelial cells, as well as in the serum of a patient with acute AMI by enzyme linked–immunoassay tests. The interaction of increasing concentrations of isolated circulating vWF+ endothelial cells and cognate T and NK cells might be investigated by lymphocyte proliferation rate, cytotoxic mediators’ expression, and cytokine production. If our hypothesis is correct, characterization of circulating vWF+ endothelial cells could grant us greater insight into their role in pathophysiology of AMI and the degree of myocardial damage.

Interdisciplinary approach at the primary healthcare level for Bolivian immigrants with Chagas disease in the city of São Paulo.

Background/MethodsIn a pioneering cross-sectional study among Bolivian immigrants in the city of São Paulo, Brazil, the epidemiological profile, clinical manifestations and morbidity of Chagas disease were described. The feasibility of the management of Chagas disease at primary healthcare clinics using a biomedical and psychosocial interdisciplinary approach was also tested. Previously, a Trypanosoma cruzi (T. cruzi) infection rate of 4.4% among 633 immigrants was reported. The samples were screened using two commercial enzyme-linked immunoassay (ELISA) tests generated with epimastigote antigens, and those with discrepant or seropositive results were analyzed by confirmatory tests: indirect immunofluorescence (IFI), TESA-blot and a commercial recombinant ELISA. PCR and blood cultures were performed in seropositive patients.ResultsThe majority of the 28 seropositive patients were women, of whom 88.89% were of child-bearing age. The predominant clinical forms of Chagas disease were the indeterminate and atypical cardiac forms. Less than 50% received the recommended antiparasitic treatment of benznidazole. An interdisciplinary team was centered on primary healthcare physicians who applied guidelines for the management of patients. Infectologists, cardiologists, pediatricians and other specialists acted as reference professionals. Confirmatory serology and molecular biology tests, as well as echocardiography, Holter and other tests, were performed for the assessment of affected organs in secondary healthcare centers. The published high performance of two commercial ELISA tests was not confirmed.ConclusionAn interdisciplinary approach including antiparasitic treatment is feasible at the primary healthcare level for the management of Chagas disease in Bolivian immigrants. The itinerant feature of immigration was associated with a lack of adherence to antiparasitic treatment and was considered a main challenge for the clinical management of this population. This approach is recommended for management of the infected population in endemic and nonendemic areas, although different strategies are needed depending on the severity of the disease and the structure of the healthcare system.

Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents

Hantaviruses infect humans via inhalation of viral particles in infected rodents' secretions such as saliva, urine and faeces or via direct contact with infected rodents. The rodent species that are known as the carriers of Dobrava (DOBV), Puumala (PUUV), Saaremaa (SAAV), Tula (TULV) and Seoul (SEOV) viruses are found in our country. The presence of specific antibodies against hantaviruses have been demonstrated in rodents collected from Black Sea and Aegean Regions of Turkey in 2004 for the first time. The first hantavirus-related hemorrhagic fever with renal syndrome (HFRS) cases were reported in Black Sea region in 2009. The determination of the hantavirus prevalence in wild life and rodent populations in the field is crucial for the information about hantavirus-related cases and to clarify the state of risk. There is no commercial product optimized for the screening of rodent serum samples in terms of HFRS agents like DOBV and PUUV that are widely seen in Eurasia as well as Turkey. In this study, the antigens belonging to the commercial enzyme-linked immunoassay (ELISA) and immunoblot tests that are produced for the screening of human sera were used for the development of antibody screening tests against hantavirus in rodent sera and were optimized. The most appropriate serum and conjugate dilutions were determined for the optimization of ELISA (Anti-Hantavirus Pool ELISA; Euroimmun, Germany) and immunoblot (Euroline Anti-Hanta Profile 1 strips; Euroimmun, Germany) methods. Optimized ELISA method was used for the screening and optimized immunoblot method was used for the confirmation. A total of 84 wild rodent sera that belonged to Apodemus and Microtus species were evaluated with this procedure and the cut-off value, sensitivity and specificity of optimized ELISA method were determined. For the optimization of ELISA 1/50, 1/100 and 1/200 serum dilutions and 1/10.000, 1/20.000 and 1/40.000 conjugate dilutions were tested. For the optimization of immunoblot, 1/50 and 1/100 serum dilutions and 1/5.000 and 1/10.000 conjugate dilutions were tested. The horseradish peroxidase conjugated goat anti-mouse IgG for ELISA and the alkaline phosphatase conjugated goat anti-mouse IgG for immunoblot were used. We followed the manufacturer's recommendations for the incubation parameters, substrate and the number of washes. 1/50 serum dilution and 1/10.000 conjugate dilution for ELISA and 1/100 serum dilution and 1/5.000 conjugate dilution for immunoblot were determined as optimal concentrations. By using the optimized ELISA, 26.2% (22/84) of rodents were found positive for hantavirus antibodies according the determined cut-off value (OD(450/620): 0.325). By using immunoblot as a confirmatory test, 20 out of 22 ELISA positive samples could be studied because of the insufficient amount of sera and 17 of them was found positive in terms of DOBV antibodies. Of these rodents 11 were Apodemus flavicollis, three were Apodemus agrarius, two were Microtus guentheri and one was Apodemus sylvaticus. When the results of ELISA were compared to immunoblot results, the optimized ELISA's sensitivity and specificity were found as 100% and 95%, respectively. In this study, a method that can be used in the screening of rodent sera was constituted which uses commercial antigens that can be provided easily, gives fast and reliable results. Similar serological methods optimized for different types of rodents are of great importance for the realization of active follow-up and monitoring of the studies in the field.

HIV testing patterns for United States Air Force personnel, 2008–2012

ObjectiveThis study evaluated 3rd generation human immunodeficiency virus (HIV) test patterns and HIV infection rates in the United States Air Force (USAF). Study designRetrospective database study. MethodsHIV enzyme-linked immunoassay (ELISA) and Western blot tests were analysed for all USAF personnel from 2008 to 2012. For new HIV cases, unadjusted and adjusted annual rates were calculated per 100,000 persons. ResultsIn total, 1,608,665 tests were performed in 626,298 individuals, with a reactive ELISA observed in 809 (0.001%) persons. Western blot (n = 1949) results included 378 (19.4%) positive, 1283 (65.8%) negative, and 288 (15.0%) indeterminate (WBi). Unadjusted annual HIV rates were between 16.7 and 20.6 per 100,000 persons during the study period. The overall age-adjusted rate was 14.8 cases per 100,000 persons tested. Blacks/African Americans had the highest risk of HIV (risk ratio 7.9 [95% confidence interval 5.78, 9.95] compared to Whites). ConclusionsWBi results, which can cause delays in determining HIV status, were relatively common with the 3rd generation assay. However, this will be mitigated by a planned transition to a 4th generation assay. Although the overall rate of HIV in the USAF is lower than US civilian adults, HIV prevention efforts targeting young Blacks/African Americans may help to reduce HIV incidence in the USAF.

Use of rapid tests for perinatal HIV infection in resource-limited settings

Early testing strategies such as human immunodeficiency virus (HIV) DNA and RNA assays have become popular, cheaper and reliable, however the measures of success of Prevention of Mother-to-Child Transmission (PMTCT) programs in predominantly breastfeeding populations often depend on long term follow-up and HIV testing of children. Diagnosis in PMTCT programs in resource-poor countries therefore remains dependent on serological assays. The use of rapid HIV tests in perinatal HIV diagnosis, as recommended by the WHO, was based on the sensitivity and specificity of the enzyme-linked immuno-assay (ELISA) in children and not guided by an independent evaluation of rapid tests. For the purpose of ensuring a reliable diagnosis of perinatal HIV infection in predominantly breastfed children and largely resource-poor settings in Sub-Saharan Africa, we evaluated a HIV rapid test against the ELISA in serial plasma samples. We tested stored plasma samples for HIV antibodies, using a rapid test (Abbott Determine HIV1/2, Abbott Diagnostics, Wiesbaden, Germany) from a cohort of children between 12 and 18 months of age, enrolled in the PETRA (perinatal transmission) study at the Durban site, and compared to pre-determined real-time ELISA results. Using the ELISA results at 18 months as gold standard, the sensitivity of the ELISA at 12 and 15 months was 100% (95% CI 79–100), and specificity improved from 90.4% (95% CI 86–94) at 12 months to 99.5% (95% CI 97–100) at 15 months. The sensitivity of the rapid HIV test when compared to ELISA results at 18 months was 100% (95% CI 46–98) at 12 and 15 months, and the specificity was 89% (95% CI 80–94) and 99% (95% CI 92–100) at 12 and 15 months respectively. The negative predictive values for the rapid test at 12, 15 and 18 months were 100% (95% CI 94–100). Using the 18 month ELISA as the gold standard, the rapid test at 12 and 15 months demonstrated similar specificities to the corresponding ELISA tests. We concluded that the sensitivity and specificity of the rapid HIV test remained optimal (100%) for the diagnosis of HIV infection in children at 18 months. The earlier use of a rapid test at 15 months correctly excluded HIV infection in 99% of the children. Any earlier use of rapid tests without subsequent confirmation could result in an estimated 10% or more children misdiagnosed as HIV infected.

Characterization of soluble antigens of Staphylococcus spp isolated from dairy farms in Venezuela

The genus Staphylococcus contains many pathogenic strains that are difficult to differentiate. Given the absence of a specific immunological test to identify autochthonous species, we have characterized soluble antigens (SAgs) using hyperimmune sera from BABL/c mice. Ten samples were taken from the farmers’ hands and cattle udders on three different farms. The isolated species were identified using the API kit (Staph) and their ability to form biofilms was determined. The species most commonly found in the isolates (90%) corresponded to the coagulase-negative bacteria and Staphylococcus sciuri (S. sciuri), which presented the ability of biofilm formation, representing the majority (60%) in this group. We produced SAgs from those Staphylococcus species present in a higher frequency, such as S. sciuri, S. aureus, and the reference strain, S. aureus ATCC 6835. Polyclonal antibodies (PAb) from mice allowed SAgs characterization by enzyme-linked immunoassay (ELISA) and immunoblotting. The humoral response obtained with the PAb by indirect ELISA tests indicated that our hyperimmune sera have a high recognition for all SAgs produced. We also evaluated the hyperimmune sera cross-reactivity between different SAgs by indirect ELISA and immunoblotting assays. The ELISA experiments showed a significant statistical difference in the recognition of S. sciuri when compared to SAgs from S. aureus. These results showed a high antigenicity and specificity from S. sciuri SAgs in immune tests. We identified a specific immunodominant polypeptide of ~31 kDa (p31) from S. sciuri SAg, which do not presented cross-reactivity between different SAgs. We concluded that the p31 polypeptide from S. sciuri SAg could be used as antigen in a differential diagnosis test for different staphylococcal species.

UPA and PAI-1 Analysis from Fixed Tissues – New Perspectives for a Known Set of Predictive Markers

The urokinase-type plasminogen activator (uPA) and its main inhibitor PAI-1 play key roles in tumor-associated processes such as the degradation of the extracellular matrix (ECM), tissue remodeling, cell adhesion and migration. Elevated expression of both molecules is known to correlate with negative outcomes in node negative breast cancer. To date, these molecules are the only prognostic markers to have reached the highest level of evidence (LOE I) in multi-centered clinical trials for prognosis of node negative breast cancer. Unfortunately, the clinical utility of these molecules as markers is limited by the use of enzyme-linked immunoassay (ELISA) tests for their detection. The ELISA relies on the use of fresh or frozen tissue, which are rarely available in routine clinical settings. In this review article, we provide an overview of the clinical relevance of uPA and PAI-1 and present alternative methods for their detection. Common uPA and PAI-1 detection methods discussed in literature include RT-PCR-based assays and classical immunohistochemistry approaches. In recent years, attempts have been made to isolate and analyze proteins of formalin fixed, paraffin embedded (FFPE) tissues. These new methods are of special interest because up to now neither RT-PCR nor immunohistochemistry are recommended for the detection of uPA and PAI-1. Here, we present an approach for the analysis of uPA and PAI-1 directly from FFPE tissues that may eventually overcome the limitations of current assays and make the use of both markers widely available for routine prognosis and therapy decisions for breast cancer patients.

Serologic detection of herpes simplex virus type 2 antibodies among pregnant women using a point-of-care test from Focus Diagnostics

Background Serologic assays that identify herpes simplex type 2 (HSV-2) type-specific antibodies have been commercially available for more than a decade. Greater acceptance of these tests is hindered by uncertainty regarding their performance in real-world clinical settings. Objectives The primary objective was to compare the test characteristics of the Focus HerpeSelect ® Express Assay (EA) versus the Focus HerpeSelect ® enzyme linked immunoassay (ELISA) for detection of HSV-2 type-specific antibodies among pregnant women enrolled from 3 geographic sites with varying prevalences of HSV-2 infection. A second objective was to evaluate the performance of a HSV-2 testing strategy in which EA screens and ELISA confirms HSV-2 serodiagnosis. Study design We enrolled 399 pregnant women from Atlanta, GA, Moorestown, NJ, and Pittsburgh, PA into this cross-sectional investigation. Capillary whole blood was obtained from study participants, and evaluated for the presence of type-specific HSV-2 antibodies using the EA. Serum samples were also obtained from all study participants for subsequent identification of HSV-2 type-specific antibodies using both ELISA and the Focus Immunoblot assays. Results We observed 96.2% agreement between results obtained with EA and ELISA. Overall, when compared to ELISA results, the sensitivity of EA for detection of HSV-2 type-specific antibodies was 94.2% and the specificity was 97.1%. Using Immunoblot results as our standard for performance calculations, the positive predictive value (PPV) of HSV-2 serodiagnosis increased from 91.7% to 98.2% when ELISA was used to confirm EA testing. Conclusions EA provides similar results to ELISA for the identification of HSV-2 type-specific antibodies among pregnant women. As use of the point-of-care (POC) EA in conjunction with confirmatory ELISA testing improves the PPV of HSV-2 serodiagnosis compared to the use of EA or ELISA testing alone, validation of this diagnostic algorithm in other at-risk populations may be warranted.

Impact of highly active antiretroviral treatment on HIV seroincidence among men who have sex with men: San Francisco.

This study assessed the countervailing effects on HIV incidence of highly active antiretroviral treatment (HAART) among San Francisco men who have sex with men (MSM). Behavioral risk was determined on the basis of responses to cross-sectional community interviews. HIV incidence was assessed through application of an enzyme-linked immunoassay testing strategy. Use of HAART among MSM living with AIDS increased from 4% in 1995 to 54% in 1999. The percentage of MSM who reported both unprotected anal intercourse and multiple sexual partners increased from 24% in 1994 to 45% in 1999. The annual HIV incidence rate increased from 2.1% in 1996 to 4.2% in 1999 among MSM who sought anonymous HIV testing, and the rate was high (5.3%) but stable in a blinded survey of MSM seeking sexually transmitted disease services. Any decrease in per contact risk of HIV transmission due to HAART use appears to have been counterbalanced or overwhelmed by increases in the number of unsafe sexual episodes.

Pseudomembranous Colitis: An Update

Clostridium difficile is the most common nosocomial infection of the gastrointestinal tract. Most cases are associated with antibiotic therapy that alters the fecal flora, allowing overgrowth of C difficile with production of its toxins. Diagnosis is made by detection of the organism or toxin in the stools. A variety of different tests can be used, but none is perfect. A stool culture can be positive in someone without diarrhea, ie, a carrier. While the cytotoxin is the gold standard, it is expensive, and there is a delay before results are available. Thus, many laboratories use the enzyme-linked immunoassay tests to detect toxin of C difficile because they are a more rapid screen. Depending on the specific test used, they can detect toxin A, toxin B or occasionally both. Sensitivity and specificity rates vary. First line therapy for C difficile disease should be metronidazole 250 mg qid for 10 days. Vancomycin should be reserved for severe cases where metronidazole has failed or where metronidazole cannot be tolerated or is contraindicated. Recurrent C difficile disease is a particularly vexing clinical problem. A variety of biotherapeutic approaches have been used. Retreatment with antibiotics is almost always necessary. In addition, the nonpathogenic yeast Saccharomyces boulardii has been showed to be of benefit as an adjunct in preventing further recurrences.

Completed hepatitis C lookback in Northern Ireland.

Hepatitis C virus screening of blood donors was introduced in September 1991 using a second-generation enzyme-linked immunoassay (ELISA) and subsequent confirmatory testing with immunoblot (RIBA) and polymerase chain reaction (PCR). In April 1995 a lookback exercise was announced by the Department of Health, the purpose of which was to trace, counsel, investigate and, if necessary, treat individuals who may have been infected with HCV through blood and blood products prior to screening. A total of 231,321 donations have been screened, of which 553 were found to be reactive. Subsequent confirmatory tests identified 24 HCV-positive donors; 13 were repeat donors who had given a total of 164 units. Ninety-three units were traced and 117 components were identified as having been issued to hospitals. Twenty-five recipients requiring follow-up were identified, of which three were assessed by their GPs as not requiring counselling. Of 22 recipients of potentially infectious units 12 showed no evidence of exposure to HCV. We discuss these results in detail.