Development of a highly sensitive chemiluminescent enzyme immunoassay for fragmented cytokeratin 18 using new antibodies.

Fragmented cytokeratin 18 (fCK18) released from epithelial cells undergoing apoptosis is widely studied in various diseases. However, fCK18 measurement is not utilized in clinical practice due to imprecise disease-state cutoff values. Therefore, we set out to generate new monoclonal antibodies (mAbs) and a recombinant fCK18 (rfCK18) calibrator in an effort to develop a highly sensitive chemiluminescent enzyme immunoassay (CLEIA). New capture mAb (K18-624) had a high binding ability compared to the current commercial antibody. New detection mAb (K18-328) recognized 323S-340G of CK18. A rfCK18 was expressed in the soluble fraction of E. coli when the N-terminal region (260 amino acid residues) of CK18 was truncated. Analysis of performance and measurement of human fCK18 were evaluated using K18-624 and K18-328 in a highly sensitive CLEIA. The coefficients of variation (CV) for within-run and between-day repeatability were below 10% and the recoveries were in the range of 15%. The detection sensitivity was 0.056 ng/mL. Serum fCK18 levels were significantly increased in non-alcoholic steatohepatitis (NASH) patients when compared to healthy individuals. Our new fCK18 mAbs showed high affinity and sensitivity. CLEIA using our new antibodies will be useful in measuring fCK18 in human blood thereby generating accurate clinical diagnoses of human liver diseases.

Comparison of 4 commercial enzyme immunoassays for serology testing of human parvovirus B19 infection.

BACKGROUND: Parvovirus B19 is a pathogenic virus often diagnosed by serology, yet little is known about analytical performance of commercial enzyme immunoassays (EIAs). OBJECTIVE: To investigate performance of 4 EIAs for parvovirus B19 IgM and IgG: Liaison, Euroimmun, Mikrogen and Virion/Serion. STUDY DESIGN: To compare 4 EIAs to Biotrin's ELISA on 168 samples and determine consensus score for discordant samples using Mikrogen's confirmatory line assay. RESULTS: Two thirds of results for IgM/IgG were identical for all 4 EIAs and Biotrin. Liaison shows the highest IgM sensitivity, but has low specificity. Euroimmun lacks IgM sensitivity. Mikrogen had a good overall performance, but had the lowest IgG specificity. Virion/Serion had variable performance with a low IgM specificity and the most borderline and cross-reactive results. CONCLUSIONS: Liaison and Mikrogen have similar performance to Biotrin's ELISA. Euroimmun lacks sensitivity and Virion/Serion produced many borderline and cross-reactive results.

A performance evaluation of chemiluminescence enzyme immunoassays on the Sysmex CN-6500 haemostasis analyser.

BACKGROUND: The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS: To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS: Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS: The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89-1.03; r2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI-C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed. CONCLUSIONS: Our data demonstrate that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser.

Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Serologic false-positive reactions for syphilis in children of adenoidal hypertrophy:2 case reports and review of the literature.

Adenoid hypertrophy (AH) is a common disease in children. We describe two cases of AH in children with syphilis enzyme-linked immunosorbent assay (EIA) for false-positive. To our knowledge, there are few reports of false-positive in syphilis EIA in children with AH. Two cases of AH children with syphilis EIA positive samples were confirmed by TPPA and LIA, both showed negative reaction. Therefore, the occurrence of syphilis EIA positive reactions in such diseases should dramatically arouse the attention of docimasters and doctors so as to avoid misdiagnosis caused by false-positive in children with AH.

Well-Based Antibody Arrays.

Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual sample all at once has been useful in developing a proteomics view of biology. This in turn has enabled the development of disease-associated immunodiagnostic panels for better prognosis and well-being. Moreover, it is well understood that such multiplexing approaches lend themselves to automation, thereby reducing labor while providing the ability to dramatically conserve both reagent and sample all of which will reduce the cost per test. Here we describe various methods to create and use multiplex immunoassays in the wells of microtiter plates or similar formats.

Performance evaluation of antibody tests for detecting infant respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is a major cause of respiratory tract disease in young children and throughout life. Infant infection is also associated with later respiratory morbidity including asthma. With a prospective birth cohort study of RSV and asthma, we evaluated the performance of an RSV antibody enzyme-linked immunoassay (EIA) for detecting prior infant RSV infection. Infant RSV infection was determined by biweekly respiratory illness surveillance plus RSV polymerase chain reaction (PCR) testing in their first RSV season and serum RSV antibodies after the season at approximately 1 year of age. RSV antibodies were detected by RSV A and B lysate EIA. Antibody and PCR results on 1707 children included 327 RSV PCR positive (PCR+) and 1380 not RSV+. Of 327 PCR+ children, 314 (96%) were lysate EIA positive and 583 out of 1380 (42%) children not PCR+ were positive. We compared the lysate EIA to RSV F, group A G (Ga), and group B G (Gb) protein antibody EIAs in a subset of 226 sera, 118 PCR+ children (97 group A and 21 group B) and 108 not PCR+. In this subset, 117 out of 118 (99%) RSV PCR+ children were positive by both the F and lysate EIAs and 103 out of 118 (87%) were positive by the Ga and/or Gb EIAs. Comparison of the two G EIAs indicated the infecting group correctly in 100 out of 118 (86%) and incorrectly in 1 out of 118 (1%). The lysate and F EIAs are sensitive for detecting infant infection and the two G EIAs can indicate the group of an earlier primary infection.

Cross-reactivity of a Histoplasma capsulatum antigen enzyme immunoassay in urine specimens from persons with emergomycosis in South Africa.

Histoplasma antigen detection in urine is a rapid diagnostic method for disseminated histoplasmosis, although cross-reactivity has been reported in specimens from patients with other thermally dimorphic fungal infections. We tested urine specimens, from persons with suspected invasive fungal infections, using a commercial monoclonal antibody Histoplasma enzyme immunoassay (EIA) at a South African national mycology reference laboratory from August 2014 through December 2018. Corresponding fungal culture and histopathology results were obtained from an electronic laboratory information system. In some cases, cultured fungal isolates were sent with the urine specimen for species-level identification by phenotypic and molecular methods. Cross-reactivity was confirmed using culture filtrates of several fungal pathogens. Of 212 referred cases, 41 (19%) were excluded since they had no recorded clinical history (n = 1), alternative diagnoses were confirmed (n = 2), or no fungal culture or histopathology results (n = 38). Eighty-seven of 212 (41%) had laboratory evidence of an invasive fungal disease, while 84 (40%) did not. Of the 87 cases, 37 (43%) were culture-confirmed mycoses: emergomycosis (n = 18), histoplasmosis (n = 8), sporotrichosis (n = 6), cryptococcosis (n = 2), talaromycosis (n = 1), and other fungi isolated (n = 2). The sensitivity and specificity of the EIA were calculated for two groups: culture-confirmed (n = 37) and histology-confirmed invasive fungal disease (n = 50). The sensitivity and specificity of the EIA for diagnosis of histoplasmosis compared to culture were 88% (7/8, 95%CI 47-100%) and 72% (21/29, 95%CI 53-87%), respectively, and for diagnosis of emergomycosis/histoplasmosis compared to histology was 83% (29/35, 95%CI 66-93%) and 93% (14/15, 95%CI 68-100%), respectively. Cross-reactions occurred in urine specimens of patients with Emergomyces africanus infection and in culture filtrates of E. africanus, T. marneffei and Blastomyces species. A commercial Histoplasma EIA had satisfactory accuracy for diagnosis of culture-confirmed histoplasmosis, but cross-reacted in urine specimens from patients with invasive disease caused by the closely-related pathogen, E. africanus and in culture filtrates of E. africanus and other related fungi. LAY SUMMARY: Emergomyces africanus and Histoplasma capsulatum are fungi that cause a multi-system disease among HIV-seropositive persons with a low CD4 cell count. Handling live cultures of these fungi to confirm a diagnosis requires specialized laboratory equipment and infrastructure which is infrequently accessible in low-resource settings. The features of the two diseases (i.e., disseminated histoplasmosis and emergomycosis) may be indistinguishable when infected tissue is prepared, stained, and examined under a microscope. Enzyme immunoassays (EIA) have been developed as rapid diagnostic tools for the detection of a cell wall component of H. capsulatum in urine specimens, although cross-reactions have been reported in specimens from patients with other fungal infections. We evaluated the accuracy of a commercial Histoplasma EIA to diagnose histoplasmosis and to assess cross-reactions in urine specimens from persons with emergomycosis and in cultures of E. africanus and related fungi. We report a sensitivity and specificity of 88% (95%CI 47-100%) and 72% (95%CI 53-87%) for diagnosis of histoplasmosis compared to culture and 83% (95%CI 66-93%) and 93% (95%CI 68-100%) for diagnosis of either histoplasmosis/emergomycosis compared to a diagnosis made by microscopic examination of infected tissue. The assay cross-reacted in urine specimens from patients with emergomycosis and in culture filtrates of related fungi. Although the EIA cross-reacted with other related fungi, this test can decrease the time to diagnosis and facilitate early treatment of emergomycosis and histoplasmosis in South Africa.

Renal Function-Adjusted D-Dimer Levels in Critically Ill Patients With Suspected Thromboembolism.

OBJECTIVES: Diagnosing thromboembolic disease typically includes D-dimer testing and use of clinical scores in patients with low to intermediate pretest probability. However, renal dysfunction is often observed in patients with thromboembolic disease and was previously shown to be associated with increased D-dimer levels. We seek to validate previously suggested estimated glomerular filtration rate-adjusted D-dimer cutoff levels. Furthermore, we strive to explore whether the type of renal dysfunction affects estimated glomerular filtration rate-adjusted D-dimer test characteristics. DESIGN: Single-center retrospective data analysis from electronic healthcare records of all emergency department patients admitted for suspected thromboembolic disease. SETTING: Tertiary care academic hospital. SUBJECTS: Exclusion criteria were as follows: age less than 16 years old, patients with active bleeding, and/or incomplete records. INTERVENTIONS: Test characteristics of previously suggested that estimated glomerular filtration rate-adjusted D-dimer cutoff levels (> 333 µg/L [estimated glomerular filtration rate, > 60 mL/min/1.73 m], > 1,306 µg/L [30-60 mL/min/1.73 m], and > 1,663 µg/L [< 30 mL/min/1.73 m]) were validated and compared with the conventional D-dimer cutoff level of 500 µg/L. MAIN RESULTS: A total of 14,477 patients were included in the final analysis, with 467 patients (3.5%) diagnosed with thromboembolic disease. Renal dysfunction was observed in 1,364 (9.4%) of the total population. When adjusted D-dimer levels were applied, test characteristics remained stable: negative predictive value (> 99%), sensitivity (91.2% vs 93.4%), and specificity (42.7% vs 50.7%) when compared with the conventional D-dimer cutoff level to rule out thromboembolic disease (< 500 µg/L). Comparable characteristics were also observed when adjusted D-dimer cutoff levels were applied in patients with acute kidney injury (negative predictive value, 98.8%; sensitivity, 95.8%; specificity, 39.2%) and/or "acute on chronic" renal dysfunction (negative predictive value, 98.0%; sensitivity, 92.9%; specificity, 48.5%). CONCLUSIONS: D-Dimer cutoff levels adjusted for renal dysfunction appear feasible and safe assessing thromboembolic disease in critically ill patients. Furthermore, adjusted D-dimer cutoff levels seem reliable in patients with acute kidney injury and "acute on chronic" renal dysfunction. In patients with estimated glomerular filtration rate less than 60 mL/min/1.73 m, the false-positive rate can be reduced when estimated glomerular filtration rate-adjusted D-dimer cutoff levels are applied.

Evaluation of accuracy of hepatitis B virus antigen and antibody detection and relationship between epidemiological factors using dried blood spot.

Dried blood spots (DBS) testing might increase the access for Hepatitis B virus (HBV) diagnosis, but little is known about the performance of these assays in real life conditions. This study aims to evaluate the diagnostic accuracy of HBsAg, anti-HBc and anti-HBs detection in DBS in clinical settings and field studies and to evaluate demographic and risk behaviour according the presence of HBsAg and anti-HBc. Paired sera and DBS samples were obtained from 2309 individuals from 3 groups, defined as follows: G1: clinical setting (n = 5-19), G2: general population (n = 1305) and G3: vulnerable individuals that could be more exposed to blood contact (n = 485). Sera and DBS were tested using commercial enzyme immunoassay (EIA), with some modifications added. Using DBS samples, the specificity values were above 90 % for HBsAg and anti-HBc in all groups and for anti-HBs range from 58.6%-85%. HBsAg testing had the best performance in GI (sensitivity = 84.4 %) and among those samples that the paired serum also presented anti-HBc marker (sensitivity = 91.6 %). High sensitivity of anti-HBc testing in DBS samples was observed in GI (80.8 %) and among HBV active cases (HBsAg+/anti-HBc+) (98.4 %). Testing of anti-HBs in DBS showed the highest sensitivity in GIII (65.5 %), in previous HBV exposed and cured individuals and when serum titers were above 100 IU/mL (86.7 %). DBS samples could be used for screening and prevalence studies for HBsAg and anti-HBc, particularly in clinical settings and among HBV active cases in field studies.

High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH-/toxin-, and 124 GDH+/toxin- samples, of which 39 were CCNA+ and 85 were CCNA- Clarity had 96.2% negative agreement with GDH-/toxin- samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin-/CCNA- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin-/CCNA+ samples, 90.0% of GDH+/toxin-/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin-/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Comparison of anti-hepatitis D virus (HDV) ETI-AB-DELTAK-2 assay and the novel LIAISON® XL MUREX anti-HDV assay in the diagnosis of HDV infection.

Hepatitis B/D virus infection leads to severe liver disease. HDV infection is not routinely investigated since the diagnosis is based on enzyme immunoassays (EIAs), which are not available in all laboratories. This study investigates the performance of new automated assay for anti HDV Ab detection: LIAISON® XL Murex anti-HDV. HBsAg-positive samples were evaluated for HDV serology using the ETI-AB-DELTAK-2 and the new LIAISON® XL Murex with a concordance of 97.5% and 2.42% discordant results. The discordant specimens reacted negatively with EIA and positively with the new test. Dilutions of HDV-purified antibodies and HDV-positive samples were tested with both assays, showing a lower detection limit for the new assay. In conclusion, LIAISON® XL Murex showed a good concordance with the reference method and allowed a more rapid HDV detection. This new diagnostic tool may be useful for a more efficient approach to the HDV diagnosis and evaluation of HDV epidemiology.

Are immunoenzymatic tests for intestinal protozoans reliable when used on archaeological material?

Intestinal protozoans found in ancient human samples have been studied primarily by microscopy and immunodiagnostic assays. However, such methods are not suitable for the detection of zoonotic genotypes. The objectives of the present study were to utilize immunoenzimatic assays for coproantigen detection of Cryptosporidium sp., Giardia duodenalis, and Entamoeba histolytica/Entamoeba dispar in sixty ancient human and animal samples collected from 14 archaeological sites in South America, and to carry out a critical analysis of G. duodenalis according to results obtained from three diagnostic methodologies: microscopy, immunodiagnostic tests (immunoenzymatic and immunofluorescence), and molecular biology (PCR and sequencing). More than half (31/60) of the samples analyzed using immunoenzymatic tests were positive for at least one of the intestinal protozoans, with 46.6% (28/60) corresponding to G. duodenalis, 26.6% (16/60) to Cryptosporidium sp., and 5% (3/60) to E. histolytica/E. dispar. Cryptosporidium sp. and G. duodenalis coinfection was observed in 15% (9/60) of the samples, whereas all three protozoans were found in 5% (3/60) of samples. In the Northeast Region of Brazil, by immunoenzymatic tests there is evidence that G. duodenlais and Cryptosporidium sp. have infected humans and rodents for at least 7150 years. However, for G. duodenalis, the results from the three diagnostic tests were discordant. Specifically, despite the efficiency of the molecular biology assay in the experimental models, G. duodenalis DNA could not be amplified from the ancient samples. These results raise the following question: Are all ancient samples positive for coproantigen of G. duodenalis by immunoenzymatic tests truly positive? This scenario highlights the importance of further studies to evaluate the sensitivity and specificity of the immunoenzymatic method in the archaeological context.

The third international standard for anti-D immunoglobulin: international collaborative study to evaluate candidate preparations.

BACKGROUND AND OBJECTIVES: The purpose of the study was to evaluate a lyophilized anti-D immunoglobulin preparation to serve as a replacement WHO International Standard for the calibration of potency assays of anti-D immunoglobulin products. Such products are used to prevent haemolytic disease of the foetus and newborn due to maternal alloanti-D. MATERIALS AND METHODS: The candidate 3rd International Standard for anti-D immunoglobulin (16/332) was evaluated and calibrated against the 2nd International Standard for anti-D immunoglobulin (01/572), along with a coded duplicate, a second candidate preparation (16/278) and a comparability sample (16/272) in an international collaborative study. Twenty of 21 laboratories in 15 countries performed one or more of the three European Pharmacopoeia reference methods. RESULTS: The overall geometric mean potency (from all methods) of the candidate 3rd International Standard, 16/332, was 296·6 IU/ampoule, with inter-laboratory variability, expressed as % GCV, of 4·7%. SE-HPLC of the immunoglobulin preparations demonstrated combined monomeric and dimeric IgG peak areas of >95% for all samples. Accelerated stability studies have shown both 16/332 and 16/278 to be very stable for long-term storage at -20°C. CONCLUSIONS: Preparation 16/332 was established by the World Health Organisation Expert Committee on Biological Standardization as the 3rd International Standard for anti-D immunoglobulin with an assigned potency of 297 IU/ampoule.

Calibration and Evaluation of Quantitative Antibody Titers for Varicella-Zoster Virus by Use of the BioPlex 2200.

Most commercially available enzyme immunoassay-based methods have limited sensitivity to detect antibody responses to varicella-zoster virus (VZV) in vaccinated individuals, who produce lower antibody levels than those with natural infection. However, more sensitive methods are either not commercially available or less amenable to high-throughput testing. The BioPlex 2200 measles, mumps, rubella, and varicella (MMRV) IgG assay (Bio-Rad Laboratories, Hercules, CA) is an automated high-throughput platform based on the microsphere Luminex technology that measures antibodies against measles, mumps, rubella, and varicella viruses simultaneously. Although it has U.S. Food and Drug Administration approval as a qualitative diagnostic test for measles, mumps, rubella, and varicella virus immunity, in this study, we have validated the assay to produce quantitative titers (off label) against the VaccZyme VZV glycoprotein (VZVgp) low-level IgG kit (The Binding Site Ltd., Birmingham, UK) using the World Health Organization international standard. Here, we show that the BioPlex 2200 MMRV IgG assay has sensitivity superior to that of the Zeus enzyme-linked immunosorbent assay (ELISA) VZV IgG assay (Zeus Diagnostics, Branchburg, NJ). Using receiver operating characteristic (ROC) analysis and adjusting the cutoff levels, we improved the sensitivity of the quantitative BioPlex 2200 MMRV IgG assay to 97.4%, while maintaining 100% specificity.

Analytical and clinical performances of the automated Lumipulse cerebrospinal fluid Aß42 and T-Tau assays for Alzheimer's disease diagnosis.

BACKGROUND: Cerebrospinal fluid (CSF) biomarkers are increasingly used to diagnose Alzheimer's disease (AD). However, important methodological and technical remain regarding measurement variability between kit providers and users. We compared the Lumipulse fully automated assays with the manual INNOTEST assays (both from Fujirebio Europe NV, Gent, Belgium) on a clinically representative sample of patients and controls. METHODS: CSF samples of 156 patients were used to quantify Amyloïd Aß1-42 peptide (Aß1-42) and Total-Tau (T-Tau) protein by chemiluminescent enzyme-immunoassay (Lumipulse). Patients were divided into several subgroups: Alzheimer (AD = 44), mild-cognitive impairment (MCI = 23), other dementias (OD = 36), non-dementing neurological conditions (ND = 11), and controls (CTRL = 42). Clinical cut-offs were determined by comparing AD and CTRL with ROC curves for the two markers and their related ratio (T-Tau/Aß1-42). Subgroups of 58 (for phosphorylated-Tau) and 115 samples (for Aß1-42 and T-Tau) were used to evaluate the concordance of this analyzer with the INNOTEST assays. RESULTS: Lumipulse and INNOTEST assays showed good concordance for all markers, but systematic bias was observed justifying the need to redefine new clinical cut-offs. To discriminate AD from CTRL subjects, T-Tau/Aß1-42 ratio was the best biomarker, with a cut-off value of 1.12 (sensitivity 81.8% and specificity 92.9%). Similar clinical performances were observed for the Lumipulse and Innotests assays on the subsample of 115 subjects. CONCLUSIONS: Our results demonstrate that the Lumipulse Aß1-42 and T-Tau assays show good analytical and clinical performances in the context of patient evaluation referred to a memory clinic. Automated analyzers should be preferred for the measurement of CSF AD biomarkers to reduce inter- and intra-laboratory variability.

Performance of commercial tests for molecular detection of Shiga toxin-producing Escherichia coli (STEC): a systematic review and meta-analysis protocol.

INTRODUCTION: Rapid detection of Shiga toxin-producing Escherichia coli (STEC) enables appropriate treatment. Numerous commercially available molecular tests exist, but they vary in clinical performance. This systematic review aims to synthesise available evidence to compare the clinical performance of enzyme immunoassay (EIA) and nucleic acid amplification tests (NAATs) for the detection of STEC. METHODS AND ANALYSIS: The following databases will be searched employing a standardised search strategy: Medline, Embase, Cochrane CENTRAL Register of Controlled Trials, Cochrane Database of Systematic Reviews, PubMed, Scopus and Web of Science. Grey literature will be searched under advice from a medical librarian. Independent reviewers will screen titles, abstracts and full texts of retrieved studies for relevant studies. Data will be extracted independently by two reviewers, using a piloted template. Quality Assessment of Diagnostic Accuracy Studies-2 will be employed to assess the risk of bias of individual studies, and the quality of evidence will be assessed with the Grading of Recommendations Assessment, Development and Evaluation approach. A bivariate random-effects model will be used to meta-analyse the sensitivity and specificity of commercial STEC diagnostic tests, and a hierarchical summary receiver operator characteristic curve will be constructed. Studies of single test accuracy of EIA and NAATs and studies of comparative accuracy will be analysed separately. ETHICS AND DISSEMINATION: Ethics approval was not required for this systematic review and meta-analysis. Findings will be disseminated in conferences, through a peer-reviewed journal and via personal interactions with relevant stakeholders. PROSPERO REGISTRATION NUMBER: CRD42018099119.

A Novel Enzyme Immunoassay for the Detection of Buprenorphine, Norbuprenorphine and Their Glucuronides in Urine.

Buprenorphine is a commonly used opioid in pain therapy as well as in opiate maintenance therapy. Immunoassays are quick and cost-effective methods for the necessary toxicological urine analysis of maintenance therapy patients. In this study a novel enzymatic immunoassay, the Thermo Fisher Scientific CEDIA Buprenorphine II assay (Bup2) was evaluated for the detection of buprenorphine, norbuprenorphine and their conjugated metabolites in human urine samples. The Bup2 assay has a cut-off of 10 ng/mL with ±25% controls, whereas the existing CEDIA Buprenorphine assay (Bup1) has a cut-off of 5 ng/mL and ±40% controls. Both assays were analyzed on a Thermo Scientific Indiko Plus benchtop analyzer. Seven-day precision studies of Bup2 assay demonstrated excellent precision of 7.2-10.6%. No crossover between control samples and the cut-off level were observed. Urine samples of 120 patients undergoing opiate maintenance therapy were collected. Immunoassay results of Bup1 and Bup2 were confirmed by gas chromatography mass spectrometry (GC/MS) for buprenorphine and norbuprenorphine as well as for their glucuronides. Comparison showed a specificity of 0.99 between the Bup2 assay and GC/MS, whereas the Bup1 assay had a specificity 0.70 due to 21 false positive samples. The reason is a known cross-reactivity of the Bup1 assay to opiate compounds. The Bup2 assay revealed one false positive result close to the cut-off value; no specific candidate possibly causing a cross-reaction was detected by GC/MS and liquid chromatography tandem mass-spectrometry (LC/MS/MS) methods. The data presented demonstrate an excellent correlation of the Bup2 assay to GC/MS, showing improved specificity and sensitivity when compared to the Bup1 assay. Thus, the Bup2 assay is highly suitable for urine testing, even for opiate maintenance patients receiving high doses of morphine.

Performance evaluation of five commercial assays for detection of acetaminophen.

BACKGROUND: To evaluate the analytical performance of five commercial acetaminophen assays and select the best method for routine use. METHODS: Imprecision, accuracy, linearity, and interferences of three enzymatic assays (Beckman Coulter AU Paracetamol, Abbott MULTIGENT Acetaminophen, and Sekisui Acetaminophen L3K) and two immunoassay-based assays (Beckman Coulter SYNCHRON ACTM (Acetaminophen) Reagent and Siemens SYVA Emit-tox Acetaminophen) were evaluated on a Beckman Coulter AU680 chemistry analyzer. Hook effect for immunoassay-based assays and recovery in ultrafiltrate for enzymatic methods were studied. RESULTS: Within-run and between-run imprecision of the enzymatic assays ranged 0.26%-0.82% and 0.53%-2.86%, respectively, while that for the immunoassay-based methods ranged 0.96%-6.34% and 1.50%-11.33%, respectively. All assays except the SYNCHRON assay fell within the program analytical performance specifications (±20 µmol/L or 10%) for external quality assurance (EQA) samples, with the highest positive bias (31.7%) observed in the SYNCHRON assay. Icteric interference was demonstrated most significantly in the Abbott assay (up to 88 µmol/L positive bias in blank serum). The lipemic interference on the SYNCHRON was significant (up to 110% positive bias at level of 100 µmol/L). The immunoassay-based methods were less susceptible to hemolytic interference, while the Abbott and AU assays were more susceptible to N-acetylcysteine interference. Both immunoassay-based methods showed no hook effect up to 18 000 µmol/L. Ultrafiltration recoveries for enzymatic methods were satisfactory, ranging from 80.0% ± 5.1% to 89.5% ± 3.0%. CONCLUSIONS: Proportional bias was observed in the SYNCHRON assay, while the Siemens and Sekisui assays were minimally affected by bilirubin interferences.