Regulation of Molecular Diagnostics.

Molecular diagnostic tests enable rapid analysis of genomic and proteomic markers. These tests are subject to diverging premarket access and postmarket surveillance requirements and mechanisms in the United States and the European Union. Each of these jurisdictions has its own challenges in keeping the regulations up to date with technological developments. A specific area of attention is that of laboratory-developed tests in the United States and health institution in-house-produced tests in the European Union, for which the United States and the European Union have markedly different regulatory approaches. Both jurisdictions have specific but differing requirements for the use of test samples and test-related data under their rules regarding the protection of (personal) health data, which can cause complexity when moving samples or sample-related data from one jurisdiction to the other.

FDA's proposed rule for the regulation of laboratory-developed tests.

In October 2023, the Food and Drug Administration (FDA) released a proposed rule that ends enforcement discretion for laboratory-developed tests (LDTs). The FDA's proposal outlines a five-stage implementation to begin regulating LDTs as they do for commercial in vitro diagnostics (IVDs), including modified FDA-approved/cleared tests. We outline here concerns from the clinical and public health microbiology laboratory perspective. It is our opinion that LDTs performed by individual Clinical Laboratory Improvement Amendments-certified diagnostic laboratories should not be regulated in the same way as commercial IVDs. This rule, if finalized, will negatively impact the diagnostic services currently offered by clinical and public health laboratories and, therefore, patients and the providers who care for them. Ending enforcement discretion will likely stifle diagnostic innovation and decrease access to diagnostic testing and health equity. Furthermore, the lack of infrastructure, including personnel and funding, at the FDA and diagnostic laboratories to support the required submissions for review is an obstacle. Like the FDA, diagnostic laboratories prioritize patient safety, accurate clinical diagnostics, and health equity. Since the scope of the LDT landscape is currently unknown, we are supportive of a registration process, along with non-burdensome adverse event reporting, to first understand the scope of clinical use of LDTs and any associated safety concerns. Any regulatory rule should be based on data that have been gathered systematically, not anecdotes or case reports. A rule must also balance the potential negative impact to patient care with realistic safety risks for infectious disease diagnostics.

Implementation and Performance of a Point-of-Care COVID-19 Test Program in 4000 California Schools.

OBJECTIVE: To evaluate the feasibility and accuracy of an unprecedented COVID-19 antigen testing program in schools, which required a healthcare provider order, laboratory director, a Clinical Laboratory Improvement Amendments certificate of waiver, as well as training of school personnel. STUDY DESIGN: Descriptive report of a point-of-care, school-based antigen testing program in California from August 1st, 2021 through May 30, 2022, in which participants grades K-12 self-swabbed and school personnel performed testing. Participants included 944 009 students, personnel, and community members from 4022 California kindergarten through high schools. Outcomes measured include sensitivity and specificity (with polymerase chain reaction [PCR] as comparator) of the Abbott BinaxNOW antigen test, number of tests performed, and active infections identified. RESULTS: Of 102 022 paired PCR/antigen tests, the overall sensitivity and specificity for the antigen test was 81.2% (95% CI: 80.5%-81.8%) and 99.6% (95% CI: 99.5%-99.6%), respectively, using cycle threshold values <30. During January through March 2022, the highest prevalence period, the positive predictive value of antigen testing was 94.7% and the negative predictive value was 94.2%. Overall, 4022 school sites were enrolled and 3 987 840 million antigen tests were performed on 944 009 individuals. A total of 162 927 positive antigen tests were reported in 135 163 individuals (14.3% of persons tested). CONCLUSIONS: Rapidly implementing a school-based testing program in thousands of schools is feasible. Self-swabbing and testing by school personnel can yield accurate results. On-site COVID-19 testing is no longer necessary in schools, but this model provides a framework for future infectious disease threats.

Lessons learned: establishing a CLIA-equivalent laboratory for targeted mass spectrometry assays - navigating the transition from research to clinical practice.

Mass spectrometry (MS) assays offer exceptional capabilities in high multiplexity, specificity, and throughput. As proteomics technologies continue advancements to identify new disease biomarkers, transition of these innovations from research settings to clinical applications becomes imperative. To meet the rigorous regulatory standards of clinical laboratories, development of a clinical protein MS assay necessitates adherence to stringent criteria. To illustrate the process, this project focused on using thyroglobulin (Tg) as a biomarker and an immuno-multiple reaction monitoring (iMRM) MS-based assay as a model for establishing a Clinical Laboratory Improvement Amendments (CLIA) compliant laboratory within the Centers of Genomic and Precision Medicine, National Taiwan University. The chosen example also illustrates the clinical utility of MS assays to complement conventional immunoassay-based methods, particularly in cases where the presence of autoantibodies in 10-30% of patients hinders accuracy. The laboratory design entails a comprehensive coordination in spatial layout, workflow organization, equipment selection, ventilation systems, plumbing, electrical infrastructure, documentation procedures, and communication protocols. Practical aspects of the transformation process, including preparing laboratory facilities, testing environments, instrument validation, assay development and validation, quality management, sample testing, and personnel competency, are discussed. Finally, concordant results in proficiency testing demonstrate the harmonization with the University of Washington Medical Center and the quality assurance of the CLIA-equivalent Tg-iMRM MS assay established in Taiwan. The realization of this model protein MS assay in Taiwan highlights the feasibility of international joint development and provides a detailed reference map to expedite the implementation of more MS-based protein assays in clinical laboratories for patient care.

Determination of optimum levels of binding antibody units (BAU) of new quantitative chemiluminescent immuno-assay (CLIA) in COVID-19 vaccinated volunteer blood donors.

BACKGROUND: For assessment of COVID-19 vaccine efficacy, neutralization activity of anti-SARS-CoV-2 antibody is measured. This study was undertaken to determine optimum levels of binding antibody units (BAU/ml) in new quantitative chemiluminescent assay (CLIA) that corresponded to neutralizing potential (30% inhibition) of sVNT assay. METHODS: Ninety-one blood samples were analyzed by CLIA and sVNT assays. Test samples (n = 75) were collected from blood donors post-2nd vaccination dose, while control samples (n = 16) were archived pre-COVID donor samples. Correlation between CLIA and sVNT was calculated and receiver operating characteristic (ROC) curve was drawn and analyzed. RESULTS: Results indicated excellent correlation between 57.5 BAU/ml on CLIA and 30%inhibition on sVNT assay. ROC curve analysis revealed that the area under the curve (AUC) was 0.971. DISCUSSION: The present study determined that 57.5 BAU/ml on CLIA corresponded to 30% inhibition on sVNT assay. Periodic quantitative analysis.

Development of plate-type and tubular chemiluminescence immunoassay against African swine fever virus p72.

African swine fever (ASF) is a highly contagious and fatal viral disease that has caused huge economic losses to the pig and related industries worldwide. At present, rapid, accurate, and sensitive laboratory detection technologies are important means of preventing and controlling ASF. However, because attenuated strains of African swine fever virus (ASFV) are constantly emerging, an ASFV antibody could be used more effectively to investigate the virus and control the disease on pig farms. The isolation of ASFV-specific antibodies is also essential for the diagnosis of ASF. Therefore, in this study, we developed two chemiluminescence immunoassays (CLIAs) to detect antibodies directed against ASFV p72: a traditional plate-type blocking CLIA (p72-CLIA) and an automatic tubular competitive CLIA based on magnetic particles (p72-MPCLIA). We compared the diagnostic performance of these two methods to provide a feasible new method for the effective prevention and control of ASF and the purification of ASFV. The cut-off value, diagnostic sensitivity (Dsn), and diagnostic specificity (Dsp) of p72-CLIA were 40%, 100%, and 99.6%, respectively, in known background serum, whereas those of p72-MPCLIA were 36%, 100%, and 99.6%, respectively. Thus, both methods show good Dsn, Dsp, and repeatability. However, when analytical sensitivity was evaluated, p72-MPCLIA was more sensitive than p72-CLIA or a commercial enzyme-linked immunosorbent assay. More importantly, p72-MPCLIA reduced the detection time to 15 min and allowed fully automated detection. In summary, p72-MPCLIA showed superior diagnostic performance and offered a new tool for detecting ASFV infections in the future. KEY POINTS: • Two chemiluminescence immunoassay (plate-type CLIA and tubular CLIA) methods based on p72 monoclonal antibody (mAb) were developed to detect ASFV antibody. • Both methods show good diagnostic performance (Dsn (100%), Dsp (99.6%), and good repeatability), and p72-MPCLIA detects antibodies against ASFV p72 with high efficiency in just 15 min.

Improving the diagnosis of active tuberculosis: a novel approach using magnetic particle-based chemiluminescence LAM assay.

OBJECTIVES: Tuberculosis (TB) is a significant global health concern, given its high rates of morbidity and mortality. The diagnosis using urine lipoarabinomannan (LAM) primarily benefits HIV co-infected TB patients with low CD4 counts. The focus of this study was to develop an ultra-sensitive LAM assay intended for diagnosing tuberculosis across a wider spectrum of TB patients. DESIGN & METHODS: To heighten the sensitivity of the LAM assay, we employed high-affinity rabbit monoclonal antibodies and selected a highly sensitive chemiluminescence LAM assay (CLIA-LAM) for development. The clinical diagnostic criteria for active TB (ATB) were used as a control. A two-step sample collection process was implemented, with the cutoff determined initially through a ROC curve. Subsequently, additional clinical samples were utilized for the validation of the assay. RESULTS: In the assay validation phase, a total of 87 confirmed active TB patients, 19 latent TB infection (LTBI) patients, and 104 healthy control samples were included. Applying a cutoff of 1.043 (pg/mL), the CLIA-LAM assay demonstrated a sensitivity of 55.2% [95%CI (44.13%~65.85%)], and a specificity of 100% [95%CI (96.52%~100.00%)], validated against clinical diagnostic results using the Mann-Whitney U test. Among 11 hematogenous disseminated TB patients, the positive rate was 81.8%. Importantly, the CLIA-LAM assay consistently yielded negative results in the 19 LTBI patients. CONCLUSION: Overall, the combination of high-affinity antibodies and the CLIA method significantly improved the sensitivity and specificity of the LAM assay. It can be used for the diagnosis of active TB, particularly hematogenous disseminated TB.

Inflated Gamma Interferon Response with QuantiFERON-TB Gold Plus Using the Automated Liaison XL Analyzer: a Testing Algorithm To Mitigate False-Positive Results in Low-Incidence Settings.

The Liaison XL chemiluminescence immunoassay (CLIA) analyzer allows total automation of gamma interferon (IFN-γ) measurement for the QuantiFERON-TB Gold Plus assay (QFT-Plus) that is used to diagnose Mycobacterium tuberculosis infection. To evaluate CLIA accuracy, plasma samples from 278 patients undergoing QFT-Plus testing were first tested with an enzyme-linked immunosorbent assay (ELISA; 150 negatives and 128 positives) and subsequently with the CLIA. Three strategies to mitigate false-positive CLIA results were investigated in 220 samples with borderline-negative ELISA results (TB1 and/or TB2, 0.1 to 0.34 IU/mL). The Bland-Altman plot of difference versus average of the two IFN-γ measurements in the Nil and antigen (TB1 and TB2) tubes showed higher IFN-γ measurements across the range of values with the CLIA than with the ELISA. Bias was 0.21 IU/mL (standard deviation, 0.61; 95% confidence interval [CI], -1.0 to 1.41). Linear regression of difference versus average had a slope of 0.08 (95% CI, 0.05 to 0.10), which was significantly nonzero (P < 0.0001). The CLIA had positive and negative percent agreement levels with the ELISA of 91.7% (121/132) and 95.2% (139/146), respectively. In borderline-negative samples tested with ELISA, CLIA was positive in 42.7% (94/220). CLIA with a standard curve resulted in 36.4% (80/220) positivity. Retesting CLIA false positives (TB1 or TB2 range, 0 to ≤1.3 IU/mL) with ELISA reduced false positives by 84.3% (59/70). Retesting with CLIA reduced the false-positive rate by 10.4% (8/77). Implementing the Liaison CLIA for QFT-Plus in low-incidence settings risks falsely elevating conversion rates and overburdening clinics and overtreating patients. Confirming borderline positives with ELISA is a viable strategy to mitigate false-positive CLIA results.

Validation of Luminex immunological and competitive Luminex immunological assays for clinical immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine.

A novel virus-like particle (VLP)-based multivalent recombinant human papillomavirus (HPV) vaccine was developed and evaluated in human, including 14 HPV-type specific VLP antigens (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). The pseudovirus-based neutralizing assay (PBNA) method is widely used for immunogenicity assessment of HPV vaccine in clinical trials. However, as many as 14 antigen-specific antibody levels need be determined, PBNA is, for many reasons, challenging and time-consuming. In this study, we developed a Luminex immunological assay (LIA) and a competitive Luminex immunological assay (cLIA). These methods increase the throughput, reproducibility and precision, as well as reduce the complexity. All assay parameters showed good characteristics in the validation of both methods, benefiting from highly purified and structurally correct VLPs, high specific antibodies, standard VLP-microspheres and PE-mAbs conjugating process, adequate assay development and stable system. Validation data support the use of both methods for immunogenicity assessment in clinical trials. LIA showed higher sensitivity than cLIA, and due to limited epitopes of mAb, cLIA detected lower antibody responses, and therefore, fewer antibodies. This work not only supports clinical trials of 14-valent HPV vaccines more efficiently and reliably, but also provides a set of validation strategies and usable standards for general vaccine immunogenicity testing.

Evaluation of Diagnostic Accuracy of Eight Commercial Assays for the Detection of Rubella Virus-Specific IgM Antibodies.

Rubella and congenital rubella syndrome are caused by the rubella virus and are preventable through vaccination, making disease eradication possible. Monitoring of progress toward global eradication and local elimination requires high-quality, sensitive disease surveillance that includes laboratory confirmation of cases. Previous evaluations of anti-rubella IgM detection methods resulted in the broad adoption of the Enzygnost (most recently manufactured by Siemens) enzyme-linked immunosorbent assay (ELISA) kits within WHO's global measles and rubella laboratory network, but they have been discontinued. This study evaluated seven comparable ELISAs from six manufacturers (Trinity Biotech, Euroimmun, Clin-Tech, NovaTec and Virion\Serion) as well as one automated chemiluminescent assay (CLIA) from DiaSorin. These assays include three IgM capture assays and five indirect ELISAs. A panel of 238 sera was used for the evaluation that included 38 archival rubella IgM-positive sera and 200 sera collected from patients with symptomatically similar diseases, such as measles, dengue, parvovirus B19 infection, and roseola. With this panel of sera, the sensitivity of the methods ranged from 63.2% to 100% and the specificity from 80.0% to 99.5%. No single method had both sensitivity and specificity of >90%, unless sera with equivocal results were considered presumptively positive. Some assays, particularly the Serion ELISA, had a large number of false positives with parvovirus B19 IgM-positive sera as well as sera from confirmed measles cases. The performance characteristics identified in this evaluation serve as a reminder to not rely solely on rubella IgM results for case confirmation in elimination settings.

Evaluation of qualitative and semi-quantitative cut offs for rapid diagnostic lateral flow test in relation to serology for the detection of SARS-CoV-2 antibodies: findings of a prospective study.

BACKGROUND: There is limited information to compare the qualitative and semi-quantitative performance of rapid diagnostic tests (RDT) and serology for the assessment of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the objective of the study was (a) to compare the efficacy of SARS-CoV-2 antibody detection between RDT and laboratory serology, trying to identify appropriate semi-quantitative cut-offs for RDT in relation with quantitative serology values and to (b) evaluate diagnostic accuracy of RDT compared to the NAAT gold standard in an unselected adult population. METHODS: SARS-CoV-2 antibodies were simultaneously measured with lateral flow immunochromatographic assays (LFA), the Cellex qSARS-CoV-2 IgG/IgM Rapid Test (by capillary blood), the iFlash-SARS-CoV-2 IgG/IgM chemiluminescent immunoassay (CLIA) (by venous blood) and the nucleic acid amplification test (NAAT) in samples from in- and out-patients with confirmed, suspected and negative diagnosis of coronavirus disease 2019 (COVID-19) attending Udine Hospital (Italy) (March-May 2020). Interpretation of RDT was qualitative (positive/negative) and semi-quantitative based on a chromatographic intensity scale (negative, weak positive, positive). RESULTS: Overall, 720 paired antibody measures were performed on 858 patients. The qualitative and semiquantitative agreement analysis performed in the whole sample between LFA and CLIA provided a Kendall's tau of 0.578 (p < 0.001) and of 0.623 (p < 0.001), respectively, for IgM and IgG. In patients with a diagnosis of COVID-19, accordance between LFA and CLIA was maintained as a function of time from the onset of COVID-19 disease and the severity of disease both for qualitative and semi-quantitative assessments. RDT compared to the NAAT gold standard in 858 patients showed 78.5% sensitivity (95% CI 75.1%-81.7%) and 94.1% specificity (95% CI 90.4%-96.8%), with variable accordance depending on the timing from symptom onset. CONCLUSION: The RDT used in our study can be a non-invasive and reliable alternative to serological tests and facilitate both qualitative and a semi-quantitative antibody detection in COVID-19.

Comparative evaluation of ADVIA Centaur® XP chemiluminescence system for screening of HBV, HCV, HIV and syphilis in Indian blood donors.

BACKGROUND: The introduction of chemiluminescent immunoassay (CLIA) in blood donor screening has led to a gradual replacement of enzyme-linked-immunosorbent assay (ELISA) as the former offers automation, higher sensitivity and lower turn-around-time. However, only a few CLIA platforms are used for blood donor screening in India. The present study evaluated one such newer platform viz., Adiva Centaur XP CLIA for screening of HBV, HCV, HIV and syphilis. MATERIAL AND METHODS: Prospective comparative study wherein 4843 whole blood donors were screened for HBsAg, Anti-HCV, HIV Ag-Ab and Anti-treponemal antibodies in both Advia Centaur XP and Architect i2000SR platforms. Additional tests were performed in samples which were reactive in only one of the platforms. The sensitivity, specificity, positive predictive value, negative predictive value, accuracy, false positive rate and false negative rate of both the platforms were compared. Kappa coefficient was calculated to determine the agreement between the testing platforms. RESULTS: The sensitivity of Advia Centaur platform for HBV, HCV, HIV and syphilis detection were 94.9 %, 100 %, 100 % and 100 % respectively as compared to 96.6 %, 100 %, 100 % and 100 % in Architect i2000SR platform. The specificity of both the platforms were 99.8-99.9 % for all the four tests. The agreement between the two platforms was almost perfect for HBV, HCV and syphilis testing; and fair for HIV testing. CONCLUSION: The Advia Centaur CLIA platform was found to be comparable with the Architect CLIA platform for blood donor screening. Unexpected finding was the occurrence of HBV false negatives in both the platforms, possibly due to HBsAg mutations.

Measurement comparability of insulin assays using conventional immunoassay kits.

BACKGROUND: The standardization of measurement aims to achieve comparability of results regardless of the analytical methods and the laboratory where analyses are carried out. In this paper, a comparison of results from several immunoassay-based insulin analysis kits is described, and the steps necessary to improve comparability are discussed. METHODS: Four manual enzyme-linked immunosorbent assay (ELISA) kits produced by Mercodia, Alpco, Epitope Diagnostics, and Abcam, and three automated chemiluminescent (CLIA) insulin assay kits (Siemens Centaur XP, Unicel Dxl800, Cobas e801) were compared by analyzing human serum samples and certified reference materials for human insulin. RESULTS: The seven evaluated assay kits showed substantial discrepancies in the results, with relative standard deviation ranges between 1.7% and 23.2%. We find that the traceability chains and the unit conversion factors are not yet harmonized, and current reference materials for insulin are not applicable for immunoassay-based method validation due to the use of different matrices. CONCLUSIONS: The findings suggest the need to fine tune insulin analysis methods, measurement traceability, and any conversion factor used in post-analysis steps in accordance with the necessity for standardization.

Validation Study of a New Random-Access Chemiluminescence Immunoassay Analyzer i-TRACK10® to Monitor Infliximab and Adalimumab Serum trough Levels and Anti-Drug Antibodies.

Background. Monitoring of biological TNF inhibitors is a very important tool to guide clinical decisions using specialized algorithms, especially in gastroenterology. A new chemiluminescent instrument (i-TRACK10® from Theradiag) could replace ELISA techniques to calculate the dosage of drugs and anti-drug antibodies. In this bi-centric study, we explored the analytical performances of i-TRACK10® using manual or automated (DS2®) ELISA Lisa-Tracker® assays, and compared the results. Patients and methods. Intra- and inter-run performances were evaluated with i-TRACK10® in two different laboratories and for two different ranges of values for infliximab, adalimumab, and their respective antibodies. Patients' samples were used in the labs to compare the results obtained between the new instrument and either the manual Lisa-Tracker® or the automated DS2. Results. Intra- and inter-run performances were satisfactory, with values between 1.8% and 16.1% (for inter-run imprecision at low/medium values of infliximab). Results were generally comparable between assays. with the lowest value of correlation at 0.59 (anti-adalimumab dosage between i-TRACK10® and manual ELISA). Most often, values of drugs and anti-drug antibodies were higher with i-TRACK10® than with manual ELISA assay, and correlation values were better with automated ELISA. Agreements were globally acceptable, and the lowest coefficients of 0.7 was obtained for adalimumab values between i-TRACK10® and the two ELISA methods, and for anti-adalimumab values between i-TRACK10® and manual ELISA. The type of assay can potentially induce a change in the class of patients and lead to divergent therapeutic decisions. Conclusions. The new random-access instrument i-TRACK10® presents many advantages in a routine laboratory: rapidity, the possibility of standardization, usability, and expansion of the measurement range. Despite the relatively good agreement of results, it is preferable to use the same assay in longitudinal follow-up of a patient, because quantitative results were not completely equivalent especially for anti-drug antibodies.

Use of Uncertainty of Measurement for Traceability of Test Results and Setting up of own Quality Goal for Methods having Lower Stability- A Tertiary Care Hospital study.

Uncertainty of measurement (UM) provides a quantitative estimate for traceability of test results. The Nordtest guide was applied for calculating UM of 26 analytes. For this, internal and external quality control data from July 2019 to April 2020 was used. UM of test results were compared to %TEa values of CLIA '2019, RiliBÄK, and Ricos. It was observed that UM for all analytes were below %TEa values of RiliBÄK. UM value of Albumin, Calcium and Sodium could not meet CLIA '2019 and Ricos guidelines. For results of Albumin, Calcium and Sodium to be traceable, more frequent quality control protocols resulted in decrease in bias. Quality goals were set for these three parameters. This helped in reduction of quality control cycles and optimum utilization of resources.

Independent Side-by-Side Validation and Comparison of 4 Serological Platforms for SARS-CoV-2 Antibody Testing.

Highly sensitive and specific platforms for the detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies are becoming increasingly important for evaluating potential SARS-CoV-2 convalescent plasma donors, studying the spread of SARS-CoV-2 infections, and identifying individuals with seroconversion. This study provides a comparative validation of 4 anti-SARS-CoV-2 platforms. A unique feature of the study is the use of a representative cohort of convalescent patients with coronavirus disease 2019 and a mild to moderate disease course. All platforms showed significant correlations with a SARS-CoV-2 plaque reduction neutralization test, with highest sensitivities for the Euroimmun and the Roche platforms, suggesting their preferential use for screening persons at increased risk of SARS-CoV-2 infections.

Anti-Human T-Cell Leukemia Virus Type 1 (HTLV-1) Antibody Assays in Cerebrospinal Fluid for the Diagnosis of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis.

The anti-human T-cell leukemia virus type 1 (HTLV-1) antibody assay in common use has changed from the particle agglutination (PA) method to chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA). These assays were validated in serum but not in cerebrospinal fluid (CSF). However, anti-HTLV-1 antibody positivity in CSF is a requisite for diagnosing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We qualitatively compared the assays in CSF from 47 HAM/TSP patients diagnosed using PA, 15 HTLV-1 carriers (HCs), and 18 negative controls. In determining the positivity or negativity of CSF anti-HTLV-1 antibodies, we used serum cutoff points for CLIA and CLEIA because CSF cutoff points had not been decided. Truth table analysis revealed that the performance of CLIA was closer to that of PA and that CLEIA had low sensitivity. CSF antibodies from HAM/TSP patients were all positive by PA and CLIA but 83.0% positive by CLEIA. CSF antibodies from HCs were positive in 73.3%, 80.0%, and 6.7% by PA, CLIA, and CLEIA, respectively. Receiver operator characteristic curve analysis for CSF revealed that with the default cutoff point used for serum, CLIA and PA had comparable performances and CLEIA was less sensitive. The best performances of CLIA and CLEIA with adjusted cutoff points were 94.8% sensitivity and 95.5% specificity and 89.7% sensitivity and 95.5% specificity, respectively. We conclude that low-sensitivity CLEIA can underdiagnose HAM/TSP and that CLIA is a better alternative to PA in anti-HTLV-1 antibody assay for CSF with the current cutoff points.

Evaluation of Diagnostic Accuracy of Eight Commercial Assays for the Detection of Measles Virus-Specific IgM Antibodies.

The World Health Organization (WHO) has targeted measles for global eradication through mass immunization. For effective monitoring of eradication targets, high-quality surveillance is needed. The detection of IgM antibodies, specific to the measles virus, with the use of commercial enzyme-linked immunosorbent assays (ELISA or EIA) is broadly used within the WHO global measles and rubella laboratory network for laboratory confirmation, and in particular, ELISA kits manufactured by Siemens (Enzygnost kits) have been primarily used. Spurred by the discontinuation of these kits, this study aims to report on the clinical sensitivity and specificity of comparable commercial ELISA kits and one automated chemiluminescent immunoassay (CLIA) method. A panel of 239 serum samples was assembled that included sera from confirmed measles cases (n = 50) and probable post-MMR vaccine response (n = 2). Measles-negative sera (n = 187) were collected from individuals presenting with other fever and rash illnesses. A total of 7 ELISA kits (Euroimmun native antigens and recombinant nucleoprotein, IBL, Clin-Tech Microimmune, NovaTec NovaLisa, Serion, and Siemens Enzygnost) and one CLIA method (DiaSorin LIAISON XL) were evaluated. The ELISA kits included two IgM capture methods and five indirect methods. Calculated sensitivities and specificities ranged from 75.0% to 98.1% and 86.6% to 99.5%, respectively. The parvovirus B19 IgM positive sera were noted to cause false-positive results, particularly for the ELISA kits from Serion and NovaLisa; specificities for this subset of samples ranged from 51.4% to 100.0%. The capture IgM ELISA methods provided the best combination of sensitivity and specificity.

Interpretive discrepancies caused by target values inter-batch variations in chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG by MAGLUMI.

Plasma specimens from coronavirus disease 2019 patients were double-tested for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies by two different batches of MAGLUMI 2019-nCov immunoglobulin M/immunoglobulin G (IgM/IgG) assays to evaluate IgM/IgG levels, qualitative interpretation, antibody kinetics, and linearity of diluted specimen. Here we show that (i) high-level IgM specimens need to be diluted with negative human plasma but not kit diluents and (ii) measured anti-SARS-CoV-2 IgM/IgG concentrations are substantially higher with later marketed immunoassay batch leading to (iii) the change of qualitative interpretation (positive vs. negative) in 12.3% of specimens measured for IgM, (iv) the informative time-course pattern of antibody production only when data from different immunoassay batches are not combined.

Evaluation of three immunochromatographic tests in COVID-19 serologic diagnosis and their clinical usefulness.

Results of three rapid immunochromatographic tests (ICTs) were compared with those obtained with two automated immunoassays for evaluation of their usefulness. One hundred fifty-nine patients and 67 healthy volunteers were included. Different assays demonstrate 41-45% of diagnostic sensitivities and 91-98% of specificities, with substantial agreement (89.3-91.2%), but a high percentage of weak positive results (13-22%) was observed with ICTs. ICTs performances were comparable to those of automated immunoassays. ICTs could have a role as screening approach due to their easy usability. Subjective interpretation, significant rate of uncertain results, uncertainty on viral antigens source are undoubtedly drawbacks.