Quality control for serological testing.

OBJECTIVES: The quality control of serological assays remains controversial. The aim of this project was to describe the problems associated with a working model for controlling these assays and solutions, including using a source of well-defined targets and acceptable limits, a process to identify lot-to-lot reagent variation and an interpretation of the result that accounted for the clinical situation. False-negative results are problematic but can be reduced by identifying and comparing reagent lot variation with previous results. METHODS: The components of the Quality Assurance strategy are the following: Lot-to-lot reagent and calibrator variation assessment; dynamic, big-data approach to determine accurate targets and acceptable limits for manufacturer-provided QC material; negative QC monitoring process; use of commutable EQA with a sufficient method subgroup size to assess bias; clinical assessment of any statistically flagged error; and provision of support to the clinician for the interpretation of results. RESULTS: The model described has been used for twelve months, and acceptable variation has been maintained. CONCLUSIONS: The paper presents a solution that emphasizes the early detection of reagent lot variation and patient risk rather than instrument control. Reducing the risk of a false result to patients requires optimal assay quality control and an effective mechanism to support the clinician's use of these results in diagnosis and monitoring. The problems of serological assays are well-known, but there remain few integrated solutions in the literature.

The serological diagnostic value of EBV-related IgA antibody panels for nasopharyngeal carcinoma: a diagnostic test accuracy meta-analysis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is diagnosed relatively late and has a poor prognosis, requiring early detection to reduce the disease burden. This diagnostic test accuracy meta-analysis evaluated the serological diagnostic value of nine EBV-related IgA antibody panels (EBNA1-IgA, VCA-IgA, EA-IgA, Zta-IgA, EBNA1-IgA + VCA-IgA, VCA-IgA + EA-IgA, VCA-IgA + Rta-IgG, EBNA1-IgA + VCA-IgA + Zta-IgA and VCA-IgA + EA-IgA + Rta-IgG), aiming to identify suitable serological detection biomarkers for NPC screening. METHODS: PubMed, Embase, China National Knowledge Infrastructure and Chinese BioMedical Literature Database were searched from January 1st, 2000 to September 30th, 2023, with keywords nasopharyngeal carcinoma, IgA, screening, early detection, early diagnosis, sensitivity and specificity. Articles on the diagnostic value of serum EBV-related IgA antibody panels for NPC were included. Study selection, data extraction, and quality assessment were performed independently by two researchers, and a third researcher was consulted in the case of disagreement. Bivariate models were used for statistical analysis. The quality of included studies was evaluated through Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2). RESULTS: A total of 70 articles were included, involving 11 863 NPC cases and 34 995 controls. Among the nine EBV-related IgA antibody panels, EBNA1-IgA + VCA-IgA [0.928 (0.898, 0.950)], VCA-IgA + Rta-IgG [0.925 (0.890, 0.949)], EBNA1-IgA + VCA-IgA + Zta-IgA [0.962 (0.909, 0.985)] and VCA-IgA + EA-IgA + Rta-IgG [0.945 (0.918, 0.964)] demonstrated higher pooled sensitivity (95%CI). In terms of diagnostic odds ratio (DOR) (95%CI), EBNA1-IgA + VCA-IgA [107.647 (61.173, 189.430)], VCA-IgA + Rta-IgG [105.988 (60.118, 186.857)] and EBNA1-IgA + VCA-IgA + Zta-IgA [344.450 (136.351, 870.153)] showed superior performance. Additionally, the SROC curves for EBNA1-IgA + VCA-IgA and VCA-IgA + Rta-IgG were more favorable. However, publication bias was detected for VCA-IgA (P = 0.005) and EBNA1-IgA + VCA-IgA (P = 0.042). CONCLUSIONS: In general, parallel detection of serum EBNA1-IgA, VCA-IgA and Zta-IgA antibodies using ELISA demonstrates better pooled sensitivity and DOR among the studied panels. In the cases where fewer indicators are used, serum VCA-IgA and EBNA1-IgA/Rta-IgG antibody panel exhibits a comparable performance. TRIAL REGISTRATION: The International Prospective Register of Systematic Reviews registration number: CRD42023426984, registered on May 28, 2023.

Cost-benefit analysis of serological and nucleic acid testing for hepatitis B virus in blood donors in southern China.

BACKGROUND: Most Chinese blood centers have implemented mini pool (MP) HBV nucleic acid testing (NAT) together with HBsAg ELISA in routine blood donor screening for HBV infection since 2015, and a few centers upgraded MP to individual donation (ID) NAT screening recently, raising urgent need for cost-benefit analysis of different screening strategies. In an effort to prevent transfusion-transmitted infections (TTIs) for HBV, cost-benefit analyses of three different screening strategies: HBsAg alone, HBsAg plus MP NAT and HBsAg plus ID NAT were performed in blood donors from southern China where HBV infection was endemic. METHODS: MP-6 HBV NAT and ID NAT were adopted in parallel to screen blood donors for further comparative analysis. On the basis of screening data and the documented parameters, the number of window period (WP) infection, HBV acute infection, chronic hepatitis B infection (CHB) and occult hepatitis B infection (OBI) was evaluated, and the potential prevented HBV TTIs and benefits of these three strategies were predicted based on cost-benefit analysis by an estimation model. RESULTS: Of 132,323 donations, the yield rate for HBsAg-/DNA + screened by ID NAT (0.12%) was significantly higher than that by MP NAT (0.058%, P < 0.05). Furthermore, the predicted transfusion-transmitted HBV cases prevented was 1.25 times more by ID NAT compared to MP-6 NAT. The cost-benefit ratio of the universal HBsAg screening, HBsAg plus ID NAT and HBsAg plus MP NAT were 1:58, 1:27 and 1:22, respectively. CONCLUSIONS: Universal HBsAg ELISA screening in combination with HBV ID NAT or MP-6 NAT strategies was highly cost effective in China. To further improve blood safety, HBsAg plus HBV DNA ID NAT screening should be considered in HBV endemic regions/countries.

Serologic screening for viral infections among blood donors: a study in a blood bank in southern Brazil.

BACKGROUND: Routine screening for viral infections at blood donation is important to avoid transfusion-transmitted infections. It also offers an opportunity to detect an asymptomatic infection. OBJECTIVE: To study changes in serology positivity for viral infections (B and C hepatitis, HTLV-1/2, and HIV) at blood donation in a blood bank from Southern Brazil, comparing two periods of 5 years: the period from 2013 to 2017 with the period from 2018 to 2022. In addition, data on the donor fidelity rate during the studied period were sought. METHODS: Retrospective study using data from 2013 to 2022 from a single blood center electronic database from Curitiba, Southern Brazil. RESULTS: A significant drop in positive serology for all studied viruses was observed: highest in HIV (OR=0.39; 95% CI=0.27-0.57) and lowest in total anti HBc (0.56; 95 CI=0.50-0.63). Anti HBc serology became more commonly seen in women in the period of 2018-2022 when compared to men. No changes in the distribution of positive serology according to donors' ages were observed. Loyalty rates had a median of 70%, with the lowest being 60% in 2013, while the highest was 73% in 2018 and 2022. CONCLUSION: A significant reduction in discarded blood bags due to viral serology was observed when the period of 2013-2017 was compared to 2018-2022 on this blood bank; the highest reduction was observed in HIV serology and the lowest in HBc serology, which became more common in women in the second period. High rates of donor fidelity were observed during the period studied.

Retrospective analysis of serologic test requests in the diagnosis of viral hepatitis: Inappropriate test requests and cost.

The rational laboratory use and implementation of test ordering procedures aim to reduce unnecessary test requests. This study aimed to determine the financial burden caused by inappropriate serological test requests for viral hepatitis and to investigate physicians' reasons for making unnecessary test requests. We performed a retrospective evaluation of inappropriate requests for hepatitis serology testing and the financial burden they caused at a tertiary care hospital over a 1-year period. The study found 2183 (3.84%) inappropriate test requests, costing $3309.00. Of these, 357 were same-day repeat requests and 1826 were requests not following diagnostic algorithms. In the logistic regression analysis of the factors affecting unnecessary test requests, a statistically significant difference was found between whether the unit was internal or surgical, whether the request came from inpatient services or outpatient clinics, and the professional titles (P < .05). Both types of inappropriate requests were more common among male physicians (P < .05). The highest rates of inappropriate test requesting were in physical therapy and rehabilitation, pediatrics, and adult emergency units. To identify the reasons behind unnecessary test requests, 135 physicians from 23 different departments participated in the survey. The main reasons for requesting tests were identified as protecting against malpractice and fears of misdiagnosis or overlooking a diagnosis. It has been observed that physicians often order tests routinely, without being fully familiar with standard test ordering procedures based on diagnostic algorithms, and lacking knowledge about rational laboratory use. The cost of tests is mostly unknown to clinicians. The study concludes that there are laboratory tests that incur much higher costs. When this assessment is applied to the entire laboratory, it becomes clear how significant a financial burden, unnecessary workload, and loss of time this situation can cause. Identifying the presence of unnecessary test requests is the first step in preventing them. Appropriate measures include highlighting these issues, providing necessary information, and offering in-service training.

Assessing the diagnostic accuracy of serological tests for hepatitis delta virus diagnosis: a systematic review and meta-analysis.

Hepatitis Delta Virus (HDV), a satellite virus of Hepatitis B virus, exacerbates liver damage in affected individuals. Screening for HDV antibodies in HBsAg positive patients is recommended, but the diagnostic accuracy of serological tests remains uncertain. This review aimed to assess the diagnostic accuracy of serological tests for HDV. We searched PubMed, Web of Science, Cochrane Central Register of Controlled Trials, Scopus etc. for relevant studies. Studies measuring the sensitivity and specificity of serological HDV tests against PCR as a reference standard were included. Pooled sensitivity and specificity for each test method and sero-marker were calculated. The review included six studies with 11 study arms, evaluating ARCHITECT immunoassay, EIA, ELISA, QMAC, RIA, and Western Blot test methods targeting Anti-HDV IgG, Total anti-HDV and Anti-HDV IgM. Sensitivities for Anti-HDV IgG, Total Anti-HDV and Anti-HDV IgM, tests were 97.4%, 51.9%, and 62.0%, respectively, with specificities of 95.3%, 80.0%, and 85.0%. Our findings, with its limited number of studies, suggest that HDV serological tests, particularly those identifying Anti IgG exhibit high accuracy and can serve as effective screening tools for HDV.

Comparison of in-house enzyme-linked immunosorbent assay (ELISA) and six commercial ELISAs for Detection of antibodies to Bordetella pertussis.

Enzyme-linked immunosorbent assays (ELISA) are currently the method of choice for the serodiagnosis of pertussis and play a key role in the diagnosis of pertussis in adolescents and adults, as well as in epidemiological studies. In the present study, the in-house developed indirect ELISA was comparatively evaluated with six commercial kits from various manufacturers. Antipertussis antibodies were measured in 40 serum samples from patients with clinical symptoms of respiratory tract infection, in two WHO standards, and in seven human ECDC control sera. IgA and IgG antibodies were detected at a diagnostically significant level by different ELISA kits of 5.0% to 27.0% and 12.0% to 70.0% of patients' sera, appropriately. The analysis of results carried out with six commercial kits showed only 17.5% consistent results in class IgG (either clearly positive or negative). The average percentage of errors in the level of antibodies determined in the control samples, reference serum samples, differed quite significantly and ranged from 9.5% to 35.4% depending on the kit. This poor correlation of the results obtained on various serological tests intended for the serodiagnosis of pertussis may cause very serious diagnostic problems, especially when examining a serum sample obtained once during the course of the disease.

Rapid single-tier serodiagnosis of Lyme disease.

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDC's LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance.

Accuracy of the Dual Path Platform (DPP) rapid test for the diagnosis of leptospirosis: A multi-center study in six Brazilian states.

Leptospirosis is a zoonotic disease with significant global impact and a challenging diagnosis. The utilization of adequately validated rapid tests is relevant for the opportune identification of the disease and for reduction in fatality rates. The present study analyzes the accuracy and reliability of the Dual Path Platform (DPP) assay -produced in Brazil by the Oswaldo Cruz Foundation (Fiocruz)- for diagnosing leptospirosis. Firstly, a serological panel was constructed in the Brazilian Reference Laboratory for Leptospirosis using samples routinely handled by reference laboratories of six Brazilian states. It consisted of 150 positive (according to MAT and IgM-ELISA) and 250 negative samples for leptospirosis. Subsequently, the panel samples were distributed to the reference laboratories for the performance of DPP assays in triplicate. Different measures were used in the assessment of diagnostic quality. Predictive values were estimated for different pre-test probability settings. Sensitivities varied between 67.33 % and 74.00 % and specificities between 93.20 % and 98.40 % in the states, and there were adequate agreements between them. Accuracies were lower for the samples of patients with less than 7 days of symptoms. In contexts of prevalence values up to around 25 %, positive and negative predictive values were around 90 %. However, in situations of high pre-test probabilities, NPVs were low. This study improves understanding of the use of DPP in diagnosing leptospirosis, particularly its application in healthcare settings. As long as the time of symptoms onset and clinical and epidemiological contexts are adequately considered for the interpretation of results, DPP is a valid option to be used in the leptospirosis diagnostic routine.

Development of two competitive ELISAs based on monoclonal antibodies for the serological detection of Bovine ephemeral fever virus.

Bovine ephemeral fever virus (BEFV) is a member of the genus Ephemerovirus in the family Rhabdoviridae. It is an arthropod-borne virus transmitted by many species of midges and mosquitoes. It can cause severe economic consequences due to losses in milk production and the general condition of cattle and water buffalo. BEF occurs in some tropical, subtropical and warm temperate regions of Africa, Australia, the Middle East and Asia with seasonal outbreaks, but its possible spread to other areas (e.g. Europe) cannot be excluded. Therefore, using and developing rapid diagnostic methods with optimal performance is essential for identifying emerging pathogens and their control. In the present study, we developed two competitive serological ELISAs based on monoclonal antibodies (mAbs), designed by using BEFV inactivated antigen and the BEF recombinant nucleoprotein (N), respectively. A panel of 77 BEF-positive and 338 BEF-negative sera was used to evaluate the two tests. With a diagnostic sensitivity of 97.4 % using the inactivated virus and 98.7 % using the recombinant N, and a diagnostic specificity of 100 % using both antigens, our results suggest that these tests are suitable for the serological diagnosis of BEF.

Discordant performance of mpox serological assays.

BACKGROUND: Since July 23, 2022, global mpox cases reached 92,546, with over 31,000 in the United States. Asymptomatic carriage is a critical mechanism influencing the global dissemination of mpox. Seroprevalence studies are crucial for determining the epidemic's true burden, but uncertainties persist in serologic assay performance and how smallpox vaccination may influence assay interpretation. OBJECTIVES: Our study aimed to assess the performance of several diagnostic assays among mpox-positive, vaccinated, and pre-outbreak negative control samples. This investigation sought to enhance our understanding and management of future mpox outbreaks. STUDY DESIGN: Serum samples from 10 mpox-positive, five vaccinated uninfected, and 137 pre-outbreak controls were obtained for serological testing. The mpox-positive samples were obtained around 100 days post symptom onset, and vaccinated patients were sampled approximately 90 days post-vaccination. Multiple diagnostic assays were employed, including four commercial ELISAs (Abbexa, RayBioTech, FineTest, ProteoGenix) and a multiplex assay (MesoScale Diagnostics (MSD)) measuring five mpox and five smallpox antigens. RESULTS: Three commercial ELISA kits had low specificity (<50 %). The Proteogenix ELISA targeting the E8L antigen had a 94 % sensitivity and 87 % specificity. The E8L antigen on the MSD assay exhibited the greatest distinction between exposure groups, with 98 % sensitivity and 93 % specificity. CONCLUSIONS: None of the assays could distinguish between mpox-positive and vaccinated samples. The MSD assay targeting the MPXV E8L antigen demonstrated the greatest differentiation between mpox-positive and pre-outbreak negative samples. Our findings underscore the imperative to identify sensitive and specific assays to monitor population-level mpox exposure and infection.

Comparative Evaluation of Commercial Test Kits Cleared for Use in Modified Two-Tiered Testing Algorithms for Serodiagnosis of Lyme Disease.

BACKGROUND: Modified 2-tiered testing (MTTT) for Lyme disease utilizes automatable, high throughput immunoassays (AHTIs) in both tiers without involving western immunoblots, offering performance and practical advantages over standard 2-tiered testing (STTT; first-tier AHTI followed by immunoglobulin M (IgM) and immunoglobulin G (IgG) western immunoblots). For MTTT, Centers for Disease Control and Prevention recommends using AHTI test kits that have been cleared by Food and Drug Administration (FDA) specifically for this intended use. We evaluated performance of FDA-cleared MTTT commercial test kits from 3 manufacturers by comparing with STTT results. METHODS: We performed MTTT (total antibody AHTI with reflex to separate IgM and IgG AHTIs) using test kits from Diasorin, Gold Standard Diagnostics (GSD), and Zeus Scientific on 382 excess serum samples submitted to the clinical laboratory for routine Lyme disease serologic testing in July 2018, measuring agreement between MTTT and STTT using the κ statistic. RESULTS: Overall agreement with STTT was 0.87 (95% confidence interval [CI], .77-.97) using Diasorin assays (almost perfect agreement), 0.80 (95% CI, .68-.93) using GSD assays (substantial agreement) and 0.79 (95% CI, .68-.90) using Zeus assays (substantial agreement). For detection of IgM reactivity, agreement between MTTT and STTT was 0.70 (.51-.90; substantial), 0.63 (95% CI, .44-.82; substantial) and 0.56 (95% CI, .38-.73; moderate), respectively. For detection of IgG reactivity, MTTT/STTT agreement was 0.73 (95% CI,.58-.88), 0.78 (95% CI, .62-.94), and 0.75 (95% CI, .60-.90), respectively (substantial agreement in all cases). CONCLUSIONS: MTTT results obtained using commercial test kits from 3 different manufacturers had substantial to almost perfect agreement with STTT results overall and moderate to substantial agreement for IgM and IgG detection independently. Commercial MTTT tests can be used broadly for the diagnosis of Lyme disease.

Production and evaluation of a new set of recombinant antigens for the serological diagnosis of human cysticercosis.

Human cysticercosis caused by Taenia soliun (T. soliun) is endemic in certain areas of Latin America, Asia and Sub-Saharan Africa. Neurocysticercosis (NCC) is mainly diagnosed by neuroimaging, which, in most cases, is unavailable in endemic areas. Due to their high sensitivity and specificity, serological tests such as enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) based on the glycosylated fraction of the cyst CS50 are widely used for the detection of the anti-cysticercus IgG antibodies despite their significant cost and the need of cysticercus material. Given their cost-effectivess and simplicity, immunoassays based on recombinant proteins could provide new alternatives for human cysticercosis diagnosis: such tests would be aimed at screening those people living in remote areas who need further examination. To date, however, no test using recombinant antigens is commercially available. Herein, five recombinant proteins (R14, R18, R93.1, R914.1, and R915.2) were produced, three of which (R93.1, R914.1, and R915.2) were newly identified from the cyst fluid. Evaluation of the diagnostic performance of these recombinant antigens by ELISA was done using sera from 200 epileptic and non-epileptic individuals in comparison with the WB-CS50 as the reference serological method. Recombinant proteins-based ELISA showed a level of diagnostic performance that is inferior than the reference serological method, but similar to that of the native antigen ELISA for human cysticercosis (commonly used for screening). Further optimization of expression conditions is still needed in order to improve proteins solubility and enhance diagnostic performance for human cysticercosis detection. However, this preliminary evaluation of the recombinant antigens has shown their potential valuable use for screening cysticercosis in patients with epilepsy attending dispensaries in remote areas. Future studies should be conducted to evaluate our recombinant antigens in a large group of patients with different stages of NCC, and in correlation with imaging findings.

Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

Sigma metrics analysis of serology screening assays to enhance quality and efficiency in New Zealand blood services.

Sigma metric analysis was conducted across two New Zealand Blood Services (NZBS) laboratories (Auckland and Christchurch) to optimize quality control (QC) procedures. We evaluated five assays (anti-HCV, HIV Ag/Ab combo, HTLV-I/II, HBsAg, and Syphilis) using internal quality control (IQC) and third-party daily QC data extracted from four Architect i2000SR instruments during Jan 2 -31st, 2023. Mean, standard deviation (SD), and coefficient of variation (CV%) were calculated, assuming zero bias. Sigma metrics were determined using the Total Allowable Error (TEa %) based on difference between positive control mean and signal-to-cutoff (s/co) cut-off. Most assays exhibited CV% values ≤10 % except for HBsAg IQC (18.5 %) and anti-HCV third-party QC (13.4 %) at Christchurch. TEa % ranged from 38 % to 90 %. Overall, the assays demonstrated Six Sigma performance (σ > 6), except for HBsAg IQC (3.97) and anti-HCV third-party QC (5.46) at Christchurch. These high-quality serology assays can benefit from simplified QC design without compromising blood safety.

A Case of Intravenous IVIg Interfering with Serological Test Results of HBV.

BACKGROUND: Since Imbach [1] first reported the use of high-dose intravenous immunoglobulin (IVIg) in the treatment of idiopathic thrombocytopenic purpura (ITP) in children, indications for IVIg therapy have been increaseing. At present, IVIg infusion has become an important means of clinical treatment. The phenomenon of anti-HBs and anti-HBc elevation caused by IVIg infusion in patients has been reported in journals, but similar reports in journals related to laboratory diagnosis are rare. METHODS: We reported a case of a patient with immune thrombocytopenia (ITP) which interfered with hepatitis B virus (HBV) serological detection after receiving intravenous IVIg. We used chemiluminescence immunoassay to detect serological markers of HBV. IU/mL was used to represent the detection data of HBsAg and HBsAb and cutoff value was used to represent the detection HBeAg, HBeAb, and HbcAb. RESULTS: The serological markers of HBV were all negative before IVIg infusion. One week after IVIG infusion, the item was tested again, and the results of HBsAb, HBeAb, and HBcAb were positive. As the time increased after infusion, HBsAb, HBeAb, and HBcAb in the patient gradually decreased. CONCLUSIONS: After IVIg infusion, the sudden positive change of HBsAb, HBeAb, and HbcAb in the patient's body was not caused by HBV infection, but caused by the infusion of foreign antibody. This case study shows that physicians should be particularly careful when interpreting results in patients treated with intravenous IVIg involving viral hepatitis B.

Investigation of atypical serological profiles for Epstein-Barr virus (EBV).

BACKGROUND: Commercial immunoassays that detect IgG and IgM directed toward VCA and IgG EBNA are used in combination to assess EBV immune status. However, this strategy does not always confirm/exclude recent/past EBV infection or absence of immunity. OBJECTIVES: The aim of our study was to perform complementary investigations on samples with atypical EBV serological profiles, in order to identify the clinical situation they correspond to. STUDY DESIGN: EBV serology was performed using EBV VCA IgM/IgG and EBNA IgG LXL® DiaSorin assay. Complementary investigations included ELISA IgM VCA, immunoblots, CMV IgM/IgG and CMV IgG avidity, and EBV PCR. RESULTS: In our study, 12810 EBV serological results were analyzed, and 3580 atypical profiles were detected (28 %). Among these latter, isolated VCA IgG represented 42.9 %, the three positive markers accounted for 29.1 %, isolated EBNA IgG represented 18.5 %, isolated VCA IgM accounted for 6.4 % and positive VCA IgM & positive EBNA IgG represented 3.1 %. VCA IgG detected alone were specific in 100 % cases and EBNA IgG detected alone were specific in 91.7 % cases. VCA IgM detected alone were false positive or due to a cross reaction with CMV in 52.8 % cases. The pattern positive VCA IgM and positive EBNA IgG correspond to a false positive in VCA IgM, EBNA IgG or both in 83.4 % cases. Positive EBV VCA IgM/IgG and EBNA IgG were unreliable to detect active EBV infection in 66.7 % cases. DISCUSSION: Atypical EBV serological profiles may correspond to several clinical situations and complementary investigations allow to determine the immune status in more than 98.5 % cases.

Comparative Evaluation of Select Serological Assays for Zika Virus Using Blinded Reference Panels.

In response to the 2015 Zika virus (ZIKV) epidemic that occurred in Brazil, numerous commercial serological assays have been developed for clinical and research applications. Diagnosis of recent infection in pregnant women remains challenging. Having standardized, comparative studies of ZIKV tests is important for implementing optimal diagnostic testing and disease surveillance. This is especially important for serology tests used to detect ZIKV infection given that antibodies against ZIKV can cross-react with other arboviruses in the same virus family, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). We looked at the sensitivity and specificity of tests detecting ZIKV antibodies (IgM, IgG) from multiple manufacturers using panels of samples previously collected with known exposure to ZIKV and other arboviruses. We found that performance of the IgM tests was highly variable, with only one test (Inbios 2.0 IgM capture ELISA) having both high sensitivity and specificity. All IgG tests showed good sensitivity; however, specificity was highly variable, with some assays giving false-positive results on samples infected by another flavivirus. Overall, the results confirmed that accurate ZIKV antibody testing is challenging, especially in specimens from regions endemic for multiple other flaviviruses, and highlight the importance of available and suitable reference samples to evaluate ZIKV diagnostics.

Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study.

BACKGROUND: Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future "screen and treat" strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. METHODS: During active screening, venous blood samples from 1095 individuals from Côte d'Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. RESULTS: One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1-99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5-88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7-84.2%); DCN HAT RDT 78.2% (95% CI: 75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n = 399) and 93.0-100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. CONCLUSIONS: Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based "screen and treat" strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. TRIAL REGISTRATION: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

Serological diagnosis of strongyloidiasis: An evaluation of three commercial assays.

BACKGROUND: Strongyloidiasis is caused by a neglected nematode, manifesting as chronic intestinal infection with potentially severe manifestations. The disease is an emerging problem in non-endemic countries affecting travelers and migrants. Diagnosis of strongyloidiasis is hampered by the lack of standardization and absence of a gold standard. Since adequate direct methods to detect the motile larvae in stool samples are not widely available, other techniques such as serology have been developed. METHODS: We evaluated three commercial ELISA kits (DRG Instruments, IVD Research, and Bordier Affinity Products) to detect IgG antibodies against Strongyloides stercoralis assays utilizing serum samples from travelers with microscopically confirmed strongyloidiasis (n = 50) and other imported helminthic infections (n = 159) as well as healthy controls (n = 50). RESULTS: The DRG, IVD, and Bordier assays showed sensitivities of 58.0%, 64.0%, and 56.0%, respectively. Specificity values were 96.0%, 96.0%, and 92.0% in healthy controls, and 67.3%, 62.9%, and 76.7% in cases with other helminth infections, respectively. Cross-reactions were mostly observed in cases with other nematodes (37.5%, 42.5%, and 20.0%, respectively), but also in trematode (33.3%, 38.1%, and 19.0%, respectively) and in cestode infections (25.0%, 30.0%, and 32.5%, respectively). CONCLUSION: The study demonstrates the diagnostic limitations of serological assays to detect or exclude cases of strongyloidiasis in returning travelers, who frequently present with recent or acute infections.