Evaluation of the New Multi-HTLV Serological Assay: Improvement for HTLV-2 Detection.

Despite the accuracy of confirmatory tests for the diagnosis of human T cell lymphotropic virus (HTLV), inconclusive or false-negative results still occur when diagnosing human T cell lymphotropic virus type 2 (HTLV-2)-positive patients. The goal of this study was to evaluate the sensitivity and accuracy of a confirmatory immunoassay, the Multi-HTLV assay. A total of 246 plasma samples were tested by real-time polymerase chain reaction (qPCR) and used to calculate the sensitivity and typing accuracy of the Multi-HTLV assay. Of the 246 plasma samples, 127 were positive for human T cell lymphotropic virus type 1 (HTLV-1), 112 were positive for HTLV-2, and 7 were positive for both HTLV-1 and HTLV-2. Thereafter, the nonparametric Mann-Whitney U test was used to calculate the concordance between the qPCR test and Multi-HTLV assay in 12 samples with discrepant and inconclusive qPCR results. The Multi-HTLV assay showed high performance in identifying HTLV-1 and HTLV-2 with sensitivities of 97% [95% confidence interval (CI): 0.92-0.98] and 94% (0.87-0.96), respectively. However, due to typing performance (98% for HTLV-1 and 94% for HTLV-2), it had 95% agreement with positive HTLV-1 qPCR results (95% CI: 90.07-97.81) and 86% (78.04-91.01) of HTLV-2 qPCR results were positive. Moreover, this test was able to recognize 80% of indeterminate samples and all HTLV-2 positive samples that showed false-negative qPCR results. Our findings, derived from a substantial number of HTLV-positive samples, underscore the inherent reliability and feasibility of the Multi-HTLV assay, regardless of the molecular testing facilities. Furthermore, the distinctive multiparametric nature of this assay, combined with its straightforward procedural execution, introduces novel perspectives for analyzing specific serological profiles in each patient, as well as the potential for immunological monitoring of disease progression.

Comparing in vivo bioluminescence imaging and the Multi-Cruzi immunoassay platform to develop improved Chagas disease diagnostic procedures and biomarkers for monitoring parasitological cure.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and is a serious public health problem throughout Latin America. With 6 million people infected, there is a major international effort to develop new drugs. In the chronic phase of the disease, the parasite burden is extremely low, infections are highly focal at a tissue/organ level, and bloodstream parasites are only intermittently detectable. As a result, clinical trials are constrained by difficulties associated with determining parasitological cure. Even highly sensitive PCR methodologies can be unreliable, with a tendency to produce "false-cure" readouts. Improved diagnostic techniques and biomarkers for cure are therefore an important medical need. METHODOLOGY/PRINCIPAL FINDINGS: Using an experimental mouse model, we have combined a multiplex assay system and highly sensitive bioluminescence imaging to evaluate serological procedures for diagnosis of T. cruzi infections and confirmation of parasitological cure. We identified a set of three antigens that in the context of the multiplex serology system, provide a rapid, reactive and highly accurate read-out of both acute and chronic T. cruzi infection. In addition, we describe specific antibody responses where down-regulation can be correlated with benznidazole-mediated parasite reduction and others where upregulation is associated with persistent infection. One specific antibody (IBAG39) highly correlated with the bioluminescence flux and represents a promising therapy monitoring biomarker in mice. CONCLUSIONS/SIGNIFICANCE: Robust, high-throughput methodologies for monitoring the efficacy of anti-T. cruzi drug treatment are urgently required. Using our experimental systems, we have identified markers of infection or parasite reduction that merit assessing in a clinical setting for the longitudinal monitoring of drug-treated patients.

A novel assessment method for COVID-19 humoral immunity duration using serial measurements in naturally infected and vaccinated subjects.

BACKGROUND: Collecting information on sustainability of immune responses after infection or vaccination is crucial to inform medical decision-making and vaccination strategies. Data on how long-lasting antibodies against SARS-COV-2 could provide a humoral and protective immunity and prevent reinfection with SARS-CoV-2 or its variants is particularly valuable. This study presents a novel method to quantitatively measure and monitor the diversity of SARS-CoV-2 specific antibody profiles over time. METHODS: Serum samples from two groups were used in this study: Samples from 20 naturally infected subjects (followed for up to 1 year) and samples from 83 subjects vaccinated with one or two doses of the Pfizer BioNtech vaccine (BNT162b2/BNT162b2) (followed for up to 6 months). The Multi-SARS-CoV-2 assay, a multiparameter serology test developed for the serological confirmation of past-infections, was used to determine the reactivity of six different SARS-CoV-2 antigens. For each subject sample, 3 dilutions (1/50, 1/400 and 1/3200) were defined as an optimal set over the six antigens and their respective linear ranges. This allowed accurate quantification of the corresponding six antibodies. Nonlinear mixed-effects modelling was applied to convert intensity readings from 3 determined dilutions to a single quantification value for each antibody. RESULTS: Median half-life for the 20 naturally infected vs 74 vaccinated subjects (two doses) was 120 vs 50 days for RBD, 127 vs 53 days for S1 and 187 vs 86 days for S2 antibodies respectively. CONCLUSION: The newly proposed method, based on a series of a limited number of dilutions, can convert a conventional qualitative assay into a quantitative assay. This conversion helps define the sustainability of specific immune responses against each relevant viral antigen and can help in defining the protection characteristics after an infection or a vaccination.

Poor Antibody Response to BioNTech/Pfizer Coronavirus Disease 2019 Vaccination in Severe Acute Respiratory Syndrome Coronavirus 2-Naive Residents of Nursing Homes.

BACKGROUND: Residents of nursing homes (NHs) are at high risk of coronavirus disease 2019 (COVID-19)-related disease and death and may respond poorly to vaccination because of old age and frequent comorbid conditions. METHODS: Seventy-eight residents and 106 staff members, naive to infection or previously infected with severe acute respiratory syndrome coronavirus (SARS-CoV-2), were recruited in NHs in Belgium before immunization with 2 doses of 30 µg BNT162b2 messenger RNA (mRNA) vaccine at days 0 and 21. Binding antibodies (Abs) to SARS-CoV-2 receptor-binding domain (RBD), spike domains S1 and S2, RBD Ab avidity, and neutralizing Abs against SARS-CoV-2 wild type and B.1.351 were assessed at days 0, 21, 28, and 49. RESULTS: SARS-CoV-2-naive residents had lower Ab responses to BNT162b2 mRNA vaccination than naive staff. These poor responses involved lower levels of immunoglobulin (Ig) G to all spike domains, lower avidity of RBD IgG, and lower levels of Abs neutralizing the vaccine strain. No naive residents had detectable neutralizing Abs to the B.1.351 variant. In contrast, SARS-CoV-2-infected residents had high responses to mRNA vaccination, with Ab levels comparable to those in infected staff. Cluster analysis revealed that poor vaccine responders included not only naive residents but also naive staff, emphasizing the heterogeneity of responses to mRNA vaccination in the general population. CONCLUSIONS: The poor Ab responses to mRNA vaccination observed in infection-naive NH residents and in some naive staff members suggest suboptimal protection against breakthrough infection, especially with variants of concern. These data support the administration of a third dose of mRNA vaccine to further improve protection of NH residents against COVID-19.

A novel high performing multiplex immunoassay Multi-HTLV for serological confirmation and typing of HTLV infections.

BACKGROUND: Human T-Cell Lymphotropic Viruses (HTLV) type 1 and type 2 account for an estimated 5 to 10 million infections worldwide and are transmitted through breast feeding, sexual contacts and contaminated cellular blood components. HTLV-associated syndromes are considered as neglected diseases for which there are no vaccines or therapies available, making it particularly important to ensure the best possible diagnosis to enable proper counselling of infected persons and avoid secondary transmission. Although high quality antibody screening assays are available, currently available confirmatory tests are costly and have variable performance, with high rates of indeterminate and non-typable results reported in many regions of the world. The objective of this project was to develop and validate a new high-performance multiplex immunoassay for confirmation and discrimination of HTLV-1 and HTLV-2 strains. METHODOLOGY/PRINCIPAL FINDINGS: The multiplex platform was used first as a tool to identify suitable antigens and in a second step for assay development. With data generated on over 400 HTLV-positive blood donors sourced from USA and French blood banks, we developed and validated a high-precision interpretation algorithm. The Multi-HTLV assay demonstrated very high performance for confirmation and strain discrimination with 100% sensitivity, 98.1% specificity and 100% of typing accuracy in validation samples. The assay can be interpreted either visually or automatically with a colorimetric image reader and custom algorithm, providing highly reliable results. CONCLUSIONS/SIGNIFICANCE: The newly developed Multi-HTLV is very competitive with currently used confirmatory assays and reduces considerably the number of indeterminate results. The multiparametric nature of the assay opens new avenues to study specific serological signatures of each patient, follow the evolution of infection, and explore utility for HTLV disease prognosis. Improving HTLV diagnostic testing will be critical to reduce transmission and to improve monitoring of seropositive patients.

Serological reactivity against T. cruzi-derived antigens: Evaluation of their suitability for the assessment of response to treatment in chronic Chagas disease.

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects more than 6 million people worldwide. Following a mostly asymptomatic acute phase, the disease progresses to a long-lasting chronic phase throughout which life-threatening disorders to the heart and/or gastrointestinal tract will manifest in about 30% of those chronically infected. During the chronic phase, the parasitemia is low and intermittent, while a high level of anti-T. cruzi antibodies persist for years. These two features hamper post-chemotherapeutic follow-up of patients with the tools available. The lack of biomarkers for timely assessment of therapeutic response discourages a greater use of the two available anti-parasitic drugs, and complicates the evaluation of new drugs in clinical trials. Herein, we investigated in a blinded case-control study the serological reactivity over time of a group of parasite-derived antigens to potentially address follow up of T. cruzi chronically infected subjects after treatment. We tested PFR2, KMP11, HSP70, 3973, F29 and the InfYnity multiplexed antigenic array, by means of serological assays on a multi-national retrospective collection of samples. Some of the antigens exhibited promising results, underscoring the need for further studies to determine their potential role as treatment response biomarkers.

Prediction of parasitological cure in children infected with Trypanosoma cruzi using a novel multiplex serological approach: an observational, retrospective cohort study.

BACKGROUND: Assessment of therapeutic response with standard serological diagnostic assays in patients with chronic Chagas disease is a major challenge due to the long persistence of parasite-specific antibodies. The current consensus for parasitological cure is to monitor conversion from positive to negative Trypanosoma cruzi serology (seroreversion). However, because of robust humoral immune response, seroreversion by standard serological tests can take years to decades. Developing novel tests of parasitological cure or surrogates is thus a priority in the Chagas disease field. We aimed to evaluate the MultiCruzi assay as a predictive tool for parasitological cure in a cohort of treated infants and children with acute and chronic Chagas disease enrolled in a long-term retrospective longitudinal study with clinical, serological, and parasitological follow-up, and to explore whether MultiCruzi could predict parasitological cure more quickly than the current reference method. METHODS: Patients from two retrospective paediatric Chagas disease cohort studies with clinical, serological, and parasitological follow-up, diagnosed and treated at the parasitology service, Hospital de Niños Ricardo Gutierrez (Buenos Aires, Argentina) were included in this retrospective cohort study. Serum samples were collected every 6 months to 12 months between Oct 22, 1990, and June 3, 2019, for cohort 1 and 1 month after birth for cohort 2 and then every 3 months for a year between July 23, 2012, and April 19, 2016. We evaluated serological follow-up with the Chagatest ELISA (Wiener Lab, Rosario, Argentina) and used this as a clinical reference method for the evaluation of seroreversion. We compared Chagatest ELISA results with results of MultiCruzi (InfYnity Biomarkers, Lyon, France), a novel antibody profiling multiplex assay, investigating seroreversion events with both of the assays and prediction of seroreversion with MultiCruzi using an interpretation formula. FINDINGS: Combining experimental data from discrete analysis of 15 T cruzi antigens efficiently predicted seroreversion at an early stage, which was later confirmed by conventional T cruzi serology. In cohort 1 (n=69), which included children of three different age groups, we observed differences 2 years after therapy. In the 27 individuals from cohort 1 who were treated within the first 12 months of age, MultiCruzi predicted early seroreversion in 21 (78%) patients whereas nine (33%) patients showed seroreversion with Chagatest ELISA (seroreversion difference 0·44, 95% CI 0·26-0·63; p=0·0005). In the 12 patients from cohort 1 treated between 1 year and 2 years of age, MultiCruzi predicted early seroreversion in six (50%) patients, whereas only one (8%) patient was confirmed to be seronegative with Chagatest ELISA (seroreversion difference 0·42, 95% CI 0·14-0·70; p=0·0253). In the 30 patients from cohort 1 who were treated between 2 years and 19 years of age, MultiCruzi predicted early seroreversion in five (6%) patients, whereas no patients were found to be seronegative with Chagatest ELISA (seroreversion difference 0·17, 0·03-0·30; p=0·0253). In cohort 2 (n=27), which included only children younger than 1 year of age and had a shorter follow up (between 5 months and 32 months), the proportion of reported events was significantly different 180 days after treatment for the T cruzi-positive group (early seroreversion predicted in nine [90%] of ten patients with MultiCruzi and confirmed seroreversion in four [40%] of ten patients with Chagatest ELISA; seroreversion difference 0·50, 95% CI 0·19-0·81; p=0·0253) and for the T cruzi-negative group 90 days (early seroreversion predicted in five [29%] of 17 patients with MultiCruzi and confirmed seroreversion in one [6%] of 17 patients with Chagatest ELISA; seroreversion difference 0·24, 0·03-0·44; p=0·0455) and 180 days (early seroreversion predicted in 17 [100%] of 17 patients with MultiCruzi and confirmed seroreversion only in seven [41%] of 17 patients with Chagatest ELISA; seroreversion difference 0·59, 0·35-0·82; p=0·0016) after treatment. INTERPRETATION: The MultiCruzi assay can be used as a predictive monitoring tool to assess parasitological cure in children. This approach might be a solution to forecast forthcoming seroreversion in treated adults infected with T cruzi, but this requires further investigation. FUNDING: Drugs for Neglected Diseases initiative. TRANSLATIONS: For the Spanish, Portuguese and French translations of the abstract see Supplementary Materials section.

The unmet medical need for Trypanosoma cruzi-infected patients: Monitoring the disease status.

Patients infected by Trypanosoma cruzi are typically diagnosed by detecting specific antibodies in serological assays. Persistence of the parasitic infection increases the risk of morbidity and mortality. There are indications that anti-parasitic therapies help to reduce these risks when comparing treated and untreated populations. However, at present, treatment efficacy cannot be properly evaluated on an individual patient basis by available laboratory methods. To monitor parasite clearance, it is essential to change the paradigm of serological methods: analyzing the broad spectrum of antibody diversity is more informative about clinical status than conventional serology tests designed merely for global detection of antibodies.

Assessment of multiplex Onchocerca volvulus peptide ELISA in non-endemic tropical regions.

BACKGROUND: Currently, serodiagnosis of infection with the helminth parasite Onchocerca volvulus is limited to the Ov-16 IgG4 test, a test that has limited sensitivity and suboptimal specificity. In previous studies, we identified several linear epitopes that have the potential to supplement the diagnostic toolbox for onchocerciasis. METHODS: In this study three peptides, bearing in total six linear epitopes were transferred to a multiplex ELISA platform. This multiplex ELISA was used to assess the clinical utility of the peptide serology markers by analyzing sample sets from both O. volvulus endemic and non-endemic regions. RESULTS: The multiplex platform was shown to be reproducible and data obtained on the multiplex platform were comparable to the singleplex ELISA data. The clinical utility assessment showed that in a population of school-aged children from western Kenya, a virtually O. volvulus-free area, significant cross-reactivity with an as-yet to be determined immunogen was detected. CONCLUSIONS: The observations made in this study invalidate the usefulness of the peptide serology markers for onchocerciasis detection. We discuss what could be the origin of this unexpected serological response, but also highlight the need for better characterized biobanks for biomarker discovery activities.

A novel antibody surrogate biomarker to monitor parasite persistence in Trypanosoma cruzi-infected patients.

BACKGROUND: Trypanosoma cruzi parasite, the causative agent of Chagas disease, infects about six million individuals in more than 20 countries. Monitoring parasite persistence in infected individuals is of utmost importance to develop and evaluate treatments to control the disease. Routine screening for infected human individuals is achieved by serological assays; PCR testing to monitor spontaneous or therapy-induced parasitological cure has limitations due to the low and fluctuating parasitic load in circulating blood. The aim of the present study is to evaluate a newly developed antibody profiling assay as an indirect method to assess parasite persistence based on waning of antibodies following spontaneous or therapy-induced clearance of the infection. METHODOLOGY/PRINCIPAL FINDINGS: We designed a multiplex serology assay, an array of fifteen optimized T. cruzi antigens, to evaluate antibody diversity in 1654 serum samples from chronic Chagas patients. One specific antibody response (antibody 3, Ab3) showed a strong correlation with T. cruzi parasite persistence as determined by T. cruzi PCR positive results. High and sustained Ab3 signal was strongly associated with PCR positivity in untreated patients, whereas significant decline in Ab3 signals was observed in BZN-treated patients who cleared parasitemia based on blood PCR results. CONCLUSION/SIGNIFICANCE: Ab3 is a new surrogate biomarker that strongly correlates with parasite persistence in chronic and benznidazole-treated Chagas patients. We hypothesize that Ab3 is induced and maintained by incessant stimulation of the immune system by tissue-based and shed parasites that are not consistently detectable by blood based PCR techniques. Hence, a simple immunoassay measurement of Ab3 could be beneficial for monitoring the infectious status of seropositive patients.

Development of a Novel Multiplex Immunoassay Multi-cruzi for the Serological Confirmation of Chagas Disease.

BACKGROUND: Chagas disease is due to the parasite Trypanosoma cruzi, a protist disseminated by a Triatome vector. This disease is endemic to Latin America and considered by WHO as one of the 17 world's neglected diseases. In Europe and in North America, imported cases are also detected, due to migration of population outside of the endemic region. Diagnosis of T. cruzi infection is usually made indirectly by the detection of specific antibodies to T. cruzi antigens. Following initial diagnostic evaluation or screening test (qualifying or discarding blood donation), a confirmation test is performed for samples initially reactive. The test presented in this study aims at the confirmation/refutation of the infectious status of human blood samples and will permit taking appropriate clinical measures. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel array of twelve antigens and printed these antigens onto 96-well plates. We tested 248 positive samples T. cruzi, 94 unscreened blood donors' samples from non-endemic area, 49 seronegative blood donors, 7 false-positive and 3 doubtful samples. The observed reactivities were analyzed to propose a decision-tree algorithm that correctly classifies all the samples, with the potential to discriminate false-positive results and sticky samples. We observed that antibodies levels (Sum of all antigens) was significantly higher for PCR positive than for PCR negative samples in all studied groups with Multi-cruzi. CONCLUSION/SIGNIFICANCE: The results described in this study indicate that the Multi-cruzi improves the serological confirmation of Chagas disease. Moreover the "sum of all antigens" detected by Multi-cruzi could reflect parasitemia level in patients-like PCR signals does-and could serve as an indicator of parasite clearance in longitudinal follow-ups. Validation of this assay is still required on an independent large collection of well characterized samples including typical false-reactive samples such as Leishmaniasis.

Trypanosoma-Cruzi Cross-Reactive Antibodies Longitudinal Follow-Up: A Prospective Observational Study in Hematopoietic Stem Cell Transplantation.

Antibodies named TcCRA "Trypanosoma cruzi Cross Reactive Antibodies" were detected in 47% of blood donors from French population unexposed to the parasite. In order to evaluate the passive or active transmissibility of TcCRA and further characterize its role and etiology, we have conducted a study in a cohort of 47 patients who underwent allogeneic Hematopoietic Stem Cell Transplantations (allo-HSCT). Donors and recipients were tested for TcCRA prior to transplantation. Recipients were further tested during follow-up after transplantation. Demographical, clinical and biological data were collected. Our primary end-point was to assess the risk of TcCRA acquisition after transplantation. During this initial analysis, we observed no seroconversion in patients receiving cells from TcCRA negative donors (n = 23) but detected seroconversion in 4 out of 24 patients who received hematopoietic stem cells from positive donors. Here, we are discussing possible scenarios to explain TcCRA-immune status in recipient after transplantation.

Anti-Trypanosoma cruzi cross-reactive antibodies detected at high rate in non-exposed individuals living in non-endemic regions: seroprevalence and association to other viral serologies.

Cross-reactive antibodies are characterized by their recognition of antigens that are different from the trigger immunogen. This happens when the similarity between two different antigenic determinants becomes adequate enough to enable a specific binding with such cross-reactive antibodies. In the present manuscript, we report the presence, at an "abnormal" high frequency, of antibodies in blood samples from French human subjects cross-reacting with a synthetic-peptide antigen derived from a Trypanosoma cruzi (T. cruzi) protein sequence. As the vector of T. cruzi is virtually confined to South America, the parasite is unlikely to be the trigger immunogen of the cross-reactive antibodies detected in France. At present, the cross-reactive antibodies are measured by using an in-house ELISA method that employs the T. cruzi -peptide antigen. However, to underline their cross-reactive characteristics, we called these antibodies "Trypanosoma cruzi Cross Reactive Antibodies" or TcCRA. To validate their cross-reactive nature, these antibodies were affinity-purified from plasma of healthy blood donor and were then shown to specifically react with the T. cruzi parasite by immunofluorescence. Seroprevalence of TcCRA was estimated at 45% in serum samples of French blood donors while the same peptide-antigen reacts with about 96% of T. cruzi -infected Brazilian individuals. In addition, we compared the serology of TcCRA to other serologies such as HSV 1/2, EBV, HHV-6, CMV, VZV, adenovirus, parvovirus B19, mumps virus, rubella virus, respiratory syncytial virus, measles and enterovirus. No association was identified to any of the tested viruses. Furthermore, we tested sera from different age groups for TcCRA and found a progressive acquisition starting from early childhood. Our findings show a large seroprevalence of cross-reactive antibodies to a well-defined T. cruzi antigen and suggest they are induced by a widely spread immunogen, acquired from childhood. The etiology of TcCRA and their clinical relevance still need to be investigated.

Evaluation of INNO-LIA syphilis assay as a confirmatory test for syphilis.

We evaluated the sensitivity and specificity of a new confirmatory test for treponemal antibodies, INNO-LIA Syphilis (Innogenetics NV, Ghent, Belgium), on a large number of sera from a clinical laboratory. This multiparameter line immunoassay (LIA) uses recombinant and synthetic polypeptide antigens derived from Treponema pallidum proteins. In a single-blinded cross-sectional retrospective study, 289 seronegative sera, 219 seropositive sera, and 23 sera with an indeterminate serological status for syphilis were analyzed. All sera were tested by the T. pallidum hemagglutination assay (TPHA), the immunoglobulin (IgG)-fluorescent T. pallidum absorption assay (IgG-FTA-ABS), and the Venereal Disease Research Laboratory (VDRL) test. In addition, some seropositive samples were analyzed by the 19S-IgM-FTA-ABS test, an enzyme immunoassay (IgM-EIA), and the MarDx immunoblotting assay. Based on a consensus diagnosis derived from conventional serology, all of the sera were classified as positive, negative, or indeterminate, and the results were compared with the findings of the INNO-LIA Syphilis assay. The sensitivity and specificity of the LIA were 100% (219 of 219) and 99.3% (286 of 288), respectively. Compared to TPHA and IgG-FTA-ABS, the new test gave a significantly higher number (P = 0.021 and P < 0.0001, respectively) of correct results than either of the other two tests. The multiparameter INNO-LIA Syphilis assay is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques.