An original multiplex method to assess five different SARS-CoV-2 antibodies.

OBJECTIVES: Accurate SARS-CoV-2 serological assays are urgently needed to help diagnose infection, determine past exposure of populations and assess the response to future vaccines. The study aims at assessing the performance of the multiplex D-tek COVIDOT 5 IgG assay for the detection of SARS-CoV-2 IgG antibodies (N, S1+S2, S1, S2 and RBD). METHODS: Sensitivity and dynamic trend to seropositivity were evaluated in 218 samples obtained from 46 rRT-PCR confirmed COVID-19 patients. Non-SARS-CoV-2 sera (n=118) collected before the COVID-19 pandemic with a potential cross-reaction to the SARS-CoV-2 immunoassay were included in the specificity analysis. RESULTS: A gradual dynamic trend since symptom onset was observed for all IgG antibodies. Sensitivities before day 14 were suboptimal. At ≥21 days, sensitivities reached 100% (93.4-100%) for N, S1+S2, S2 and RBD-directed IgG and 96.3% (87.3-99.6%) for S1-directed IgG. In 42 out of 46 patients (91.3%), all five antibodies were detected at ≥14 days. The four remaining patients had between 2 and 4 positive antibodies at their respective maximal follow-up period. The specificity was 100 % for S1+S2, S2 and RBD, 98.3% for N and 92.4% (86.0-96.5%) for S1-directed IgG. The combined use of antigens increases the early sensitivity whilst enforcing high specificity. CONCLUSIONS: Sensitivities at ≥21 days and specificities were excellent, especially for N, S1+S2, S2 and RBD-directed IgG. Caution is however required when interpreting single S1-directed reactivities. Using a multiplex assay complies with the orthogonal testing algorithm of the CDC and allows a better and critical interpretation of the serological status of a patient.

Long-Term Dynamic of Anti-TrimericS and Anti-RBD Antibodies in Naive and COVID-19 Recovered mRNA-1273 Vaccine Recipients.

OBJECTIVE: Patients and physicians are increasingly requesting their clinical laboratory to provide SARS-CoV-2 serology interpretation. Our study aimed to assess the evolution of SARS-CoV-2 antibodies in Moderna-vaccinated health care workers. METHODS: We analyzed the evolution of mRNA-1273 (Moderna)-elicited antibodies by 2 high-throughput assays, TrimericS IgG (Diasorin) and SARS-CoV-2 IgG-II (Abbott). RESULTS: After the first injection, the COVID-19-recovered vaccinees showed a serological response as strong as that observed 1 month after the second injection in participants without COVID-19 history. Although remaining above the positivity thresholds, the TrimericS immunoglobulin G (IgG) and anti-RBD (receptor-binding domain) IgG levels fell considerably between 1 and 7 months postvaccination, dropping to 10.6% and 13% for the COVID-19 recovered subgroup and to 11.7% and 9.3% for the COVID-19 naive subgroup. CONCLUSION: Regardless of the test used, a decrease in circulating anti-SARS-CoV-2 IgG levels should be expected a few months after vaccination. As this decline does not preclude the efficacy of immune response, caution is necessary when interpretating postvaccination serological data.

Flow cytometry for basophil activation markers: the measurement of CD203c up-regulation is as reliable as CD63 expression in the diagnosis of cat allergy.

The flow cytometric basophil activation test (BAT), based on the detection of allergen-induced CD63 expression, has been proved effective in the diagnosis of various IgE-mediated allergies. However, there is not yet consensus about the suitability of CD203c expression as a specific basophil activation marker and its diagnostic reliability. The goal of the present study was to compare measurement of CD63 and CD203c expression using BAT in a model of cat allergy and to determine optimal experimental conditions for both markers. Heparinized whole blood samples from 20 cat allergic patients and 19 controls were incubated with Fel d1 (relevant allergen) or anti-FcepsilonRI (positive control) either in IL-3 or IL-3-free conditions. An optimal gating of basophils was achieved in triple staining protocols: anti-IgE PE/anti-CD45 PerCP/anti-CD63 FITC or anti-IgE FITC/anti-CD45 PerCP/anti-CD203c PE. We demonstrated that IL-3 significantly enhanced CD63-induced expression by basophils obtained from cat allergic patients in response to Fel d1. Sensitivity was found to be 100%. The CD203c protocol, when performed under IL-3-free conditions, also demonstrated 100% sensitivity. Only one of the control subjects was positive in both tests. In conclusion, using well-defined experimental conditions, the measurement of CD203c up-regulation on basophils in response to specific allergens is as reliable as CD63-BAT for the in vitro diagnosis of patients with IgE-mediated allergy.

Value of non-identified ANCA (non-PR3, non-MPO) in the diagnosis of granulomatosis with polyangiitis (Wegener's granulomatosis).

OBJECTIVE: Determine the frequency of granulomatosis with polyangiitis (GPA) associated with non-identified ANCA (non-MPO, non-PR3 ANCA) and secondarily compare their clinic with GPA associated with MPO-positive or PR3-positive ANCA. METHODS: In a monocentric retrospective observational study, clinical data of 398 patients with non-identified ANCA (titer of ANCA at least 1/80 by immunofluorescence on ethanol fixed PMN) was gathered over a period of 6 years. GPA patients from this population were compared with GPA patients with identified ANCA on the basis of clinical, biological, immunological and histological features. RESULTS: The most common diseases associated with non-identified ANCA were inflammatory bowel diseases accounting for 17% of diseases. GPA accounted for only 1.8% of cases. There were no significant differences in terms of clinical and histological characteristics between GPA with non-identified ANCA and GPA with identified ANCA, but significantly higher CRP levels were observed in GPA patients with identified ANCA (p = 0.005). Localized disease (ear, nose and throat and/or lung involvement without any other systemic involvement) was more frequent in the group of GPA with nonidentified ANCA (p = 0.047) as compared to GPA with identified ANCA. This explains that the former group of patients was less frequently treated by cyclophosphamide than the latter (p = 0.016). CONCLUSION: GPA with non-MPO, non-PR3 ANCAs is relatively rare. Our study suggests that GPA with nonidentified ANCA differs from GPA with identified ANCA by the frequency of localized forms.

Predictive markers for development of severe organ involvement in patients with systemic sclerosis.

Prognosis of systemic sclerosis (SSc) is associated with the extent of skin involvement and the presence of lung, heart, kidney, and/or digestive tract damage. Most patients do not require disease-modifying therapy. However, to avoid irreversible tissue injury, early detection of visceral involvement is crucial for prompt initiation of therapy. The aim of this study is to identify, among initial investigations performed at time of diagnosis, predictive markers for the development of severe organ involvement (SOI). We retrospectively reviewed the medical records of 41 patients with SSc who were followed for 5 years after diagnosis, including those who died because of SSc within this 5-year period. We considered 68 clinical, biological, and technical parameters obtained at time of diagnosis and studied their association with development of SOI during this 5-year period, using a multivariate logistic regression. Eighteen patients (44%) developed SOI, with a median delay of 1.5 years following diagnosis. Three independent markers were identified: reduced vital capacity, fulfilment of American College of Rheumatology criteria at diagnosis, and presence of rheumatoid factor. To identify patients at risk for developing severe disease, given the short delay between diagnosis of SSc and development of SOI, we recommend monitoring these markers at diagnosis.

Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study.

OBJECTIVE: To investigate the value of serum antitissue transglutaminase IgA antibodies (IgA-TTG) and IgA antiendomysial antibodies (IgA-EMA) in the diagnosis of coeliac disease in cohorts from different geographical areas in Europe. The setting allowed a further comparison between the antibody results and the conventional small-intestinal histology. METHODS: A total of 144 cases with coeliac disease [median age 19.5 years (range 0.9-81.4)], and 127 disease controls [median age 29.2 years (range 0.5-79.0)], were recruited, on the basis of biopsy, from 13 centres in nine countries. All biopsy specimens were re-evaluated and classified blindly a second time by two investigators. IgA-TTG were determined by ELISA with human recombinant antigen and IgA-EMA by an immunofluorescence test with human umbilical cord as antigen. RESULTS: The quality of the biopsy specimens was not acceptable in 29 (10.7%) of 271 cases and a reliable judgement could not be made, mainly due to poor orientation of the samples. The primary clinical diagnosis and the second classification of the biopsy specimens were divergent in nine cases, and one patient was initially enrolled in the wrong group. Thus, 126 coeliac patients and 106 controls, verified by biopsy, remained for final analysis. The sensitivity of IgA-TTG was 94% and IgA-EMA 89%, the specificity was 99% and 98%, respectively. CONCLUSIONS: Serum IgA-TTG measurement is effective and at least as good as IgA-EMA in the identification of coeliac disease. Due to a high percentage of poor histological specimens, the diagnosis of coeliac disease should not depend only on biopsy, but in addition the clinical picture and serology should be considered.

Clinical significance of Cryofibrinogenemia: possible pathophysiological link with Raynaud's phenomenon.

OBJECTIVE: To describe the clinical findings and prevalence of patients with cryofibrinogenemia (CF) and to determine whether CF is associated with primary Raynaud's phenomenon. METHODS: Between June 2006 and December 2009, 227 patients were tested for CF in a single university hospital. Forty-five patients with primary Raynaud's phenomenon were tested for CF. RESULTS: A total of 117 patients with CF without cryoglobulinemia were included. The main clinical manifestations included skin manifestations (50%) and arthralgia (35%). There were 67 patients with primary CF and 50 patients with secondary CF. There was no significant difference in the mean concentration of the cryoprecipitate in primary CF as compared to the secondary form (172 ± 18.6 vs 192 ± 20.9 mg/dl, respectively; p = 0.41). Highest concentrations of cryoprecipitate were observed in those containing fibrinogen only as compared to cryoprecipitates containing fibrinogen and fibronectin (301 ± 43.5 vs 125 ± 10.6 mg/dl; p < 0.001). Patients having skin necrosis (n = 3) had significantly higher values of cryofibrinogen compared to those without necrosis (638 ± 105 vs 160 ± 10.2 mg/dl; p = 0.0046). Among the 45 patients with primary Raynaud's phenomenon, 36 had associated CF. There was no significant difference in the mean concentration of the cryoprecipitate in these patients compared to those with primary CF. CONCLUSION: There seems to be a significant correlation between cryofibrinogen concentration and the severity of the clinical signs, particularly when cryoprecipitate is composed of fibrinogen alone. CF might have a possible pathophysiological role in primary Raynaud's phenomenon.

Betaine homocysteine methyl transferase 1, a novel auto-antigen associated with anti-Golgi immune reactivity.

Anti-Golgi antibodies are rare autoantibodies that have been described in systemic autoimmune diseases. Not all Golgi auto-antigens are known. The objective of this study was to identify a novel auto-antigen associated with anti-Golgi immune reactivity. Sera from a patient with Golgi immune reactivity and from a control individual were used for Western blotting after 2-dimensional gel separation of a rat Golgi-enriched extract. Betaine homocysteine S-methyltransferase 1 (BHMT1) was identified as an auto-antigen by MALDI-TOF/TOF mass spectrometry. Using human recombinant BHMT1, a strong positive blotting signal was obtained with serum from the patient but not from a control. Pre-absorption of the serum sample with reactivity to BHMT1 with recombinant human BHMT1 resulted in decreased reactivity on Western blotting and in disappearance of the Golgi-like pattern on indirect immunofluorescence. Using immunocytochemistry, we confirmed the subcellular localization of BHMT1 to the Golgi apparatus. Antibodies to BHMT1 were found in four of 80 samples with a Golgi-pattern on indirect immunofluorescence. The antibodies were not associated with a specific clinical condition. We identified BHMT1 as a novel auto-antigen associated with anti-Golgi immune reactivity.

IL-4 and IL-13 mRNA real-time PCR quantification on whole blood to assess allergic response.

Although routinely used in clinical practice, skin prick tests and serum specific IgE often fail to distinguish between IgE-sensitization and symptomatic IgE-mediated allergy. There is therefore a need for new laboratory tests relating allergic symptoms to the offending agent. In this way, we evaluated the diagnostic reliability of a new whole blood quantitative real-time PCR assay for IL-4 and IL-13 mRNAs. We compared the response of cat allergic patients and non-cat allergic controls upon anti-IgE and cat allergen (Fel d1) stimulation of whole blood. Allergen addition led to a significant increase of IL-4 and IL-13 mRNAs in allergic patients compared to non-allergic controls (p<0.0001). Both cytokine mRNA levels were strongly correlated and peaked within 2 h after Fel d1 or anti-IgE addition. This rapid increase as well as purification experiments led us to the conclusion that basophils represent an important if not the main source of both transcripts in this setting. The effect was allergen-specific since not observed when stimulating blood from cat allergic patients with birch pollen. Interestingly, we found that IFN-gamma mRNA, contrary to IL-4 mRNA, reached higher levels in response to Fel d1 with control individuals than with patients allergic to cat. This study shows that whole blood real-time PCR is a valuable method for IL-4 and IL-13 mRNA measurement after in vitro allergen challenge. We suggest that it might be useful, complementing conventional markers, for the diagnosis and the follow-up of allergic diseases. Further assessments are required to evaluate the clinical potential of this technique.

Reliability of antitransglutaminase antibodies as predictors of gluten-free diet compliance in adult celiac disease.

OBJECTIVE: Strict lifelong compliance to a gluten-free diet (GFD) minimizes the long-term risk of mortality, especially from lymphoma, in adult celiac disease (CD). Although serum IgA antitransglutaminase (IgA-tTG-ab), like antiendomysium (IgA-EMA) antibodies, are sensitive and specific screening tests for untreated CD, their reliability as predictors of strict compliance to and dietary transgressions from a GFD is not precisely known. We aimed to address this question in consecutively treated adult celiacs. METHODS: In a cross-sectional study, 95 non-IgA deficient adult (median age: 41 yr) celiacs on a GFD for at least 1 yr (median: 6 yr) were subjected to 1) a dietician-administered inquiry to pinpoint and quantify the number and levels of transgressions (classified as moderate or large, using as a cutoff value the median gluten amount ingested in the overall noncompliant patients of the series) over the previous 2 months, 2) a search for IgA-tTG-ab and -EMA, and 3) perendoscopic duodenal biopsies. The ability of both antibodies to discriminate celiacs with and without detected transgressions was described using receiver operating characteristic curves and quantified as to sensitivity and specificity, according to the level of transgressions. RESULTS: Forty (42%) patients strictly adhered to a GFD, 55 (58%) had committed transgressions, classified as moderate (< or = 18 g of gluten/2 months; median number 6) in 27 and large (>18 g; median number 69) in 28. IgA-tTG-ab and -EMA specificity (proportion of correct recognition of strictly compliant celiacs) was 0.97 and 0.98, respectively, and sensitivity (proportion of correct recognition of overall, moderate, and large levels of transgressions) was 0.52, 0.31, and 0.77, and 0.62, 0.37, and 0.86, respectively. IgA-tTG-ab and -EMA titers were correlated (p < 0.001) to transgression levels (r = 0.560 and R = 0.631, respectively) and one to another (p < 0.001) in the whole patient population (r = 0.834, N = 84) as in the noncompliant (r = 0.915, N = 48) group. Specificity and sensitivity of IgA-tTG-ab and IgA-EMA for recognition of total villous atrophy in patients under a GFD were 0.90 and 0.91, and 0.60 and 0.73, respectively. CONCLUSIONS: In adult CD patients on a GFD, IgA-tTG-ab are poor predictors of dietary transgressions. Their negativity is a falsely secure marker of strict diet compliance.