Antinuclear antibodies in individuals with COVID-19 reflect underlying disease: Identification of new autoantibodies in systemic sclerosis (CDK9) and malignancy (RNF20, RCC1, TRIP13).

A high prevalence of antinuclear antibodies (ANA) in COVID-19 has been insinuated, but the nature of the target antigens is poorly understood. We studied ANA by indirect immunofluorescence in 229 individuals with COVID-19. The target antigens of high titer ANA (≥1:320) were determined by immunoprecipitation (IP) combined with liquid-chromatography-mass spectrometry (MS). High titer ANA (≥1:320) were found in 14 (6%) of the individuals with COVID-19. Of the 14 COVID-19 cases with high titer ANA, 6 had an underlying autoimmune disease and 5 a malignancy. IP-MS revealed known target antigens associated with autoimmune disease as well as novel autoantigens, including CDK9 (in systemic sclerosis) and RNF20, RCC1 and TRIP13 (in malignancy). The novel autoantigens were confirmed by IP-Western blotting. In conclusion, in depth analysis of the targets of high titer ANA revealed novel autoantigens in systemic sclerosis and in malignant disease.

Complementarity determining regions in SARS-CoV-2 hybrid immunity.

Pre-vaccination SARS-CoV-2 infection can boost protection elicited by COVID-19 vaccination and post-vaccination breakthrough SARS-CoV-2 infection can boost existing immunity conferred by COVID-19 vaccination. Such 'hybrid immunity' is effective against SARS-CoV-2 variants. In order to understand 'hybrid immunity' at the molecular level we studied the complementarity determining regions (CDR) of anti-RBD (receptor binding domain) antibodies isolated from individuals with 'hybrid immunity' as well as from 'naive' (not SARS-CoV-2 infected) vaccinated individuals. CDR analysis was done by liquid chromatography/mass spectrometry-mass spectrometry. Principal component analysis and partial least square differential analysis showed that COVID-19 vaccinated people share CDR profiles and that pre-vaccination SARS-CoV-2 infection or breakthrough infection further shape the CDR profile, with a CDR profile in hybrid immunity that clustered away from the CDR profile in vaccinated people without infection. Thus, our results show a CDR profile in hybrid immunity that is distinct from the vaccination-induced CDR profile.

Immunovirological and environmental screening reveals actionable risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks.

Coronavirus Disease 2019 (COVID-19) vaccination has resulted in excellent protection against fatal disease, including in older adults. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases among residents) by combining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aerosol monitoring, whole-genome phylogenetic analysis and immunovirological profiling of nasal mucosa by digital nCounter transcriptomics. Phylogenetic investigations indicated that each outbreak stemmed from a single introduction event, although with different variants (Delta, Gamma and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 d after the initial infection. Combining demographic, immune and viral parameters, the best predictive models for mortality comprised IFNB1 or age, viral ORF7a and ACE2 receptor transcripts. Comparison with published pre-vaccine fatal COVID-19 transcriptomic and genomic signatures uncovered a unique IRF3 low/IRF7 high immune signature in post-vaccine fatal COVID-19 outbreaks. A multi-layered strategy, including environmental sampling, immunomonitoring and early antiviral therapy, should be considered to prevent post-vaccination COVID-19 mortality in nursing homes.

IgG anti-spike antibody levels in healthcare workers with and without prior COVID-19 up to 3 months after BNT162b2 vaccination.

We report IgG anti-spike levels up to 3 months after vaccination with 2 doses of BNT162b2 mRNA vaccine in healthcare workers (HCW). The antibody response was significantly stronger in previously infected vaccinated HCW compared to uninfected HCW, and stronger after vaccination compared to (mostly) mild natural infection.

Added value of anti-SARS-CoV-2 antibody testing in a Flemish nursing home during an acute COVID-19 outbreak in April 2020.

OBJECTIVES: To examine the added value of anti-SARS-CoV-2 antibody testing in a nursing home during an acute COVID-19 outbreak. RT-PCR is the gold standard, but can be false-negative. METHODS: 119 residents and 93 staff members were tested with RT-PCR test and/or a rapid IgM/IgG test. Of these participants, 176 had both tests, 24 only RT-PCR, and 12 only IgM/IgG in the period April 14 to 16 April 2020. RESULTS: 40 (34%) residents and 11 (13%) staff were PCR-positive. Using a rapid IgM/IgG test, 17 (17%) residents and 18 (20%) staff were positive for IgM and/or IgG (IgM/IgG). Thirty-two PCR-positive residents had an IgM/IgG test: 9 (28%), 11 (34%), and 13 (41%) were positive for IgM, IgG, and IgM/IgG. Ten PCR-positive staff had an IgM/IgG test: 3 (30%), 6 (60%), and 6 (60%) were positive for IgM, IgG, and IgM/IgG. Additional IgM/IgG tests were performed in 9 residents 11 to 14 days after the positive RT-PCR test. Of those, 7 (78%) tested positive for IgM/IgG. When retested 3 weeks later, the 2 remaining residents also tested positive. Of the 134 PCR-negative participants who had an IgM/IgG test, 15 were positive for IgM/IgG (8% of the 200 participants tested with RT-PCR). CONCLUSIONS: During an acute outbreak in a nursing home, 26% of residents and staff were PCR-positive. An additional 8% was diagnosed using IgM/IgG antibody testing. The use of RT-PCR alone as the sole diagnostic method for surveillance during an acute outbreak is insufficient to grab the full extent of the outbreak.

Three Cases of Atypical Pneumonia with Chlamydia psittaci: The Role of Laboratory Vigilance in the Diagnosis of Psittacosis.

Chlamydia psittaci is an established zoonotic agent causing respiratory disease in humans. An infection often remains asymptomatic but can also result in flu-like illness, pneumonia or even multi-organ failure. This paper describes three patients, hospitalised at AZ Sint-Lucas Hospital, with atypical pneumonia who were diagnosed with C. psittaci after an in-depth anamnesis and laboratory investigation in the midst of the COVID pandemic. All three infections were confirmed with PCR and serology, whereas viable bacteria were only present for one patient. Genotyping revealed the presence of genotype B for patient 1 and 2 whereas ompA genotyping was unsuccessful for patient 3. This case report demonstrates the importance of a thorough patient history as close contact with birds is one of the main risk factors to contract the pathogen. Once exposure to birds has been confirmed, a diagnosis by a combination of PCR and serology is essential in order to initiate a treatment with the proper antibiotics. As psittacosis is still an underestimated and underdiagnosed disease, communication between laboratory, clinicians and bird fanciers is encouraged.

IgG Anti-Spike Antibodies and Surrogate Neutralizing Antibody Levels Decline Faster 3 to 10 Months After BNT162b2 Vaccination Than After SARS-CoV-2 Infection in Healthcare Workers.

Background: IgG anti-spike (S) antibodies arise after SARS-CoV-2 infection as well as vaccination. Levels of IgG anti-S are linked to neutralizing antibody titers and protection against (re)infection. Methods: We measured IgG anti-S and surrogate neutralizing antibody kinetics against Wild Type (WT) and 4 Variants of Concern (VOC) in health care workers (HCW) 3 and 10 months after natural infection ("infection", n=83) or vaccination (2 doses of BNT162b2) with ("hybrid immunity", n=17) or without prior SARS-CoV-2 infection ("vaccination", n=97). Results: The humoral immune response in the "vaccination" cohort was higher at 3 months, but lower at 10 months, compared to the "infection" cohort due to a faster decline. The "hybrid immunity" cohort had the highest antibody levels at 3 and 10 months with a slower decline compared to the "vaccination" cohort. Surrogate neutralizing antibody levels (expressed as %inhibition of ACE-2 binding) showed a linear relation with log10 of IgG anti-S against WT and four VOC. IgG anti-S corresponding to 90% inhibition ranged from 489 BAU/mL for WT to 1756 BAU/mL for Beta variant. Broad pseudoneutralization predicted live virus neutralization of Omicron BA.1 in 20 randomly selected high titer samples. Conclusions: Hybrid immunity resulted in the strongest humoral immune response. Antibodies induced by natural infection decreased more slowly than after vaccination, resulting in higher antibody levels at 10 months compared to vaccinated HCW without prior infection. There was a linear relationship between surrogate neutralizing activity and log10 IgG anti-S for WT and 4 VOC, although some VOC showed reduced sensitivity to pseudoneutralization.

Lower persistence of anti-nucleocapsid compared to anti-spike antibodies up to one year after SARS-CoV-2 infection.

We retrospectively compared the long-term evolution of IgG anti-spike (S) and anti-nucleocapsid (N) levels (Abbott immunoassays) in 116 non-severe and 115 severe SARS-CoV-2 infected patients from 2 university hospitals up to 365 days post positive RT-PCR. IgG anti-S and anti-N antibody levels decayed exponentially up to 365 days after a peak 0 to 59 days after positive RT-PCR. Peak antibody level/cut-off ratio 0 to 59 days after positive RT-PCR was more than 70 for anti-S compared to less than 6 for anti-N (P < 0.01). Anti-S and anti-N were significantly higher in severe compared to non-severe patients up to 180 to 239 days and 300 to 365 days, respectively (P < 0.05). Despite similar half-lives, the estimated time to 50% seronegativity was more than 2 years for anti-S compared to less than 1 year for anti-N in non-severe and severe COVID-19 patients, due to the significantly higher peak antibody level/cut-off ratio for anti-S compared to anti-N.

Liver or Kidney Transplantation After SARS-CoV-2 Infection: Prevalence, Short-term Outcome, and Kinetics of Serum IgG Antibodies.

BACKGROUND: There is a paucity of data on the prevalence, adequate timing, and outcome of solid organ transplantation after severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and the kinetics of immunoglobulin G (IgG) antibodies in these patients. METHODS: SARS-CoV-2 antinucleocapsid (N) IgG and polymerase chain reaction via a nasopharyngeal swab were analyzed in all patients within 24 h before liver or kidney transplantation. Kinetics of IgG antibodies were analyzed and compared with an immunocompetent cohort. RESULTS: Between May 1, 2020, and March 18, 2021, 168 patients underwent liver or kidney transplantation in our center, of which 11 (6.54%) patients with a previous SARS-CoV-2 infection were identified. The median interval between SARS-CoV-2 infection and transplantation was 4.5 mo (range, 0.9-11). After a median posttransplant follow-up of 4.9 mo, 10 out of 11 patients were alive without clinical signs of viral shedding or recurrent or active infection. One patient without symptom resolution at time of transplantation died after combined liver-kidney transplantation. In 9 out of 11 patients with previously polymerase chain reaction-confirmed infection, SARS-CoV-2 anti-N and antispike (S) IgG were detectable at day of transplantation. Absolute levels of anti-N and anti-S IgG were positively correlated, declined over time in all patients, and were significantly lower compared with immunocompetent individuals. All patients remained anti-S IgG positive until the last posttransplant follow-up, whereas 3 patients became anti-N negative. CONCLUSIONS: We observed an uncomplicated course of liver or kidney transplantation after SARS-CoV-2 infection in selected patients. Although having lower absolute IgG antibody levels than immunocompetent individuals, all seroconverted patients remained anti-S IgG positive. These encouraging data need validation in larger studies.

Corrigendum: IgG anti-spike antibodies and surrogate neutralizing antibody levels decline faster 3 to 10 months after BNT162b2 vaccination than after SARS-CoV-2 infection in healthcare workers.

[This corrects the article DOI: 10.3389/fimmu.2022.909910.].

Two Separate Clusters of SARS-CoV-2 Delta Variant Infections in a Group of 41 Students Travelling from India: An Illustration of the Need for Rigorous Testing and Quarantine.

We report two clusters of SARS-CoV-2 B.1.617.2 (Delta variant) infections in a group of 41 Indian nursing students who travelled from New Delhi, India, to Belgium via Paris, France. All students tested negative before departure and had a second negative antigen test upon arrival in Paris. Upon arrival in Belgium, the students were quarantined in eight different houses. Four houses remained COVID-free during the 24 days of follow-up, while all 27 residents of the other four houses developed an infection during quarantine, including the four residents who were fully vaccinated and the two residents who were partially vaccinated. Genome sequencing revealed two distinct clusters affecting one and three houses, respectively. In this group of students, vaccination status did not seem to prevent infection nor decrease the viral load. No severe symptoms were reported. Extensive contact tracing and 3 months of nationwide genomic surveillance confirmed that these outbreaks were successfully contained and did not contribute to secondary community transmission in Belgium. These clusters highlight the importance of repeated testing and quarantine measures among travelers coming from countries experiencing a surge of infections, as all infections were detected 6 days or more after arrival.

Delayed diagnosis and treatment of extreme hypertriglyceridemia due to rejection of a lipemic sample.

INTRODUCTION: Most laboratories routinely determine haemolysis, icterus and lipemia indices to identify lipemic samples and reject potentially affected results. Hypertriglyceridemia is the most common cause of lipemia and severe hypertriglyceridemia (≥ 11.3 mmol/L) is a major risk factor of acute pancreatitis. LABORATORY ANALYSIS: A 56-year-old woman attended the outpatient clinic for a follow-up visit 1 month after a kidney transplantation. Her immunosuppressive therapy consisted of corticosteroids, cyclosporine, and mycophenolic acid. The routine clinical chemistry sample was rejected due to extreme lipemia. The comment "extreme lipemic sample" was added on the report, but the requesting physician could not be reached. The Cobas 8000 gave a technical error (absorption > 3.3) for the HIL-indices (L-index: 38.6 mmol/L) which persisted after high-speed centrifugation. The patient was given a new appointment 2 days later. The new sample was also grossly lipemic and gave the same technical error (L-index: 35.9 mmol/L). WHAT HAPPENED: The second sample was manually diluted 20-fold after centrifugation to obtain a result for triglycerides within the measuring range (0.10-50.0 mmol/L). Triglycerides were 169.1 mmol/L, corresponding to very severe hypertriglyceridemia. This result was communicated to the nephrologist and the patient immediately recalled to the hospital. She received therapeutic plasma exchange the next day and did not develop acute pancreatitis. MAIN LESSON: This case illustrates the delicate balance between avoiding the release of unreliable results due to lipemia and the risk of delayed diagnosis when results are rejected. Providing an estimate of the degree of hypertriglyceridemia might be preferable to rejecting the result.

Longitudinal follow-up of IgG anti-nucleocapsid antibodies in SARS-CoV-2 infected patients up to eight months after infection.

BACKGROUND: Most SARS-CoV-2 infected patients develop IgG antibodies within 2-3 weeks after symptom onset. Antibody levels have been shown to gradually decrease in the first months after infection, but few data are available at six months or later. METHODS: A retrospective multi-center study was performed using 652 samples of 236 PCR-confirmed SARS-CoV-2 infected patients from 2 Belgian University hospitals. Patients were included if at least two samples were available (range 2-7 samples); including at least one sample collected 30 days or later after first positive PCR (range 0-240 days). Of those 236 patients, 19.1 % were classified as mild/asymptomatic (mild) and 80.9 % as moderate to critical (severe). IgG anti-nucleocapsid antibodies (anti-N) were measured using the Abbott Architect immunoassay. RESULTS: 22.2 % of mild and 2.6 % of severe COVID-19 cases never seroconverted (p < 0.001). Of the mild patients who seroconverted 0-59 days after PCR; 18.8 %, 40.0 % and 61.1 % were seronegative in the windows 60-119 days, 120-179 days and 180-240 days after PCR, respectively. In severe patients, these numbers were 1.9 %, 10.8 % and 29.4 % respectively (p < 0.05 each). Antibody levels were significantly higher in severe patients compared to mild patients in each 60 day window (p < 0.001 each). CONCLUSIONS: SARS-CoV-2 anti-N IgG antibody levels steadily decreased after 2 months up to 8 months post PCR. Of severe COVID-19 patients, 70.6 % remained positive up to eight months after infection. Antibody levels were significantly lower in mild SARS-CoV-2 infected patients and 61.1 % became seronegative within 6 months after the first positive PCR.

A case of severe pseudohyperkalaemia due to muscle contraction.

INTRODUCTION: Severe hyperkalaemia is a serious medical condition requiring immediate medical attention. Before medical treatment is started, pseudohyperkalaemia has to be ruled out. CASE DESCRIPTION: A 10-month old infant presented to the emergency department with fever and coughing since 1 week. Routine venous blood testing revealed a severe hyperkalaemia of 6.9 mmol/L without any indication of haemolysis. Reanalysis of the plasma sample confirmed the hyperkalaemia (7.1 mmol/L). Based on these results, the clinical pathologist suggested to perform a venous blood gas analysis and electrocardiogram (ECG) which revealed a normal potassium of 3.7 mmol/L and normal ECG, ruling out a potentially life-treating hyperkalaemia. The child was diagnosed with pneumonia. The paediatrician had difficulty to perform the first venous blood collection due to excessive movement of the infant during venipuncture. The muscle contractions of the child in combination with venous stasis most probably led to a local increase of potassium in the sampled limbs. The second sample collected under optimal preanalytical circumstances had a normal potassium. Since muscle contraction typically does not cause severe hyperkalaemia, other causes of pseudohyperkalaemia were excluded. K3-EDTA contamination and familial hyperkalaemia were ruled out and the patient did not have extreme leucocytosis or thrombocytosis. By exclusion a diagnosis of pseudohyperkalaemia due to intense muscle movement and venous stasis was made. CONCLUSION: This case suggests that intense muscle contraction and venous stasis can cause severe pseudohyperkalemia without hemolysis. Once true hyperkalemia has been ruled out, a laboratory work-up can help identify the cause of pseudohyperkalaemia.

Münchhausen Syndrome: A Case Report of an Unusual Cause of Vitamin K Antagonist Intoxication.

Psychiatric disorders must be considered in the differential diagnosis of patients presenting to the emergency department with unexplainable somatic symptoms. Physicians should be aware of Münchhausen syndrome as a possible diagnosis. A 46-year-old female patient presented at the emergency department with signs of coagulopathy. She denied taking any anticoagulant drugs as well as rat poison. Urine toxicology revealed the presence of vitamin K antagonists (VKAs). After an extensive workup, she was diagnosed with Münchhausen syndrome. Intentional intoxication with VKA is rare.