PLASMA-BASED ANTIGEN PERSISTENCE IN THE POST-ACUTE PHASE OF SARS-CoV-2 INFECTION

Background: There is mounting evidence regarding the frequency and spectrum of post-acute sequelae of SARS-CoV-2 infection (PASC), but a search for causes has been elusive. Recently, a plasma-based assay for SARS-CoV-2 antigen has been developed, which in initial use revealed that a high fraction of severely affected patients with PASC had circulating antigen. It is unknown whether detectable SARS-CoV-2 antigen is specific for PASC or how the assay performs in a broader clinical spectrum of patients with PASC. Method(s): We evaluated a cohort of patients with RNA-confirmed SARS-CoV-2 infection enrolled >=3 weeks following initial symptoms. Participants, both with and without PASC at enrollment, were identified via facility- and communitybased advertising and examined every 4 months. An interviewer-administered questionnaire ascertained presence of 30 different symptoms (new or worse compared to pre-COVID) in the prior 2 days at each exam. Using the single molecule array (Simoa) assay, we measured spike, S1, and nucleocapsid SARSCoV- 2 antigens in plasma collected at time of symptom assessment. Result(s): We examined 172 participants (50% men, 46% non-white, median age 46 years) who contributed 667 timepoints from 0.7 to 15.4 months following infection, at which 66% featured report of >=1 symptom. Sixty-one of 667 timepoints (9.1%) representing 24% of persons had >=1 detectable SARSCoV- 2 antigen. Among the 437 timepoints at which >=1 symptom was present, 9.8% had >=1 detectable antigen;this compares to 7.8% of timepoints at which symptoms were absent. In comparison to those without symptoms, individuals with several specific symptom complexes (gastrointestinal, musculoskeletal, and central neurologic) more commonly had detectable antigen (Figure). Hospitalization during acute COVID-19 was strongly related to antigen detection. Conclusion(s): Among a diverse group of SARS-CoV-2-infected persons in the post-acute phase of infection, SARS-CoV-2 antigen is detectable in plasma in both those with and without symptoms but more commonly in those with gastrointestinal, musculoskeletal, and central neurologic complaints. The findings indicate that antigen persists in at least some persons and suggest (but do not prove) that antigen is causally related to symptoms. That antigen is found in only a fraction of those with PASC indicates either that not all symptoms are driven by antigen, current plasma antigen detection is insensitive relative to tissue, or nominal PASC symptoms are sometimes unrelated to SARS-CoV-2. (Figure Presented).

Evaluation of the EUROIMMUN Aspergillus antigen immunoenzyme assay in serum and bronchoalveolar lavage fluid samples

Introduction: The most widely used marker for the diagnosis of invasive aspergillosis (IA) is the detection of galactomannan by ELISA. This study describes the evaluation of the results obtained by Euroimmun Aspergillus antigen ELISA (EIA-GM-E) in serum samples and bronchoalveolar lavage fluid (BAL) from patients at risk of IA, and compares these results with those obtained by Bio-Rad Galactomannan EIA (EIA-GM-BR). Method(s): Anonymous retrospective case-control comparative study in 64 serum samples and 28 BAL from 51 patients. Result(s): Overall agreement of the results of the two assays was observed in 72 of 92 samples (78.3%). The sensitivity of EIA-GM-BR and EIA-GM-E in serum samples was 88.9% and 43.2%, respectively, and 100% and 88.9% for BAL. The specificity of EIA-GM-BR and EIA-GM-E in serum samples was 91.9% for both assays, and 68.4% and 84.2% in BAL. There were no statistically significant differences in the results of both assays. Conclusion(s): Both methods show good results for the discrimination of patients with IA when BAL is tested, or serum in case of EIA-GM-BR.Copyright © 2021 Sociedad Espanola de Enfermedades Infecciosas y Microbiologia Clinica

Detection of Aspergillus spp. and Candida albicans antigenemia in a sample of Iraqi patients with severe COVID-19

Introduction: Since December 2019, coronavirus disease 2019 (COVID-19), which is caused by SARSCoV- 2, has spread locally in Wuhan, China, and later on, a worldwide outbreak occurred. Invasive fungal infections can cause complications in critically ill immunocompromised patients of COVID-19, especially those admitted to intensive care units and who required mechanical ventilation. Candida albicans have been the most common pathogenic species, followed by other Candida spp. Mannan is a major component of the Candida cell wall and can be detected by the enzyme-linked immunosorbent assay (ELISA) in blood and other fluids. Invasive pulmonary aspergillosis is considered a lifethreatening infection, especially among immunocompromised patients. COVID-19-associated pulmonary aspergillosis has emerged as an important complication among patients in the intensive care units. Galactomannan (GM) is a major cell-wall component of Aspergillus spp. and can be found in body fluids. Blood GM can be detected by the enzyme immunoassay. The aim of the current study is to assess the frequency of aspergillosis and candidiasis among COVID-19 patients in some hospitals in Baghdad by using GM and mannan biomarkers. Methods: During the period from February 2020 to May 2021, 175 COVID-19 blood samples of patients were collected and a sandwich ELISA test was performed to detect GM Ag of Aspergillus spp. and mannan Ag of Candida spp. Results: Regarding C-reactive protein (CRP), significant differences were seen among Aspergillus/- COVID-19 patients ( p 0.029). Regarding sex and age group, the results indicated that of a total of 175 adult patients with positive COVID-19, more than half of the patients were males. Regarding the distribution of mannan Ag and GM Ag in COVID-19 patients, it was seen that out of the 175 patients, 167 (95.43%) Candida mannan Ag were negative and only 8 (4.57%) were positive, and 170 (79.14%) Aspergillus GM Ag were negative and only 5 (2.86%) were positive. It was also seen that 2 patients (1.14%) who had both Candida mannan and Aspergillus GM were positive and 173 (98.6%) were negative. No statistically significant difference was seen in candidiasis and aspergillosis among patients with COVID-19 regarding age group, sex, underlying chronic diseases (hypertension and diabetes mellitus), and biochemical tests. Conclusion: COVID-19 infections increased with age and were seen more in males than in females. The percentage of infection with C. albicans and Aspergillus spp. among COVID-19 patients was not significant, and this may come from the random collection of samples from patients with different stages of illness. A significant correlation was found between Aspergillus GM Ag in COVID-19 patients and the CRP test.

Baseline Plasma Viral Antigen Levels Are Associated with Clinical Deterioration in Hospitalized Covid-19: Potential Value for Stratifying Patients for Therapeutic Interventions

RATIONALE: Some biomarkers of host response to viral infection are associated with COVID-19 outcomes, but these biomarkers do not directly measure viral burden. The association between plasma viral antigen levels and clinical outcomes has not been previously studied. Our aim was to investigate the relationship between plasma SARS-CoV-2 viral antigen concentration and proximal clinical deterioration in hospitalized patients. METHODS: SARS-CoV-2 nucleocapsid antigen concentrations were measured using a validated microbead immunoassay (Quanterix, NIH/NIAID laboratory) in plasma collected at enrollment from 256 subjects in a prospective observational cohort of hospitalized patients with COVID-19 from 3 hospitals, admitted between March 2020 and August 2021. Relationships between viral antigen concentration and clinical status at 1 week as measured by the World Health Organization (WHO) ordinal scale as well as ICU admission were assessed. Models were adjusted for age and sex, baseline comorbidities including immunosuppression, endogenous neutralizing antibodies, baseline COVID-19 severity, smoking status, remdesivir therapy, steroid therapy, and vaccine status. Missing covariate data were imputed using multiple imputation by chained equations. RESULTS: The median viral antigen concentration for the 35 subjects who deteriorated by 1 week was 4507 (IQR 1225-9665) pg/mL compared to 494 (IQR 18-3882) pg/mL in the 212 subjects who did not (p = 0.0004 Figure a). Using ordinal regression, each doubling in viral antigen concentration was significantly associated with a worse WHO ordinal scale at 1 week (unadjusted OR 1.07, 95% CI 1.02-1.13;adjusted OR 1.10, 95% CI 1.02-1.18). Among 168 patients not in the ICU at baseline, the median viral antigen concentration for the 40 patients who progressed to the ICU was 4697 (IQR 482- 10410) pg/mL vs. 459 (IQR 15-3062) pg/mL in the 128 patients who did not progress to require ICU care (p = 0.0001 Figure b). Using logistic regression, each doubling in viral antigen concentration was significantly associated with ICU admission (unadjusted OR 1.18, 95% CI 1.06-1.32, adjusted OR 1.40, 95% CI 1.11-1.76). CONCLUSIONS: Higher plasma viral antigen concentration at hospital admission is independently associated with a significantly worse clinical status at 1 week and a higher odds of ICU admission among hospitalized patients with COVID-19. This novel finding indicates that plasma viral antigen concentration may identify hospitalized COVID-19 patients at highest risk of short-term clinical deterioration in both clinical practice and research. Results of plasma antigen tests are available within 2-3 hours and could be integrated for identifying hospitalized COVID-19 patients who might benefit from early intervention.

SARS-CoV-2 N-ANTIGEN SERA LEVELS ARE ASSOCIATED with COVID-19 PROGNOSTIC VALUE

Background: Small studies have reported that high levels of free SARS-CoV-2 nucleocapsid-antigen in sera (N-antigenemia) was associated with prognostic. Here, we assessed the association between N-antigenemia levels and patient's outcome in a large cohort of hospitalized patients. Methods: We analysed data from patients with at least one sera sample that were included in the French COVID cohort between January, 25 2020 and September, 2 2020. N-antigenemia levels were determined with the COV-Quanto® assay (AAZ) with a limit of detection of 2.98 pg/mL. IgG (anti-N, anti-S and anti-RBD) levels were assessed using the V-PLEX panel assay (MSD). Patient's outcome was classified in three groups: death, recovery with ICU transfer (ICU) and recovery without ICU transfer (Hospital). Results: We included 1166 samples from 357 patients, with 66% of male and a median age at 63 years [IQR: 52-71]. A total of 82, 96 and 142 patients were in the Death, ICU and Hospital groups, respectively. The sensitivity of N-antigenemia was 79% (131/165) within the first 10 days SSO (since symptoms onset) and 62% (365/589) from 11 to 30 days SSO. Positivity rates were significantly different across severity groups from 0 to 15 days SSO: 95% (95/100), 64% (118/183), 79% (83/105) (p<0.001) for Death, ICU and Hospital groups, respectively. Among positive patients, a significant gradient was found in the levels of N-antigenemia according to disease severity, with median levels of 302, 134 and 89 in Death, ICU and hospital groups, respectively. Similar relationships were found when stratifying on the time SSO (see figure). Overall, 95, 80, 43 and 22% of N-antigenemia >10,000, >5,000, >1,000 and <1,000 pg/mL corresponded to patients who died. IgG antibodies titers were not correlated to severity and the presence of both sera N-antigen and anti-N IgG was observed for 42% (490/1166) samples. Conclusion: We observed, on a large prospective cohort, a strong relationship between N-antigenemia and COVID-19 severity. This new diagnostic tool should help to prognostic evaluation of COVID-19 patients. To our knowledge, COVID-19 is the first demonstration of the presence of free antigenemia in a viral pneumonia, and its association with prognostic.

COMPARAÇÃO DE PERFIL ANTIGÊNICO DOS SISTEMAS RH E KELL EM DOADORES X RECEPTORES NO MUNICÍPIO DO RIO DE JANEIRO DURANTE PERÍODO DE PANDEMIA COVID 19

Introdução: Durante a pandemia de COVID-19 tivemos um aumento do consumo de hemocomponentes com redução do número de doações voluntárias, prejudicando a gestão de hemocomponentes. Pacientes em programas de hemotransfusão crônica ou com aloimunização necessitam de concentrados de hemácias com fenótipo idênticos para os sistemas Rh e antígeno K foram impactados pela menor disponibilidade de hemocomponentes seguindo essas premissas. Objetivo: Comparar a frequência dos antígenos E, e, C, c, K entre doadores e receptores de sangue em banco de sangue privado na cidade do Rio de Janeiro durante a pandemia de COVID-19, avaliar se o município possui perfil similar entre essas 2 populações e seu impacto para gestão de hemocomponentes. Materiais e métodos: Foram analisados resultados de identificação de antígenos E, e, C, c e K de doadores voluntários e de receptores com indicação de transfusão com protocolo de fenotipagem para sistema RH e K no período de janeiro 2020 até junho 2021.Os doadores com tipagem O e A foram 100% fenotipados para os antígenos em questão. Os demais doadores foram fenotipados de acordo com a demanda do serviço de hemoterapia. Os receptores foram fenotipados conforme a indicação de aplicação do protocolo de fenotipagem. Número de indivíduos testados: Doadores 17.159 e Receptores 3.292. Resultados: Entre os doadores avaliados: 59% eram do sexo masculino, encontramos o seguinte perfil antigênico antígeno D=88%, antígeno E=25%, antígeno e=98%, antígeno C=60%, antígeno c=83%, antígeno K=6%. Importante ressaltar que dentre os doadores D negativos o percentual de antígeno c e antígeno e negativos era de 0%. Entre receptores avaliados: 45% eram do sexo masculino, encontramos o seguinte perfil antigênico antígeno D=85%, antígeno E=23%, antígeno e=98%, antígeno C=64%, antígeno c=83%, antígeno K=6%. Discussão: Investigamos o perfil fenotípico de doadores comparativo ao de receptores com base no município do Rio tomando como base o período de pandemia de COVID-19. Avaliamos sexo, naturalidade e fenótipos de doadores e receptores. Realizamos um estudo preliminar com população de doadores no Brasil em 2018, sem comparação com receptores, e encontramos aproximadamente o mesmo resultado percentual de antigenemia deste estudo. Percebemos que a antigenemia dos receptores segue a mesma proporcionalidade que a dos doadores. Conclusão: Este estudo sugere que a população da cidade do Rio de Janeiro durante a pandemia de COVID-19 possui uma frequência de antígenos do sistema Rh e Kell semelhante entre doadores × receptores, e ao mesmo tempo semelhante ao nosso estudo realizado pré pandemia apenas com doadores da região sudeste e nordeste. Podemos então até o momento supor que a metrópole Rio de janeiro possui população representativa do perfil antigênico brasileiro, com potencial para autossutentabilidade caso a cultura de doação de repetição fosse ampliada na população, reduzindo a dificuldade de atendimento principalmente para pacientes D negativos com antígenos raros como c ou e negativos. Estudos comparativos de todas as regiões do país podem auxiliar a compreensão mais fidedigna do perfil populacional a auxiliar nas estratégias de captação e retenção de doadores.

POS-760 PULMONARY ANGIOINVASIVE ASPERGILLOSIS IN A KIDNEY RECIPIENT: AN ATYPICAL CLINICAL PRESENTATION

Introduction: Angioinvasive aspergillosis is a rare opportunistic infection. its occurrence increases the mortality and morbidity of organ transplant recipients. Methods: It is about a 27-year-old patient with a history of end-stage renal failure due to an apparent mineralocorticoid deficiency, hemodialysis for 10 months until he received a kidney transplant from a related living donor sharing 4 HLA identities. He received induction therapy with globulin antithymocyte and methylprednisolone followed by maintenance therapy with Mycophenolate Mofetil, prednisolone, tacrolimus. Since the recipient was not immune to cytomegalovirus (CMV), he received ganciclovir prophylaxis immediately after transplant. Post-transplant evolution was marked by the immediate resumption of diuresis, creatinine figures stagnating between 200 and 250 µmol/l. The remainder of her usual treatment consists of a proton pump inhibitor, β-blocker, cotrimoxazole. Our patient presented 2 months post transplant with febrile neutropenia. Clinically, apart from a fever of 38 °, the examination was without abnormalities. Biologically, pancytopenia, inflammatory syndrome (high CRP and procalcitonin), hypokalaemia, hypophosphatemia and hypomagnesemia, hyperferritinemia and hypertriglyceridemia were noted. A post-infectious macrophagic activation syndrome was suspected, confirmed by a sternal puncture. An etiological investigation has been launched;viral serologies (HBV, HCV, HIV, EBV, CMV, COVID 19, HSV) were negative, a cardiac ultrasound ruled out infective endocarditis, a thoracoabdominal CT scan showed multifocal sub-segmental parenchymal condensations of the right lung surrounded by a halo, an appearance suggesting angio-invasive pulmonary aspergillosis and intracortical graft hematomas. Sputum bacteriological examination did not show pneumocystis but isolated two types of candida crucei and tropicalis. A weakly positive aspergillus antigenemia (index : 0.53) was noticed. Our patient received triple antibiotic therapy (vancomycin / imipenem / levofloxacin) and an antifungal (voriconazole) after adaptation according to the antibiogram for a total duration of 15 days with daily dosage of tacrolimus. In addition, he received venoglobulins for 5 days at a dose of (0.4 mg / kg / day). The clinical and biological course was favorable with apyrexia and improvement in the blood count from the third day of treatment. Aspergillus antigenemia and a follow-up chest CT scan were scheduled after the end of treatment. Results: Initially, In view of the intensity of the induction treatment received, leuconeutropenia and in order of frequency, the 2 most evoked causes were CMV et covid19 but the CT aspect straightened the diagnosis, which was supported by aspergillary antigenemia. The emerging interest of this case report is the clinical and computed tomography discordance and the unusual rapidly favorable outcome to an aspect of angioinvasive pulmonary aspergillosis which usually show respiratory signs such as cough, hemoptysis or even respiratory distress. Conclusions: Invasive mycoses, associated with significant morbidity and mortality, are a frequent difficulty in solid organ transplantation. The clinical pictures differ and we could be faced with an atypical presentation which affects the therapeutic management. No conflict of interest

The association of cytokine profiles and SARS-CoV-2 viral load with COVID-19 severity and mortality

Background: Markedly elevated levels of pro-inflammatory cytokines and defective type-I interferon responses were reported in COVID-19 patients. This study aimed to determine whether particular profiles of cytokine combinations and SARS-CoV-2 viral loads are associated with COVID-19 severity and mortality. Method: Cytokine concentrations and SARS-CoV-2 antigen were measured at hospital admission in serum of symptomatic COVID-19 patients (N = 115), classified at hospitalization into three respiratory severity groups: moderate severity with no need for mechanical ventilatory support (No-MVS), intermediate severity requiring mechanical ventilatory support (MVS) and critical severity requiring extracorporeal membrane oxygenation (ECMO). Principal component analysis was used to characterize cytokine profiles associated with severity and mortality. The results were thereafter confirmed in an independent validation cohort (N = 86). Results: At time of hospitalization, moderately severe No-MVS patients presented an elevated type-I interferon response involving IFN-α and IFN-β, with relatively low levels of pro-inflammatory cytokines. In contrast, critically severe ECMO patients presented a dominant pro-inflammatory response and relatively low type-I interferon response. Intermediate severity MVS patients exhibited a mix of the above cytokine combination profiles. SARS-CoV-2 antigen levels correlated with type-I interferon levels, but not with pro-inflammatory response. Mortality at one month after hospitalization was accurately (87%) predicted by higher levels of IFN-α, IFN-β, and Il-10 in combination with higher SARS-CoV-2 antigenemia and older patients' age, irrespectively of the oxygen support modality. Conclusion: Different levels of two distinct cytokine profiles are observed in different patients in association with COVID-19 severity. Furthermore, by measuring three cytokines and SARS-CoV-2 antigen at time of hospital admission, mortality one month later can be accurately predicted. These results warrant personalized treatment of COVID-19 patients based on cytokine profiling and viral load measurement.

2021 ACLPS Annual Meeting Abstracts

The proceedings contain 38 papers. The topics discussed include: longitudinal assessment of SARS-CoV-2 nucleocapsid antigenemia in patients hospitalized with COVID-19;machine learning-based automated selection of regions for analysis on bone marrow aspirate smears;direct detection of beta-lactamase mediated antibiotic resistance in clinical specimens;convalescent plasma does not provide adequate replacement of C1-Inhibitor and complements in COVID-19 patients;mapping cell-to-cell mitochondria transfer in obesity using high-dimensional spectral flow cytometry;capture the tag: mitigation of biotin interference in ELISA and automated immunoassays by pre-conjugating biotinylated antibodies to the streptavidin surface;and assessment of coagulation tests in hospitalized COVID19 patients;challenging coagulopathies.