Diagnostic performance of four SARS-CoV-2 antibody assays in patients with COVID-19 or with bacterial and non-SARS-CoV-2 viral respiratory infections

European Journal of Clinical Microbiology & Infectious Diseases - SARS-CoV-2 antibody assays are used for epidemiological studies and for the assessment of vaccine responses in highly...     

Long-term coexistence of SARS-CoV-2 with antibody response in COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has spread worldwide. Whether antibodies are important for the adaptive immune responses against SARS-CoV-2 infection needs to be determined. Here, 26 cases of COVID-19 in Jinan, China, were examined and shown to be mild or with common clinical symptoms, and no case of severe symptoms was found among these patients. Strikingly, a subset of these patients had SARS-CoV-2 and virus-specific IgG coexist for an unexpectedly long time, with two cases for up to 50 days. One COVID-19 patient who did not produce any SARS-CoV-2–bound IgG successfully cleared SARS-CoV-2 after 46 days of illness, revealing that without antibody-mediated adaptive immunity, innate immunity alone may still be powerful enough to eliminate SARS-CoV-2. This report may provide a basis for further analysis of both innate and adaptive immunity in SARS-CoV-2 clearance, especially in nonsevere cases.     

Antibody dynamics to SARS-CoV-2 in asymptomatic COVID-19 infections

Background

The missing asymptomatic COVID-19 infections have been overlooked because of the imperfect sensitivity of the nucleic acid testing (NAT). Globally understanding the humoral immunity in asymptomatic carriers will provide scientific knowledge for developing serological tests, improving early identification, and implementing more rational control strategies against the pandemic.

Measure

Utilizing both NAT and commercial kits for serum IgM and IgG antibodies, we extensively screened 11 766 epidemiologically suspected individuals on enrollment and 63 asymptomatic individuals were detected and recruited. Sixty-three healthy individuals and 51 mild patients without any preexisting conditions were set as controls. Serum IgM and IgG profiles were further probed using a SARS-CoV-2 proteome microarray, and neutralizing antibody was detected by a pseudotyped virus neutralization assay system. The dynamics of antibodies were analyzed with exposure time or symptoms onset.

Results

A combination test of NAT and serological testing for IgM antibody discovered 55.5% of the total of 63 asymptomatic infections, which significantly raises the detection sensitivity when compared with the NAT alone (19%). Serum proteome microarray analysis demonstrated that asymptomatics mainly produced IgM and IgG antibodies against S1 and N proteins out of 20 proteins of SARS-CoV-2. Different from strong and persistent N-specific antibodies, S1-specific IgM responses, which evolved in asymptomatic individuals as early as the seventh day after exposure, peaked on days from 17 days to 25 days, and then disappeared in two months, might be used as an early diagnostic biomarker. 11.8% (6/51) mild patients and 38.1% (24/63) asymptomatic individuals did not produce neutralizing antibody. In particular, neutralizing antibody in asymptomatics gradually vanished in two months.

Conclusion

Our findings might have important implications for the definition of asymptomatic COVID-19 infections, diagnosis, serological survey, public health, and immunization strategies.

     

Diagnostic usefulness of subgenomic RNA detection of viable SARS-CoV-2 in patients with COVID-19

ObjectivesThe development of a rapid diagnostic test for viable SARS-CoV-2 is important for infection control. Real-time RT-PCR assays detect non-viable virus, and cell culture differentiates viable virus but it takes several weeks and is labour-intensive. Subgenomic RNAs may reflect replication-competent virus. We therefore evaluated the usefulness of subgenomic RNAs for diagnosing viable SARS-CoV-2 in patients with COVID-19.MethodsPatients with various severities of confirmed COVID-19 were enrolled at a tertiary hospital between February and December 2020. RT-PCR assay results for genomic and subgenomic RNA of SARS-CoV-2 from nasopharyngeal swab, sputum and saliva specimens were compared with cell culture results.ResultsA total 189 specimens from 20 COVID-19 patients were tested in genomic and subgenomic PCR assays and cultured on Vero cells. Of these 189 samples, 62 (33%) gave positive culture results, 93 (49%) negative results and the remaining 34 (18%) indeterminate results. Compared with cell culture results, the sensitivities of genomic RNA and subgenomic RNA of the N and S genes were comparable at 100%, but the specificity of subgenomic RNA (N, 65% and S, 68%) was higher than that of genomic RNA (N, 23% and S, 17%, p < 0.001). The mean durations of positive culture and subgenomic RNA were 11.39 ± 10.34 and 13.75 ± 11.22 days after symptom onset (p 0.437), respectively, while that of genomic RNA was 22.85 ± 11.83 days after symptom onset (p < 0.001).DiscussionOur comparison of subgenomic RNA detection with symptom duration and SARS-CoV-2 culture positivity provides a significant advancement on the transmissibility-based approach beyond the detection of SARS-CoV-2 genomic RNA, and warrants further studies on the development of better diagnostic strategy.     

Viable SARS-CoV-2 in various specimens from COVID-19 patients

ObjectivesThe aim was to determine whether various clinical specimens obtained from COVID-19 patients contain the infectious virus.MethodsTo demonstrate whether various clinical specimens contain the viable virus, we collected naso/oropharyngeal swabs and saliva, urine and stool samples from five COVID-19 patients and performed a quantitative polymerase chain reaction (qPCR) to assess viral load. Specimens positive with qPCR were subjected to virus isolation in Vero cells. We also used urine and stool samples to intranasally inoculate ferrets and evaluated the virus titres in nasal washes on 2, 4, 6 and 8 days post infection.ResultsSARS-CoV-2 RNA was detected in all naso/oropharyngeal swabs and saliva, urine and stool samples collected between days 8 and 30 of the clinical course. Notably, viral loads in urine, saliva and stool samples were almost equal to or higher than those in naso/oropharyngeal swabs (urine 1.08 ± 0.16–2.09 ± 0.85 log₁₀ copies/mL, saliva 1.07 ± 0.34–1.65 ± 0.46 log₁₀ copies/mL, stool 1.17 ± 0.32 log₁₀ copies/mL, naso/oropharyngeal swabs 1.18 ± 0.12–1.34 ± 0.30 log₁₀ copies/mL). Further, viable SARS-CoV-2 was isolated from naso/oropharyngeal swabs and saliva of COVID-19 patients, as well as nasal washes of ferrets inoculated with patient urine or stool.DiscussionViable SARS-CoV-2 was demonstrated in saliva, urine and stool samples from COVID-19 patients up to days 11–15 of the clinical course. This result suggests that viable SARS-CoV-2 can be secreted in various clinical samples and respiratory specimens.     

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19

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COVID-19 (SARS-CoV-2 infection) in lymphoma patients: A review

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection now has a global resonance and represents a major threat for several patient populations. Observations from initial case series suggested that cancer patients in general might have an unfavorable outcome following coronavirus disease 2019 (COVID-19), due to their underlying conditions and cytotoxic treatments. More recently, data regarding the incidence and clinical evolution of COVID-19 in lymphomas have been reported with the aim to identify those more frequently associated with severe complications and death. Patients with lymphoma appear particularly vulnerable to SARS-CoV-2 infection, only partly because of the detrimental effects of the anti-neoplastic regimens (chemotherapy, pathway inhibitors, monoclonal antibodies) on the immune system. Here, we systematically reviewed the current literature on COVID-19 in adult patients with lymphoma, with particular emphasis on disease course and prognostic factors. We also highlighted the potential differences in COVID-19 clinical picture according to lymphoma subtype, delivered treatment for the hematological disease and its relationship on how these patients have been managed thus far.     

The nasopharyngeal microbiota in patients with viral respiratory tract infections is enriched in bacterial pathogens

The nasopharynx is the primary site of colonization by respiratory pathogen that constitutes the port of entrance in the respiratory tract. The role of mucosal respiratory microbiota in infection has been recently emphasized; therefore, we aimed to assess if a specific respiratory microbiota profile was associated with symptomatic infection and/or with presence of respiratory viruses. We performed a case-control study to characterize the healthy respiratory microbiota and its alteration during acute viral infections. Next-generation sequencing of the 16S rRNA gene was applied to 225 nasopharyngeal samples from 177 patients with viral respiratory infection and 48 matched healthy controls. We evidenced an important decrease of bacterial alpha-diversity in patients with symptomatic respiratory infection and a loss of the healthy core microbiota, specifically anaerobes and Prevotella spp. Moreover, eight respiratory pathogens were enriched in these patients, including Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Dol osigranulum pigrum and Corynebacterium propinquum/pseudodiphtheriticum, whose role in respiratory infection is unclear. The asymptomatic carrier of influenza harbors a microbiota similar to healthy subjects, suggesting a critical role of microbiota in the clinical expression of viruses. These data suggest that the commensal microbiota plays a significant role in susceptibility to viral infection. The frequent co-detection of virus and bacteria raises the question of a strategy to prevent bacterial disease, focusing on the prevention of nasopharyngeal colonization through effective antibiotic treatment. In addition to antibiotics, further studies should test preventive or therapeutic interventions for maintaining or restoring a healthy nasopharyngeal microbiota.     

SARS-CoV-2 antibody dynamics and B-cell memory response over time in COVID-19 convalescent subjects

ObjectivesThe worldwide spread of coronavirus disease 2019 (COVID-19) highlights the need for assessment of long-term humoral immunity in convalescent subjects. Our objectives were to evaluate long-term IgG antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and B-cell memory response in COVID-19 convalescent subjects.MethodsBlood samples were collected from a cohort of subjects recovering from COVID-19 and from healthy subjects who donated blood. SARS-CoV-2 IgG antibodies were quantitatively detected by ELISA using anti-S1 spike IgG. SARS-CoV-2 spike-specific IgG memory B cells were evaluated by reversed B-cell FluroSpot based on human IgG SARS-CoV-2 receptor-binding domain in a randomly selected group of subjects recovering from COVID-19. Statistical analysis was performed with clinical variables and time post COVID-19 infection.ResultsAntibody response was not detected in 26 of 392 COVID-19 convalescent subjects (6.6%). Over a period of 9 months, the level of antibodies decreased by 50% but stabilized at 6 months, and a protective level prevailed for up to 9 months. No differences were found regarding IgG SARS-CoV-2 antibody levels for age, gender, and major blood types over time. Over time, asymptomatic COVID-19 subjects did not differ in antibody level from subjects with mild to severe disease. Repeated paired IgG SARS-CoV-2 antibody level analyses disclosed that, over 6 and 9 months, 15.3% (nine of 59) and 15.8% (three of 19) of subjects became SARS-CoV-2 IgG-seronegative, respectively, all with a low antibody level at 3 months. Rate of antibody decline was not affected by age, gender, or clinical symptomatology. In a subgroup of recovering subjects, memory B-cell response up to 9 months post-COVID-19 infection was undetectable in 31.8% of subjects (14/44), and there was no correlation with age, SARS-CoV-2 antibody level, or time post infection.ConclusionsThe majority of convalescent COVID-19 subjects develop an IgG SARS-CoV-2 antibody response and a protective level prevails over a period of up to 9 months, regardless of age, gender, major blood types or clinical symptomatology.     

A whole blood test to measure SARS-CoV-2-specific response in COVID-19 patients

ObjectivesTo examine whether specific T-cell-responses to SARS-CoV-2 peptides can be detected in COVID-19 using a whole-blood experimental setting, which may be further explored as a potential diagnostic tool.MethodsWe evaluated interferon (IFN)-γ levels after stimulating whole-blood with spike and remainder-antigens peptides megapools (MP) derived from SARS-CoV-2 sequences; interleukin (IL)-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, eotaxin, basic fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, Interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, Platelet-derived growth factor (PDGF), RANTES (regulated on activation, normal T cell expressed and secreted), tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) were also evaluated.ResultsIFN-γ-response to spike and remainder-antigens MPs was significantly increased in 35 COVID-19 patients compared with 29 ‘no COVID-19’ individuals (medians spike-MP: 0.26 vs 0, p = 0.0002; medians remainder-antigens-MP: 0.07 vs 0.02; p = 0.02).This response was detected independently of patients' clinical parameters. IFN-γ-response to SARS-CoV-2-unrelated antigens cytomegalovirus (CMV) and Staphylococcal Enterotoxin B (SEB) was similar in COVID-19 compared with ‘no COVID-19’ individuals (median CMV: 3.46 vs 5.28, p = 0.16; median SEB: 12.68 vs 15.05; p = 0.1). In response to spike-MPs in COVID-19- compared with ‘no COVID-19’ -individuals, we found significant higher median of IL-2 (50.08 vs 0, p = 0.0018), IFN-γ (90.16 vs 0, p = 0.01), IL-4 (0.52 vs 0, p = 0.03), IL-13 (0.84 vs 0, p = 0.007) and MCP-1 (4602 vs 359.2, p = 0.05).ConclusionsImmune response to SARS-CoV-2 peptides in a whole-blood assay is associated with COVID-19 and it is characterized by both Th1 and Th2 profile. This experimental approach may be useful for developing new T-cell based diagnostic tests for disease and vaccine settings.     

Correction to: The Unyvero Hospital-Acquired pneumonia panel for diagnosis of secondary bacterial pneumonia in COVID-19 patients

European Journal of Clinical Microbiology & Infectious Diseases -     

The kinetics of SARS-CoV-2 viremia in COVID-19 patients receiving remdesivir

Detection of SARS-CoV-2 RNA in serum, viremia, has been linked to disease severity and outcome. The kinetics of viremia in patients receiving remdesivir has not been thoroughly studied and could help predict treatment response and outcome. We investigated the kinetics of SARS-CoV-2 viremia and factors associated with baseline viremia, viral clearance and 30-day mortality in patients receiving remdesivir. An observational study including 378 hospitalised patients (median age 67 years, 67% male) sampled with serum SARS-CoV-2 RT-PCR within ± 24 h of initiation of remdesivir treatment. Baseline viremia was present in 206 (54%) patients with a median Ct value of 35.3 (IQR = 33.3–37.1). In patients with baseline viremia, the estimated probability of viral clearance was 72% by day 5. Ct values decreased significantly during remdesivir treatment for viremic patients, indicating an increase in viral load. In total, 44 patients (12%) died within 30 days, and mortality was significantly associated with viremia at baseline (OR = 2.45, p = 0.01) and lack of viral clearance by day 5 (OR = 4.8, p =  < 0.01). Viral clearance was not associated with any individual risk factor. Viremia appears to be a prognostic marker before and during remedesivir treatment. The resolution of viremia was similar to patients not receiving remdesivir in other studies, and the decrease in Ct values during treatment questions the antiviral capacity of remdesivir in vivo. Prospective studies are warranted to confirm our findings.