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Secondary bacterial pneumonia (2°BP) is associated with significant morbidity following respiratory viral infection, yet remains incompletely understood. In a prospective cohort of 112 critically ill adults intubated for COVID-19, we comparatively assess longitudinal airway microbiome dynamics and the pulmonary transcriptome of patients who developed 2°BP versus controls who did not. We find that 2°BP is significantly associated with both mortality and corticosteroid treatment. The pulmonary microbiome in 2°BP is characterized by increased bacterial RNA mass and dominance of culture-confirmed pathogens, detectable days prior to 2°BP clinical diagnosis, and frequently also present in nasal swabs. Assessment of the pulmonary transcriptome reveals suppressed TNFα signaling in patients with 2°BP, and sensitivity analyses suggest this finding is mediated by corticosteroid treatment. Further, we find that increased bacterial RNA mass correlates with reduced expression of innate and adaptive immunity genes in both 2°BP patients and controls. Taken together, our findings provide fresh insights into the microbial dynamics and host immune features of COVID-19-associated 2°BP, and suggest that suppressed immune signaling, potentially mediated by corticosteroid treatment, permits expansion of opportunistic bacterial pathogens., (© 2024. The Author(s).)

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection 1,2 , yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases., (© 2024. The Author(s).)

Life-threatening "breakthrough" cases of critical COVID-19 are attributed to poor or waning antibody (Ab) response to SARS-CoV-2 vaccines in individuals already at risk. Preexisting auto-Abs neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; their contribution to hypoxemic breakthrough cases in vaccinated people is unknown. We studied a cohort of 48 individuals (aged 20 to 86 years) who received two doses of a messenger RNA (mRNA) vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Ab levels to the vaccine, neutralization of the virus, and auto-Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal Ab response to the vaccine. Among them, 10 (24%) had auto-Abs neutralizing type I IFNs (aged 43 to 86 years). Eight of these 10 patients had auto-Abs neutralizing both IFN-α2 and IFN-ω, whereas two neutralized IFN-ω only. No patient neutralized IFN-β. Seven neutralized type I IFNs at 10 ng/ml and three at 100 pg/ml only. Seven patients neutralized SARS-CoV-2 D614G and Delta efficiently, whereas one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only type I IFNs at 100 pg/ml neutralized both D614G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating Abs capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a notable proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population.

Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells., (© 2023. Springer Nature Limited.)

Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as long COVID. The underlying pathophysiology of long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of SARS-CoV-2 infection and certain other post-COVID sequelae, their potential role in long COVID is important to investigate. Here, we apply a well-established, unbiased, proteome-wide autoantibody detection technology (T7 phage-display assay with immunoprecipitation and next-generation sequencing, PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected that separated individuals with prior SARS-CoV-2 infection from those never exposed to SARS-CoV-2, we did not detect patterns of autoreactivity that separated individuals with long COVID from individuals fully recovered from COVID-19. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and long COVID was apparent by this assay.

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false-negative viral PCR test results. Such tests are also susceptible to false-positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses, and nonviral conditions ( n  = 318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to diagnose COVID-19. We find that optimal classifiers include an interferon-stimulated gene that is strongly induced in COVID-19 compared with nonviral conditions, such as IFI6 , and a second immune-response gene that is more strongly induced in other viral infections, such as GBP5 . The IFI6 + GBP5 classifier achieves an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort ( n  = 553). We further provide proof-of-concept demonstration that the classifier can be implemented in a clinically relevant RT-qPCR assay. Finally, we show that its performance is robust across common SARS-CoV-2 variants and is unaffected by cross-contamination, demonstrating its utility for improved accuracy of COVID-19 diagnostics. IMPORTANCE In this work, we study upper respiratory tract gene expression to develop and validate a 2-gene host-based COVID-19 diagnostic classifier and then demonstrate its implementation in a clinically practical qPCR assay. We find that the host classifier has utility for mitigating false-negative results, for example due to SARS-CoV-2 variants harboring mutations at primer target sites, and for mitigating false-positive viral PCR results due to laboratory cross-contamination. Both types of error carry serious consequences of either unrecognized viral transmission or unnecessary isolation and contact tracing. This work is directly relevant to the ongoing COVID-19 pandemic given the continued emergence of viral variants and the continued challenges of false-positive PCR assays. It also suggests the feasibility of pan-respiratory virus host-based diagnostics that would have value in congregate settings, such as hospitals and nursing homes, where unrecognized respiratory viral transmission is of particular concern.

Interferon (IFN)-specific autoantibodies have been implicated in severe coronavirus disease 2019 (COVID-19) and have been proposed as a potential driver of the persistent symptoms characterizing "long COVID," a type of postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report that only 2 of 215 participants with convalescent SARS-CoV-2 infection tested over 394 time points, including 121 people experiencing long COVID symptoms, had detectable IFN-α2 antibodies. Both had been hospitalized during the acute phase of the infection. These data suggest that persistent anti-IFN antibodies, although a potential driver of severe COVID-19, are unlikely to contribute to long COVID symptoms in the postacute phase of the infection., Competing Interests: Potential conflicts of interest. T. J. H. reports grants from Merck, Gilead Biosciences, and Bristol-Myers Squibb, outside the submitted work. T. J. H. has provided consulting for Roche. S. G. D. reports grants and/or personal fees from Gilead Sciences, Merck, ViiV, AbbVie, Eli Lilly, ByroLogyx, and Enochian Biosciences, outside the submitted work. JD reports paid compensation for consulting for the Public Health Company and Allen & Co. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work (Vazquez et al., 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies., Competing Interests: SV, SM, AB, AK, ZQ, EF, NL, DE, PB, SZ, JL, AM, IP, DY, CM, CW, BM, GS, KZ, AC, VT, CS, AT, KL, MW, OK, KD, OD, RB, LN, JB, JC, ML, TT, MA, JD No competing interests declared

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults whereas disease burden in children is lower. To investigate whether differences in the upper airway immune response may contribute to this disparity, we compare nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 older adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes is robustly activated in both children and adults with SARS-CoV-2 infection compared to the respective non-viral groups, with only subtle distinctions. Children, however, demonstrate markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including response to TNF and production of IFNγ, IL-2 and IL-4. Cell type deconvolution confirms greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibit a decrease in proportions of ciliated cells, among the primary targets of SARS-CoV-2, upon infection. These findings demonstrate that children elicit a more robust innate and especially adaptive immune response to SARS-CoV-2 in the upper airway that likely contributes to their protection from severe disease in the lower airway., (© 2022. The Author(s).)

Background: Pneumonia is a leading cause of death worldwide and is a major health-care challenge in people living with HIV. Despite this, the causes of pneumonia in this population remain poorly understood. We aimed to assess the feasibility of metatranscriptomics for epidemiological surveillance of pneumonia in patients with HIV in Uganda., Methods: We performed a retrospective observational study in patients with HIV who were admitted to Mulago Hospital, Kampala, Uganda between Oct 1, 2009, and Dec 31, 2011. Inclusion criteria were age 18 years or older, HIV-positivity, and clinically diagnosed pneumonia. Exclusion criteria were contraindication to bronchoscopy or an existing diagnosis of tuberculosis. Bronchoalveolar lavage fluid was collected within 72 h of admission and a combination of RNA sequencing and Mycobacterium tuberculosis culture plus PCR were performed. The primary outcome was detection of an established or possible respiratory pathogen in the total study population., Findings: We consecutively enrolled 217 patients during the study period. A potential microbial cause for pneumonia was identified in 211 (97%) patients. At least one microorganism of established respiratory pathogenicity was identified in 113 (52%) patients, and a microbe of possible pathogenicity was identified in an additional 98 (45%). M tuberculosis was the most commonly identified established pathogen (35 [16%] patients; in whom bacterial or viral co-infections were identified in 13 [37%]). Streptococcus mitis, although not previously reported as a cause of pneumonia in patients with HIV, was the most commonly identified bacterial organism (37 [17%] patients). Haemophilus influenzae was the most commonly identified established bacterial pathogen (20 [9%] patients). Pneumocystis jirovecii was only identified in patients with a CD4 count of less than 200 cells per mL., Interpretation: We show the feasibility of using metatranscriptomics for epidemiologic surveillance of pneumonia by describing the spectrum of respiratory pathogens in adults with HIV in Uganda. Applying these methods to a contemporary cohort could enable broad assessment of changes in pneumonia aetiology following the emergence of SARS-CoV-2., Funding: US National Institutes of Health, Chan Zuckerberg Biohub., Competing Interests: Declaration of interests LH reports grants from the US National Institutes of Health. SI is employed by Second Genome. SVL reports fees from Siolta Therapeutics, outside of the submitted work. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: During the COVID-19 pandemic within the United States, much of the responsibility for diagnostic testing and epidemiologic response has relied on the action of county-level departments of public health. Here we describe the integration of genomic surveillance into epidemiologic response within Humboldt County, a rural county in northwest California., Methods: Through a collaborative effort, 853 whole SARS-CoV-2 genomes were generated, representing ~58% of the 1,449 SARS-CoV-2-positive cases detected in Humboldt County as of March 12, 2021. Phylogenetic analysis of these data was used to develop a comprehensive understanding of SARS-CoV-2 introductions to the county and to support contact tracing and epidemiologic investigations of all large outbreaks in the county., Results: In the case of an outbreak on a commercial farm, viral genomic data were used to validate reported epidemiologic links and link additional cases within the community who did not report a farm exposure to the outbreak. During a separate outbreak within a skilled nursing facility, genomic surveillance data were used to rule out the putative index case, detect the emergence of an independent Spike:N501Y substitution, and verify that the outbreak had been brought under control., Conclusions: These use cases demonstrate how developing genomic surveillance capacity within local public health departments can support timely and responsive deployment of genomic epidemiology for surveillance and outbreak response based on local needs and priorities., (© 2022. The Author(s).)

Bats are natural reservoirs for both Alpha - and Betacoronaviruses and the hypothesized original hosts of five of seven known zoonotic coronaviruses. To date, the vast majority of bat coronavirus research has been concentrated in Asia, though coronaviruses are globally distributed; indeed, SARS-CoV and SARS-CoV-2-related Betacoronaviruses in the subgenus Sarbecovirus have been identified circulating in Rhinolophid bats in both Africa and Europe, despite the relative dearth of surveillance in these regions. As part of a long-term study examining the dynamics of potentially zoonotic viruses in three species of endemic Madagascar fruit bat ( Pteropus rufus, Eidolon dupreanum, Rousettus madagascariensis ), we carried out metagenomic Next Generation Sequencing (mNGS) on urine, throat, and fecal samples obtained from wild-caught individuals. We report detection of RNA derived from Betacoronavirus subgenus Nobecovirus in fecal samples from all three species and describe full genome sequences of novel Nobecoviruses in P. rufus and R. madagascariensis . Phylogenetic analysis indicates the existence of five distinct Nobecovirus clades, one of which is defined by the highly divergent ancestral sequence reported here from P. rufus bats. Madagascar Nobecoviruses derived from P. rufus and R. madagascariensis demonstrate, respectively, Asian and African phylogeographic origins, mirroring those of their fruit bat hosts. Bootscan recombination analysis indicates significant selection has taken place in the spike, nucleocapsid, and NS7 accessory protein regions of the genome for viruses derived from both bat hosts. Madagascar offers a unique phylogeographic nexus of bats and viruses with both Asian and African phylogeographic origins, providing opportunities for unprecedented mixing of viral groups and, potentially, recombination. As fruit bats are handled and consumed widely across Madagascar for subsistence, understanding the landscape of potentially zoonotic coronavirus circulation is essential for mitigation of future zoonotic threats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kettenburg, Kistler, Ranaivoson, Ahyong, Andrianiaina, Andry, DeRisi, Gentles, Raharinosy, Randriambolamanantsoa, Ravelomanantsoa, Tato, Dussart, Heraud and Brook.)

Importance: Neuropsychiatric manifestations of COVID-19 have been reported in the pediatric population., Objective: To determine whether anti-SARS-CoV-2 and autoreactive antibodies are present in the cerebrospinal fluid (CSF) of pediatric patients with COVID-19 and subacute neuropsychiatric dysfunction., Design, Setting, and Participants: This case series includes 3 patients with recent SARS-CoV-2 infection as confirmed by reverse transcriptase-polymerase chain reaction or IgG serology with recent exposure history who were hospitalized at the University of California, San Francisco Benioff Children's Hospital and for whom a neurology consultation was requested over a 5-month period in 2020. During this period, 18 total children were hospitalized and tested positive for acute SARS-CoV-2 infection by reverse transcriptase-polymerase chain reaction or rapid antigen test., Main Outcomes and Measures: Detection and characterization of CSF anti-SARS-CoV-2 IgG and antineural antibodies., Results: Of 3 included teenaged patients, 2 patients had intrathecal anti-SARS-CoV-2 antibodies. CSF IgG from these 2 patients also indicated antineural autoantibodies on anatomic immunostaining. Autoantibodies targeting transcription factor 4 (TCF4) in 1 patient who appeared to have a robust response to immunotherapy were also validated., Conclusions and Relevance: Pediatric patients with COVID-19 and prominent subacute neuropsychiatric symptoms, ranging from severe anxiety to delusional psychosis, may have anti-SARS-CoV-2 and antineural antibodies in their CSF and may respond to immunotherapy.

Neutralizing autoantibodies against type I interferons (IFNs) have been found in some patients with critical coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the prevalence of these antibodies, their longitudinal dynamics across the disease severity scale, and their functional effects on circulating leukocytes remain unknown. Here, in 284 patients with COVID-19, we found type I IFN–specific autoantibodies in peripheral blood samples from 19% of patients with critical disease and 6% of patients with severe disease. We found no type I IFN autoantibodies in individuals with moderate disease. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 patients with COVID-19 and 26 non–COVID-19 controls revealed a lack of type I IFN–stimulated gene (ISG-I) responses in myeloid cells from patients with critical disease. This was especially evident in dendritic cell populations isolated from patients with critical disease producing type I IFN–specific autoantibodies. Moreover, we found elevated expression of the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) on the surface of monocytes isolated from patients with critical disease early in the disease course. LAIR1 expression is inversely correlated with ISG-I expression response in patients with COVID-19 but is not expressed in healthy controls. The deficient ISG-I response observed in patients with critical COVID-19 with and without type I IFN–specific autoantibodies supports a unifying model for disease pathogenesis involving ISG-I suppression through convergent mechanisms.

The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a "cytokine storm," we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients reveals distinct immunological features of COVID-19 ARDS., (© 2021. The Author(s).)

Background: Following the first detection of SARS-CoV-2 in passengers arriving from Europe on 19 March 2020, Madagascar took several mitigation measures to limit the spread of the virus in the country., Methods: Nasopharyngeal and/or oropharyngeal swabs were collected from travellers to Madagascar, suspected SARS-CoV-2 cases and contact of confirmed cases. Swabs were tested at the national reference laboratory using real-time RT-PCR. Data collected from patients were entered in an electronic database for subsequent statistical analysis. All distribution of laboratory-confirmed cases were mapped, and six genomes of viruses were fully sequenced., Results: Overall, 26,415 individuals were tested for SARS-CoV-2 between 18 March and 18 September 2020, of whom 21.0% (5,553/26,145) returned positive. Among laboratory-confirmed SARS-CoV-2-positive patients, the median age was 39 years (IQR: 28-52), and 56.6% (3,311/5,553) were asymptomatic at the time of sampling. The probability of testing positive increased with age with the highest adjusted odds ratio of 2.2 [95% CI: 1.9-2.5] for individuals aged 49 years and more. Viral strains sequenced belong to clades 19A, 20A and 20B indicative of several independent introduction of viruses., Conclusions: Our study describes the first wave of the COVID-19 in Madagascar. Despite early strategies in place Madagascar could not avoid the introduction and spread of the virus. More studies are needed to estimate the true burden of disease and make public health recommendations for a better preparation to another wave., (© 2021 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

The role of children in the spread of the SARS-CoV-2 coronavirus has become a matter of urgent debate as societies in the US and abroad consider how to safely reopen schools. Small studies have suggested higher viral loads in young children. Here we present a multicenter investigation on over five thousand SARS-CoV-2 cases confirmed by real-time reverse transcription (RT) PCR assay. Notably, we found no discernable difference in amount of viral nucleic acid among young children and adults.

Comprehensive understanding of the serological response to SARS-CoV-2 infection is important for both pathophysiologic insight and diagnostic development. Here, we generate a pan-human coronavirus programmable phage display assay to perform proteome-wide profiling of coronavirus antigens enriched by 98 COVID-19 patient sera. Next, we use ReScan, a method to efficiently sequester phage expressing the most immunogenic peptides and print them onto paper-based microarrays using acoustic liquid handling, which isolates and identifies nine candidate antigens, eight of which are derived from the two proteins used for SARS-CoV-2 serologic assays: spike and nucleocapsid proteins. After deployment in a high-throughput assay amenable to clinical lab settings, these antigens show improved specificity over a whole protein panel. This proof-of-concept study demonstrates that ReScan will have broad applicability for other emerging infectious diseases or autoimmune diseases that lack a valid biomarker, enabling a seamless pipeline from antigen discovery to diagnostic using one recombinant protein source., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)