Anorectal and oropharyngeal exposures are implicated in sexual transmission of mpox, but authorized assays in the United States are only validated with cutaneous lesion swabs. Diagnostic assays for anorectal and oropharyngeal swabs are needed to address potential future outbreaks. The Cepheid Xpert® Mpox is the first point-of-care assay to receive FDA emergency use authorization in the United States and would be a valuable tool for evaluating these sample types. Our exploratory study demonstrates 100 % positive agreement with our in-house PCR assay for natural positive anorectal and oropharyngeal specimens and 92 % sensitivity with low-positive spiked specimens. The Xpert® assay detected viral DNA in specimens not detected by our reference PCR assay from four participants with mpox DNA at other sites, suggesting it may be more sensitive at low viral loads. In conclusion, the validation of the Xpert® for oropharyngeal and anorectal sample types can be rapidly achieved if clinical need returns and prospective samples become available.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) Alpha variant in 2020 demonstrated the need for reanalysis of diagnostic tests to ensure detection of emerging variants. Here, we present a protocol for creating and characterizing SARS-CoV-2 variant testing panels using remnant clinical samples for diagnostic assay testing. We describe steps for characterizing SARS-CoV-2 remnant clinical samples and preparing them into pools and their use in preparing varying quantities of virus. We then detail procedures for verifying variant detection using the resulting sample panel. For complete details on the use and execution of this protocol, please refer to Rao et al.1,2

The 2022 mpox outbreak primarily involved sexual transmission among men who have sex with men and disproportionately affected persons with human immunodeficiency virus (HIV). We examined viral dynamics and clinical features in a cohort evaluated for mpox infection at a comprehensive HIV clinic in Atlanta, Georgia. Viral DNA was found in 8 oropharyngeal and 5 anorectal specimens among 10 mpox cases confirmed by lesion swab polymerase chain reaction. Within-participant anatomic site of lowest cycle threshold (Ct) value varied, and lower Ct values were found in oropharyngeal and anorectal swabs when corresponding symptoms were present. This provides insight into mpox infection across multiple anatomic sites among people with HIV.

Limited data highlight the need to understand differences in SARS-CoV-2 omicron (B.1.1.529) variant viral load between the gold standard nasopharyngeal (NP) swab, mid-turbinate (MT)/anterior nasal swabs, oropharyngeal (OP) swabs, and saliva. MT, OP, and saliva samples from symptomatic individuals in Atlanta, GA, in January 2022 and longitudinal samples from a small familial cohort were tested by both RT-PCR and ultrasensitive antigen assays. Higher concentrations in the nares were observed in the familial cohort, but a dominant sample type was not found among 39 cases in the cross-sectional cohort. The composite of positive MT or OP assay for both RT-PCR and antigen assay trended toward higher diagnostic yield but did not achieve significant difference. Our data did not identify a singular preferred sample type for SARS-CoV-2 testing, but higher levels of saliva nucleocapsid, a trend toward higher yield of composite OP/MT result, and association of apparent MT or OP predominance with symptoms warrant further study.

IntroductionSwab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described.MethodsWe tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens.ResultsWe observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit.ConclusionClinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized.

Abstract Background Nasopharyngeal qualitative reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is not practical or sufficient in every clinical scenario due to its inability to distinguish active from resolved infection. Alternative or adjunct testing may be needed to guide isolation precautions and treatment in patients admitted to the hospital. Methods We performed a single-center, retrospective analysis of residual clinical specimens and medical record data to examine blood plasma nucleocapsid antigen as a candidate biomarker of active SARS-CoV-2. Adult patients admitted to the hospital or presenting to the emergency department with SARS-CoV-2 ribonucleic acid (RNA) detected by RT-PCR from a nasopharyngeal swab specimen were included. Both nasopharyngeal swab and a paired whole blood sample were required to be available for analysis. Results Fifty-four patients were included. Eight patients had positive nasopharyngeal swab virus cultures, 7 of whom (87.5%) had concurrent antigenemia. Nineteen (79.2%) of 24 patients with detectable subgenomic RNA and 20 (80.0%) of 25 patients with N2 RT-PCR cycle threshold ≤ 33 had antigenemia. Conclusions Most individuals with active SARS-CoV-2 infection are likely to have concurrent antigenemia, but there may be some individuals with active infection in whom antigenemia is not detectable. The potential for high sensitivity and convenience of a blood test prompts interest in further investigation as a screening tool to reduce reliance on nasopharyngeal swab sampling and as an adjunct diagnostic test to aid in clinical decision making during the period after acute coronavirus disease 2019.

Abstract Background The global spread of Mpox in 2022 largely affected men who have sex with men and exposures via the oropharynx and anorectum are thought to contribute to sexual transmission. New molecular platforms including point-of-care assays for detection of Mpox DNA from cutaneous lesion samples have received emergency use authorization in the United States but these technologies are not validated for use with other sample types, including oropharyngeal or anorectal swabs. Methods Anorectal, oropharyngeal and cutaneous lesion swabs swabs were initially collected and frozen dry at -80C during clinical evaluation for suspected Mpox at a large HIV care center in Atlanta, GA. Swabs were thawed to room temperature and eluted in 3 mL of transport medium prior to testing on the Cepheid Xpert platform, which was performed according to the assay's instructions for use. DNA extracted from 140 μL of the same sample was used for non-variola orthopoxvirus (E9L-NVAR) PCR as a reference test and RNaseP PCR as a sample adequacy control. Results Positive agreement between the Xpert Mpox assay and in-house PCR was 100% for 9 cutaneous lesion swabs, 8 oropharyngeal swabs, and 5 anorectal swabs (see Figure). There were no false-negative results returned by the Xpert. Overall negative agreement was 86.7% (26/30). The Xpert assay was positive in 6 specimens where viral DNA was not detected by the NVOG assay including two specimens that also did not have detectable RNAseP. 4 of these 6 specimens with discordant results were from patients with viral DNA detected at other anatomic sites. Results of the Xpert Mpox assay, NVAR PCR, and sample adequacy controls for cutaneous lesion, oropharyngeal and anorectal specimens. The Xpert assay was positive for 100% of specimens when the non-variola orthopoxvirus (NVAR) target detected by our reference assay and there were no false negative results. The Xpert assay also detected viral targets in 6 specimens where no viral DNA was detected by the NVAR assay. However, four of these specimens were collected from patients with Mpox DNA detected at other anatomic sites. Conclusion The Xpert Mpox assay had perfect positive agreement with the NVAR PCR assay in anorectal, oropharyngeal and cutaneous lesion swab specimens suggesting it has high sensitivity for diagnosis of Mpox infection at the point-of-care, including with not-yet validated sample types. Specificity of the assay is difficult to estimate from our data, as a likely explanation of our results is that the Xpert Mpox assay has a lower limit of detection compared to our reference assay. This is evidenced by detection of Mpox DNA with high Ct values in samples from patients with viral DNA confirmed by both assays at other anatomic sites. The Xpert Mpox assay can be expected to perform well with oropharyngeal and anorectal swab specimens in clinical settings. Disclosures Jonathan Colasanti, MD, MSPH, DKB MED LLC: Honoraria|Prime Education LLC: Advisor/Consultant

Abstract Background Current routine methodologies for monoclonal immunoglobulin measurements may not be sufficiently sensitive to reflect the depth of response seen in patients since the introduction of novel therapies; mass spectrometry may offer a valuable, sensitive alternative approach. Here we describe the preliminary analytical performance characteristics of the EXENT® solution (in development by The Binding Site, part of Thermo Fisher scientific) that combines specific immunoprecipitation steps and mass spectrometry for the identification and quantification of IgG, IgA and IgM intact monoclonal immunoglobulins. Each intact monoclonal immunoglobulin clone can be tracked using its unique m/z value. Methods The Lower Limit of Measuring Interval (LLMI) was established for each immunoglobulin type following EP17-A2:2012. Linearity studies were performed according to CLSI EP06-A2:2020 using high and low pools of IgG, IgA, and IgM monoclonal samples and with additional linearity testing below 1 g/L for each specificity to more effectively demonstrate low-end linearity. Within run, between run, between analyzer, between lot and total precision for M protein concentrations and for molecular mass (m/z) of the monoclonal peaks were assessed according to CLSI EP5-A3-2014. Interference was tested following ED3:2018 using 20 potential interferents against 7 samples including high and low IgG, IgA and IgM monoclonal samples. Results The initial indication of the EXENT solution performance, in development, are set out below. Results suggest an analytical sensitivity of the assays around 15 mg/L at the LLMI for all specificities. Linearity over a range of 0.014–88.9 g/L for IgG, 0.011–68.4 g/L for IgA, and 0.11–74.2 g/L for IgM. Coefficients of variation (CVs) for M protein concentrations in precision studies were <15% for all specificities and samples. Mass/charge values were within ±1.1 to ±1.5 m/z in total precision studies, and within ±2.7 to ±3.9 m/z in between lot precision studies, respectively, for M proteins with an m/z value ranging from 11 360.6 to 11 698.5 m/z. No significant interference effects were observed when testing the 20 interferents including intralipid (20 g/L), triglyceride (15 g/L), bilirubin (400 mg/L), rheumatoid factor (200 IU/mL) and haemoglobin (10 g/L). Conclusion The new EXENT solution demonstrates the potential for a wide measuring interval and the ability to detect M proteins with very low concentration. Also, it could provide stable and reproducible performance for the detection and typing of monoclonal immunoglobulins.

Rapid Antigen Tests (RAT) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of eight commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR Cycle threshold (C T ) value (a proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Abstract Background Advances in diagnostics since the emergence of COVID-19 has resulted in easy availability of rapid antigen tests (RAT). Building on this, multiplex tests for simultaneous detection of Flu A, Flu B, and SARS-CoV-2 are being evaluated as part of NIH’s Rapid Acceleration of Diagnostics (RADx) program. Critically important for assay development and assessment are viral panels prepared under validated conditions to evaluate new tests. These panels may contain live virus or may be inactivated to promote biosafety. They also may undergo freeze-thaw cycles during storage and transport. We characterized the effect of heat inactivation (HI) on detection of Flu A and B using two RATs, and the effect of freeze-thaw cycles on a point-of-care (PoC) molecular assay already on the market. Methods Panels were prepared by serially diluting Flu A or B strains obtained from BEI Resources. Both live and HI panels were prepared. HI was achieved in-house (60°C, 30 minutes). Panels with and without HI were used to assess two RATs by performing triplicate tests and determining limit of detection (LOD) as the lowest TCID50 at which 3 of 3 replicates were positive. Additionally, a molecular-based PoC assay was evaluated by recording cycle threshold (Ct) values before and after freeze-thaw of specimens. All assays were performed according to the manufacturer’s instructions. Results Both RATs detected Flu A with similar sensitivity (2-fold increase in LOD) before and after heat inactivation (Figure 1), whereas Flu B was not detected by either RAT after heat inactivation. These results were consistent across both RATs and two strains of each virus. Live Flu A and B were stable and consistently detected up to four freeze-thaw cycles in the molecular assay with minor attrition of Ct values (Figure 2). Results of LOD characterization for two independent RATs with influenza A and B with and without heat inactivation. Correlation of Flu A and Flu B Ct values for fresh samples versus after four cycles of freeze thaw measured using a PoC molecular assay. Conclusion Pre-treatment and storage of testing material plays a crucial role in the assessment of diagnostic tests used for Flu A and B detection. Loss of reactivity in Flu B antigen tests after inactivation suggests that heating may denature a critical epitope recognized by immunoassays. Selection of live versus HI samples and degradational effects of freeze thaw cycles must be considered by laboratorians and engineers creating or validating diagnostic tests. Disclosures All Authors: No reported disclosures

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of human angiotensin converting enzyme 2 (hACE-2) binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using Wuhan-WT (vaccine strain), delta (B.1.167.2), omicron BA1 and BA2 variant viral strains showed strong correlation with cell-based pseudovirus neutralization activity (PNA) and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta and omicron variant resistance to neutralization in samples with paired vaccine strain and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. Importantly, this completely automated assay can be performed in 4 h to measure neutralizing antibody titers for 16 samples over 8 serial dilutions or, 128 samples at a single dilution with replicates. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

AbstractBackgroundAntibodies induced by COVID-19 vaccination have been shown to wane over time. Current tests for assessing virus-neutralizing antibodies are complex and time-intensive. There is a need for a simple diagnostic test that measures levels of protective antibodies to help monitor immunity status.MethodUsing a commercially available FDA-authorized semi-quantitative SARS-CoV-2 IgG test, we monitored the duration of the immune response in dried blood microsamples (DBS) and saliva to vaccination by 3 different vaccines across prospective cohorts of 8 COVID-19 naïve and 29 COVID-19 recovered individuals over a six-month period. We correlated the results to a binding blockade assay validated to a live virus neutralization assay to validate the test for measurement of protective antibodies.ResultsThe immune response characteristics between the two mRNA vaccines were similar over the 6-month period in both the COVID-19 naïve and recovered cohorts. IgG titers in DBS were generally 3-4 orders of magnitude higher than in saliva, and longitudinal profiles were highly correlated between the two matrices (Rm = 0.80). Median IgG concentrations post-vaccination declined to <10% neutralization capacity with all vaccines by six months.ConclusionsThe potential of a simple, fully automated high throughput anti-SARS-CoV-2 IgG test to quantitatively measure protective antibodies in samples collected remotely or at the point of care was demonstrated. The IgG immune response and protective immunity was shown to decline significantly by six months.Plain Language SummaryIn response to infection the immune system produces proteins called antibodies that recognize and bind to foreign invaders. Vaccines train the immune system to recognize and produce antibodies against specific invaders, such as SAR-CoV-2. Measurement of antibody levels in blood help monitor a person’s response to vaccination and have been shown to correlate with protection against disease, which wanes over time following vaccination. It is desirable to have an easy test that predicts protection against infection and measuring antibody levels may provide a solution, however different tests report results differently hindering the establishment of a cutoff for protected vs. not. We quantified antibody levels in saliva and dried blood microsamples (DBS) following vaccination using an automated semi-quantitative IgG test. By reporting concentration of antibodies, and if anchored to an international standard, this test could help establish a cutoff of protection that would be transferable across the multiple different test types. Furthermore, by measuring in saliva and DBS we demonstrate an easy path to at-home or point-of-care sample collection, which could allow wide-scale monitoring of immune protection against SARS-CoV-2.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for diagnosis, treatment, and infection control. Polymerase chain reaction (PCR) fails to distinguish acute from resolved infections, as RNA is frequently detected after infectiousness. We hypothesized that nucleocapsid in blood marks acute infection with the potential to enhance isolation and treatment strategies. In a retrospective serosurvey of inpatient and outpatient encounters, we categorized samples along an infection timeline using timing of SARS-CoV-2 testing and symptomatology. Among 1860 specimens from 1607 patients, the highest levels and frequency of antigenemia were observed in samples from acute SARS-CoV-2 infection. Antigenemia was higher in seronegative individuals and in those with severe disease. In our analysis, antigenemia exhibited 85.8% sensitivity and 98.6% specificity as a biomarker for acute coronavirus disease 2019 (COVID-19). Thus, antigenemia sensitively and specifically marks acute SARS-CoV-2 infection. Further study is warranted to determine whether antigenemia may aid individualized assessment of active COVID-19.

Background: Nucleocapsid antigenemia in adults has demonstrated high sensitivity and specificity for acute infection, and antigen burden is associated with disease severity. Data regarding SARS-CoV-2 antigenemia in children are limited. Methods: We retrospectively analyzed blood plasma specimens from hospitalized children with COVID-19 or MIS-C. Nucleocapsid and spike were measured using ultrasensitive immunoassays. Results: We detected nucleocapsid antigenemia in 62% (50/81) and spike antigenemia in 27% (21/79) of children with acute COVID-19 but 0% (0/26) and 15% (4/26) with MIS-C from March 2020–March 2021. Higher nucleocapsid levels were associated with radiographic infiltrates and respiratory symptoms in children with COVID-19. Conclusions: Antigenemia lacks the sensitivity to diagnose acute infection in children but is associated with signs and symptoms of lower respiratory tract involvement. Further study into the mechanism of antigenemia, its association with specific organ involvement, and the role of antigenemia in the pathogenesis of COVID-19 is warranted.

Immunocompromised patients with prolonged coronavirus disease 2019 symptoms present diagnostic and therapeutic challenges. We measured viral nucleocapsid antigenemia in 3 patients treated with anti-CD20 immunotherapy who acquired severe acute respiratory syndrome coronavirus 2 infection and experienced protracted symptoms. Our results support nucleocapsid antigenemia as a marker of persistent infection and therapeutic response.

BACKGROUND:Upper respiratory samples for SARS-CoV-2 detection include the gold standard nasopharyngeal (NP) swab, and mid-turbinate (MT) nasal swabs, oropharyngeal (OP) swabs, and saliva. Following the emergence of the omicron (B.1.1.529) variant, limited preliminary data suggest that OP swabs or saliva samples may be more sensitive than nasal swabs, highlighting the need to understand differences in viral load across different sites.METHODS:MT, OP, and saliva samples were collected from symptomatic individuals presenting for evaluation in Atlanta, GA, in January 2022. Longitudinal samples were collected from a family cohort following COVID-19 exposure to describe detection of viral targets over the course of infection.RESULTS:SARS-CoV-2 RNA and nucleocapsid antigen measurements demonstrated a nares-predominant phenotype in a familial cohort. A consistent dominant location for SARS-CoV-2 was not found among 54 individuals. Positive percent agreement for virus detection in MT, OP and saliva specimens were 66.7 [54.1-79.2], 82.2 [71.1-93.4], and 72.5 [60.3-84.8] by RT-PCR, respectively, and 46.2 [32.6-59.7], 51.2 [36.2-66.1], and 72.0 [59.6-84.4] by ultrasensitive antigen assay. The composite of positive MT or OP assay was not significantly different than either alone for both RT-PCR and antigen assay (PPA 86.7 [76.7-96.6] and 59.5 [44.7-74.4], respectively).CONCLUSIONS:Our data suggest that SARS-CoV-2 nucleocapsid and RNA exhibited similar kinetics and diagnostic yield in three upper respiratory sample types across the duration of symptomatic disease. Collection of OP or combined nasal and OP samples does not appear to increase sensitivity versus validated nasal sampling for rapid detection of viral antigen.

Viability of saliva samples stored for longer than 28 days has not been reported in the literature. The COVID-19 pandemic has spawned new research evaluating various sample types, thus large biobanks have been started. Residual saliva samples from university student surveillance testing were retested on SalivaDirect and compared with original RT-PCR (cycle threshold values) and quantitative antigen values for each month in storage. We conclude that saliva samples stored at -80°C are still viable in detecting SARS-CoV-2 after 12 months of storage, establishing the validity of these samples for future testing.