Reduced control of SARS-CoV-2 infection associates with lower mucosal antibody responses in pregnancy.

Pregnant patients are at greater risk of hospitalization with severe COVID-19 than non-pregnant people. This was a retrospective observational cohort study of remnant clinical specimens from patients who visited acute care hospitals within the Johns Hopkins Health System in the Baltimore, MD-Washington DC, area between October 2020 and May 2022. Participants included confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected pregnant people and matched non-pregnant people (the matching criteria included age, race/ethnicity, area deprivation index, insurance status, and vaccination status to ensure matched demographics). The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant patients were at increased risk of hospitalization (odds ratio [OR] = 4.2; confidence interval [CI] = 2.0-8.6), intensive care unit admittance (OR = 4.5; CI = 1.2-14.2), and being placed on supplemental oxygen therapy (OR = 3.1; CI = 1.3-6.9). Individuals infected during their third trimester had higher mucosal anti-S IgG titers and lower viral RNA levels (P < 0.05) than those infected during their first or second trimesters. Pregnant individuals experiencing breakthrough infections due to the Omicron variant had reduced anti-S IgG compared to non-pregnant patients (P < 0.05). The observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity through booster vaccines may be important for the protection of this at-risk population.IMPORTANCEIn this retrospective observational cohort study, we analyzed remnant clinical samples from non-pregnant and pregnant individuals with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections who visited the Johns Hopkins Hospital System between October 2020 and May 2022. Disease severity, including intensive care unit admission, was greater among pregnant than non-pregnant patients. Vaccination reduced recovery of infectious virus and viral RNA levels in non-pregnant patients, but not in pregnant patients. In pregnant patients, increased nasopharyngeal viral RNA levels and recovery of infectious virus were associated with reduced mucosal IgG antibody responses, especially among women in their first trimester of pregnancy or experiencing breakthrough infections from Omicron variants. Taken together, this study provides insights into how pregnant patients are at greater risk of severe COVID-19. The novelty of this study is that it focuses on the relationship between the mucosal antibody response and its association with virus load and disease outcomes in pregnant people, whereas previous studies have focused on serological immunity. Vaccination status, gestational age, and SARS-CoV-2 omicron variant impact mucosal antibody responses and recovery of infectious virus from pregnant patients.

Evolution of Influenza A(H3N2) Viruses in 2 Consecutive Seasons of Genomic Surveillance, 2021-2023.

Background: The circulation and the genomic evolution of influenza A(H3N2) viruses during the 2021/2022 and 2022/2023 seasons were studied and associated with infection outcomes. Methods: Remnant influenza A-positive samples following standard-of-care testing from patients across the Johns Hopkins Health System (JHHS) were used for the study. Samples were randomly selected for whole viral genome sequencing. The sequence-based pEpitope model was used to estimate the predicted vaccine efficacy (pVE) for circulating H3N2 viruses. Clinical data were collected and associated with viral genomic data. Results: A total of 121 683 respiratory specimens were tested for influenza at JHHS between 1 September 2021 and 31 December 2022. Among them, 6071 (4.99%) tested positive for influenza A. Of these, 805 samples were randomly selected for sequencing, with hemagglutinin (HA) segments characterized for 610 samples. Among the characterized samples, 581 were H3N2 (95.2%). Phylogenetic analysis of HA segments revealed the exclusive circulation of H3N2 viruses with HA segments of the 3C.2a1b.2a.2 clade. Analysis of a total of 445 complete H3N2 genomes revealed reassortments; 200 of 227 of the 2022/2023 season genomes (88.1%) were found to have reassorted with clade 3C.2a1b.1a. The pVE was estimated to be -42.53% for the 2021/2022 season and 30.27% for the 2022/2023 season. No differences in clinical presentations or admissions were observed between the 2 seasons. Conclusions: The increased numbers of cases and genomic diversity of influenza A(H3N2) during the 2022/2023 season were not associated with a change in disease severity compared to the previous influenza season.

Reduced control of SARS-CoV-2 infection is associated with lower mucosal antibody responses in pregnant women.

Importance: Pregnant women are at increased risk of severe COVID-19, but the contribution of viral RNA load, the presence of infectious virus, and mucosal antibody responses remain understudied. Objective: To evaluate the association of COVID-19 outcomes following confirmed infection with vaccination status, mucosal antibody responses, infectious virus recovery and viral RNA levels in pregnant compared with non-pregnant women. Design: A retrospective observational cohort study of remnant clinical specimens from SARS-CoV-2 infected patients between October 2020-May 2022. Setting: Five acute care hospitals within the Johns Hopkins Health System (JHHS) in the Baltimore, MD-Washington, DC area. Participants: Participants included confirmed SARS-CoV-2 infected pregnant women and matched non-pregnant women (matching criteria included age, race/ethnicity, and vaccination status). Exposure: SARS-CoV-2 infection, with documentation of SARS-CoV-2 mRNA vaccination. Main Outcomes: The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. Clinical outcomes were compared using odds ratios (OR), and measures of virus and antibody were compared using either Fisher's exact test, two-way ANOVA, or regression analyses. Results were stratified according to pregnancy, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant. Resultss: A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant women were at increased risk of hospitalization (OR = 4.2; CI = 2.0-8.6), ICU admittance, (OR = 4.5; CI = 1.2-14.2), and of being placed on supplemental oxygen therapy (OR = 3.1; CI =13-6.9). An age-associated decrease in anti-S IgG titer and corresponding increase in viral RNA levels (P< 0.001) was observed in vaccinated pregnant, but not non-pregnant, women. Individuals in their 3rd trimester had higher anti-S IgG titers and lower viral RNA levels (P< 0.05) than those in their 1st or 2nd trimesters. Pregnant individuals experiencing breakthrough infections due to the omicron variant had reduced anti-S IgG compared to non-pregnant women (P< 0.05). Conclusions and Relevance: In this cohort study, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant were each identified as drivers of differences in mucosal anti-S IgG responses in pregnant compared with non-pregnant women. Observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity may be important for protection of this at-risk population.

Enterovirus characterized from cerebrospinal fluid in a cohort from the Eastern United States.

BACKGROUND: Enteroviruses (EVs) are predominant causes of a spectrum of neurological diseases. To better understand the origins of the outbreaks of disease associated with EV, it is essential to develop an efficient surveillance system that identifies the circulating EVs and correlate their genomic evolution with the disease presentations. METHODS: The clinical presentations of patients with positive EV from cerebrospinal fluid (CSF) between 2014 and 2022, diagnosed at the Johns Hopkins Medical Microbiology Laboratory, were compared from year to year. EV typing and whole genome sequencing were performed and correlated to the spectrum of disease. RESULTS: A total of 95 CSF specimens were positive for EV between 2014 and 2022. The percentage positivity ranged from the lowest of 1.1% in 2020 to the highest of 3.2% in 2015. The median ages declined from 22 years in 2014 to less than one year starting in 2016 to 34 in 2022. Typing using VP1 sequencing revealed that E30 and E6 were associated with meningitis in adults but coxsackieviruses (CVs-B3 and B5) were detected from pediatric patients with fever. Whole genome sequencing revealed multiple recombination events. In 2020, a recombinant CV-A9 was detected in a CSF sample associated with unusual presentation of sepsis, profound acute bilateral sensory neural hearing loss, and myofasciitis. CONCLUSIONS: EV genomic surveillance is needed for a better understanding of the genetic determinants of neurovirulence. Whole genome sequencing can reveal recombination events missed by traditional molecular surveillance methods.

An increase in enterovirus D68 circulation and viral evolution during a period of increased influenza like illness, The Johns Hopkins Health System, USA, 2022.

BACKGROUND: An increase in influenza like illness in children and adolescents at the Johns Hopkins Health system during summer 2022 was associated with increased positivity for enterovirus/ rhinovirus. We sought to characterize the epidemiology and viral evolution of enterovirus D68 (EV-D68). METHODS: A cohort of remnant respiratory samples tested at the Johns Hopkins Microbiology Laboratory was screened for EV-D68. EV-D68 positives were characterized by whole genome sequencing and viral loads were assessed by droplet digital PCR (ddPCR). Genomic changes and viral loads were analyzed along with patients' clinical presentations. RESULTS: Of 566 screened samples, 126 were EV-D68 (22.3%). The median age of EV-D68 infected patients was four years, a total of 52 required supplemental oxygen (41.3%), and 35 (27.8%) were admitted. Lung disease was the most frequent comorbidity that was associated with hospitalization. A total of 75 complete and 32 partial genomes were characterized that made a new cluster within the B3 subclade that was closest to US genomes from 2018. Amino acid changes within the BC and DE loops were identified from 31 genomes (29%) which correlated with an increase in average viral load in respiratory specimens and the need for supplemental oxygen. CONCLUSIONS: EV-D68 outbreaks continue to cause influenza like illness that could be overwhelming for the health system due to a significant demand for high flow oxygen. Viral evolution and an increase in the susceptible population are likely driving the trends of the increased EV-D68 infections.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Variants on Inpatient Clinical Outcome.

BACKGROUND: Prior observation has shown differences in COVID-19 hospitalization risk between SARS-CoV-2 variants, but limited information describes hospitalization outcomes. METHODS: Inpatients with COVID-19 at 5 hospitals in the eastern United States were included if they had hypoxia, tachypnea, tachycardia, or fever, and SARS-CoV-2 variant data, determined from whole-genome sequencing or local surveillance inference. Analyses were stratified by history of SARS-CoV-2 vaccination or infection. The average effect of SARS-CoV-2 variant on 28-day risk of severe disease, defined by advanced respiratory support needs, or death was evaluated using models weighted on propensity scores derived from baseline clinical features. RESULTS: Severe disease or death within 28 days occurred for 977 (29%) of 3369 unvaccinated patients and 269 (22%) of 1230 patients with history of vaccination or prior SARS-CoV-2 infection. Among unvaccinated patients, the relative risk of severe disease or death for Delta variant compared with ancestral lineages was 1.30 (95% confidence interval [CI]: 1.11-1.49). Compared with Delta, the risk for Omicron patients was .72 (95% CI: .59-.88) and compared with ancestral lineages was .94 (.78-1.1). Among Omicron and Delta infections, patients with history of vaccination or prior SARS-CoV-2 infection had half the risk of severe disease or death (adjusted hazard ratio: .40; 95% CI: .30-.54), but no significant outcome difference by variant. CONCLUSIONS: Although risk of severe disease or death for unvaccinated inpatients with Omicron was lower than with Delta, it was similar to ancestral lineages. Severe outcomes were less common in vaccinated inpatients, with no difference between Delta and Omicron infections.

Omicron Subvariants: Clinical, Laboratory, and Cell Culture Characterization.

BACKGROUND: The variant of concern Omicron has become the sole circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant for the past several months. Omicron subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 evolved over the time, with BA.1 causing the largest wave of infections globally in December 2021-January 2022. This study compared the clinical outcomes in patients infected with different Omicron subvariants and the relative viral loads and recovery of infectious virus from upper respiratory specimens. METHODS: SARS-CoV-2-positive remnant clinical specimens, diagnosed at the Johns Hopkins Microbiology Laboratory between December 2021 and July 2022, were used for whole-genome sequencing. The clinical outcomes of infections with Omicron subvariants were compared with infections with BA.1. Cycle threshold (Ct) values and the recovery of infectious virus on the VeroTMPRSS2 cell line from clinical specimens were compared. RESULTS: BA.1 was associated with the largest increase in SARS-CoV-2 positivity rate and coronavirus disease 2019 (COVID-19)-related hospitalizations at the Johns Hopkins system. After a peak in January, cases decreased in the spring, but the emergence of BA.2.12.1 followed by BA.5 in May 2022 led to an increase in case positivity and admissions. BA.1 infections had a lower mean Ct value when compared with other Omicron subvariants. BA.5 samples had a greater likelihood of having infectious virus at Ct values <20. CONCLUSIONS: Omicron subvariants continue to be associated with a relatively high rate of polymerase chain reaction (PCR) positivity and hospital admissions. The BA.5 infections are more while BA.2 infections are less likely to have infectious virus, suggesting potential differences in infectibility during the Omicron waves.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) exposure investigations using genomic sequencing among healthcare workers and patients in a large academic center.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) transmissions among healthcare workers and hospitalized patients are challenging to confirm. Investigation of infected persons often reveals multiple potential risk factors for viral acquisition. We combined exposure investigation with genomic analysis confirming 2 hospital-based clusters. Prolonged close contact with unmasked, unrecognized infectious, individuals was a common risk.

Sinonasal Amoebiasis: An Unexpected Cause of Sinonasal Necroinflammatory Disease.

While amoebic infection is widely known as a cause of gastroenteritis, keratitis, and meningoencephalitis, amoebae are challenging to recognize at unexpected sites. Despite multiple case reports of sinonasal amoebiasis, amoebic infection is not regularly considered in the differential diagnosis of sinonasal necroinflammatory disease. Here, we aim to characterize the pathologic features of sinonasal amoebiasis to facilitate better recognition. We identified sinonasal amoebiasis in 4 men, median age of 67 years (range: 37 to 71 y). All were immunocompromised, including 2 with chronic lymphocytic leukemia, 1 with human immunodeficiency virus, and 1 with human immunodeficiency virus and kidney transplant. Patients presented with nasal mucosal necrosis or polypoid masses, with facial ulceration in 1 patient and distant dermal nodules in another. Biopsies displayed extensive necrotic debris and inflammation. Although amoebic cysts were abundant in 3 cases, they were mistaken for yeast at frozen section in 1 case; 1 case showed only rare trophozoites that were not recognized on initial biopsy. Periodic acid Schiff and Grocott Methenamine Silver stains highlighted the organisms, and polymerase chain reaction confirmed Acanthamoeba species in 3 cases tested. 2 patients responded well to antiprotozoal medications, but 2 died of disease. Overall, sinonasal amoebiasis presents as a necroinflammatory process in patients immunocompromised for various reasons. Amoebae can mimic other organisms or be incredibly scarce, requiring active consideration to recognize amoebiasis and differentiate it from overlapping conditions like invasive fungal sinusitis, granulomatosis with polyangiitis, and natural killer/T-cell lymphoma. Because sinonasal amoebiasis is highly treatable when diagnosed promptly, pathologists play a critical role in the recognition of this rare necroinflammatory disease.

SARS-CoV-2 reinfections during the Delta and Omicron waves.

BACKGROUNDIncreased SARS-CoV-2 reinfection rates have been reported recently, with some locations basing reinfection on a second positive PCR test at least 90 days after initial infection. In this study, we used Johns Hopkins SARS-CoV-2 genomic surveillance data to evaluate the frequency of sequencing-validated, confirmed, and inferred reinfections between March 2020 and July 2022.METHODSPatients who had 2 or more positive SARS-CoV-2 tests in our system, with samples sequenced as a part of our surveillance efforts, were identified as the cohort for our study. SARS-CoV-2 genomes of patients' initial and later samples were compared.RESULTSA total of 755 patients (920 samples) had a positive test at least 90 days after the initial test, with a median time between tests of 377 days. Sequencing was attempted on 231 samples and was successful in 127. Rates of successful sequencing spiked during the Omicron surge; there was a higher median number of days from initial infection in these cases compared with those with failed sequences. A total of 122 (98%) patients showed evidence of reinfection; 45 of these patients had sequence-validated reinfection and 77 had inferred reinfections (later sequencing showed a clade that was not circulating when the patient was initially infected). Of the 45 patients with sequence-validated reinfections, 43 (96%) had reinfections that were caused by the Omicron variant, 41 (91%) were symptomatic, 32 (71%) were vaccinated prior to the second infection, 6 (13%) were immunosuppressed, and only 2 (4%) were hospitalized.CONCLUSIONSequence-validated reinfections increased with the Omicron surge but were generally associated with mild infections.FUNDINGFunding was provided by the Johns Hopkins Center of Excellence in Influenza Research and Surveillance (HHSN272201400007C), CDC (75D30121C11061), Johns Hopkins University President's Fund Research Response, Johns Hopkins Department of Pathology, and the Maryland Department of Health.

Omicron Subvariants: Clinical, Laboratory, and Cell Culture Characterization.

Background: The variant of concern, Omicron, has become the sole circulating SARS-CoV-2 variant for the past several months. Omicron subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 evolved over the time, with BA.1 causing the largest wave of infections globally in December 2021- January 2022. In this study, we compare the clinical outcomes in patients infected with different Omicron subvariants and compare the relative viral loads, and recovery of infectious virus from upper respiratory specimens. Methods: SARS-CoV-2 positive remnant clinical specimens, diagnosed at the Johns Hopkins Microbiology Laboratory between December 2021 and July 2022, were used for whole genome sequencing. The clinical outcomes of infections with Omicron subvariants were compared to infections with BA.1. Cycle threshold values (Ct) and the recovery of infectious virus on VeroTMPRSS2 cell line from clinical specimens were compared. Results: The BA.1 was associated with the largest increase in SARS-CoV-2 positivity rate and COVID-19 related hospitalizations at the Johns Hopkins system. After a peak in January cases fell in the spring, but the emergence of BA.2.12.1 followed by BA.5 in May 2022 led to an increase in case positivity and admissions. BA.1 infections had a lower mean Ct when compared to other Omicron subvariants. BA.5 samples had a greater likelihood of having infectious virus at Ct values less than 20. Conclusions: Omicron subvariants continue to associate with a relatively high positivity and admissions. The BA.5 infections are more while BA.2 infections are less likely to have infectious virus, suggesting potential differences in infectibility during the Omicron waves. Funding: Centers for Disease Control and Prevention contract 75D30121C11061, NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, and The Modeling Infectious Diseases in Healthcare Network (MInD) under awards U01CK000589.

Combination Therapy With Casirivimab/Imdevimab and Remdesivir for Protracted SARS-CoV-2 Infection in B-cell-Depleted Patients.

Profoundly B-cell-depleted patients can have prolonged severe acute respiratory syndrome coronavirus 2 infections with evidence of active viral replication, due to inability to mount an adequate humoral response to clear the virus. We present 3 B-cell-depleted patients with prolonged coronavirus disease 2019 infection who were successfully treated with a combination of casirivimab/imdevimab and remdesivir.

Sequence Proven Reinfections with SARS-CoV-2 at a Large Academic Center.

Background: Increased reinfection rates with SARS-CoV-2 have recently been reported, with some locations basing reinfection on a second positive PCR test at least 90 days after initial infection. Methods: We identified cases where patients had two positive tests for SARS-CoV-2 and evaluated which of these had been sequenced as part of our surveillance efforts, and evaluated sequencing and clinical data. Results: 750 patients (920 samples) had a positive test at least 90 days after the initial test. The median time between tests was 377 days, and 724 (79%) of the post 90-day positives were collected after the emergence of the Omicron variant in November 2021. Sequencing was attempted on 231 samples and successful in 127. Successful sequencing spiked during the Omicron surge and showed higher median days from initial infection compared to failed sequences (median 398 days compared to 276 days, p<0.0005). A total of 122 (98%) patients showed evidence of reinfection, 45 of which had sequence proven reinfection and 77 had inferred reinfections (later sequence showed a clade that was not circulating when the patient was initially infected). Children accounted for only 4% of reinfections. 43 (96%) of 45 infections with sequence proven reinfection were caused by the Omicron variant, 41 (91%) were symptomatic, 32 (71%), were vaccinated prior to the second infection, and 6 (13%) were Immunosuppressed. Only 2 (4%) were hospitalized, and both had underlying conditions. Conclusion: Sequence proven reinfections increased with the Omicron variant but generally caused mild infections.

Circulation of Enterovirus D68 during Period of Increased Influenza-Like Illness, Maryland, USA, 2021.

We report enterovirus D68 circulation in Maryland, USA, during September-October 2021, which was associated with a spike in influenza-like illness. The characterized enterovirus D68 genomes clustered within the B3 subclade that circulated in 2018 in Europe and the United States.

The displacement of the SARS-CoV-2 variant Delta with Omicron: An investigation of hospital admissions and upper respiratory viral loads.

BACKGROUND: The increase in SARS-CoV-2 infections in December 2021 was driven primarily by the Omicron variant, which largely displaced the Delta over a three-week span. Outcomes from infection with Omicron remain uncertain. We evaluated whether clinical outcomes and viral loads differed between Delta and Omicron infections during the period when both variants were co-circulating. METHODS: In this retrospective observational cohort study, remnant clinical specimens, positive for SARS-CoV-2 after standard of care testing at the Johns Hopkins Microbiology Laboratory, between the last week of November and the end of December 2021, were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. FINDINGS: The Omicron variant displaced Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N = 1,119) were more likely to be vaccinated compared to patients with Delta (N = 908), but were less likely to be admitted (0.33 CI 0.21-0.52), require ICU level care (0.38 CI 0.17-0.87), or succumb to infection (0.26 CI 0.06-1.02) regardless of vaccination status. There was no statistically significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. INTERPRETATION: Compared to Delta, Omicron was more likely to cause breakthrough infections of vaccinated individuals, yet admissions were less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing Omicron transmission are required as, though the admission risk might be lower, the increased numbers of infections cause large numbers of hospitalizations. FUNDING: NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061, and The Modeling Infectious Diseases in Healthcare Network (MInD) under awards U01CK000589.

SARS-CoV-2 infections in mRNA vaccinated individuals are biased for viruses encoding spike E484K and associated with reduced infectious virus loads that correlate with respiratory antiviral IgG levels.

INTRODUCTION: COVID-19 large scale immunization in the US has been associated with breakthrough positive molecular testing. In this study, we investigated whether a positive test is associated with a high anti-viral IgG, specific viral variant, recovery of infectious virus, or symptomatic infection during an early phase after vaccination rollout. METHODS: We identified 133 SARS-CoV-2 positive patients who had received two doses of either Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) vaccines, the 2nd of which was received between January and April of 2021. The positive samples were collected between January and May of 2021. Samples were sequenced to characterize the whole genome and Spike protein changes and cycle thresholds that reflect viral loads were determined using a single molecular assay. Respiratory SARS-CoV-2 IgG antibodies were examined using ELISA and specimens were grown on cell culture to assess the recovery of infectious virus as compared to a control unvaccinated cohort. RESULTS: Of 133 specimens, 24 failed sequencing and yielded a negative or very low viral load on the repeat PCR. Of 109 specimens that were used for further genome analysis, 68 (62.4%) were from symptomatic infections, 11 (10.1%) were admitted for COVID-19, and 2 (1.8%) required ICU admission with no associated mortality. The predominant virus variant was the Alpha (B.1.1.7), however a significant association between lineage B.1.526 and amino acid change S: E484K with positives after vaccination was noted. A significant reduction of the recovery of infectious virus on cell culture was accompanied by an increase in localized IgG levels in respiratory samples of vaccinated individuals. CONCLUSIONS: Vaccination reduces the recovery of infectious virus in breakthrough infections caused primarily by the Alpha variant accompanied by an increase in upper respiratory tract IgG levels.

Large Scale SARS-CoV-2 Molecular Testing and Genomic Surveillance Reveal Prolonged Infections, Protracted RNA shedding, and Viral Reinfections.

Large-scale SARS-CoV-2 molecular testing coupled with whole genome sequencing in the diagnostic laboratories is instrumental for real-time genomic surveillance. The extensive genomic, laboratory, and clinical data provide a valuable resource for understanding cases of reinfection versus prolonged RNA shedding and protracted infections. In this study, data from a total of 22,292 clinical specimens, positive by SARS-CoV-2 molecular diagnosis at Johns Hopkins clinical virology laboratory between March 11th 2020 to September 23rd 2021, were used to identify patients with two or more positive results. A total of 3,650 samples collected from 1,529 patients who had between 2 and 20 positive results were identified in a time frame that extended up to 403 days from the first positive. Cycle threshold values (Ct) were available for 1,622 samples, the median of which was over 30 by 11 days after the first positive. Extended recovery of infectious virus on cell culture was notable for up to 70 days after the first positive in immunocompromised patients. Whole genome sequencing data generated as a part of our SARS-CoV-2 genomic surveillance was available for 1,027 samples from patients that had multiple positive tests. Positive samples collected more than 10 days after initial positive with high quality sequences (coverage >90% and mean depth >100), were more likely to be from unvaccinated, or immunosuppressed patients. Reinfections with viral variants of concern were found in 3 patients more than 130 days from prior infections with a different viral clade. In 75 patients that had 2 or more high quality sequences, the acquisition of more substitutions or deletions was associated with lack of vaccination and longer time between the recovered viruses. Our study highlights the value of integrating genomic, laboratory, and clinical data for understanding the biology of SARS-CoV-2 as well as for setting a precedent for future epidemics and pandemics.

Bma-LAD-2, an Intestinal Cell Adhesion Protein, as a Potential Therapeutic Target for Lymphatic Filariasis.

Lymphatic filariasis is a debilitating disease that afflicts over 70 million people worldwide. It is caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Despite substantial success, efforts to eliminate LF will likely require more time and resources than predicted. Identifying new drug and vaccine targets in adult filariae could help elimination efforts. This study's aim was to evaluate intestinal proteins in adult Brugia malayi worms as possible therapeutic targets. Using short interfering RNA (siRNA), we successfully targeted four candidate gene transcripts: Bma-Serpin, Bma-ShTK, Bma-Reprolysin, and Bma-LAD-2. Of those, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was determined to be essential for adult worm survival. We observed a 70.42% knockdown in Bma-LAD-2 transcript levels 1 day post-siRNA incubation and an 87.02% reduction in protein expression 2 days post-siRNA incubation. This inhibition of Bma-LAD-2 expression resulted in an 80% decrease in worm motility over 6 days, a 93.43% reduction in microfilaria release (Mf) by day 6 post-siRNA incubation, and a dramatic decrease in (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Transmission electron microscopy revealed the loss of microvilli and unraveling of mitochondrial cristae in the intestinal epithelium of Bma-LAD-2 siRNA-treated worms. Strikingly, Bma-LAD-2 siRNA-treated worms exhibited an almost complete loss of pseudocoelomic fluid. A luciferase immunoprecipitation system assay did not detect anti-Bma-LAD-2 IgE in the serum of 30 LF patients, indicating that LF exposure does not result in IgE sensitization to this antigen. These results indicate that Bma-LAD-2 is an essential protein for adult Brugia malayi and may be an effective therapeutic target. IMPORTANCE Brugia malayi is a parasitic nematode that can cause lymphatic filariasis, a debilitating disease prevalent in tropical and subtropical countries. Significant progress has been made toward eliminating the disease. However, complete eradication may require new therapeutics such as drugs or a vaccine that kill adult filariae. In this study, we identified an immunoglobulin superfamily cell adhesion molecule (Bma-LAD-2) as a potential drug and vaccine candidate. When we knocked down Bma-LAD-2 expression, we observed a decrease in worm motility, fecundity, and metabolism. We also visualized the loss of microvilli, destruction of the mitochondria in the intestinal epithelium, and loss of pseudocoelomic fluid contents after Bma-LAD-2 siRNA treatment. Finally, we demonstrated that serum from filaria-infected patients does not contain preexisting IgE to Bma-LAD-2, which indicates that this antigen would be safe to administer as a vaccine in populations where the disease is endemic.

A Quick Displacement of the SARS-CoV-2 variant Delta with Omicron: Unprecedented Spike in COVID-19 Cases Associated with Fewer Admissions and Comparable Upper Respiratory Viral Loads.

BACKGROUND: The increase in SARS-CoV-2 infections in December 2021 in the United States was driven primarily by the Omicron variant which largely displaced the Delta over a three week span. Outcomes from infection with the Omicron remain uncertain. We evaluate whether clinical outcomes and viral loads differ between Delta and Omicron infections during the period when both variants were co-circulating. METHODS: Remnant clinical specimens from patients that tested positive for SARS-CoV-2 after standard of care testing between the last week of November and the end of December 2021were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. RESULTS: The Omicron variant displaced the Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N= 1121) were more likely to be vaccinated compared to patients with Delta (N = 910), but were less likely to be admitted, require ICU level care, or succumb to infection regardless of vaccination status. There was no significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. CONCLUSIONS: Omicron infections of vaccinated individuals are expected, yet admissions are less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing the Omicron transmission are required as even though the admission risk is lower, the numbers of infections continue to be high. RESEARCH IN CONTEXT EVIDENCE BEFORE THIS STUDY: The unprecedented increase in COVID-19 cases in the month of December 2021, associated with the displacement of the Delta variant with the Omicron, triggered a lot of concerns. An understanding of the disease severity associated with infections with Omicron is essential as well as the virological determinants that contributed to its widespread predominance. We searched PubMed for articles published up to January 23, 2022, using the search terms ("Omicron") AND ("Disease severity") as well as ("Omicron") AND ("Viral load") And/ or ("Cell culture"). Our search yielded 3 main studies that directly assessed the omicron's clinical severity in South Africa, its infectious viral load compared to Delta, and the dynamics of viral RNA shedding. In South Africa, compared to Delta, Omicron infected patients showed a significant reduction in severe disease. In this study, Omicron and non-Omicron variants were characterized based on S gene target failure using the TaqPath COVID-19 PCR (Thermo Fisher Scientific). In the study from Switzerland that assessed the infectious viral load in Omicron versus Delta, the authors analyzed only 18 Omicron samples that were all from vaccinated individuals to show that compared to Delta, Omicron had equivalent infectious viral titers. The third study that assessed the Omicron viral dynamics showed that the peak viral RNA in Omicron infections is lower than Delta. No published studies assessed the clinical discrepancies of Omicron and Delta infected patients from the US, nor comprehensively assessed, by viral load and cell culture studies, the characteristics of both variants stratified by vaccination status. ADDED VALUE OF THIS STUDY: To the best of our knowledge, this is the only study to date to compare the clinical characteristics and outcomes after infection with the Omicron variant compared to Delta in the US using variants characterized by whole genome sequencing and a selective time frame when both variant co-circulated. It is also the first study to stratify the analysis based on the vaccination status and to compare fully vaccinated patients who didn't receive a booster vaccination to patients who received a booster vaccination. In addition, we provide a unique viral RNA and infectious virus load analyses to compare Delta and Omicron samples from unvaccinated, fully vaccinated, and patients with booster vaccination. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: Omicron associated with a significant increase in infections in fully and booster vaccinated individuals but with less admissions and ICU level care. Admitted patients showed similar requirements for supplemental oxygen and ICU level care when compared to Delta admitted patients. Viral loads were similar in samples from Omicron and Delta infected patients regardless of the vaccination status. The recovery of infectious virus on cell culture was reduced in samples from patients infected with Delta who received a booster dose, but this was not the case with Omicron. The recovery of infectious virus was equivalent in Omicron infected unvaccinated, fully vaccinated, and samples from patients who received booster vaccination. FUNDING: NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061.

Impact of SARS-CoV-2 variants on inpatient clinical outcome.

BACKGROUND: Prior observation has shown differences in COVID-19 hospitalization rates between SARS-CoV-2 variants, but limited information describes differences in hospitalization outcomes. METHODS: Patients admitted to 5 hospitals with COVID-19 were included if they had hypoxia, tachypnea, tachycardia, or fever, and data to describe SARS-CoV-2 variant, either from whole genome sequencing, or inference when local surveillance showed ≥95% dominance of a single variant. The average effect of SARS-CoV-2 variant on 14-day risk of severe disease, defined by need for advanced respiratory support, or death was evaluated using models weighted on propensity scores derived from baseline clinical features. RESULTS: Severe disease or death within 14 days occurred for 950 of 3,365 (28%) unvaccinated patients and 178 of 808 (22%) patients with history of vaccination or prior COVID-19. Among unvaccinated patients, the relative risk of 14-day severe disease or death for Delta variant compared to ancestral lineages was 1.34 (95% confidence interval [CI] 1.13-1.55). Compared to Delta variant, this risk for Omicron patients was 0.78 (95% CI 0.62-0.97) and compared to ancestral lineages was 1.04 (95% CI 0.84-1.24). Among Omicron and Delta infections, patients with history of vaccination or prior COVID-19 had one-half the 14-day risk of severe disease or death (adjusted hazard ratio 0.46, IQR 0.34-0.62) but no significant outcome difference between Delta and Omicron infections. CONCLUSIONS: Although the risk of severe disease or death for unvaccinated patients with Omicron was lower than Delta, it was similar to ancestral lineages. Severe outcomes were less common in vaccinated patients, but there was no difference between Delta and Omicron infections.