Mpox Recurrence and Tecovirimat Resistance in a Patient With Advanced Human Immunodeficiency Virus Disease.

We present a case of mpox recurrence in a transgender woman with AIDS. Her recurrent lesions required several courses of antiviral therapy over a 5-month period and her monkeypox viral genome was subsequently noted to have tecovirimat resistance mutations. Interestingly, she developed a robust orthopoxvirus-specific T-cell response.

SARS-CoV-2 Variants from Long-Term, Persistently Infected Immunocompromised Patients Have Altered Syncytia Formation, Temperature-Dependent Replication, and Serum Neutralizing Antibody Escape.

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

Respiratory virus disease and outcomes at a large academic medical center in the United States: a retrospective observational study of the early 2023/2024 respiratory viral season.

Respiratory disease, attributed to influenza, respiratory syncytial virus (RSV), and SARS-CoV-2, was reported nationally during the 2023/2024 respiratory viral season. The emergence of novel SARS-CoV-2 variants was considered a significant factor contributing to the rise in COVID-19 cases. Data from the Johns Hopkins Hospital System (JHHS) showed that enterovirus/rhinovirus had also been circulating at high rates. Analyzing clinical outcomes of the most prevalent respiratory viruses is crucial for understanding the role of circulating viral genotypes. A retrospective cohort of patients who tested positive for SARS-CoV-2, influenza, RSV, or enterovirus/rhinovirus between 1 June and 31 December 2023 was included in the study. Remnant clinical samples were utilized for targeted viral whole-genome sequencing and genotyping. Patients' metadata and outcomes following infection were studied, stratified by viral variants and genotypes. The increase of SARS-CoV-2 positivity in December was associated with the predominance of JN.1. Admissions for patients under 18 years old were primarily associated with enterovirus/rhinovirus and RSV, while older age groups were mainly linked to SARS-CoV-2 and influenza infections. SARS-CoV-2-related admissions increased with the predominance of the JN.1 variant in December. No significant difference in admissions for influenza subtypes, rhinovirus species, or SARS-CoV-2 variants was observed. RSV A was associated with slightly higher odds of admission compared with RSV B. Our data highlight the importance of systematically analyzing respiratory viral infections to inform public health strategies and clinical management, especially as SARS-CoV-2 becomes endemic. The findings highlight the value of expanded genomic surveillance in elucidating the clinical significance of viral evolution.IMPORTANCEThe analysis of the epidemiology and clinical outcomes of multiple co-circulating respiratory viruses in the early 2023/2024 respiratory virus season highlights the emergence of the SARS-CoV-2 JN.1 variant as well as underscores the importance of enterovirus/rhinovirus in respiratory infections. Understanding these dynamics is essential for refining public health strategies and clinical management, especially as SARS-CoV-2 transitions to an endemic status. This work emphasizes the need for ongoing surveillance, robust diagnostic algorithms, and detailed genomic analyses to anticipate and mitigate the burden of respiratory viral infections, ultimately contributing to more informed decision-making in healthcare settings and better patient outcomes.

Genomic evolution of influenza during the 2023-2024 season, the johns hopkins health system.

Influenza, a human disease caused by viruses in the Orthomyxoviridae family, is estimated to infect 5% -10 % of adults and 20% -30 % of children annually. Influenza A (IAV) and Influenza B (IBV) viruses accumulate amino acid substitutions (AAS) in the hemagglutinin (HA) and neuraminidase (NA) proteins seasonally. These changes, as well as the dominating viral subtypes, vary depending on geographical location, which may impact disease prevalence and the severity of the season. Genomic surveillance is crucial for capturing circulation patterns and characterizing AAS that may affect disease outcomes, vaccine efficacy, or antiviral drug activities. In this study, whole-genome sequencing of IAV and IBV was attempted on positive remnant clinical samples (587) collected from 580 patients between June 2023 and February 2024 in the Johns Hopkins Health System (JHHS). Full-length HA segments were obtained from 424 (72.2 %) samples. H1N1pdm09 (71.7 %) was the predominant IAV subtype, followed by H3N2 (16.7 %) and IBV-Victoria clade V1A.3a.2 (11.6 %). Within H1N1pdm09 HA sequences, the 6B1A.5a.2a.1 (60.5 %) clade was the most represented. Full-length NA segments were obtained from 421 (71.7 %) samples. Within H1N1pdm09 and IBV, AAS previously proposed to change susceptibility to NA inhibitors were infrequently detected. Phylogeny of HA and NA demonstrated heterogeneous HA and NA H1N1pdm09 and IBV subclades. No significant differences were observed in admission rates or use of supplemental oxygen between different subtypes or clades. Influenza virus genomic surveillance is essential for understanding the seasonal evolution of influenza viruses and their association with disease prevalence and outcomes.

Genomic Evolution and Surveillance of Respiratory Syncytial Virus during the 2023-2024 Season.

Respiratory syncytial virus (RSV) is a significant cause of morbidity, particularly in infants. This study describes RSV genomic diversity and disease outcomes during the 2023-2024 season in the Johns Hopkins Hospital System (JHHS). Between August and December 2023, 406 patient samples were sequenced, showing that RSV-B GB5.0.5a was the dominant genotype detected. RSV-A genotype GA2.3.5 was detected less frequently. Metadata analysis of patient data revealed that, although RSV-B was more commonly detected, patients with RSV-A infections were more frequently hospitalized. Analysis of both the G- and F-genes revealed multiple amino acid substitutions in both RSV-A and RSV-B, with some positions within the F-protein that could be associated with evasion of antibody responses. Phylogenetic analysis revealed the genetic diversity of circulating GB5.0.5a and GA2.3.5 genotypes. This study serves as an important baseline for genomic surveillance of RSV within the JHHS and will assist in characterizing the impact of the newly approved RSV vaccines on RSV genomic evolution and the emergence of escape mutations.

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.

Increased circulation of human adenovirus in 2023: an investigation of the circulating genotypes, upper respiratory viral loads, and hospital admissions in a large academic medical center.

IMPORTANCE: The circulation of human adenoviruses (HAdV) increased in 2023. In this manuscript, we show that HAdV-B3 was predominant in 2023 in a cohort characterized by the Johns Hopkins Hospital System. We also show that HAdV-B3 was associated with an increase in viral loads in respiratory samples and provide a correlation with the clinical presentations and outcomes.

Risk Factors for Hospitalization and Effect of Immunosuppression on Clinical Outcomes Among an Urban Cohort of Patients With Mpox.

Background: During the 2022 mpox outbreak most patients were managed as outpatients, but some required hospitalization. Uncontrolled human immunodeficiency virus (HIV) has been identified as a risk factor for severe mpox. Methods: Patients with mpox diagnosed or treated within the Johns Hopkins Health System between 1 June and 15 December 2022 were included. The primary outcome of interest was risk of hospitalization. Demographic features, comorbid conditions, treatment, and clinical outcomes were determined. Results: A total of 353 patients were tested or treated for mpox; 100 had mpox diagnosed or treated (median age, 35.3 years; 97.0% male; 57.0% black and 10.0% Hispanic; 46.0% people with HIV [PWH]). Seventeen patients (17.0%) required hospitalization, 10 of whom were PWH. Age >40 years, race, ethnicity, HIV status, insurance status, and body mass index >30 (calculated as weight in kilograms divided by height in meters squared) were not associated with hospitalization. Eight of 9 patients (88.9%) with immunosuppression were hospitalized. Immunosuppression was associated with hospitalization in univariate (odds ratio, 69.3 [95% confidence interval, 7.8-619.7]) and adjusted analysis (adjusted odds ratio, 94.8 [8.5-1060.1]). Two patients (11.8%) who were hospitalized required intensive care unit admission and died; both had uncontrolled HIV infection and CD4 T-cell counts <50/µL. Median cycle threshold values for the first positive mpox virus sample did not differ between those who were hospitalized and those who were not. Conclusions: Immunosuppression was a significant risk factor for hospitalization with mpox. PWH with CD4 T-cell counts <50/µL are at high risk of death due to mpox infection. Patients who are immunosuppressed should be considered for early and aggressive treatment of mpox, given the increased risk of hospitalization.

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.

Influenza A Virus in Pigs in Senegal and Risk Assessment of Avian Influenza Virus (AIV) Emergence and Transmission to Human.

We conducted an active influenza surveillance in the single pig slaughterhouse in Dakar to investigate the epidemiology and genetic characteristics of influenza A viruses (IAVs) and to provide serologic evidence of avian influenza virus (AIV) infection in pigs at interfaces with human populations in Senegal. Nasal swab and blood samples were collected on a weekly basis from the same animal immediately after slaughter. Influenza A viruses were diagnosed using RT-qPCR and a subset of positive samples for H3 and H1 subtypes were selected for full genome amplification and NGS sequencing. Serum samples were tested by HI assay for the detection of antibodies recognizing four AIVs, including H9N2, H5N1, H7N7 and H5N2. Between September 2018 and December 2019, 1691 swine nasal swabs were collected and tested. Influenza A virus was detected in 30.7% (520/1691), and A/H1N1pdm09 virus was the most commonly identified subtype with 38.07% (198/520), followed by A/H1N2 (16.3%) and A/H3N2 (5.2%). Year-round influenza activity was noted in pigs, with the highest incidence between June and September. Phylogenetic analyses revealed that the IAVs were closely related to human IAV strains belonging to A/H1N1pdm09 and seasonal H3N2 lineages. Genetic analysis revealed that Senegalese strains possessed several key amino acid changes, including D204 and N241D in the receptor binding site, S31N in the M2 gene and P560S in the PA protein. Serological analyses revealed that 83.5% (95%CI = 81.6-85.3) of the 1636 sera tested were positive for the presence of antibodies against either H9N2, H5N1, H7N7 or H5N2. Influenza H7N7 (54.3%) and H9N2 (53.6%) were the dominant avian subtypes detected in Senegalese pigs. Given the co-circulation of multiple subtypes of influenza viruses among Senegalese pigs, the potential exists for the emergence of new hybrid viruses of unpredictable zoonotic and pandemic potential in the future.

Dissemination and genome characterization of a human adenovirus F41 in a patient with B-Cell lymphoma.

Adenovirus (HAdV) F41 is a common cause of gastroenteritis and has rarely been reported associated with disseminated disease. In this report, an adult patient with a history of ulcerative colitis, cryptogenic cirrhosis, stage III adenocarcinoma, high-grade diffuse large B-cell lymphoma on chemotherapy was diagnosed with disseminated adenovirus infection. HAdV DNA was quantified in stool, plasma, and urine with viral loads of 7, 4, and 3 log10 copies/mL, respectively. The patient's course was rapidly progressive and he passed away 2 days after initiation of antiviral therapy. The patient's infecting virus was characterized as HAdV-F41 by whole genome sequencing.

Severity outcomes associated with SARS-CoV-2 XBB variants, an observational analysis.

The rapidity with which SARS-CoV-2 XBB variants rose to predominance has been alarming. We used a large cohort of patients diagnosed with Omicron infections between September 2022 and mid-February 2023 to evaluate the likelihood of admission or need for supplemental oxygen in patients infected with XBB variants. Our data showed no significant association between XBB or XBB.1.5 infections and admissions. Older age groups, lack of vaccination, immunosuppression and underlying heart, kidney, and lung disease showed significant associations with hospitalization.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Respiratory Adenovirus Quantification with a Droplet Digital Polymerase Chain Reaction (ddPCR) Assay.

Human adenoviruses (HAdVs) are double-stranded DNA viruses that can cause a wide spectrum of disease, including respiratory infections. Little is known about the value of respiratory HAdV quantification and its correlation with disease severity. In this study, we developed a quantitative HAdV droplet digital PCR (ddPCR) assay to study the association between viral loads, circulating types, and clinical outcomes. Remnant respiratory specimens positive for HAdV after the standard of care testing were collected from December 2020 to April 2022. A total of 129 samples were tested by a ddPCR method. Typing was performed using Nanopore sequencing of the hexon gene hypervariable region. Clinical chart reviews were performed to correlate the viral loads with the disease severity. The ddPCR assay showed an analytical sensitivity and a lower limit of quantification below 100 copies/mL. Of 129 positive clinical samples, 100 were quantified by ddPCR, 7 were too concentrated to be quantified, and 22 were negative. Of the 22 false negatives, only 3 were successfully typed; however, 99 of the 107 positive samples had a characterized genotype. The main HAdV types identified in this cohort were C1 (49.5%) followed by C2 (34.3%). No significant difference in HAdV loads was noted between patients who were admitted, those who required supplemental oxygen, and outpatients or between different HAdV types. HAdV ddPCR is a reliable absolute quantification approach for HAdV from respiratory samples. HAdV loads at initial presentation does not appear to differ between patients who require hospitalization versus outpatients. IMPORTANCE Measuring viral load using droplet digital PCR (ddPCR) is an absolute quantification approach that can facilitate comparability between different laboratories. This approach could prove valuable in studies that focus on the clinical utility of quantification. In this study, we evaluate a human adenovirus (HAdV) ddPCR assay and study the relationship between viral loads and outcomes after HAdV respiratory infections.

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.

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.

Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.