Enhanced throughput of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR panel by assay multiplexing and specimen pooling.

A multiplex real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed based on the same primer and probe sequences of an existing U.S. CDC Emergency Use authorized test panel, targeting SARS-CoV-2 N1, N2 and human RNase P genes in singleplex. Both singleplex and multiplex assays demonstrated linear dynamic ranges of 8 orders of magnitude and analytical limits of detection of 5 RNA transcript copies/reaction. Both assays showed 100 % agreement with 364 previously characterized clinical specimens (146 positive and 218 negative) for detection of SARS-CoV-2 RNA. To further increase testing throughput, 40 positive and 20 negative four-specimen pools were tested by the multiplex assay and showed 97.75 % and 100 % congruence with individual specimen tests, respectively. rRT-PCR assay multiplexing and sample pooling, individually or in combination, can substantially increase throughput of SARS-CoV-2 testing.

Outbreaks of Adenovirus-associated Respiratory Illness on 5 College Campuses in the United States, 2018-2019.

BACKGROUND: Human adenoviruses (HAdVs) are commonly associated with acute respiratory illness. HAdV outbreaks are well documented in congregate military training settings, but less is known about outbreaks on college campuses. During fall 2018 and spring 2019, 5 United States (US) colleges reported increases in HAdV-associated respiratory illness. Investigations were performed to better understand HAdV epidemiology in this setting. METHODS: A case was defined as a student at one of the 5 colleges, with acute respiratory illness and laboratory-confirmed HAdV infection during October 2018-December 2018 or March-May 2019. Available respiratory specimens were typed by HAdV type-specific real-time polymerase chain reaction assays, and for a subset, whole genome sequencing was performed. We reviewed available medical records and cases were invited to complete a questionnaire, which included questions on symptom presentation, social history, and absenteeism. RESULTS: We identified 168 HAdV cases. Median age was 19 (range, 17-22) years and 102 cases (61%) were male. Eleven cases were hospitalized, 10 with pneumonia; 2 cases died. Among questionnaire respondents, 80% (75/94) missed ≥ 1 day of class because of their illness. Among those with a type identified (79%), HAdV types 4 and 7 were equally detected, with frequency of each varying by site. Genome types 4a1 and 7d were identified, respectively, by whole genome sequence analysis. CONCLUSIONS: HAdV respiratory illness was associated with substantial morbidity and missed class time among young, generally healthy adults on 5 US college campuses. HAdVs should be considered a cause of respiratory illness outbreaks in congregate settings such as college campuses.

Severe Acute Respiratory Syndrome Coronavirus 2 Prevalence, Seroprevalence, and Exposure among Evacuees from Wuhan, China, 2020.

To determine prevalence of, seroprevalence of, and potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a cohort of evacuees returning to the United States from Wuhan, China, in January 2020, we conducted a cross-sectional study of quarantined evacuees from 1 repatriation flight. Overall, 193 of 195 evacuees completed exposure surveys and submitted upper respiratory or serum specimens or both at arrival in the United States. Nearly all evacuees had taken preventive measures to limit potential exposure while in Wuhan, and none had detectable SARS-CoV-2 in upper respiratory tract specimens, suggesting the absence of asymptomatic respiratory shedding among this group at the time of testing. Evidence of antibodies to SARS-CoV-2 was detected in 1 evacuee, who reported experiencing no symptoms or high-risk exposures in the previous 2 months. These findings demonstrated that this group of evacuees posed a low risk of introducing SARS-CoV-2 to the United States.

US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10-1.5 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.

Outbreak of Respiratory Illness Associated With Human Adenovirus Type 7 Among Persons Attending Officer Candidates School, Quantico, Virginia, 2017.

A respiratory outbreak associated with human adenovirus type 7 (HAdV-7) occurred among unvaccinated officer candidates attending initial military training. Respiratory infections associated with HAdV-7 can be severe, resulting in significant morbidity. Genomic sequencing revealed HAdV-7d, a genome type recently remerging in the United States as a significant respiratory pathogen, following reports from Southeast Asia. Twenty-nine outbreak cases were identified; this likely represents an underestimate. Although the HAdV type 4 and 7 vaccine is currently given to US military enlisted recruit trainees, it is not routinely given to officer candidates. Administration of the HAdV type 4 and 7 vaccine may benefit this cohort.

Isolation and growth characterization of novel full length and deletion mutant human MERS-CoV strains from clinical specimens collected during 2015.

Middle East respiratory syndrome (MERS) is a viral respiratory illness first reported in Saudi Arabia in September 2012 caused by the human coronavirus (CoV), MERS-CoV. Using full-genome sequencing and phylogenetic analysis, scientists have identified three clades and multiple lineages of MERS-CoV in humans and the zoonotic host, dromedary camels. In this study, we have characterized eight MERS-CoV isolates collected from patients in Saudi Arabia in 2015. We have performed full-genome sequencing on the viral isolates, and compared them to the corresponding clinical specimens. All isolates were clade B, lineages 4 and 5. Three of the isolates carry deletions located on three independent regions of the genome in the 5'UTR, ORF1a and ORF3. All novel MERS-CoV strains replicated efficiently in Vero and Huh7 cells. Viruses with deletions in the 5'UTR and ORF1a exhibited impaired viral release in Vero cells. These data emphasize the plasticity of the MERS-CoV genome during human infection.

Respiratory viral surveillance of healthcare personnel and patients at an adult long-term care facility.

We conducted active surveillance of acute respiratory viral infections (ARIs) among residents and healthcare personnel (HCP) at a long-term care facility during the 2015-2016 respiratory illness season. ARIs were observed among both HCP and patients, highlighting the importance of including HCP in surveillance programs.

Middle East Respiratory Syndrome Coronavirus Infection Dynamics and Antibody Responses among Clinically Diverse Patients, Saudi Arabia.

Middle East respiratory syndrome coronavirus (MERS-CoV) shedding and antibody responses are not fully understood, particularly in relation to underlying medical conditions, clinical manifestations, and mortality. We enrolled MERS-CoV-positive patients at a hospital in Saudi Arabia and periodically collected specimens from multiple sites for real-time reverse transcription PCR and serologic testing. We conducted interviews and chart abstractions to collect clinical, epidemiologic, and laboratory information. We found that diabetes mellitus among survivors was associated with prolonged MERS-CoV RNA detection in the respiratory tract. Among case-patients who died, development of robust neutralizing serum antibody responses during the second and third week of illness was not sufficient for patient recovery or virus clearance. Fever and cough among mildly ill patients typically aligned with RNA detection in the upper respiratory tract; RNA levels peaked during the first week of illness. These findings should be considered in the development of infection control policies, vaccines, and antibody therapeutics.

Respiratory Illness Associated With Emergent Human Adenovirus Genome Type 7d, New Jersey, 2016-2017.

BACKGROUND: Human adenoviruses (HAdVs) are known causes of respiratory illness outbreaks in congregate settings, but cases and clusters are less well described from community settings in the United States. During December 2016-February 2017, the New Jersey Department of Health received reports of HAdV infections from 3 sources in 3 adjacent counties. We investigated to characterize the epidemiologic, laboratory, and clinical features of this HAdV outbreak. METHODS: A case was defined as a New Jersey resident with acute respiratory illness during December 1, 2016-March 31, 2017 with laboratory identification of HAdV genome type 7d (HAdV-7d). Human adenovirus was detected by real-time and conventional polymerase chain reaction and molecular typed by partial hexon capsid protein gene sequencing. The HAdV genome type was identified by whole genome sequencing analysis. Available medical, public health, and surveillance records were reviewed. RESULTS: We identified 12 cases, including 3 treatment facility patients, 7 college students, and 2 cases at a tertiary-care hospital. Four cases died; all had underlying comorbidities. Nine HAdV-7d whole genome sequences obtained from all 3 sites were nearly identical. CONCLUSIONS: Transmission of HAdV-7d occurred in community and congregate settings across 3 counties and resulted in severe morbidity and mortality in some cases with underlying comorbidities. Clinicians and local and state health departments should consider HAdV in patients with severe respiratory infection.

Outbreak of Acute Respiratory Illness Associated with Adenovirus Type 4 at the U.S. Naval Academy, 2016.

Human adenoviruses (HAdVs) are known to cause respiratory illness outbreaks at basic military training (BMT) sites. HAdV type-4 and -7 vaccines are routinely administered at enlisted BMT sites, but not at military academies. During August-September 2016, U.S. Naval Academy clinical staff noted an increase in students presenting with acute respiratory illness (ARI). An investigation was conducted to determine the extent and cause of the outbreak. During 22 August-11 September 2016, 652 clinic visits for ARI were identified using electronic health records. HAdV-4 was confirmed by realtime polymerase chain reaction assay in 18 out of 33 patient specimens collected and 1 additional HAdV case was detected from hospital records. Two HAdV-4 positive patients were treated for pneumonia including 1 hospitalized patient. Molecular analysis of 4 HAdV-4 isolates identified genome type 4a1, which is considered vaccine-preventable. Understanding the impact of HAdV in congregate settings other than enlisted BMT sites is necessary to inform discussions regarding future HAdV vaccine strategy.

Scope and extent of healthcare-associated Middle East respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in Riyadh, Saudi Arabia, 2017.

OBJECTIVE: To investigate a Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak event involving multiple healthcare facilities in Riyadh, Saudi Arabia; to characterize transmission; and to explore infection control implications. DESIGN: Outbreak investigation. SETTING: Cases presented in 4 healthcare facilities in Riyadh, Saudi Arabia: a tertiary-care hospital, a specialty pulmonary hospital, an outpatient clinic, and an outpatient dialysis unit. METHODS: Contact tracing and testing were performed following reports of cases at 2 hospitals. Laboratory results were confirmed by real-time reverse transcription polymerase chain reaction (rRT-PCR) and/or genome sequencing. We assessed exposures and determined seropositivity among available healthcare personnel (HCP) cases and HCP contacts of cases. RESULTS: In total, 48 cases were identified, involving patients, HCP, and family members across 2 hospitals, an outpatient clinic, and a dialysis clinic. At each hospital, transmission was linked to a unique index case. Moreover, 4 cases were associated with superspreading events (any interaction where a case patient transmitted to ≥5 subsequent case patients). All 4 of these patients were severely ill, were initially not recognized as MERS-CoV cases, and subsequently died. Genomic sequences clustered separately, suggesting 2 distinct outbreaks. Overall, 4 (24%) of 17 HCP cases and 3 (3%) of 114 HCP contacts of cases were seropositive. CONCLUSIONS: We describe 2 distinct healthcare-associated outbreaks, each initiated by a unique index case and characterized by multiple superspreading events. Delays in recognition and in subsequent implementation of control measures contributed to secondary transmission. Prompt contact tracing, repeated testing, HCP furloughing, and implementation of recommended transmission-based precautions for suspected cases ultimately halted transmission.

Adenovirus-Associated Influenza-Like Illness among College Students, Pennsylvania, USA.

Among students with influenza-like illness at a Pennsylvania college student health center during 2016-2017, 44 (15%) of 288 with respiratory specimens tested positive for human adenovirus (HAdV). HAdV-3, -7, and -4 predominated, and types clustered temporally. HAdV infection should be considered among college students with acute respiratory illness.

Infectious MERS-CoV Isolated From a Mildly Ill Patient, Saudi Arabia.

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with a wide range of clinical presentations, from asymptomatic or mildly ill to severe respiratory illness including death. We describe isolation of infectious MERS-CoV from the upper respiratory tract of a mildly ill 27-year-old female in Saudi Arabia 15 days after illness onset.

Severe Respiratory Illness Outbreak Associated with Human Coronavirus NL63 in a Long-Term Care Facility.

We describe an outbreak of severe respiratory illness associated with human coronavirus NL63 in a long-term care facility in Louisiana in November 2017. Six of 20 case-patients were hospitalized with pneumonia, and 3 of 20 died. Clinicians should consider human coronavirus NL63 for patients in similar settings with respiratory disease.

Non-mumps Viral Parotitis During the 2014-2015 Influenza Season in the United States.

Background: During the 2014-2015 US influenza season, 320 cases of non-mumps parotitis (NMP) among residents of 21 states were reported to the Centers for Disease Control and Prevention (CDC). We conducted an epidemiologic and laboratory investigation to determine viral etiologies and clinical features of NMP during this unusually large occurrence. Methods: NMP was defined as acute parotitis or other salivary gland swelling of >2 days duration in a person with a mumps- negative laboratory result. Using a standardized questionnaire, we collected demographic and clinical information. Buccal samples were tested at the CDC for selected viruses, including mumps, influenza, human parainfluenza viruses (HPIVs) 1-4, adenoviruses, cytomegalovirus, Epstein-Barr virus (EBV), herpes simplex viruses (HSVs) 1 and 2, and human herpes viruses (HHVs) 6A and 6B. Results: Among the 320 patients, 65% were male, median age was 14.5 years (range, 0-90), and 67% reported unilateral parotitis. Commonly reported symptoms included sore throat (55%) and fever (48%). Viruses were detected in 210 (71%) of 294 NMP patients with adequate samples for testing, ≥2 viruses were detected in 37 samples, and 248 total virus detections were made among all samples. These included 156 influenza A(H3N2), 42 HHV6B, 32 EBV, 8 HPIV2, 2 HPIV3, 3 adenovirus, 4 HSV-1, and 1 HSV-2. Influenza A(H3N2), HHV6B, and EBV were the most frequently codetected viruses. Conclusions: Our findings suggest that, in addition to mumps, clinicians should consider respiratory viral (influenza) and herpes viral etiologies for parotitis, particularly among patients without epidemiologic links to mumps cases or outbreaks.

Severe Respiratory Illness Associated With Rhinovirus During the Enterovirus D68 Outbreak in the United States, August 2014-November 2014.

Background: In 2014, a nationwide outbreak of severe respiratory illness occurred in the United States, primarily associated with enterovirus D68 (EV-D68). A proportion of illness was associated with rhinoviruses (RVs) and other enteroviruses (EVs), which we aimed to characterize further. Methods: Respiratory specimens from pediatric and adult patients with respiratory illness were submitted to the Centers for Disease Control and Prevention during August 2014-November 2014. While initial laboratory testing focused on identification of EV-D68, the negative specimens were typed by molecular sequencing to identify additional EV and RV types. Testing for other pathogens was not conducted. We compared available clinical and epidemiologic characteristics among patients with EV-D68 and RV species A-C identified. Results: Among 2629 typed specimens, 1012 were EV-D68 (39%) and 81 (3.1%) represented 24 other EV types; 968 were RVs (37%) covering 114 types and grouped into 3 human RV species (RV-A, 446; RV-B, 133; RV-C, 389); and 568 (22%) had no RV or EV detected. EV-D68 was more frequently identified in patients who presented earlier in the investigation period. Among patients with EV-D68, RV-A, RV-B, or RV-C, the age distributions markedly differed. Clinical syndromes and intensive care unit admissions by age were largely similar. Conclusions: RVs were commonly associated with severe respiratory illness during a nationwide outbreak of EV-D68, and most clinical. Characteristics were similar between groups. A better understanding of the epidemiology of RVs and EVs is needed to help inform development and use of diagnostic tests, therapeutics, and preventive measures.

Serology Enhances Molecular Diagnosis of Respiratory Virus Infections Other than Influenza in Children and Adults Hospitalized with Community-Acquired Pneumonia.

Both molecular and serological assays have been used previously to determine the etiology of community-acquired pneumonia (CAP). However, the extent to which these methods are correlated and the added diagnostic value of serology for respiratory viruses other than influenza virus have not been fully evaluated. Using data from patients enrolled in the Centers for Disease Control and Prevention (CDC) Etiology of Pneumonia in the Community (EPIC) study, we compared real-time reverse transcription-PCR (RT-PCR) and serology for the diagnosis of respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus 1 to 3 (PIV1, PIV2, and PIV3), and adenovirus (AdV) infections. Of 5,126 patients enrolled, RT-PCR and serology test results were available for 2,023, including 1,087 children below the age of 18 years and 936 adults. For RSV, 287 (14.2%) patients were positive by RT-PCR and 234 (11.6%) were positive by serology; for HMPV, 172 (8.5%) tested positive by RT-PCR and 147 (7.3%) by serology; for the PIVs, 94 (4.6%) tested positive by RT-PCR and 92 (4.6%) by serology; and for AdV, 111 (5.5%) tested positive by RT-PCR and 62 (3.1%) by serology. RT-PCR provided the highest number of positive detections overall, but serology increased diagnostic yield for RSV (by 11.8%), HMPV (by 25.0%), AdV (by 32.4%), and PIV (by 48.9%). The method concordance estimated by Cohen's kappa coefficient (κ) ranged from good (for RSV; κ = 0.73) to fair (for AdV; κ = 0.27). Heterotypic seroresponses observed between PIVs and persistent low-level AdV shedding may account for the higher method discordance observed with each of these viruses. Serology can be a helpful adjunct to RT-PCR for research-based assessment of the etiologic contribution of respiratory viruses other than influenza virus to CAP.

Elevation of Alanine Aminotransferase Activity Occurs after Activation of the Cell-Death Signaling Initiated by Pattern-Recognition Receptors but before Activation of Cytolytic Effectors in NK or CD8+ T Cells in the Liver During Acute HCV Infection.

Pattern-recognition receptors (PRRs) promote host defenses against HCV infection by binding to their corresponding adapter molecules leading to the initiation of innate immune responses including cell death. We investigated the expression of PRR genes, biomarkers of liver cell-death, and T cell and NK cell activation/inhibition-related genes in liver and serum obtained from three experimentally infected chimpanzees with acute HCV infection, and analyzed the correlation between gene expression levels and clinical profiles. Our results showed that expression of hepatic RIG-I, TLR3, TLR7, 2OAS1, and CXCL10 mRNAs was upregulated as early as 7 days post-inoculation and peaked 12 to 83 days post-inoculation. All of the three HCV infected chimpanzees exhibited significant elevations of serum alanine aminotransferase (ALT) activity between 70 and 95 days after inoculation. Elevated levels of serum cytokeratin 18 (CK-18) and caspases 3 and 7 activity coincided closely with the rise of ALT activity, and were preceded by significant increases in levels of caspase 3 and caspase 7 mRNAs in the liver. Particularly we found that significant positive auto-correlations were observed between RIG-I, TLR3, CXCL10, 2OAS1, and PD-L1 mRNA and ALT activity at 3 to 12 days before the peak of ALT activity. However, we observed substantial negative auto-correlations between T cell and NK cell activation/inhibition-related genes and ALT activity at 5 to 32 days after the peak of ALT activity. Our results indicated cell death signaling is preceded by early induction of RIG-I, TLR3, 2OAS1, and CXCL10 mRNAs which leads to elevation of ALT activity and this signaling pathway occurs before the activation of NK and T cells during acute HCV infection. Our study suggests that PRRs and type I IFN response may play a critical role in development of liver cell injury related to viral clearance during acute HCV infection.