Development of an Accessible and Scalable Quantitative Polymerase Chain Reaction Assay for Monkeypox Virus Detection.

During the 2022 monkeypox (MPX) outbreak, testing has been limited and results delayed, allowing ongoing transmission. Gold-standard quantitative polymerase chain reaction (qPCR) diagnostics are difficult to obtain. This research adapted the June 2022 CDC MPX qPCR assay for broad implementation. Validated using MPX stocks in a matrix with multiple sample types, MPX was detected with cycle threshold (Ct) values 17.46-35.59 and titer equivalents 8.01 × 106 to 2.45 × 100 plaque-forming unit (PFU)/mL. The detection limit was 3.59 PFU/mL. Sensitivity and specificity were both 100%. This qPCR assay can be quickly and broadly implemented in research and public health laboratories to increase diagnostic capacity amid the growing MPX outbreak.

Interferon α/ß Decoy Receptor Encoded by a Variant in the Dryvax Smallpox Vaccine Contributes to Virulence and Correlates with Severe Vaccine Side Effects.

Although providing long-lasting immunity, smallpox vaccination was associated with local and systemic reactions and rarely with severe complications, including progressive vaccinia and postvaccinia encephalitis. As the Dryvax smallpox vaccine consists of a population of variants, we investigated a particularly pathogenic isolate called clone 3 (CL3). Virus replication was monitored by inserting the gene encoding firefly luciferase (Luc) into the genomes of CL3 and ACAM2000, the second-generation smallpox vaccine derived from a less virulent clone. Greater luminescence occurred following intranasal or intraperitoneal inoculation of mice with CL3-Luc than ACAM2000-Luc. Previous genome sequencing of CL3 and ACAM2000 revealed numerous differences that could affect pathogenicity. We focused on a 4.2-kbp segment, containing several open reading frames, in CL3 that is absent from ACAM2000 and determined that lower virulence of the latter was associated with a truncation of the interferon α/ß (IFN-α/ß) decoy receptor. Truncation of the decoy receptor in CL3-Luc and repair of the truncated version in ACAM2000-Luc decreased and increased virulence, respectively. Blockade of the mouse type 1 IFN receptor increased the virulence of ACAM2000-Luc to that of CL3-Luc, consistent with the role of IFN in attenuating the former. The severities of disease following intracranial inoculation of immunocompetent mice and intraperitoneal inoculation of T cell-depleted mice were also greater in viruses expressing the full-length decoy receptor. Previous evidence for the low affinity of a similarly truncated decoy receptor for IFN and the presence of a full-length decoy receptor in virus isolated from a patient with progressive vaccinia support our findings. IMPORTANCE Attenuated live viruses make effective vaccines, although concerns exist due to infrequent complications, particularly in individuals with immunological defects. Such complications occurred with smallpox vaccines, which were shown to be comprised of populations of variants. Clone 3, isolated from Dryvax, the vaccine most widely used in the United States during the smallpox eradication campaign, was particularly pathogenic in animal models. We demonstrated that the full-length IFN-α/ß decoy receptor in CL3 and a truncation of the receptor in the clone used for the second-generation smallpox vaccine ACAM2000 account for their difference in pathogenicity. Viruses expressing the full-length decoy receptor were more virulent following intranasal, intraperitoneal, or intracranial inoculation of mice than ACAM2000, and disease was exacerbated following T cell depletion. Correspondingly, the full-length decoy receptor is present in smallpox vaccines with high rates of side effects and in a Dryvax clone obtained from a lesion in a patient with progressive vaccinia.

COVID-19 infection among emergency department healthcare providers in a large tertiary academic medical center following the peak of the pandemic.

The COVID-19 pandemic has spread through the US during the last few months exposing healthcare providers to possible infection. Here we report testing of emergency department (ED) healthcare providers (HCP) for exposure to COVID-19 through lateral flow point of care (POC) and lab-based enzyme-linked immunosorbent assay (ELISA), and RTq-PCR for evidence of acute infection. 138 ED HCP were tested between May 26th (approximately one month after the peak of COVID-19 first wave of cases) and June 14th. Enrolled ED HCP represented about 70% of the total ED HCP workforce during the study period. Subjects were tested with a POC COVID-19 antibody test, and standard ELISA performed by a university-based research lab. Subjects also provided a mid-turbinate swab and a saliva specimen for RTq-PCR. All subjects provided demographic information, past medical history, information about personal protective equipment (PPE) use, COVID-19 symptoms, as well as potential COVID-19 exposures during the previous 4 weeks, both in the ED, and outside the clinical setting. None of the HCP had positive RT-PCR results; 7 HCP (5%) had positive IgG for COVID-19; there was strong agreement between the lab-based ELISA (reference test) and the POC Ab test (P ≤ 0.0001). For the POC Ab test there were no false negatives and only one false positive among the 138 participants. There was no significant difference in demographic/ethnic variables, past medical history, hours worked in the ED, PPE use, or concerning exposures between seropositive and seronegative individuals. Moreover, there was no significant difference in reported symptoms between the two groups during the previous four weeks. The rate of COVID-19 seroconversion in our ED was 5% during the month following the pandemic's first wave. Based on questionnaire responses, differences in demographics/ethnicity, medical history, COVID-19 exposures, and PPE use were not associated with ED HCP having been infected with SARS-CoV-2. In the setting of our limited cohort of subjects the COVID-19 POC Ab test performed comparably to the ELISA lab-based standard.