Development and Validation of an Enzyme Immunoassay for Detection and Quantification of SARS-CoV-2 Salivary IgA and IgG.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

Factors influencing environmental sampling recovery of healthcare pathogens from non-porous surfaces with cellulose sponges.

Results from sampling healthcare surfaces for pathogens are difficult to interpret without understanding the factors that influence pathogen detection. We investigated the recovery of four healthcare-associated pathogens from three common surface materials, and how a body fluid simulant (artificial test soil, ATS), deposition method, and contamination levels influence the percent of organisms recovered (%R). Known quantities of carbapenemase-producing KPC+ Klebsiella pneumoniae (KPC), Acinetobacter baumannii, vancomycin-resistant Enterococcus faecalis, and Clostridioides difficile spores (CD) were suspended in Butterfield's buffer or ATS, deposited on 323cm2 steel, plastic, and laminate surfaces, allowed to dry 1h, then sampled with a cellulose sponge wipe. Bacteria were eluted, cultured, CFU counted and %R determined relative to the inoculum. The %R varied by organism, from <1% (KPC) to almost 60% (CD) and was more dependent upon the organism's characteristics and presence of ATS than on surface type. KPC persistence as determined by culture also declined by >1 log10 within the 60 min drying time. For all organisms, the %R was significantly greater if suspended in ATS than if suspended in Butterfield's buffer (p<0.05), and for most organisms the %R was not significantly different when sampled from any of the three surfaces. Organisms deposited in multiple droplets were recovered at equal or higher %R than if spread evenly on the surface. This work assists in interpreting data collected while investigating a healthcare infection outbreak or while conducting infection intervention studies.

Recovery efficiency of two glove-sampling methods.

Two methods to sample pathogens from gloved hands were compared: direct imprint onto agar and a sponge-wipe method. The sponge method was significantly better at recovering Clostridiodes difficile spores, and no difference was observed between the methods at 101 inoculum for carbapenemase-producing KPC+ Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii.

Sampling efficiency of Candida auris from healthcare surfaces: culture and nonculture detection methods.

Sponges and swabs were evaluated for their ability to recover Candida auris dried 1 hour on steel and plastic surfaces. Culture recovery ranged from <0.1% (sponges) to 8.4% (swabs), and cells detected with an esterase activity assay revealed >50% recovery (swabs), indicating that cells may enter a viable but nonculturable state.

Descriptive evaluation of antibody responses to severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection in plasma and gingival crevicular fluid in a nursing home cohort-Arkansas, June-August 2020.

OBJECTIVE: To characterize and compare severe acute respiratory coronavirus virus 2 (SARS-CoV-2)-specific immune responses in plasma and gingival crevicular fluid (GCF) from nursing home residents during and after natural infection. DESIGN: Prospective cohort. SETTING: Nursing home. PARTICIPANTS: SARS-CoV-2-infected nursing home residents. METHODS: A convenience sample of 14 SARS-CoV-2-infected nursing home residents, enrolled 4-13 days after real-time reverse transcription polymerase chain reaction diagnosis, were followed for 42 days. After diagnosis, plasma SARS-CoV-2-specific pan-Immunoglobulin (Ig), IgG, IgA, IgM, and neutralizing antibodies were measured at 5 time points, and GCF SARS-CoV-2-specific IgG and IgA were measured at 4 time points. RESULTS: All participants demonstrated immune responses to SARS-CoV-2 infection. Among 12 phlebotomized participants, plasma was positive for pan-Ig and IgG in all 12 participants. Neutralizing antibodies were positive in 11 participants; IgM was positive in 10 participants, and IgA was positive in 9 participants. Among 14 participants with GCF specimens, GCF was positive for IgG in 13 participants and for IgA in 12 participants. Immunoglobulin responses in plasma and GCF had similar kinetics; median times to peak antibody response were similar across specimen types (4 weeks for IgG; 3 weeks for IgA). Participants with pan-Ig, IgG, and IgA detected in plasma and GCF IgG remained positive throughout this evaluation, 46-55 days after diagnosis. All participants were viral-culture negative by the first detection of antibodies. CONCLUSIONS: Nursing home residents had detectable SARS-CoV-2 antibodies in plasma and GCF after infection. Kinetics of antibodies detected in GCF mirrored those from plasma. Noninvasive GCF may be useful for detecting and monitoring immunologic responses in populations unable or unwilling to be phlebotomized.

Development and validation of an enzyme immunoassay for detection and quantification of SARS-CoV-2 salivary IgA and IgG.

Oral fluids offer a non-invasive sampling method for the detection of antibodies. Quantification of IgA and IgG antibodies in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG antibodies against the prefusion-stabilized form of the SARS-CoV-2 spike protein. Normalization against total antibody isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 pre-pandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA: 95.5%; IgG: 89.7%) without compromising specificity (IgA: 99%; IgG: 97%). No cross reactivity with seasonal coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/mL and 0.30 ng/mL, respectively. Salivary IgA and IgG antibodies were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary antibody titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 weeks in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

A Comparison of Less Invasive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Diagnostic Specimens in Nursing Home Residents-Arkansas, June-August 2020.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing remains essential for early identification and clinical management of cases. We compared the diagnostic performance of 3 specimen types for characterizing SARS-CoV-2 in infected nursing home residents. METHODS: A convenience sample of 17 residents were enrolled within 15 days of first positive SARS-CoV-2 result by real-time reverse transcription polymerase chain reaction (RT-PCR) and prospectively followed for 42 days. Anterior nasal swabs (AN), oropharyngeal swabs (OP), and saliva specimens (SA) were collected on the day of enrollment, every 3 days for the first 21 days, and then weekly for 21 days. Specimens were tested for presence of SARS-CoV-2 RNA using RT-PCR and replication-competent virus by viral culture. RESULTS: Comparing the 3 specimen types collected from each participant at each time point, the concordance of paired RT-PCR results ranged from 80% to 88%. After the first positive result, SA and OP were RT-PCR-positive for ≤48 days; AN were RT-PCR-positive for ≤33 days. AN had the highest percentage of RT-PCR-positive results (21/26 [81%]) when collected ≤10 days of participants' first positive result. Eleven specimens were positive by viral culture: 9 AN collected ≤19 days following first positive result and 2 OP collected ≤5 days following first positive result. CONCLUSIONS: AN, OP, and SA were effective methods for repeated testing in this population. More AN than OP were positive by viral culture. SA and OP remained RT-PCR-positive longer than AN, which could lead to unnecessary interventions if RT-PCR detection occurred after viral shedding has likely ceased.

Infectious Period of Severe Acute Respiratory Syndrome Coronavirus 2 in 17 Nursing Home Residents-Arkansas, June-August 2020.

BACKGROUND: To estimate the infectious period of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in older adults with underlying conditions, we assessed duration of coronavirus disease 2019 (COVID-19) symptoms, reverse-transcription polymerase chain reaction (RT-PCR) positivity, and culture positivity among nursing home residents. METHODS: We enrolled residents within 15 days of their first positive SARS-CoV-2 test (diagnosis) at an Arkansas facility from July 7 to 15, 2020 and instead them for 42 days. Every 3 days for 21 days and then weekly, we assessed COVID-19 symptoms, collected specimens (oropharyngeal, anterior nares, and saliva), and reviewed medical charts. Blood for serology was collected on days 0, 6, 12, 21, and 42. Infectivity was defined by positive culture. Duration of culture positivity was compared with duration of COVID-19 symptoms and RT-PCR positivity. Data were summarized using measures of central tendency, frequencies, and proportions. RESULTS: We enrolled 17 of 39 (44%) eligible residents. Median participant age was 82 years (range, 58-97 years). All had ≥3 underlying conditions. Median duration of RT-PCR positivity was 22 days (interquartile range [IQR], 8-31 days) from diagnosis; median duration of symptoms was 42 days (IQR, 28-49 days). Of 9 (53%) participants with any culture-positive specimens, 1 (11%) severely immunocompromised participant remained culture-positive 19 days from diagnosis; 8 of 9 (89%) were culture-positive ≤8 days from diagnosis. Seroconversion occurred in 12 of 12 (100%) surviving participants with ≥1 blood specimen; all participants were culture-negative before seroconversion. CONCLUSIONS: Duration of infectivity was considerably shorter than duration of symptoms and RT-PCR positivity. Severe immunocompromise may prolong SARS-CoV-2 infectivity. Seroconversion indicated noninfectivity in this cohort.

Clinical Course of SARS-CoV-2 Infection in Adults with ESKD Receiving Outpatient Hemodialysis.

Background: Patients with ESKD on maintenance dialysis receive dialysis in common spaces with other patients and have a higher risk of severe SARS-CoV-2 infections. They may have persistently or intermittently positive SARS-CoV-2 RT-PCR tests after infection. We describe the clinical course of SARS-CoV-2 infection and the serologic response in a convenience sample of patients with ESKD to understand the duration of infectivity. Methods: From August to November 2020, we enrolled patients on maintenance dialysis with SARS-CoV-2 infections from outpatient dialysis facilities in Atlanta, Georgia. We followed participants for approximately 42 days. We assessed COVID-19 symptoms and collected specimens. Oropharyngeal (OP), anterior nasal (AN), and saliva (SA) specimens were tested for the presence of SARS-CoV-2 RNA, using RT-PCR, and sent for viral culture. Serology, including neutralizing antibodies, was measured in blood specimens. Results: Fifteen participants, with a median age of 58 (range, 37‒77) years, were enrolled. Median duration of RT-PCR positivity from diagnosis was 18 days (interquartile range [IQR], 8‒24 days). Ten participants had at least one, for a total of 41, positive RT-PCR specimens ≥10 days after symptoms onset. Of these 41 specimens, 21 underwent viral culture; one (5%) was positive 14 days after symptom onset. Thirteen participants developed SARS-CoV-2-specific antibodies, 11 of which included neutralizing antibodies. RT-PCRs remained positive after seroconversion in eight participants and after detection of neutralizing antibodies in four participants; however, all of these samples were culture negative. Conclusions: Patients with ESKD on maintenance dialysis remained persistently and intermittently SARS-CoV-2-RT-PCR positive. However, of the 15 participants, only one had infectious virus, on day 14 after symptom onset. Most participants mounted an antibody response, including neutralizing antibodies. Participants continued having RT-PCR-positive results in the presence of SARS-CoV-2-specific antibodies, but without replication-competent virus detected.

Initial and Repeated Point Prevalence Surveys to Inform SARS-CoV-2 Infection Prevention in 26 Skilled Nursing Facilities - Detroit, Michigan, March-May 2020.

Skilled nursing facilities (SNFs) are focal points of the coronavirus disease 2019 (COVID-19) pandemic, and asymptomatic infections with SARS-CoV-2, the virus that causes COVID-19, among SNF residents and health care personnel have been described (1-3). Repeated point prevalence surveys (serial testing of all residents and health care personnel at a health care facility irrespective of symptoms) have been used to identify asymptomatic infections and have reduced SARS-CoV-2 transmission during SNF outbreaks (1,3). During March 2020, the Detroit Health Department and area hospitals detected a sharp increase in COVID-19 diagnoses, hospitalizations, and associated deaths among SNF residents. The Detroit Health Department collaborated with local government, academic, and health care system partners and a CDC field team to rapidly expand SARS-CoV-2 testing and implement infection prevention and control (IPC) activities in all Detroit-area SNFs. During March 7-May 8, among 2,773 residents of 26 Detroit SNFs, 1,207 laboratory-confirmed cases of COVID-19 were identified during three periods: before (March 7-April 7) and after two point prevalence surveys (April 8-25 and April 30-May 8): the overall attack rate was 44%. Within 21 days of receiving their first positive test results, 446 (37%) of 1,207 COVID-19 patients were hospitalized, and 287 (24%) died. Among facilities participating in both surveys (n = 12), the percentage of positive test results declined from 35% to 18%. Repeated point prevalence surveys in SNFs identified asymptomatic COVID-19 cases, informed cohorting and IPC practices aimed at reducing transmission, and guided prioritization of health department resources for facilities experiencing high levels of SARS-CoV-2 transmission. With the increased availability of SARS-CoV-2 testing, repeated point prevalence surveys and enhanced and expanded IPC support should be standard tools for interrupting and preventing COVID-19 outbreaks in SNFs.