Practical strategies for SARS-CoV-2 RT-PCR testing in resource-constrained settings

Abstract

Background: Standard nasopharyngeal swab testing for SARS-CoV-2 detection by PCR is not always feasible due to limitations in trained personnel, personal protective equipment, swabs, PCR reagents, and access to cold chain and biosafety hoods. Methods: We piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including both standard polyester flocked swabs as well as 3D printed plastic lattice swabs, placed into either viral transport media or an RNA stabilization agent. Quantitative SARS-CoV-2 viral detection by RT-qPCR was compared to that obtained by nasopharyngeal sampling as the reference standard. Pooling specimens in the lab versus pooling swabs at the point of collection was also evaluated. Results: Among 275 participants, flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), mostly missing those with low viral load (<10^3 viral copies/uL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into an RNA stabilizer, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. Pooling sample specimens or swabs both successfully detected samples >102 viral copies/uL. Conclusions: Nasal swabs are likely adequate for clinical diagnosis of acute infections to help expand testing capacity in resource-constrained settings. When collected into an RNA preservative that also inactivates infectious virus, nasal swabs yielded quantitative viral loads approximating those obtained by nasopharyngeal sampling.

High household transmission of SARS-CoV-2 in the United States: living density, viral load, and disproportionate impact on communities of color

“…Nasopharyngeal and nasal swab samples were tested using a CDC RT-qPCR protocol authorized for emergency use that consists of three unique assays: two targeting regions of the virus’ nucleocapsid gene (N1, N2) and one targeting human RNase P gene (RP) (Catalog # 2019-nCoVEUA-01, Integrated DNA Technologies) [11]. Details of assay implementation and calculation of the limit of detection are described elsewhere [12]. Briefly, samples were designated positive if all three PCRs were positive (N1 and N2 for virus, RP for adequate sampling).…”

“…For those participants who did not complete an NP swab on study day 1, we used a transformed NMT viral load to impute the missing NP value. The transformation formula was derived from a linear regression equation generated from >100 study participants with positive viral load from both NP and NMT swabs on study day 1 [12]. To determine whether NP viral load in index cases was associated with secondary cases in the same household, we dichotomized the NP viral load with a cutoff of 1×10^6 viral copies/ul and compared the proportion of transmission events.…”

Household Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in the United States: Living Density, Viral Load, and Disproportionate Impact on Communities of Color

“…Details for all laboratory methods are found in the Supplementary Materials. Quantitative PCR testing for SARS-CoV-2 was performed using a Centers for Disease Control and Prevention reverse-transcription quantitative-PCR protocol authorized by the US Food and Drug Administration for emergency use, as previously described [11]. Serology was performed using enzyme-linked immunoassay (ELISA) that detects antibodies to the receptor binding domain of the spike protein with high sensitivity and specificity [12].…”

Assessment of the Field Utility of a Rapid Point-of-Care Test for SARS-CoV-2 Antibodies in a Household Cohort

“…These were tested using a CDC reverse transcriptase quantitative PCR protocol authorized for emergency use. 12 We defined asymptomatic cases as those who reported no or one cumulative symptoms in daily symptom diaries collected over 14 days, as long as the one symptom was not fever, shortness of breath, or anosmia. Among those who were SARS-CoV-2 PCR-positive but asymptomatic, the sensitivity of the LFA at D28 was 74% (14/19, 95% CI: 49-91%), compared with 92% (11/12, 95% CI: 62-100%) for the ELISA (Figure 3).…”