Geographically skewed recruitment and COVID-19 seroprevalence estimates: a cross-sectional serosurveillance study and mathematical modelling analysis.

OBJECTIVES: Convenience sampling is an imperfect but important tool for seroprevalence studies. For COVID-19, local geographic variation in cases or vaccination can confound studies that rely on the geographically skewed recruitment inherent to convenience sampling. The objectives of this study were: (1) quantifying how geographically skewed recruitment influences SARS-CoV-2 seroprevalence estimates obtained via convenience sampling and (2) developing new methods that employ Global Positioning System (GPS)-derived foot traffic data to measure and minimise bias and uncertainty due to geographically skewed recruitment. DESIGN: We used data from a local convenience-sampled seroprevalence study to map the geographic distribution of study participants' reported home locations and compared this to the geographic distribution of reported COVID-19 cases across the study catchment area. Using a numerical simulation, we quantified bias and uncertainty in SARS-CoV-2 seroprevalence estimates obtained using different geographically skewed recruitment scenarios. We employed GPS-derived foot traffic data to estimate the geographic distribution of participants for different recruitment locations and used this data to identify recruitment locations that minimise bias and uncertainty in resulting seroprevalence estimates. RESULTS: The geographic distribution of participants in convenience-sampled seroprevalence surveys can be strongly skewed towards individuals living near the study recruitment location. Uncertainty in seroprevalence estimates increased when neighbourhoods with higher disease burden or larger populations were undersampled. Failure to account for undersampling or oversampling across neighbourhoods also resulted in biased seroprevalence estimates. GPS-derived foot traffic data correlated with the geographic distribution of serosurveillance study participants. CONCLUSIONS: Local geographic variation in seropositivity is an important concern in SARS-CoV-2 serosurveillance studies that rely on geographically skewed recruitment strategies. Using GPS-derived foot traffic data to select recruitment sites and recording participants' home locations can improve study design and interpretation.

Temporary Regulatory Deviations and the Coronavirus Disease 2019 (COVID-19) PCR Labeling Update Study Indicate What Laboratory-Developed Test Regulation by the US Food and Drug Administration (FDA) Could Look Like.

The coronavirus disease 2019 (COVID-19) response necessitated innovations and a series of regulatory deviations that also affected laboratory-developed tests (LDTs). To examine real-world consequences and specify regulatory paradigm shifts, legislative proposals were aligned on a common timeline with Emergency Use Authorization (EUA) of LDTs and the US Food and Drug Administration (FDA)-orchestrated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) labeling update study. The initial EUA adoption by LDT developers shows that the FDA can have oversight over LDTs. We used efficiency-corrected microcosting of our EUA PCR assay to estimate the national cost of the labeling update study to $0.3 to $1.4 million US dollars. Labeling update study performance data showed lower average detection limits in commercial in vitro diagnostic (IVD) assays versus LDTs (32,000 ± 75,000 versus 71,000 ± 147,000 nucleic acid amplification tests/mL; P = 0.04); however, comparison also shows that FDA review of IVD assays and LDTs did not prevent differences between initial and labeling update performance (IVD assay, P < 0.0001; LDT, P = 0.003). The regulatory shifts re-emphasized that both commercial tests and LDTs rely heavily on laboratory competence and procedures; however, lack of performance data on authorized tests, when clinically implemented, precludes assessment of the benefit related to regulatory review. Temporary regulatory deviations during the pandemic and regulatory science tools (ie, reference material) have generated valuable real-world evidence to inform pending legislation regarding LDT regulation.

Development of a qualitative real-time RT-PCR assay for the detection of SARS-CoV-2: a guide and case study in setting up an emergency-use, laboratory-developed molecular microbiological assay.

Developing and deploying new diagnostic tests are difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important. During a pandemic, laboratories play a key role in helping healthcare providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, PCR remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular-based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to establishing fast and accurate diagnostic testing in crisis conditions.

Remote Fingerstick Blood Collection for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Testing.

The rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has propelled the rapid development of serologic tests that can detect anti-SARS-CoV-2 antibodies. These have been used for studying the prevalence and spread of infection in different populations, and helping establish a recent diagnosis of coronavirus disease 2019 (COVID-19), and will likely be used to confirm humoral immunity after infection or vaccination. However, nearly all lab-based high-throughput SARS-CoV-2 serologic assays require a serum sample from venous blood draw, limiting their applications and scalability. Here, we present a method that enables large-scale SARS-CoV-2 serologic studies by combining self or office collection of fingerprick blood with a volumetric absorptive microsampling device (Mitra, Neoteryx LLC) with a high-throughput electrochemiluminescence-based SARS-CoV-2 total antibody assay (Roche Elecsys, Roche Diagnostics Inc) that is emergency use authorization approved for use on serum samples and widely used by clinical laboratories around the world. We found that the Roche Elecsys assay has a high dynamic range that allows for accurate detection of SARS-CoV-2 antibodies in serum samples diluted 1:20 as well as contrived dried blood extracts. Extracts of dried blood from Mitra devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies compared with neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of Mitra dried blood collection device with the Roche Elecsys SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies.

Presymptomatic Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Among Residents and Staff at a Skilled Nursing Facility: Results of Real-time Polymerase Chain Reaction and Serologic Testing.

High rates of asymptomatic coronavirus disease 2019 infection suggest benefits to routine testing in congregate care settings. Screening was undertaken in a single nursing facility without a known case of coronavirus disease 2019, demonstrating an 85% prevalence among residents and 37% among staff. Serology was not helpful in identifying infections.

High Seroprevalence of Anti-SARS-CoV-2 Antibodies in Chelsea, Massachusetts.

SARS-CoV-2 antibody testing allows quantitative determination of disease prevalence, which is especially important in high-risk communities. We performed anonymized convenience sampling of 200 currently asymptomatic residents of Chelsea, the epicenter of COVID-19 illness in Massachusetts, by BioMedomics SARS-CoV-2 combined IgM-IgG point-of-care lateral flow immunoassay. The seroprevalence was 31.5% (17.5% IgM+IgG+, 9.0% IgM+IgG-, and 5.0% IgM-IgG+). Of the 200 participants, 50.5% reported no symptoms in the preceding 4 weeks, of which 24.8% (25/101) were seropositive, and 60% of these were IgM+IgG-. These data are the highest seroprevalence rates observed to date and highlight the significant burden of asymptomatic infection.

COVID-19-neutralizing antibodies predict disease severity and survival.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

Evaluation of Three Commercial SARS-CoV-2 Serologic Assays and Their Performance in Two-Test Algorithms.

Sensitive and specific severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologic assays are needed to inform diagnostic, therapeutic, and public health decision-making. We evaluated three commercial serologic assays as stand-alone tests and as components of two-test algorithms. Two nucleocapsid antibody tests (Abbott IgG and Roche total antibody) and one spike protein antibody test (DiaSorin IgG) were included. We assessed sensitivity using 128 serum samples from symptomatic PCR-confirmed coronavirus disease 2019 (COVID-19)-infected patients and specificity using 1,204 samples submitted for routine serology prior to COVID-19's emergence, plus 64 pandemic-era samples from SARS-CoV-2 PCR-negative patients with respiratory symptoms. Assays were evaluated as stand-alone tests and as components of a two-test algorithm in which positive results obtained using one assay were verified using a second assay. The two nucleocapsid antibody tests were more sensitive than the spike protein antibody test overall (70% and 70% versus 57%; P ≤ 0.003), with pronounced differences observed using samples collected 7 to 14 days after symptom onset. All three assays were comparably sensitive (≥89%; P ≥ 0.13) using samples collected >14 days after symptom onset. Specificity was higher using the nucleocapsid antibody tests (99.3% and 99.7%) than using the spike protein antibody test (97.8%; P ≤ 0.002). When any two assays were paired in a two-test algorithm, the specificity was 99.9% (P < 0.0001 to 0.25 compared with the individual assays), and the positive predictive value (PPV) improved substantially, with a minimal effect on the negative predictive value (NPV). In conclusion, two nucleocapsid antibody tests outperformed a spike protein antibody test. Pairing two different serologic tests in a two-test algorithm improves the PPV, compared with the individual assays alone, while maintaining the NPV.

Clinical Assessment and Validation of a Rapid and Sensitive SARS-CoV-2 Test Using Reverse Transcription Loop-Mediated Isothermal Amplification Without the Need for RNA Extraction.

BACKGROUND: Amid the enduring pandemic, there is an urgent need for expanded access to rapid, sensitive, and inexpensive coronavirus disease 2019 (COVID-19) testing worldwide without specialized equipment. We developed a simple test that uses colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect severe acute resrpiratory syndrome coronavirus 2 (SARS-CoV-2) in 40 minutes from sample collection to result. METHODS: We tested 135 nasopharyngeal specimens from patients evaluated for COVID-19 infection at Massachusetts General Hospital. Specimens were either added directly to RT-LAMP reactions, inactivated by a combined chemical and heat treatment step, or inactivated then purified with a silica particle-based concentration method. Amplification was performed with 2 SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. RESULTS: Direct RT-LAMP testing of unprocessed specimens could only reliably detect samples with abundant SARS-CoV-2 (>3 000 000 copies/mL), with sensitivities of 50% (95% CI, 28%-72%) and 59% (95% CI, 43%-73%) in samples collected in universal transport medium and saline, respectively, compared with quantitative polymerase chain reaction (qPCR). Adding an upfront RNase inactivation step markedly improved the limit of detection to at least 25 000 copies/mL, with 87.5% (95% CI, 72%-95%) sensitivity and 100% specificity (95% CI, 87%-100%). Using both inactivation and purification increased the assay sensitivity by 10-fold, achieving a limit of detection comparable to commercial real-time PCR-based diagnostics. CONCLUSIONS: By incorporating a fast and inexpensive sample preparation step, RT-LAMP accurately detects SARS-CoV-2 with limited equipment for about US$6 per sample, making this a potentially ideal assay to increase testing capacity, especially in resource-limited settings.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.