A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP.

The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.

Comparative Performance of SARS-CoV-2 Detection Assays using Seven Different Primer/Probe Sets and One Assay Kit.

More than 100,000 people worldwide are known to have been infected with SARS-CoV-2 beginning in December 2019. The virus has now spread to over 93 countries including the United States, with the largest cluster of US cases to date in the Seattle metropolitan area in Washington. Given the rapid increase in the number of local cases, the availability of accurate, high-throughput SARS-CoV-2 testing is vital to efforts to manage the current public health crisis. In the course of optimizing SARS-CoV-2 testing performed by the University of Washington Clinical Virology Lab (UW Virology Lab), we tested assays using seven different primer/probe sets and one assay kit. We found that the most sensitive assays were those the used the E-gene primer/probe set described by Corman et al. (Eurosurveillance 25(3), 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045) and the N2 set described by the CDC (Division of Viral Diseases, Centers for Disease Control and Prevention, 2020, https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf). All assays tested were found to be highly specific for SARS-CoV-2, with no cross-reactivity with other respiratory viruses observed in our analyses regardless of the primer/probe set or kit used. These results will provide invaluable information to other clinical laboratories who are actively developing SARS-CoV-2 testing protocols at a time when increased testing capacity is urgently needed worldwide.

Validation of SARS-CoV-2 detection across multiple specimen types.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused considerable disruption across the world, resulting in more than 235,000 deaths since December 2019. SARS-CoV-2 has a wide tropism and detection of the virus has been described in multiple specimen types, including various respiratory secretions, cerebrospinal fluid, and stool. OBJECTIVE: To evaluate the accuracy and sensitivity of a laboratory modified CDCbased SARS-CoV-2 N1 and N2 assay across a range of sample types. Study Design We compared the matrix effect on the analytical sensitivity of SARS-CoV-2 detection by qRT-PCR in nasal swabs collected in viral transport medium (VTM), bronchoalveolar lavage (BAL), sputum, plasma, cerebral spinal fluid (CSF), stool, VTM, phosphate buffered saline (PBS), and Hanks' Balanced Salt Solution (HBSS). Initial limits of detection (LoD) were subsequently narrowed to confirm an LoD for each specimen type and target gene. RESULTS: LoDs were established using a modified CDC-based laboratory developed test and ranged from a mean CT cut-off of 33.8-35.7 (10-20 copies/reaction) for the N1 gene target, and 34.0-36.2 (1-10 copies/reaction) for N2. Alternatives to VTM such as PBS and HBSS had comparable LoDs. The N2 gene target was found to be most sensitive in CSF. CONCLUSION: A modified CDC-based laboratory developed test is able to detect SARSCoV- 2 accurately with similar sensitivity across all sample types tested.

Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit.

Nearly 400,000 people worldwide are known to have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beginning in December 2019. The virus has now spread to over 168 countries including the United States, where the first cluster of cases was observed in the Seattle metropolitan area in Washington. Given the rapid increase in the number of cases in many localities, the availability of accurate, high-throughput SARS-CoV-2 testing is vital to efforts to manage the current public health crisis. In the course of optimizing SARS-CoV-2 testing performed by the University of Washington Clinical Virology Lab (UW Virology Lab), we evaluated assays using seven different primer-probe sets and one assay kit. We found that the most sensitive assays were those that used the E-gene primer-probe set described by Corman et al. (V. M. Corman, O. Landt, M. Kaiser, R. Molenkamp, et al., Euro Surveill 25:2000045, 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045) and the N2 set developed by the CDC (Division of Viral Diseases, Centers for Disease Control and Prevention, 2020, https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf). All assays tested were found to be highly specific for SARS-CoV-2, with no cross-reactivity with other respiratory viruses observed in our analyses regardless of the primer-probe set or kit used. These results will provide valuable information to other clinical laboratories who are actively developing SARS-CoV-2 testing protocols at a time when increased testing capacity is urgently needed worldwide.