Simultaneous monitoring of HIV viral load and screening of SARS-CoV-2 employing a low-cost RT-qPCR test workflow.

The COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of virologic failure and HIV-associated illness. Often this population is at high risk for exposure to SARS-CoV-2 infection, and once infected, for severe disease. Therefore, close monitoring of HIV plasma viral load (VL) and screening for SARS-CoV-2 infection are needed. We developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. The extracted RNA is then submitted to RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV virologic failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). In contrived samples, the in-house RNA extraction workflow achieved a detection limit of 200-copies per mL for HIV RNA in plasma and 100-copies per mL for SARS-CoV-2 RNA in NS. Similar detection limits were observed for HIV and SARS-CoV-2 in pooled plasma/NS contrived samples. When comparing in-house with standard extraction methods, we found high agreement (>0.91) between input and measured RNA copies for HIV LTR in contrived plasma; SARS-CoV-2 N1/N2 in contrived NS; and LTR, N1, and N2 in pooled plasma/NS samples. We further evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-positive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 combined plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house RNA extraction to those using a commercial kit (standard extraction method). The in-house extraction and standard extraction of clinical specimens were positively correlated: plasma HIV VL (R2 of 0.81) and NS SARS-CoV-2 VL (R2 of 0.95 and 0.99 for N1 and N2 genes, respectively); and pooled plasma/NS HIV VL (R2 of 0.71) and SARS-CoV-2 VL (R2 of 1 both for N1 and N2 genes). Our low-cost molecular test workflow ($1.85 per pooled sample extraction) for HIV RNA and SARS-CoV-2 RNA could serve as an alternative to current standard assays ($12 per pooled sample extraction) for laboratories in low-resource settings.

Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection.

RNA amplification tests sensitively detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but their complexity and cost are prohibitive for expanding coronavirus disease 2019 (COVID-19) testing. We developed "Harmony COVID-19," a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device. Our ready-to-use, multiplexed reverse transcription, loop-mediated isothermal amplification (RT-LAMP) can detect down to 0.38 SARS-CoV-2 RNA copies/µl and can report in 17 min for high­viral load samples (5000 copies/µl). Harmony detected 97 or 83% of contrived samples with ≥0.5 viral particles/µl in nasal matrix or saliva, respectively. Evaluation in clinical nasal specimens (n = 101) showed 100% detection of RNA extracted from specimens with ≥0.5 SARS-CoV-2 RNA copies/µl, with 100% specificity in specimens positive for other respiratory pathogens. Extraction-free analysis (n = 29) had 95% success in specimens with ≥1 RNA copies/µl. Usability testing performed first time by health care workers showed 95% accuracy.

Multiplexed Functional Assessment of Genetic Variants in CARD11.

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Cross-Sectional Prevalence of SARS-CoV-2 Among Skilled Nursing Facility Employees and Residents Across Facilities in Seattle.

BACKGROUND: Skilled nursing facilities (SNFs) are high-risk settings for SARS-CoV-2 transmission. Infection rates among employees are infrequently described. OBJECTIVE: To describe SARS-CoV-2 rates among SNF employees and residents during a non-outbreak time period, we measured cross-sectional SARS-CoV-2 prevalence across multiple sites in the Seattle area. DESIGN: SARS-CoV-2 testing was performed for SNF employees and residents using quantitative real-time reverse transcription polymerase chain reaction. A subset of employees completed a sociodemographic and symptom questionnaire. PARTICIPANTS: Between March 29 and May 13, 2020, we tested 1583 employees and 1208 residents at 16 SNFs for SARS-CoV-2. MAIN MEASURE: SARS-CoV-2 testing results and symptom report among employees and residents. KEY RESULTS: Eleven of the 16 SNFs had one or more resident or employee test positive. Overall, 46 (2.9%) employees had positive or inconclusive testing for SARS-CoV-2, and among those who completed surveys, most were asymptomatic and involved in direct patient care. The majority of employees tested were female (934, 73%), and most employees were Asian (392, 30%), Black (360, 28%), or white (360, 28%). Among the 1208 residents tested, 110 (9.1%) had positive or inconclusive results. There was no association between the presence of positive residents and positive employees within a SNF (p = 0.62, McNemar's test). CONCLUSIONS: In the largest study of SNFs to date, SARS-CoV-2 infections were detected among both employees and residents. Employees testing positive were often asymptomatic and involved in direct patient care. Surveillance testing is needed for SNF employees and residents during the pandemic response.

Multiplex assessment of protein variant abundance by massively parallel sequencing.

Determining the pathogenicity of genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes requires generalizable, scalable assays. We describe variant abundance by massively parallel sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance simultaneously. We apply VAMP-seq to quantify the abundance of 7,801 single-amino-acid variants of PTEN and TPMT, proteins in which functional variants are clinically actionable. We identify 1,138 PTEN and 777 TPMT variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and show that p.Pro38Ser, which accounts for ~10% of PTEN missense variants in melanoma, functions via a dominant-negative mechanism. Finally, we demonstrate that VAMP-seq is applicable to other genes, highlighting its generalizability.