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

Abstract

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.

Fig. 1.. Workflows for SARS-CoV-2 screening and HIV viral load testing

(a) Schema of the workflows for analysis of only plasma specimens or the mixtures of contrived plasma and NS samples. Specimens are extracted using our low-cost, in-house extraction method and analyzed using RT-qPCR assays which target HIV LTR gene, SARSCoV-2 N gene (N1, N2), and human RP gene (RP). (b) Assay interpretation of the results based on HIV and SARS-CoV-2 RT-qPCR results (c) Analysis of HIV and SARS-CoV-2 RNA spiked in lysed negative plasma at 0 or 200 copies/mL. (d) Analysis of HIV and SARS-CoV-2 RNA spiked in lysed pooled NS and plasma at 0 or 200 copies/mL. Individual Cq values of three technical replicates (individually extracted and RT-qPCR assayed) are plotted.

Fig. 2.. Quantitation and classification of HIV and SARS-CoV-2 RNA obtained from inhouse and standard extraction methods.

Scatter plots of (a) measured HIV VL (n = 3) vs input RNA in contrived plasma samples (lysed plasma and spiked with synthetic HIV RNA at 0, 100, 200, 600, 6K, or 60K copies/mL), and (b) measured SARS-Cov-2 VL (n = 3) vs input RNA in contrived NS (lysed and spiked with synthetic SARS-CoV-2 RNA at 0, 100, 200, 600, 6K, or 60K copies/mL). Diagonal lines represent 100% theoretical RNA recovery from the extraction method based on the spiked-in RNA input. Dashed diagonal lines indicate the bound for precise measurement (i.e., measured VL within ±0.3 log10(VL, copies/mL of input)). Efficiency is the slope of the linear regression fit, reported along with its R2. Summary of HIV VL classification and SARS-CoV-2 detection using both extraction methods using (c) OPTION 1 workflow to analyze either NS or plasma, and (d) OPTION 2 workflow to analyze pooled plasma/NS samples. For HIV samples, we classified results as viral load suppressed (VLS) or virological failure (VF). For SARS-CoV-2, we classified results as positive or negative based on whether they are detected or not detected by both N1 and N2 assays. Supplementary figure 6 is the scatter plots of the OPTION 2 workflow. Data in Fig. 2 and Supplementary figure 2 is collected by a single assay operator.

Fig. 3.. Quantitation and classification of HIV RNA in clinical plasma specimens.

(a) Schema of experimental design to compare the performance of standard vs in-house extraction methods. Plasma RNA extracted by standard or in-house extraction was evaluated using RT-qPCR. The RT-qPCR results were compared in terms of measured viral load and classifications. All HIV seropositive specimens had VL ≥1000 copies/mL when tested by the reference lab using Abbott m2000. (b) Cq values of the HIV long-terminal repeat (LTR) assay and human ribonuclease P (RP) assay (control for negative specimens) from the plasma RNA extracted by the in-house method. Extracted RNA from HIV-seropositive specimens were analyzed by LTR and RP RT-qPCR assays in duplicate. HIV-seronegative specimens (as tested by the vendor) were each extracted twice, and each extracted RNA aliquot was analyzed by LTR and RP RT-qPCR assays. The average RP Cq values are plotted. (c) Scatter plot of measured HIV VL in specimens extracted by standard vs in-house methods. The diagonal line represents a theoretical 100% correlation of measured HIV VL of specimens extracted by both extraction methods. The shaded box around the diagonal lines indicates a precise measurement bound (±0.3 log10(VL measured in RNA extracted by standard method, copies/mL)). Diamonds are samples with measured VL outside precise measurement bound. (d) Classifications correspond to VL results. Negative results are labeled as “-” in white boxes. Virologic failure results were classified using 200 copies/mL (dark purple boxes) and 1000 copies/mL (light purple boxes) thresholds. RNA extracted by both methods was collected by one assay operator and analyzed by RT-qPCR by another assay operator.

Fig. 4.. Quantification and detection of SARS-CoV-2 RNA in clinical NS specimens.

(a) Schema of experimental design to compare the performance of standard vs in-house extraction methods. NS RNA obtained from standard or in-house extraction were evaluated using RT-qPCR. The RT-qPCR results were compared in terms of measured viral load and classifications. Discordant classifications were ruled out based on previous results from the CLIA-certified lab. (b) Cq values of the SARS-CoV-2 N1, N2, and human RP assay (control for negative specimens) from the in-house method. Each specimen was extracted, followed by RT-qPCR, and represented as individual data points. (c) Classifications of specimens based on the results from the two extraction methods. Specimens detected by N1 and N2 (regardless of RP detection) are classified as positive; specimens detected by either N1 or N2 assays are classified as presumptive positive; specimens not detected by N1 and N2 but detected by RP assay are classified as negative; specimens not detected by any assays are classified as indeterminate (IND). Scatter plot of measured SARS-CoV-2 in specimens extracted by standard vs in-house methods in (d) N1 assay (e) N2 assay. The diagonal lines represent a theoretical 100% correlation of SARS-CoV-2 VL measured in RNA extracted from both extraction methods. Dashed lines indicate RT-qPCR precise measurement bound (i.e., ±0.3log10(VL measured in RNA extracted from standard method, copies/mL). Diamonds are samples with measured VL that significantly deviates from those obtained via standard extraction. (f) Summary table for classifications by the two methods. Data were collected by two assay operators.

Fig. 5.. Comparison of HIV LTR and SARS-CoV-2 Cq values obtained from RNA co-extracted from NS/plasma specimens using in-house vs standard extraction protocols.

(a) Schema of experimental design to compare the performance of standard vs in-house extraction methods. NS/plasma RNA extracted by standard or in-house extraction were evaluated using RT-qPCR. The RT-qPCR results were compared in terms of measured VL and classification of infection status as shown in Fig 1b. (b) Cq values of the SARS-CoV-2 LTR, N1, N2, and human RP assay (control for negative specimens) from the in-house method. Each specimen was extracted, followed by RT-qPCR, and represented as individual data points. (c) Classifications of specimens based on the results obtained from the two extraction methods. Scatter plots showing correlation between the in-house and standard extraction methods for HIV VL by LTR assay (d), SARS-CoV-2 VL by N1 assay (e) and SARS-CoV2 VL by N2 assay (f) in pooled NS/Plasma specimens. The diagonal lines represent a theoretical 100% correlation of VL measured from both methods. Dashed lines indicate precise measurement bound. Diamonds are samples with measured VL that significantly deviates from those obtained via standard extraction.