Diagnostic performance of RT-PCR-based sample pooling strategy for the detection of SARS-CoV-2

Study design and population

We carried out a cross-sectional study of diagnostic tests to evaluate the diagnostic performance of the strategy of grouping NS samples to detect SARS-CoV-2 employing RT-PCR, compared with the individual RT-PCR of samples of NS. NS were collected during the period from september to december 2020.

People diagnosed at Hospital III Daniel Alcides Carrión and Hospital Base Hipolito Unanue, located in the province and region of Tacna in Peru, were evaluated. Participants attended to rule out SARS-CoV-2 infection by taking an NS sample that RT-PCR then analyzed; patients with symptoms of up to 7 days of evolution and who also signed the informed consent were included in the study (Fig. 1).

Fig. 1figure 1

Distribution of nasopharyngeal swab samples for subsequent analysis

Transportation, handling, and pooling of samples

After taking the individual NS samples, they were transferred in 2 mL of the viral transport medium (VTM). Later, they were analyzed by RT-PCR of the SARS-CoV-2 virus following the manufacturer's instructions [13]. Forty-five of these samples were positive and were classified according to their Ct values as high (H: ≤ 25), moderate (M: 26–30), and low (L: 31–35) concentration of viral RNA [14]; the rest (375) had a negative result. From these NS samples (positive and negative), 42 clusters were formed, each containing ten samples. These pools contained a total of 1.0 mL of NS-VTM (0.1 mL for each sample) and were categorized into three groups. Fifteen clusters were formed from the combination of 0.1 mL of NS-VTM from a positive sample for SARS-CoV-2 with nine negative samples, each with 0.1 mL of NS-VTM (total, 0.9 mL), simulating a positivity of 10%. These first 15 clusters were subdivided into 5 clusters with a high, moderate, and low concentration of viral RNA, respectively. The second block of 15 clusters was formed considering two positive samples for SARS-CoV-2, each with 0.1 mL of NS-VTM (total, 0.2 mL), with eight negative samples, each with 0.1 mL of NS-VTM (total, 0.8 mL), simulating a positivity of 20%. This second set of 15 clusters was subdivided similarly to the first (high, moderate, and low). Finally, the third group evaluated 12 clusters formed with ten negative samples for SARS-CoV-2, each with 0.1 mL of NS-VMT (total, 1.0 mL). These distributions were made using the study by Wacharapluesadee et al. [14] (Fig. 2).

Fig. 2figure 2

Pooling of nasopharyngeal swab samples according to positivity and concentration of SARS-CoV-2 viral RNA

SARS-CoV-2 detection

RNA extractions were performed from 100 μL of VTM pooled using the RADI COVID-19 Detection Kit from KH medical Co. For the RT-PCR, the SD Biosensor brand Standard M nCoV real-time PCR kit was used, which detects the RdRp and E genes of the virus, following the procedures specified by the manufacturer [13]. The reaction was run in the QIAGEN Model RotorGene™ brand real-time Thermal Cycler. The individual and group samples were considered positive when the SARS-CoV-2 target (RdRp gene and E gene) had a Ct < 36; otherwise (Ct ≥ 36), they were deemed to be negative, as indicated by the instructions for use [13].

Statistic analysis

All data were analyzed using the Stata version 16 statistical program (StataCorp., Texas, USA). The individual and grouping Ct values were presented in their median and interquartile range (IQR) due to their non-symmetric distribution. The bivariate analysis was performed using the Wilcoxon statistical test of signs and ranges to compare the median of the Ct values of the samples before being grouped with the Ct values after the grouping.

We evaluate the NS sample grouping strategy using the RT-PCR test results of individual samples were taken as a reference. First, we calculated the area under the ROC curve (Receiver Operating Characteristic) the Youden index, as well as sensitivity, specificity, likelihood ratio (LR), positive and negative predictive value with their respective global 95% CI and by subgroups according to their Ct values that were classified as high (H: ≤ 25), moderate (M: 26–30) and low (L: 31–35) concentration of viral RNA [14]. However, the positive and negative LR could not be calculated in all the subgroups since the sensitivity, and specificity parameters in some of these reached 100%, generating a calculation of LR that was not estimable.

Ethical aspects

This research adheres to the Helsinki standards for research on human subjects. The protocol was approved by the institutional research ethics committee of the Private University of Tacna (Registration Code: 045-FACSA-UI) and by the research committees of the Hospitals: Daniel Alcides Carrión EsSalud-Tacna and Hipolito Unanue-Tacna. Likewise, it was entered with code EI00000001461 to the national registry of health research projects (PRISA) developed by the National Institute of Health of Peru (INS-Peru). Informed consent was requested from all patients who participated in the research, providing their nasopharyngeal swab samples for RT-PCR analysis.

The Universidad Privada de Tacna funded the study. Support was received from the Regional Directorate of Health of Tacna (DIRESA-Tacna) for molecular analysis through its molecular biology laboratory.

留言 (0)

沒有登入
gif