Real-time RT-PCR for SARS-CoV-2
In all cases, nasopharyngeal samples were collected and tested for
SARS-CoV-2 with the LightMix® Modular Wuhan CoV RdRP-gene Kit
(53-0777-96) by TIB MOLBIOL, Berlin, Germany (https://www.roche-as.es).
The rRT-PCR assay was performed according to the manufacturer’s protocol
using probe P1 from the WHO protocol (RdRP_SARSr-P1:
FAM-CCAGGTGGWACRTCATCMGGTGATGC-BBQ). The rRT-PCR assay was performed
following recommended cycling conditions: reverse transcription at 55 °C
for 5 min and 95 °C for 5 min, followed by 45 cycles of PCR at 95 °C for
15 sec and 60 °C for 15 sec. The cycle threshold (Ct) value above 39.0
was considered negative. The tested sample was considered SARS-CoV-2
positive if LightMix showed positive results for the RdRP gene.
Flow Cytometry for determination of lymphocyte
subsets
Lymphocyte subsets were detected by flow
cytometry analysis. Samples of EDTA anticoagulated peripheral blood (2
mL) were collected from patients with COVID-19 before initiation of
treatment. All samples were tested within 6 hours of being obtained, and
CD4+ and CD8+ T-cell counts
(cells/μL) were measured by multiple-color flow cytometry. The cells
were analyzed on a BD FACS Canto II flow cytometry system (BD
Biosciences).
Statistical analysis
Statistical analysis was performed with the Statistical Package for the
Social Sciences (SPSS version 18.0, SPSS Inc., Chicago, IL, USA).
Categorical data were described as percentages and continuous data as
median with interquartile range (IQR). Normally distributed continuous
variables were presented as means with standard deviations (SD).
Comparison of the differences between the two groups was conducted using
the t-test, Chi-square test, or Mann–Whitney U test. Variables with a
two-tailed p-value < 0.05 were considered statistically
significant.