Based on the combination of the results of rRT-PCR and the antibody titers, three groups of people were created retrospectively, as below.

In group A, were allocated people, with the following results: (a) decrease of Ct for the N gene from 2nd run NPS-1 to NPS-2 and (b) increase of anti-N antibody titers from BS-1 (negative) to BS-2 (positive).

In group B, were allocated people, with the following results: (a) increase of Ct for the N gene from 2nd run NPS-1 to NPS-2 to ≥40 and (b) further increase of anti-N antibody titers from BS-1 (positive) to BS-2 (positive).

In group C, were allocated people, with the following results: (a) Ct for the N gene (a1) ≥ 40 at 2nd run NPS-1 or (a2) > 35 at 2nd run NPS-1 and ≥40 at NPS-2 and (b) no increase of anti-N antibody titers from BS-1 (negative) to BS-2 (negative).

During evaluation of Ct for the rRT-PCR, values were rounded to the nearest unit (half unit values (i.e., *.5) were rounded to the higher unit). For the analysis, Ct values for the N gene were used, as previous studies have indicated that these had a higher specificity compared to Ct values for the E gene [14]. For the purposes of the statistical analysis only, in rRT-PCR, Ct values found to be ≥40, were given the arithmetic value of 40 to facilitate the computations; as this was the lowest possible negative value, statistical differences were not increased artificially.

The change in Ct values between NPS-1 and NPS-2 was calculated as the result of subtraction of Ct value obtained during the 2nd run NPS-1 from the Ct value obtained during the NPS-2 of rRT-PCR for the N gene. The change in antibody titers was calculated as the result of subtraction of the titers obtained for BS-1 from that obtained for BS-2; separate changes were calculated for anti-N and anti-S antibodies.

Data were entered into Microsoft Excel and analyzed using SPSS v. 25 (IBM Analytics, Armonk, NY, USA). Basic descriptive analysis was performed.

Frequencies were compared by using cross-tabulation with Pearson’s chi-square test or Fisher exact test, as appropriate.

Ct values for the N gene obtained from 1st run NPS-1 and 2nd run NPS-1 were compared between them by using the Wilcoxon signed-rank test. The same test was also used to compare Ct values obtained in 2nd run NPS-1 and NPS-2, as well as anti-N and anti-S antibody titers obtained in BS-1 and BS-2. Differences between groups were evaluated by means of the Kruskal-Wallis test.

Correlation analysis was performed between changes in Ct values and antibody titers.

The potential association of changes in Ct values with changes in antibody titers was assessed by analysis of correlation. Correlations and correlation coefficients are those of Pearson.

In all analyses, statistical significance was defined at p < 0.05.

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