Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection

Abstract

Background: The assessment of antibody responses to severe acute respiratory syndrome coronavirus-2 is potentially confounded by exposures to flaviviruses. The aims of the present research were to determine whether anti-dengue antibodies affect the viral load and the detection of anti-coronavirus nucleocapsid (N)-protein antibodies in coronavirus infectious disease 2019 (COVID-19) in Bangladesh.

Methods: Viral RNA was evaluated in swab specimens from 115 COVID-19 patients by real-time reverse transcription polymerase chain reaction (rT-PCR). The anti-N-protein antibodies, anti-dengue virus E-protein antibodies and the dengue non-structural protein-1 were determined in serum from 115 COVID-19 patients, 30 acute dengue fever pre-COVID-19 pandemic and nine normal controls by ELISA.

Results: The concentrations of viral RNA in the nasopharyngeal; Ct median (95% CI); 22 (21.9-23.3) was significantly higher than viral RNA concentrations in oropharyngeal swabs; and 29 (27-30.5) p < 0.0001. Viral RNA concentrations were not correlated with-dengue IgG levels. The anti-nucleocapsid antibodies were IgA 27% positive and IgG 35% positive at days 1 to 8 post-onset of COVID-19 symptoms versus IgA 0% and IgG 0% in dengue patients, p < 0.0001. The levels of anti- nucleocapsid IgA or IgG versus the levels of anti-dengue IgM or IgG revealed no significant correlations.

Conclusions: Viral RNA and anti-nucleocapsid antibodies were detected in COVID-19 patients from dengue-endemic regions of Bangladesh, independently of the dengue IgG levels.

Keywords: COVID-19; DENV; ELISA; IgA; IgG; IgM; N-protein; SARS-CoV-2; dengue fever; rT-PCR.

Figure 1

Study Plan. Elisa reactivity was assessed in four independent patient groups. Group 1; n = 48 COVID-19 patients in Dhaka were confirmed to be SARS-CoV-2 rT-PCR positive, Group 2; COVID-19 in two neighboring districts that tested negative SARS-CoV-2 by rT-PCR; Dhaka (n = 45) district boundary outlined in red and Nayaranganj (n = 22) district boundary outlined in blue. Group 3; Pre-pandemic 2019 Dengue patients and Group 4; Control subjects in Dhaka during the 2020 COVID-19 pandemic. District boundaries adapted from the map of Bangladesh [29].

Figure 2

SARS-CoV-2 RNA levels and correlations with dengue IgG levels. The boxplot corresponds to the 25th and 75th interquartile range (IQR), the horizontal line inside the box to the median and the bars outside the box to the minimum and maximum RNA concentrations (Ct values). The Ct values in n = 19 nasopharyngeal specimens (NP, circles) with a mean of 22.6 (95% CI 22–23) versus n = 29 oropharyngeal specimens (OP, triangles) median of 29 (95% CI 28–30) *** p < 0.0001. The Ct values of NP specimens from COVID-19 patients reporting symptoms (sympt) with a mean of 22 (95% CI 21.8–23) versus no symptoms (none) with a mean of 23.6 (21.5–25.6) p = 0.06. The filled symbols represent severe cases (A). The scatter plots represented by open circles and the curve fit of linear regression analysis of Ct values and anti-dengue (DENV) IgG levels are represented by the solid line (B and C); n = 19 NP specimens and matched serum; r = −0.11 (95% CI −0.54–0.36), p = 0.65 (B) and n = 17 OP specimens and matched serum; r = 0.38 (95% CI −0.73–0.12, p = 0.3.

Figure 3

Kinetics of anti-SARS-CoV-2 N-protein antibodies in rT-PCR positive COVID-19. The IgA (A) and IgG (B) levels in the course of n = 48 COVID-19 patients. The horizontal dotted line represents the 10 U/L cut-off for positive reactivity in ELISA. Serial measurements of n = 17 patients (open circles) and single time point measurements of n = 12 patients between day 1 to day 8 (open triangles) or n = 19 patients between day 65 to day 177 (open squares). ** p = 0.001 paired student t-test comparing the mean IgA level and the mean IgG level at interval day 65 to day 177.

Figure 4

Levels of anti-SARS-CoV-2 N-protein antibodies and anti-dengue antibodies. The N-protein IgA and IgG and the dengue IgM and IgG in rT-PCR negative COVID-19 (A) and in pre-pandemic hospitalized dengue fever (B). The difference in mean antibody activities between the two districts were compared by independent student t-tests. In Dhaka city, n = 45 (circles) anti-N-protein specific antibody levels IgA; mean 16 [95% CI 0.8–32], 14% positive and IgG; mean 9 (95% CI 6.7–11.8), 25% positive versus Narayanganj district n = 22 (squares), IgA; mean activity 43 (95% CI 20–66), 73% positive ** p < 0.001 and IgG; mean activity 25 (95% CI 18–32), 73% positive ** p < 0.001. The anti-dengue IgM in Dhaka city mean activity 4.3 (95% CI 3.8–4.9) 2.3%positive versus Narayanganj district; mean activity 5.3 (95% CI 4.4–6.2) 4.5% positive, p = 0.07, and anti-dengue IgG; mean activity 34 [95% CI 28–40] 72% positive versus mean activity 46 (95% CI 39–53), 86% positive, respectively, * p = 0.01. NS = not significant.

Figure 5

Correlations of SARS CoV-2 N protein IgA or IgG and Dengue IgG. The anti-N-protein IgA (A) and (B) and IgG (C) and (D) at 14–90 days after COVID-19 symptoms in n = 36 rT−PCR positive (A) and (C) and at 1–8 days after COVID-19 symptoms in n = 67 rT-PCR negative patients (B,D). The values within the area of the box are below the 10 U/L cut-off for positive reactivity in ELISA. The coefficient of correlations between dengue IgG and N-protein IgA in rT-PCR pos COVID-19; r = 0.26 95% CI −0.07–0.54), p = 0.127 and in rT-PCR neg COVID-19; r = −0.28 (95% CI −0.53–0.03), p = 0.07. The coefficient of correlations between dengue IgG and N-protein IgG in rT-PCR pos COVID-19; 0.44 (95% CI 0.03–0.73), p = 0.04 and in rT-PCR neg COVID-19; r = −0.33 (95% CI −0.64–0.08), p = 0.11.