Antibodies against dengue virus in COVID-19 patients in Japan: a dengue virus non-endemic country

Abstract

Background

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was associated with a global pandemic in 2020. Infection and vaccination induced a variety of immune responses in humans that differed from those observed before the pandemic. Several reports indicated that SARS-CoV-2 infection induced the production of antibodies cross-reactive to dengue virus. However, many of these reports emerged from dengue-endemic regions, and the possibility that induction of cross-reactive antibodies was affected by memory immunity induced by previous dengue virus infection could not be ruled out.

Main text 

In the present study, we investigated the levels of cross-reactive antibodies against dengue virus in 118 COVID-19 patients in Japan, a dengue non-endemic country. Serum samples collected from each patient during the acute and convalescent phases were analyzed using commercial IgG and IgM ELISA kits. The analysis revealed two dengue IgM-positive cases in both the acute and convalescent phases of COVID-19. These two cases showed an increasing trend of dengue IgM antibody titer in the convalescent phase. One additional case was dengue IgM-negative in the acute phase and IgM-borderline in the convalescent phase. By contrast, no cases of positive or elevated dengue IgG were observed. All 118 cases investigated in the present study lacked a history of recent travel to dengue-endemic countries. These findings suggest that SARS-CoV-2 infection induces IgM that cross-reacts with dengue virus antigens, even in the absence of immunological memory against dengue virus.

Conclusions

Certain COVID-19 patients in Japan produced IgM that cross-reacted with dengue virus. Therefore, the presence of false positives should be considered in the serodiagnosis of dengue fever and COVID-19.

Background

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in China in 2019 and quickly spread worldwide. The World Health Organization declared a public health emergency of international concern in January 2020, which ended in May 2023 due to factors, such as the emergence of less-virulent strains, such as the Omicron variant, the development of acquired immunity through infection, availability and use of therapeutic drugs and vaccines, and the establishment of better methods to treat patients. Nevertheless, the COVID-19 epidemic has continued to the present. As an emerging infectious disease, COVID-19 induced previously undescribed immune responses in humans through viral infection and vaccination. A number of studies reported that some antibodies induced by SARS-CoV-2 cross-react with other pathogens [1], such as dengue virus [1–12], which causes dengue fever. Subsequent studies reported that antibodies against SARS-CoV-2 spike protein and its receptor-binding domain (S1-RBD) also cross-react with dengue virus [13, 14]. However, as most of the reports of cross-reactive antibodies emerged from dengue-endemic countries, co-infection with SARS-CoV-2 and dengue virus and previous dengue virus infection could not be completely ruled out as the cause of cross-reactive antibody production. The mechanism by which these cross-reactive antibodies are induced within acute infection and convalescent SARS-CoV-2 patients also remains unclear. In the present study, therefore, preserved serum collected from COVID-19 patients in Japan (a dengue virus non-endemic country) during both the acute (initial visit) and convalescent (2–4 weeks after the initial visit) phases of infection was used to quantify anti-dengue IgG and IgM by ELISA to evaluate changes in antibody titers during the acute and convalescent phases.

Main text

The study examined preserved serum samples collected from 118 patients (mean age and standard deviation, 59.7 ± 14.4 years; 93 males and 25 females) who visited Osaka City General Hospital between April 2020 and September 2021 for treatment in the acute and convalescent phases of COVID-19 (average 22.9 days later, minimum 17 days, maximum 31 days). Serum titers of anti-dengue virus IgG and IgM were measured using a Dengue virus IgG ELISA kit (Abnova, Taiwan) and a Dengue virus IgM ELISA kit (TECAN, Hamburg, Germany) according to the respective instructions.

According to the kit criteria, all samples were negative for dengue IgG in both the acute and convalescent phases. However, samples derived from two patients (patients A and B) were positive for IgM in both the acute and convalescent phases (Fig. 1). In addition, a sample from one patient (patient C) showed borderline positive/negative results in the convalescent phase, although the result was negative in the acute phase. The dengue ELISA actual values exhibited a mean for all samples of 0.42 ± 0.15 Results in Units (RIU) in the acute phase and 0.42 ± 0.13 RIU in the convalescent phase for IgG and 3.71 ± 2.33 RIU in the acute phase and 3.86 ± 2.64 RIU in the convalescent phase for IgM. Both patient A and patient B showed higher values in the convalescent phase than the acute phase (patient A: 11.74 RIU [acute phase], 15.87 RIU [convalescent phase]; patient B: 18.24 RIU [acute phase], 20.58 RIU [convalescent phase]). The values for patient C were 1.72 RIU higher in the convalescent phase (10.14 RIU) than the acute phase (8.42 RIU). This degree of increase was lower than that of patients A (4.13 RIU) and B (2.34 RIU) (Fig. 1).

Fig. 1.

Dynamic changes in titers of IgG and IgM against dengue virus among COVID-19 patients between the acute and convalescent phases (n = 118). Data points for dengue virus IgM in Patients A–C are indicated by orange circles

These three patients had no confirmed history of travel to dengue-endemic areas. In addition, these patients had not been vaccinated against SARS-CoV-2 at the time of their first visit for COVID-19 treatment, suggesting that the patients were infected with SARS-CoV-2 in the absence of immunological exposure to the virus. In addition, none of these patients had a chronic infection associated with hypergammaglobulinemia.

SARS-CoV-2 is a positive-sense, single-stranded RNA virus of the family Coronaviridae; genus Betacoronavirus [15]. By contrast, dengue virus is a positive-sense, single-stranded RNA virus of the family Flaviviridae; genus Flavivirus [16]. Thus, both viruses are taxonomically distinct and not usually thought to induce immune cross-reactivity. However, the results of the present study showed that 1.7% of the cases (2 of 118) were dengue IgM-positive in both the acute and convalescent phases of COVID-19, and 0.85% of cases (1 of 118) were dengue IgM-negative in the acute phase and IgM-borderline in the convalescent phase of COVID-19, indicating that some COVID-19 patients in Japan—a dengue non-endemic country—produced antibodies cross-reactive to dengue virus. However, no cases of positive or elevated dengue IgG were observed, indicating that IgM to IgG maturation had not progressed. Although induction of dengue IgG in COVID-19 patients has been reported in dengue-endemic countries, it is less frequent than induction of dengue IgM [3, 4, 6, 10]. Nevertheless, the presence of genuinely cross-reactive antibodies would generally be expected to involve IgG class switching. One potential explanation for the observed findings is polyclonal B-cell activation. Chronic infections caused by viruses, bacteria, or parasites—as well as autoimmune disorders—are known to induce transient or sustained increases in IgM levels through nonspecific polyclonal B-cell activation. Similarly, in COVID-19, such nonspecific activation has been suggested to promote the production of nonspecific antibodies [17]. Indeed, elevated rheumatoid factor (RF) levels have been reported in patients with acute COVID-19 [18], and another study demonstrated that increased RF levels can lead to the detection of dengue IgM antibodies, thereby contributing to false-positive results in IgM assays [19]. In the present study, no chronic infections were documented, and no data regarding RF levels were available; RF absorption or other interference-elimination procedures were not performed prior to testing. In addition, patient A had a past medical history of rheumatoid arthritis. Therefore, the possibility that the detected dengue IgM reactivity was attributable to nonspecific IgM produced through COVID-19-associated polyclonal B-cell activation cannot be ruled out. As the presence of antibodies cross-reactive between dengue virus and SARS-CoV-2 has been detected in stored serum collected from dengue patients prior to the COVID-19 pandemic, there is concern regarding the impact of these antibodies on serological diagnosis of dengue fever and COVID-19.

The present study has several limitations. First, the analysis was performed using only a single commercially available ELISA kit each for IgG and IgM. In addition, controls such as serum samples collected from healthy individuals prior to the COVID-19 pandemic were not included, nor were serum samples from individuals with other infectious or autoimmune diseases. Therefore, the possibility that the observed results represent kit-related false-positive reactions cannot be ruled out.

Conclusions

Serum obtained from 118 COVID-19 patients in the acute and convalescent phases of infection in Japan, where dengue is not endemic, showed no cross-reactivity for dengue IgG. However, two patients tested positive for anti-dengue IgM in the acute and convalescent phases. The presence of false positives should be considered in the serodiagnosis of dengue fever and COVID-19.

Author contributions

EEN, TSh, and TSa conceptualized and designed the study methodology. NT, RM, EEN, RK, TG, MS, and TSa participated in sample and data collection and analysis. EEN, TSh, TG, MS, and PL supervised the entire project and edited the manuscript. NT and TSa performed the data analysis and prepared the final version of the manuscript. All authors read and approved the final version of the manuscript.

Funding

Not applicable.

Availability of data and materials

The datasets generated and analyzed during the current study are not publicly available due to ethical and privacy restrictions but are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of the Research Institute for Microbial Diseases, Osaka University (approval number: 31-14-1). Written comprehensive informed consent was obtained from each patient.

Consent for publication

Not applicable.

Competing interest

The authors declare no competing interest.