Potential Misdiagnosis between COVID-19 and Dengue Infection Using Rapid Serological Test

Round 1

Reviewer 1 Report

The manuscript raises an important issue with regard to cross antibody reactions between SARS-CoV-2 and Dengue. This may give rise to misdiagnosis with serious consequences. Besides extensive English editing a few ponts need to be addressed.

1. The authors raise point in discussion about lack of seroconversion in some patients who have had a positve PCR test. Do they have any data as to when the PCR tests were carried out in relation to the serology which they carried out as this may explain that serum samples may have been taken too soon after infection for sero-conversion. This information should be in the material and mehtods or If not available it should be stated and reason why.
2. Details of all the assays and the manufacturers need included in the materials and method including the NS1 test which isn't described at all.
3. Why are there not any quantitative results for the PCR to compare with the serology? This is a qPCR test so Ct values should be available. If not again reason needs to be given. 
4. Line 132 and Table 1. Plus and minus values are given only with no actual results. Also a comment about faint appearance is give. The actual quantitative data from the test should be shown as well as for the positive control. 
5. Was it possible to carry out virus neutralisation test and this may distinguish the viruses? At least this should be discussed as a possible back up test.
6. The authors need to discuss the need for an additional SARS-2-CoV serum test in Dengue regions. e.g. The gold standard Roche electrochemiluminescence assay uses the SARS-2-CoV nucleocapsid rather than the spike protein. This may distinguish from Dengue.

Author Response

Thank you for the opportunity to revise our manuscript, idr-1210659 entitled "Antibody Cross Reactivity: Potential Misdiagnosis between COVID-19 and Dengue" by Khairunisa, S. Q. et al. We appreciate the detailed review and constructive suggestions. We consider the quality of the manuscript to have been markedly improved by the suggested edits.

We here in present the reviewers’ comments, followed by our point-by-point responses (in red color), including their locations in the modified text. The page and line numbers described here refer to those in the revised manuscript. Changes made in the manuscript are marked using by using yellow highlight and track changes in MS Word.

Response to Reviewer 1 Comments

Point 1: The authors raise point in discussion about lack of seroconversion in some patients who have had a positve PCR test. Do they have any data as to when the PCR tests were carried out in relation to the serology which they carried out as this may explain that serum samples may have been taken too soon after infection for sero-conversion. This information should be in the material and methods or If not available it should be stated and reason why.

Response 1: Thank you very much for your suggestion. Actually we already mention that “the blood was taken approximately within one week after positive COVID-19 confirmation” in materials and method section in originally in page 3, lines 14. We would like to add the table in discussion section in page 6-9. The table with detail data of PCR test and correlation with serological test. We also revised the sentences in page 6 lines 23-24 as follows: “To date, the response of the host immune system and variation of antibodies profile toward COVID-19 are still not fully understood, the data showed PCR test and correlation with serological test in Table 2”.

Point 2: Details of all the assays and the manufacturers need included in the materials and method including the NS1 test which isn't described at all.

Response 2: Thank you for the suggestion. Actually we already mention that the NS1 is from SD Biosensor in material and method. We would like to add the sentences in page 3, lines 24-25, as follows: “All serological tests were performed according to the manufactured instruction’s kit”

Point 3: Why are there not any quantitative results for the PCR to compare with the serology? This is a qPCR test so Ct values should be available. If not again reason needs to be given. 

Response 3: Thank you for the comments. As we mention in our answer for feedback point 1. We would like to add the table in discussion section in page 6-9 . The table with detail data of PCR test including the Ct value.

Point 4: Line 132 and Table 1. Plus and minus values are given only with no actual results. Also a comment about faint appearance is give. The actual quantitative data from the test should be shown as well as for the positive control. 

Response 4: Thank you for the comments. The serological tests in this study were done using rapid kits (lateral flow assay) which gave qualitative result. Therefore there is no quantitative data that can be given. We can only provided whether the result is positive or negative by marking with plus minus values. We would like to add the information in Table 1 in page 4 as follows: “**+ sign denotes positive results and *** - sign denotes negative results”. We would like to add the sentences in page 4 lines 36-39 and page 5 lines 1-3 as follows: “We mention faint appearance since the bands intensity, indicating the presence of dengue IgG, were not high. Nevertheless it is still counted as positive result. Another limitation of the dengue rapid kit which we used (SD Biosensor, Korea) is that the limit of detection (LOD) is not mentioned by manufacturer. However according to the manual the specificity and sensitivity of IgG, IgM and NS1 detection show high correlation compared to RT-PCR and ELISA (≥92.9%)”.

Point 5: Was it possible to carry out virus neutralisation test and this may distinguish the viruses? At least this should be discussed as a possible back up test.

Response 5: Thank you for your suggestion. This is one of our limitations.. We would like to add the sentences in the end of discussion section in page 10, lines 30-31, as follows: “Third, unfortunately we couldn’t do neutralization assay due to unavailability of the materials”. We would like to revise the sentences in the end of discussion section in page 10, lines 31-32, as follows: “Therefore, future study should document the viral load, serologic response in the body and do neutralization assay”.

Point 6: The authors need to discuss the need for an additional SARS-2-CoV serum test in Dengue regions. e.g. The gold standard Roche electrochemiluminescence assay uses the SARS-2-CoV nucleocapsid rather than the spike protein. This may distinguish from Dengue.

Response 6: Thank you very much for your suggestion. We understand that the standard method for COVID-19 detection is qPCR. In fact, all of our samples were first confirmed as COVID-19 positive by qPCR detection. With this study, we would like to emphasize that the usage of COVID-19 Ab rapid tests should be taken with caution especially in dengue region. On the other hand, the use of Ab (IgG/IgM) rapid test for dengue detection is common during diagnostic. Thus we would like to raise awareness that for determining acute infection, rapid Ab test which has potential of cross reactivity is not recommended. At least the rapid test should not be used as standalone for diagnostic.

Reviewer 2 Report

An important manuscript in the iht against COVID-19, especially since now the pandemic has high impact in countries with Dengue.

It is clear that Dengue and COVID-19 can "interfere" and make the diagnostic procedure very complex.

The is just one comment, contributing to the complexity

The authors have not discussed the performance of the various diagnostic tests. i.e. it was not presented the sensitivity and specificity of the diagnostic test. Such diagnostic errors may contribute more to the complex situation. Thus I propose, at least to indicate the specificity and sensitivity of the techniques and optimally discuss the probability of diagnostic errors due to false-positive and false-negative results.

Author Response

Thank you for the opportunity to revise our manuscript, idr-1210659 entitled "Antibody Cross Reactivity: Potential Misdiagnosis between COVID-19 and Dengue" by Khairunisa, S. Q. et al. We appreciate the detailed review and constructive suggestions. We consider the quality of the manuscript to have been markedly improved by the suggested edits.

We here in present the reviewers’ comments, followed by our point-by-point responses (in red color), including their locations in the modified text. The page and line numbers described here refer to those in the revised manuscript. Changes made in the manuscript are marked using by using yellow highlight and track changes in MS Word.

Response to Reviewer 2 Comments

Point 1: An important manuscript in the iht against COVID-19, especially since now the pandemic has high impact in countries with Dengue.

It is clear that Dengue and COVID-19 can "interfere" and make the diagnostic procedure very complex.

The is just one comment, contributing to the complexity

The authors have not discussed the performance of the various diagnostic tests. i.e. it was not presented the sensitivity and specificity of the diagnostic test. Such diagnostic errors may contribute more to the complex situation. Thus I propose, at least to indicate the specificity and sensitivity of the techniques and optimally discuss the probability of diagnostic errors due to false-positive and false-negative results.

Response 1: Thank you for your suggestion. We would like to add the sentences in page 5 lines 39 and page 6 lines 1-18 as follows: “According to the kit manual (SD biosensor) for dengue, the kit performance had been tested to determine the sensitivity and specificity. The IgM detection was compared with ELISA indicating 97.5% and 96.6% sensitivity and specificity respectively. Likewise IgG was also comparable to ELISA showing sensitivity 97.2% and specificity 96.2%. NS1 detection was compared to RT-PCR giving sensitivity and specificity of 92.9% and 98.7% respectively. COVID-19 detection kit (Vazyme) has sensitivity 91.54% (95% CI: 86.87%, 94.65%) and specifity 97.02% (95% CI: 94.74%, 98.33%) according to the manual.

COVID-19 detection kit (UNscience) has a clinical sensitivity of 98.511% (95% CI: 96.788%, 99.452%) and specificity of 88.208% (95% CI: 83.086%, 92.221%) according to the manual.

Similar study was done by Marsha et al using the same SD Biosensor to assessed antibody indicating 2 samples were dengue IgG positive among 33 positive COVID-19 samples from asymptomatic people. In addition, 4 samples also positive for dengue IgG among 19 samples that tested positive for COVID-19 IgM.[14] The sensitivity and specificity of different dengue antibody rapid diagnostic tests (RDT) has been done by Kok-Siang Yow et al. The result indicated that Standard Q (SD Biosensor) had the highest sensitivity in detection of IgM and NS1 compare to Multisure, Bioline, and careUS. All RDTs had high specificity for dengue NS1 detection (100%). IgM detection was also high at 100% except for Multisure (96.7%).[15]”

Reviewer 3 Report

Authors found that four among 120 patients with covid-19 were also serologically reactive to dengue virus and suggested the possibility of cross reactivity of antibody cross reactivity between covid-19 and dengue infection because of undetectable dengue virus in the patients. It’s interesting hypothesis but includes potential to mislead. In the dengue endemic area, there would be many people who have already experienced dengue and had immunity against dengue virus. A presence of the antibody against a virus in blood does not mean a presence of the virus in blood. It, therefore, makes sense that the four people have IgG antibody to dengue virus even they are not infected “now” with the virus. They should assess positive rate of IgG against dengue virus in healthy donors “especially free from covid-19” in the same country or area if they want to say antibody cross reactivity between covid-19 and dengue infection. I believe that some people are detected for IgG as well as the covid-19 patients.

Author Response

Thank you for the opportunity to revise our manuscript, idr-1210659 entitled "Antibody Cross Reactivity: Potential Misdiagnosis between COVID-19 and Dengue" by Khairunisa, S. Q. et al. We appreciate the detailed review and constructive suggestions. We consider the quality of the manuscript to have been markedly improved by the suggested edits.

We here in present the reviewers’ comments, followed by our point-by-point responses (in red color), including their locations in the modified text. The page and line numbers described here refer to those in the revised manuscript. Changes made in the manuscript are marked using by using yellow highlight and track changes in MS Word.

Response to Reviewer 3 Comments

Point 1: Authors found that four among 120 patients with covid-19 were also serologically reactive to dengue virus and suggested the possibility of cross reactivity of antibody cross reactivity between covid-19 and dengue infection because of undetectable dengue virus in the patients. It’s interesting hypothesis but includes potential to mislead. In the dengue endemic area, there would be many people who have already experienced dengue and had immunity against dengue virus. A presence of the antibody against a virus in blood does not mean a presence of the virus in blood. It, therefore, makes sense that the four people have IgG antibody to dengue virus even they are not infected “now” with the virus. They should assess positive rate of IgG against dengue virus in healthy donors “especially free from covid-19” in the same country or area if they want to say antibody cross reactivity between covid-19 and dengue infection. I believe that some people are detected for IgG as well as the covid-19 patients.

Response 1: Thank you for your suggestion.  However the present of IgG against Dengue virus in population of dengue endemic area as Indonesia indicates the possibility of antibody cross reactivity between COVID-19 and dengue in serological testing.

Round 2

Reviewer 1 Report

The authors hace considerably improved the manuscript with additonal information and explainations.

Author Response

Thank you for the opportunity to revise our manuscript, idr-1210659 entitled "Antibody Cross Reactivity: Potential Misdiagnosis between COVID-19 and Dengue" by Khairunisa, S. Q. et al. We appreciate the detailed review and constructive suggestions. We consider the quality of the manuscript to have been markedly improved by the suggested edits.

We herein present the reviewers’ comments, followed by our point-by-point responses (in red color), including their locations in the modified text. The page and line numbers described here refer to those in the revised manuscript. Changes made in the manuscript are marked using by using yellow highlight and track changes in MS Word.

Response to Reviewer 1 Comments

Point 1: English language and style are Moderate English changes required.

Response 1: Thank you for your suggestion.  According to reviewer 1’s suggestions, we agree with reviewer 1 to proofread our manuscript. The manuscript was proofread by Medical English Service, Universitas Airlangga, Surabaya, Indonesia.

Point 2: The authors hace considerably improved the manuscript with additonal information and explainations.

Response 2: Thank you for your comments and suggestions, we modified the sentences and added the sentences in the discussion section on page 6 until page 11 to improve our manuscript.

Reviewer 3 Report

Authors seem not to understand my point. And I cannot understand why authors think that the presence of IgG against Dengue virus indicates the possibility of antibody cross reactivity between COVID-19 and dengue in serological testing.

Authors should demonstrate that the four patients (serologically positive for both COVID-19 and dengue) have never infected with dengue virus at least last one or two years if authors want to say the possibility of antibody cross reactivity between them.

Also, if antibody against dengue virus has cross reactivity to COVID-19,  does the antibody protect the person from COVID-19?  

If so, many people in the dengue virus endemic area do not infected with COVID-19?

Expectation should not be a conclusion without any scientific assessment.

Author Response

Thank you for the opportunity to revise our manuscript, idr-1210659 entitled "Antibody Cross Reactivity: Potential Misdiagnosis between COVID-19 and Dengue" by Khairunisa, S. Q. et al. We appreciate the detailed review and constructive suggestions. We consider the quality of the manuscript to have been markedly improved by the suggested edits.

We here in present the reviewers’ comments, followed by our point-by-point responses (in red color), including their locations in the modified text. The page and line numbers described here refer to those in the revised manuscript. Changes made in the manuscript are marked using by using yellow highlight and track changes in MS Word.

Response to Reviewer 3 Comments

Point 1: Authors seem not to understand my point. And I cannot understand why authors think that the presence of IgG against Dengue virus indicates the possibility of antibody cross reactivity between COVID-19 and dengue in serological testing.

Authors should demonstrate that the four patients (serologically positive for both COVID-19 and dengue) have never infected with dengue virus at least last one or two years if authors want to say the possibility of antibody cross reactivity between them.

Also, if antibody against dengue virus has cross reactivity to COVID-19,  does the antibody protect the person from COVID-19?  

If so, many people in the dengue virus endemic area do not infected with COVID-19?

Expectation should not be a conclusion without any scientific assessment.

Response 1: Thank you for your suggestion.  According to the Reviewer 3’s suggestions, we agree with reviewer 3 to do the assessment of positive rate of IgG, IgM and NS1 against dengue virus in healthy donors and we got the interesting results. We would like to add the table 2 showed The characteristics of healthy individuals (pre COVID-19 date) tested by dengue RDT (n = 38) in Surabaya, Indonesia in page 5 and we added the sentences in results section in page 5 line 25 – 30 as follows: “As many as 38 sera from healthy individuals in Surabaya were collected using the dengue RDT (pre-COVID-19 date). According to Table 2, one of the samples showed positive NS1 and positive DENV IgG. The results indicated that the individual could be in the early phase of dengue infection. The other sample showed positive DENV IgG but negative NS1. This result indicated the individual had the dengue antibody as a result of the previous infection.”

We added the sentences in method section (serological test) in page 3 line 19 – 21 as follows: “In addition, sera from healthy individuals were analyzed using dengue rapid diagnostic test (RDT).”

 In addition, we also added the explanation in discussion section in page 10, lines 9 – 34, as follows: “The four among 120 samples were serologically positive to dengue IgG while the NS1 test and RT-PCR test showed a negative result. There are two possibilities that could explain the results. Firstly, these four patients ever had dengue infection before the hospital admission due to COVID-19. Therefore, the antibody remained circulating in the blood although the virus had already gone. Dengue antibody response in the post infection can last for long period. IgM circulates in the body up to 2 to 6 months, while IgG persists longer generally up to 6 months to 2 years after dengue primary infection. Furthermore, upon secondary infection IgG reacts earlier with higher levels and longer deployment period.

https://www.mdpi.com/2076-393X/8/2/174/htm (1)

Thus the presence of higher dengue IgG compared to IgM in healthy residents of dengue endemic area is plausible. One study showed from 910 healthy adult donors in Saudi Arabia 38.9% were seropositive to IgG, while positive IgM and NS1 were found in  5.5% and 5.3%, respectively.

https://pubmed.ncbi.nlm.nih.gov/28469422/ (2)

Another seroprevalance screening in Guangzhou observed among 2085 serum samples, IgG and IgM positive rates were 11.80% and 3.98% respectively. (Liu et al., 2018) To the best of the researcher’s knowledge, there is no seroprevalance study yet to assess the positive rate of healthy/ asymptomatic people in Indonesia. Therefore we conducted additional RDT analysis to healthy samples from 2014 (pre-COVID-19 date) which have been kept in our laboratories. Due to limitation of samples and materials, only 38 samples were included. The results indicated 2 samples were reactive to IgG detection, which one of them showed positive NS1. Based on the data this study suggested the rate of dengue IgG among healthy people in Indonesia was 5.26%. However to get more reliable data this study suggests performing  a similar experiment with a higher number of samples and repeating testing in future studies.

The second possible reason is the presence of antibody cross reactivity between COVID-19 and dengue infection in the samples.”

We also changed the sentences in conclusion section in page 12, line 4 – 14 as follows: “In conclusion, using the serological RDT to determine COVID-19 or dengue infection might lead to misdiagnosis, while the gold standard of COVID-19 detection is Real-Time PCR. However the usage of serological RDT for dengue infection is popular  due to its simplicity and affordability particularly in developing countries. Thus, IgG/IgM RDT cannot be used standalone. We recommend priotitize the NS1 result for detection of dengue infection during the COVID-19 pandemic. Ideally patients are examined for both diseases using the PCR-based method. Overall, the findings alert the health care in dengue endemic regions to improve awareness and accurate diagnoses. The potential of concomitant infection should also be considered to prevent dangerous impacts on  patients. This also emphasizes that a rapid serological method with high sensitivity and specificity is required to distinguish between SARS-CoV-2 and dengue infections.”

We also added the sentences in abstract in page 1 line 16 – 18 as follows: “In addition, 38 sera from healthy individuals (pre-COVID-19 date) were analyzed using a dengue rapid test.”, and page 1 line 19 – 22 as follows: “Interestingly, regarding seropositivity of NS1 and DENV IgG from healthy individuals (pre COVID-19 infection), 2 samples were positive DENV IgG, while one of them was positive NS1. This suggested that in the dengue endemic area, many people have already experienced dengue and have immunity against dengue virus.”

According to the Reviewer 3’s suggestions, the results of the assessment of positive rate of IgG, IgM and NS1 against dengue virus in healthy donors, we would like to change the title from “Antibody Cross Reactivity: Potential Misdiagnosis between COVID-19 and Dengue” to

“Potential Misdiagnosis between COVID-19 and Dengue Infection using Rapid Serological Test”.