Seroprevalence of hospital staff in a province with zero COVID-19 cases.

BACKGROUND: COVID-19 seroprevalence data, particularly in less developed countries with a relatively low incidence, has been scant. We aimed to explore the seroprevalence of hospital staff in the area with zero confirmed COVID-19 case to shed light on the situation of COVID-19 infection in zero or low infection rate countries where mass screening was not readily available.
METHODS: A locally developed rapid immunoglobulin M (IgM)/immunoglobulin G (IgG) test kit was used for hospital staff screening of Ranong hospital which is located in a province with zero COVID-19 prevalence in Thailand from 17th April to 17th May 2020. All staff was tested, 100 of which were randomly invited to have a repeating antibody test in one month. (Thai Clinical Trials Registry: TCTR20200426002).
RESULTS: Of 844 hospital staff, 82 were tested twice one month apart (response rate for repeating antibody test 82%). Overall, 0.8% of the participants (7 of 844) had positive IgM, none had positive IgG. Female staff had 1.0% positive IgM (95% CI: 0.5-2.1%) while male had 0.5% positive IgM (95% CI: 0.1-2.6%). No participants with a history of travel to the high-risk area or close contact with PCR-confirmed COVID-19 case developed SARS-CoV-2 antibodies. Among 844 staff, 811 had no symptoms and six of them developed IgM seropositive (0.7%) while 33 had minor symptoms and only one of them developed IgM seropositive (3.0%). No association between SARS-CoV-2 IgM status and gender, history of travel to a high-risk area, close contact with PCR-confirmed or suspected COVID-19 case, presence of symptoms within 14 days, or previous PCR status was found. None of the hospital staff developed SARS-CoV-2 IgG.
CONCLUSIONS: COVID-19 antibody test could detect a considerable number of hospital staff who could be potential silent spreaders in a province with zero COVID-19 cases. Accurate antibody testing is a valuable screening tool, particularly in asymptomatic healthcare workers. Trial registration: This study was approved by the Institutional Review Board of Chulalongkorn University (IRB No.236/63) and the Institutional Review Board of Ranong Hospital. (Thai Clinical Trials Registry: TCTR20200426002).

Introduction

Seroprevalence data has been scarce in Asian countries besides China. Along with the gold-standard polymerase chain reaction (PCR) testing, antibody testing is beneficial for epidemiological investigation and epidemic control of infectious diseases including the present coronavirus disease 2019 (COVID-19). In Singapore, serological evidence was used to trace and identify missing spreaders for three clusters [1]. Asymptomatic silent spreaders have been of major concern as suggested by a systematic review—could be as low as 1.6% of COVID-19 confirmed cases in China or as high as 51.7% of confirmed cases in Diamond Princess cruise [2]. An estimated proportion of asymptomatic COVID-19 patients was 13.34% and was substantially higher in healthcare workers at 36.96% [3] with the transmission rate of asymptomatic patients to close contact individuals at 18.8% [4].

Early COVID-19 prevalence studies were based only on PCR for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals. However, recent studies tended to report PCR along with serological test results. An early report of an overall seroprevalence of 2.5% in a hospital setting in China, in which 1.8% and 3.5% were among healthcare workers and asymptomatic patients, respectively [5]. The recent meta-analysis of evidence up to 24th August 2020 reported an estimated overall seroprevalence in healthcare workers at 8.7% [6]. Additionally, China studied the development of antibodies against SARS-CoV-2 in symptomatic confirmed COVID-19 cases and found that immunoglobulin M (IgM) reached its peak 20–22 days after onset while immunoglobulin G (IgG) reached its peak 17–19 days after onset [7]. A more recent systematic review reported IgM median seroconversion time ranged from four to 14 days, peak at two to five weeks, and undetectable at six weeks after onset while IgG median seroconversion time range from 12–15 days, peak at three to seven weeks, and decline after eight weeks post-onset [8]. Some works emphasized antibody testing for the hospital workforce and policy issues. Another seroprevalence study in Belgium conducted on healthcare personnel who worked in a tertiary hospital found 6.4% seroprevalence and identified some risk factors for developing antibodies against SARS-CoV-2 [9].

There were inter-regional variations of seroprevalence in healthcare personnel with an estimated seroprevalence of 12.7% in North America, 8.5% in Europe, 8.2% in Africa, and 4.0% in Asia [6] with a range of seroprevalence among healthcare workers from 0% in Alzintan, Libya [10] to 45.3% in London, UK [11]. Moreover, intra-regional variations of seroprevalence were observed in healthcare workers, for example, seroprevalence among hospital staff in China range from 1.8% [5] to 17.1% [12] whereas seroprevalence of healthcare workers in Asia besides China and Southeast Asia ranged from 0.4% [13] to 9.1% [14]. In Southeast Asia, there were only two studies on the seroprevalence of COVID-19 in healthcare workers: one from Thailand reported a 3.7% seroprevalence [15], and one from Vietnam reported a zero seroprevalence [16]. Seroprevalences in healthcare personnel varied from 1.3% [17] to 45.3% [11] in Europe, from 0.8% [18] to 44.0% [19] in North America, from 0.9% [20] to 14.1% [21] in South America, and from zero [10] to 45.1% [22] in Africa whereas no seroprevalence studies were from Antarctica or Australia.

Ideally, both PCR and antibody testing provided complementing information to shape the picture of the situation in a specific hospital, area, or country. However, most low- to middle-income countries could not afford the cost of laboratory tests and had to develop criteria-based policies for resource-use optimization. In Thailand, for instance, the PCR is reserved for individuals who meet the national criteria for COVID-19 PCR testing.

Ranong is one of 77 provinces in Thailand with zero cumulative confirmed COVID-19 cases from 17th April to 17th May 2020, and still had no case as of December 5, 2020 (Fig 1). Hospital is one of the highest risk areas for receiving and spreading pathogens—healthcare workers developed a higher chance of getting infected by co-workers or patients and vice versa. This study aims to estimate the hospital-wide seroprevalence in healthcare workers who worked in the largest public hospital in Ranong to develop the strategies to slow down the pandemic and ensure the safety of healthcare workers who come to work and patients who visit the hospital.

Fig 1

Numbers of confirmed COVID-19 cases across geographical areas of Thailand as of July 4, 2020.

Bangkok metropolitan had the most cumulative confirmed COVID-19 cases, 1,609 cases, while Ranong, the province of focus in this study, had no confirmed COVID-19 case.

Methods

Participant

All 844 healthcare workers including the physician, nurse, medical assistance, medical technician, and non-medical officers of the largest public hospital in Ranong province that reported zero cumulative PCR-confirmed COVID-19 cases during the entire study period (17th April to 17th May 2020), were invited to participate in this study. All of them accepted to participate with written informed consent. Participants with active symptoms suiting national criteria for polymerase chain reaction testing were quarantined and excluded. Participants were asked to answer a survey about risk history for COVID-19, recent symptoms in the past two weeks, and previous PCR tests if available.

Antibody testing

The locally developed Baiya Rapid IgG/IgM test kit (Baiya Phytopharm, Thailand) which reported the presence of IgM and IgG qualitatively, was used for antibody testing in this study. The internal validation of the test kit using the serum of 51 PCR confirmed COVID-19 cases and 150 controls showed sensitivity 94.1% (48 of 51) and specificity 98.0% (147 of 150) of IgM or IgG antibody. Participants with positive IgM were encouraged to have a PCR test if available.

Study procedures

On 17th April 2020, of 844 participants, 100 were randomly selected to have their first antibody testing. On 17th May 2020, all the remaining participants were tested for serological immunity whereas the 100 healthcare workers who tested in April had their second antibody testing.

Statistical analysis

Categorical data were presented with counts and percentages while continuous data were reported with median and interquartile range. Association between categorical variables and the status of immunoglobulin was analyzed using Fisher’s exact test. The 95% confidence interval (CI) of the seroprevalence was calculated by Wilson’s method using binomial probabilities. Missing data were excluded. All data were analyzed using Stata 16.1 (College Station, TX).

Ethics committee approval

This study was approved by the Institutional Review Board of Chulalongkorn University (IRB No.236/63) and the Institutional Review Board of Ranong Hospital. (Thai Clinical Trials Registry: TCTR20200426002)

Patient and public involvement

Patients and the public have not been directly involved in the design, conduct, or reporting of this study. However, we believe that our study would provide additional information for patients who would visit the hospital in zero or relatively low COVID-19 prevalence area and for the public to have more accessible real-world experience.

Results

Healthcare workers’ demographic

All 844 Thai hospital staff were invited to participate in the study and tested for IgM and IgG antibodies against SARS-CoV-2 (100% participation rate). Their median age was 42 years (interquartile range 32–50). Most of them (71.7%) were female and 96.1% had no symptoms. The 33 symptomatic participants (3.9%) reported cough (1.7%), rhinitis (1.5%), sore throat (1.4%), dyspnea (0.5%), and fever (0.2%). History of travel to the high-risk area was 2.5%, history of close contact PCR confirmed COVID-19 case was 2.0%, history of close contact suspected case was 38.1%. Only 1% of participants had previous negative PCR results while the rest never got tested (Table 1).

Table 1

Demographic and clinical characteristics of Ranong hospital staff.

		IgM+
	Total	n (%)	95% CI
Total	844	7 (0.8%)	0.4–1.7
Median age, years (25th–75th percentile)	42 (32–50)
Gender
Male, n (%)	211 (25.0%)	1 (0.5%)	0.1–2.6
Female, n (%)	605 (71.7%)	6 (1.0%)	0.5–2.1
Unspecified, n (%)	28 (3.3%)	0 (0.0%)	NA
Ethnicity
Thai	844 (100.0%)	7 (0.8%)	0.4–1.7
Non-Thai	0 (0.0%)	0 (0.0%)	NA
History of travel to high risk area
Yes	21 (2.5%)	0 (0.0%)	NA
No	823 (97.5%)	7 (0.9%)	0.4–1.7
History of close contact confirmed case
Yes	17 (2.0%)	0 (0.0%)	NA
No	827 (98.0%)	7 (0.8%)	0.4–1.7
History of close contact suspected case
Yes	322 (38.1%)	3 (0.9%)	0.3–2.7
No	522 (61.9%)	4 (0.8%)	0.3–2.0
Asymptomatic	811 (96.1%)	6 (0.7%)	0.3–1.6
Symptomatic	33 (3.9%)	1 (3.0%)	0.5–15.3
Fever	2 (0.2%)	0 (0.0%)	NA
Cough	14 (1.7%)	0 (0.0%)	NA
Rhinitis	13 (1.5%)	0 (0.0%)	NA
Sore throat	12 (1.4%)	1 (8.3%)	1.5–35.4
Dyspnea	4 (0.5%)	0 (0.0%)	NA
Previous PCR status
Negative	8 (1.0%)	0 (0.0%)	NA
Never tested	836 (99.0%)	7 (0.8%)	0.4–1.7

Data were presented in counts and percentages unless otherwise specified.

IgM+, immunoglobulin M positive; NA, not available; PCR, polymerase chain reaction.

Serological results of healthcare workers in Ranong hospital

Overall, seven hospital staff tested positive for COVID-19 IgM (0.8%, 95% CI: 0.4–1.7%) while none of the participants developed IgG. Female staff had 1.0% positive IgM (95% CI: 0.5–2.1%) while male staff had 0.5% positive IgM (95% CI: 0.1–2.6%). None of the participants with a history of travel to the high-risk area or a history of close contact with PCR-confirmed COVID-19 case developed antibodies against SARS-CoV-2. There was no statistical difference in IgM seroprevalence between staff with and without a history of close contact with suspected COVID-19 cases. Among 844 staff, 811 had no symptoms and six of them developed IgM seropositive (0.7%, 95% CI: 0.3–1.6%) while 33 had minor symptoms and only one of them developed immunoglobulin M (3.0%, 95% CI: 0.5–15.3%). Of 12 staff with a sore throat, one had positive IgM (8.3%, 95% CI: 1.5–35.4%). There was zero IgM seroprevalence in staff with fever, cough, rhinitis, or dyspnea. Of 844 participants, eight had previous negative PCR results and none has developed the antibody for SARS-CoV-2 (Table 1). No association between IgM antibody against SARS-CoV-2 status and gender, history of travel to a high-risk area, history of close contact with PCR-confirmed COVID-19 case, history of close contact with suspected COVID-19 case, presence of symptoms within 14 days, or previous PCR status was found. None of the hospital staff developed IgG against SARS-CoV-2.

Characteristics of seropositive participants

Seven participants developed IgM antibodies in May. Their age ranged from 20 to 49 years. Six of them were female while only one staff was male. All of them were Thai, had no history of travel to a high-risk area, no history of contact with PCR-confirmed COVID-19 case, and no previous PCR status. Three had a history of contact with suspected COVID-19 cases while the other four did not. Participant 7 was the only one with IgM positive who had a symptom (i.e. sore throat) within 14 days before antibody testing. All seven staff with a positive antibody subsequently got nasopharyngeal swabs for PCR and resulted in negative for SARS-CoV-2 on PCR (Table 2).

Table 2

Characteristics of hospital staff who developed immunoglobulin M antibody and subsequent PCR status.

	Age Range, years	Gender	Ethnicity	History of travel	History of contact with a confirmed case	History of contact with a suspected case	Previous PCR status	Symptoms	Subsequent PCR status
Participant 1	30–39	Female	Thai	No	No	Yes	Never tested	No	Negative
Participant 2	20–29	Female	Thai	No	No	No	Never tested	No	Negative
Participant 3	40–49	Female	Thai	No	No	No	Never tested	No	Negative
Participant 4	40–49	Female	Thai	No	No	Yes	Never tested	No	Negative
Participant 5	30–39	Female	Thai	No	No	No	Never tested	No	Negative
Participant 6	40–49	Male	Thai	No	No	No	Never tested	No	Negative
Participant 7	40–49	Female	Thai	No	No	Yes	Never tested	Sore throat	Negative

PCR, polymerase chain reaction.

Repeating antibody tests in random sampling participants

Of 844 participants, 100 were randomly selected to get antibody testing in April and then repeat in May at hospital-wide antibody testing. None of them had any immunoglobulin developed in April. Of 100 randomly selected participants, 82 participated in antibody testing in May (response rate for repeating antibody test 82%) which showed no antibody against COVID-19.

Discussion

In a small seaside province, Ranong, which had 193,370 inhabitants, located in the south of Thailand closed to the Andaman Sea and had a border close to Myanmar, there was zero officially PCR-confirmed COVID-19 case compared to the current situation in Thailand (0.048 per thousand). This zero prevalence could be either from good compliance to public health recommendations or a low number of individuals who get PCR testing due to the stringent national criteria.

In this study, we reported a 0.8% IgM seroprevalence in Ranong hospital staff while their PCR tests were negative. While critics might argue that the antibody test had a high false-positive rate, especially in a population with high pre-test probability such as hospital staff, the PCR test could have a high false-negative rate, especially without a highly sensitive diagnostic test [23].

Of seven seropositive hospital staff, only one was symptomatic and none of them had a history of travel to high-risk areas or history of close contact with PCR-confirmed COVID-19 case. The current national criteria and policies for PCR testing mostly rely on risk histories and symptoms so asymptomatic individuals or those with minor symptoms were left out, resulting in an underestimation of the actual prevalence of COVID-19. However, the lack of association with risk history might be because of the small number of staff with positive IgM.

Our study reported a lower seroprevalence in healthcare workers than hospitals in China (0.8% vs. 1.8%) [5], a tertiary hospital in Belgium (0.8% vs. 6.4%) [9], and a rural hospital located in low COVID-19 prevalence county of Germany (0.8% vs 2.7%) [24]. Unlike China, Belgium, and Germany where the seroprevalences were mostly from positive IgG, our study revealed mostly positive IgM. Comparison with Belgium and German hospitals should be interpreted with caution due to the unknown PCR status of subjects of the two studies.

All seropositive healthcare workers in our study had positive IgM and negative IgG. We speculated two possible explanations that might associate with the negative IgG result. First, the time frame of antibody testing from the onset of the disease might play a crucial role [25]. While IgM and IgG can be detected in some patients as early as 4–6 and 5–10 days after symptom onset, respectively [26], 70% of symptomatic patients developed positive IgM by days 8–14 and 90% of total antibody tests positive by days 11–24 [27]. Moreover, COVID-19 confirmed patients had higher SARS-CoV-2 IgM concentration than IgG before the 15th-day post symptom onset and vice versa after the 15th-day post symptom onset [28], and patients developed peak IgM of 94.1% approximately 20–22 days and all of them finally have positive IgG approximately 17–19 days after symptom onset [7]. Thus, antibody testing during the window period could lead to a false-negative result of IgG antibody [29]. For patients with no or minor symptoms, the onset might not be possible to determine. Moreover, IgM can be persisted for 42 days post symptom [30]. Nonetheless, the time of antibody detection might not reflect the time of infection and disease transmission. Secondly, the difference in patients’ immune response might be a relevant factor. Some patients recover from COVID-19 infection without any production of SAR-CoV-2 IgG, reflecting that strong innate immunity might be sufficient to eradicate SARS-CoV-2 [31].

In this study, we used a qualitative antibody test kit so the relatively lower antibody level in early infected persons might not be detectable. Repeating PCR or quantitative antibody tests for suspected individuals was practiced in many countries. However, the cost would be doubled or more, and might not be possible for low- or middle-income countries to cover all suspected individuals. We prospectively designed the workflow to get participants with positive IgM, which related to acute infection of SARS-CoV-2, subsequently tested with a nasopharyngeal swab for PCR which was the gold standard for diagnosis COVID-19 during the study period. We also attempted to repeat the antibody test by prospectively randomly selected 100 participants to get antibody testing in April and then participate in hospital-wide antibody testing in May for repeating test; however, only 82 of them could participate in the follow-up test and none of them developed antibodies against SARS-CoV-2 in both first and second tests. All IgM positive participants were tested in hospital-wide antibody testing in May and we did not have a chance to repeat the antibody test in seropositive participants; however, all of them were tested for PCR after IgM positive as planned. Rapid antibody tests should be more affordable to permit multiple tests among high-risk asymptomatic individuals who could be silent spreaders.

While the data is relatively limited, this data is the best evidence we can obtain in the current situation in Thailand where the appropriate COVID-19 test in population has not been conducted. Moreover, as the hospital underwent a major significant change in its organizational structure and management team, along with the compliance to the ethical approval we received, our request to conduct repeat surveillance of the same cohort has not been granted.

Serological testing provides some crucial epidemiological information and would have been more effective when combined with other diagnostic tests such as PCR. With immunoglobulin status and PCR results, we can shape the situation more precisely for both individual and regional views. The antibody testing should be used as a screening tool, not a diagnostic tool, and should be conducted with consideration of the current situation in each area. Hopefully, with this and other vigorous and dedicated studies on antibody status around the globe, antibody testing would provide useful information for pandemic control.

Conclusions

The COVID-19 antibody test could detect a considerable number of hospital staff who could be potential silent spreaders in areas with zero COVID-19 cases. Accurate antibody testing is a valuable screening tool, particularly in asymptomatic healthcare workers.

(XLSX)

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17 Nov 2020

PONE-D-20-25321

Seroprevalence of Hospital Staff in Province with Zero COVID-19 Cases

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Reviewer #1: This manuscript by Nopsopon et al described the SARS-CoV-2 seroprevalence of the hospital staff of one of the largest hospitals in the Ranong province of Thailand, where no confirmed COVID-19 was found by between April-May, 2020. The testing was carried out using a homemade IgM/IgG detection test. The authors found that among the 844 members tested only 7 cases were found to be positive for IgM but negative for IgG. Only 1 of the positive cases has reported sore throat. All the positive cases were PCR negative. This is the first report of the testing of hospital staff in the region, however, based on numerous reports it is hardly surprising that there are individuals who have prior exposure to the virus but have yet remained asymptomatic. The data presented is very limited and for only a one month period, way back in April-May. With the fast changing pandemic more recent surveillance of the same cohort will be preferable and compared to the results obtained from the earlier time point. The addition data will substantially strengthen the manuscript. In addition, there are several other concerns.

1. It is not clear why only IgM and not IgG was detected. This is quite surprising. This needs to be followed up and explain whether this could be due to experimental errors. In addition, were the cases being followed up and retested for Ab and for IgG?

2. There is a concern on the test sensitivity of 94.1% (48 of 51), some positive cases could have been missed.

3. Was second Ab tested carried out with the IgM positive cases?

4. It is not clear whether any hospital staff were excluded. It was indicated in line 106 that “Participants with active symptoms suiting national criteria for polymerase chain reaction testing were quarantined and excluded.

5. It is not clear whether 84 or 100 individuals were tested twice, conflicting numbers were provided in the abstract versus the text.

Reviewer #2: Burden of disease deals with a range of medical statistics aimed to give a comprehensive picture of how diseases impact on society or to allocate health care and research resources. Diagnostic tests are an important part in this regard. A perfect test, usually referred to as the gold standard implies being positive in all patients with the disease and negative in all patients who do not have the disease. However, most tests are imperfect. In the work by Pongpirul et al. the authors carried out a survey assessing the presence of anti-COVID-19 antibodies in hospital workers from a Thailand area in which COVID-19 had not been reported during the study period. Among 844 participants few of them (<1%) revealed the presence of IgM antibodies bearing no close relationship with their clinical or personal characteristics. The study adds some information to the local features of COVID-19 infection.

It would be interesting to know whether there existed a different degree of exposure risk among staff members.

It is intriguing that people yielding an IgM positive test at the beginning failed to develop IgG response a month later. Any tentative explanation for this?

Information is needed on whether validation studies were carried in recent COVID-19 cases, or not?

The same applies when talking about the amount of missing data

In discussing results please note that lack of association may depend on the reduced number of people yielding a positive IgM response

Page 8, lines 191-192, there are more studies in this regard which should be mentioned.

Please also note that the time when Ab are detected may have nothing to do with transmission

The Bangkok Metropolitan area might be indicated in the Figure.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

8 Dec 2020

Dear Editor,

We thank you and the reviewers for the comments and suggestions. Please find our point-by-point responses below:

Editor: Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. The data presented is very limited (only a one month period). More recent surveillance of the same cohort will be preferable and compared to the results obtained from the earlier time point. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised during the review process.

Response: Thank you for your time and consideration. The manuscript was revised to meet PLOS ONE’s publication criteria. While the data is relatively limited, this data is the best evidence we can obtain in the current situation in Thailand where the appropriate COVID-19 test in population has not been conducted. Moreover, as the hospital underwent a major significant change in its organizational structure and management team, along with the compliance to the ethical approval we initially received, our request to conduct repeat surveillance of the same cohort has not been granted yet and might take several months. Nonetheless, please note that, as of December 7, 2020, this province still reports zero cases. We added these limitation statements to the Discussion section accordingly.

Editor: 2.We note that [Figure(s) 1] in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth).

Response: As Figure 1 contains original data from our research, we provide a replacement figure with an original image from our colleague whom we acknowledged in the revised manuscript so the copyright issue should no longer be a concern. As of December 7, 2020, this province still reports zero cases so we updated the data in Figure 1 as well.

Editor: 3.Thank you for stating the following in the Acknowledgments Section of your manuscript:

[We thank Baiya Phytopharm, Thailand for supporting the Baiya Rapid COVID-19 IgG/IgM test kit. The company did not involve in the data analysis, interpretation, or manuscript preparation.]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

[The author(s) received no specific funding for this work.]

Additionally, because some of your funding information pertains to [commercial funding//patents], we ask you to provide an updated Competing Interests statement, declaring all sources of commercial funding.

Response: We did not mean to provide funding information in the Acknowledgment Section. None of the authors receive any funding from the company mentioned in the Acknowledgment Section. We only wanted to thank them for their contribution to developing the local COVID-19 IgG/IgM test kit for Thailand upon request free of charge, which resulted in an opportunity for us to conduct this observational study. We revised the choice of words in the Acknowledgment Section accordingly.

Editor: In your Competing Interests statement, please confirm that your commercial funding does not alter your adherence to PLOS ONE Editorial policies and criteria by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests. If this statement is not true and your adherence to PLOS policies on sharing data and materials is altered, please explain how. Please include the updated Competing Interests Statement and Funding Statement in your cover letter. We will change the online submission form on your behalf.

Response: Authors have no competing interests according to the PLOS definition. The suggested statement is added in the Competing Interests statement. The updated Competing Interests Statement and Funding Statement were included in the cover letter.

Editor: 4.We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: https://www.medrxiv.org/content/10.1101/2020.06.24.20139188v1?versioned=true

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

[Note: HTML markup is below. Please do not edit.]

Response: All sources used in this manuscript are already cited in the previously submitted manuscript (including the concerning publication mentioned above – Ref. 12). All overlapping text outside the methods section is rephrased. Please advise if we still have to address this concern differently.

Reviewer #1: This manuscript by Nopsopon et al described the SARS-CoV-2 seroprevalence of the hospital staff of one of the largest hospitals in the Ranong province of Thailand, where no confirmed COVID-19 was found by between April-May, 2020. The testing was carried out using a homemade IgM/IgG detection test. The authors found that among the 844 members tested only 7 cases were found to be positive for IgM but negative for IgG. Only 1 of the positive cases has reported sore throat. All the positive cases were PCR negative. This is the first report of the testing of hospital staff in the region, however, based on numerous reports it is hardly surprising that there are individuals who have prior exposure to the virus but have yet remained asymptomatic. The data presented is very limited and for only a one month period, way back in April-May. With the fast changing pandemic more recent surveillance of the same cohort will be preferable and compared to the results obtained from the earlier time point. The addition data will substantially strengthen the manuscript. In addition, there are several other concerns.

Response: Thank you for your time in reviewing the manuscript. Despite the limitation of data in this manuscript, we provide the best evidence available in the current situation in Thailand where COVID-19 testing is low, given the limited resources and national screening policy. With the volatile nature of the pandemic, we would like to provide evidence of an actual situation where the zero or low number of COVID-19 confirmed cases in Thailand might come from a low number of people tested. Although we wanted to conduct a repeat survey as suggested, the hospital underwent a major significant change in its organizational structure and management team, along with the compliance to the ethical approval we received; hence, our request to conduct repeat surveillance of the same cohort has not been granted and might take several months. We added these limitation statements to the Discussion section accordingly (Line 194-198). We believe that the data we provided would bring attention to the overlooked problem of the low number of testing in this region, thus encourage the governing body to take more action and change the perspective that only high-risk or symptomatic patients should get tested for mass screening to get the actual situation in Thailand.

Reviewer #1: 1. It is not clear why only IgM and not IgG was detected. This is quite surprising. This needs to be followed up and explain whether this could be due to experimental errors. In addition, were the cases being followed up and retested for Ab and for IgG?

Response: More discussion on predominant IgM positive compared to IgG positive is provided in Discussion section. Mass screening or antibody testing has not been readily supported in Thailand, especially in the current situation where the national policy allows people to get tested as low as possible to keep the low number of confirmed cases. We really would like to conduct a follow-up test to provide the most recent situation in Thailand; however, without the first piece of evidence to demonstrate how mass screening would provide benefit and reality of COVID-19 situation in Thailand, the follow-up test is currently not possible. The cases were not retested for Antibody but instead get PCR testing which, at that time, was considered the gold standard for COVID-19 confirmation in Thailand.

Reviewer #1: 2. There is a concern on the test sensitivity of 94.1% (48 of 51), some positive cases could have been missed.

Response: We concern about some positive cases would be missed by this rapid IgM/IgG test kit we used; however, this is the best readily available test that could be used as mass screening in Thailand at the time we conducted the study. As of December 7, 2020, this province still reports zero cases.

Reviewer #1: 3. Was second Ab tested carried out with the IgM positive cases?

Response: The cases were not retested for Antibody but instead get PCR testing which, at that time, was considered the gold standard for COVID-19 confirmation in Thailand.

Reviewer #1: 4. It is not clear whether any hospital staff were excluded. It was indicated in line 106 that “Participants with active symptoms suiting national criteria for polymerase chain reaction testing were quarantined and excluded.

Response: We are sorry for the confusion and thank you very much for pointing that out. The statement “Participants with active symptoms suiting national criteria for polymerase chain reaction testing were quarantined and excluded.” was in the Methods section to describe how our observational study concurs with the current COVID-19 screening policy in Thailand. Hence, no staff was excluded from our study per se.

Reviewer #1: 5. It is not clear whether 82 or 100 individuals were tested twice, conflicting numbers were provided in the abstract versus the text.

Response: Again, we are sorry for the confusion. We revised both the “Repeating antibody tests in random sampling participants” subsection of the Method section as follows: “Of 844 participants, 100 were randomly selected to get repeating antibody testing in April and May. None of them had any immunoglobulin developed in April. Of 100 randomly selected participants, 82 participated in antibody testing in May (response rate for repeating antibody test 82%) which showed no antibody against COVID-19.” The Abstract has also been revised accordingly.

Reviewer #2: Burden of disease deals with a range of medical statistics aimed to give a comprehensive picture of how diseases impact on society or to allocate health care and research resources. Diagnostic tests are an important part in this regard. A perfect test, usually referred to as the gold standard implies being positive in all patients with the disease and negative in all patients who do not have the disease. However, most tests are imperfect. In the work by Pongpirul et al. the authors carried out a survey assessing the presence of anti-COVID-19 antibodies in hospital workers from a Thailand area in which COVID-19 had not been reported during the study period. Among 844 participants few of them (<1%) revealed the presence of IgM antibodies bearing no close relationship with their clinical or personal characteristics. The study adds some information to the local features of COVID-19 infection.

Response: Thank you very much. We are pleased to know that our study is useful.

Reviewer #2: It would be interesting to know whether there existed a different degree of exposure risk among staff members.

Response: Staff members consist of many occupations which have various degree of exposure risk, thus we provide the history of close contact confirmed COVID-19 case and close contact suspected COVID-19 case to assess association with a history of risk exposure and seroprevalence.

Reviewer #2: It is intriguing that people yielding an IgM positive test at the beginning failed to develop IgG response a month later. Any tentative explanation for this?

Response: Staffs with IgM positive did not have an antibody test a month later, instead they have PCR testing which was considered the gold standard at that time. For repeating antibody testing, we randomly selected 100 from 844 staff in Ranong hospital to get antibody testing twice in April and May, which none of them developed antibodies against SARS-CoV-2.

Reviewer #2: Information is needed on whether validation studies were carried in recent COVID-19 cases, or not?

Response: Yes, the validation study for the rapid test kit was conducted in recent COVID-19 cases.

Reviewer #2: In discussing results please note that lack of association may depend on the reduced number of people yielding a positive IgM response.

Response: Thank you very much. The discussion about lack of association may depend on the reduced number of people yielding a positive IgM response is provided in the Discussion section as advised (Line 174-175).

Reviewer #2: Page 8, lines 191-192, there are more studies in this regard which should be mentioned.

Response: Additional studies are added as suggested (Line 182-193).

Reviewer #2: Please also note that the time when Ab are detected may have nothing to do with transmission.

Response: The discussion about the time when Ab is detected may have nothing to do with the transmission is provided in the Discussion Section.

Reviewer #2: The Bangkok Metropolitan area might be indicated in the Figure.

Response: The Bangkok Metropolitan area is now indicated in the revised Figure.

We hope that our responses are satisfactory. Should there be anything that might improve our work, please kindly inform us. Thank you very much for your kind consideration.

Best Regards,

Assist. Prof. Dr. Krit Pongpirul, MD, MPH, PhD.

On behalf of the authors

Submitted filename: Response to Reviewer Ranong.docx

Click here for additional data file.

4 Jan 2021

PONE-D-20-25321R1

Seroprevalence of Hospital Staff in Province with Zero COVID-19 Cases

PLOS ONE

Dear Dr. Krit Pongpirul,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised during the review process.

Please submit your revised manuscript by January 30. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Daniela Flavia Hozbor

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. The information in the introduction is outdated and needs to be updated, for example lines 61-64.

2. The weakness of the paper is the failing to resolve the cases with IgM and no IgG, with a second Ab test to resolve whether the test is missing these cases during very early phases of infection before the development of IgG or the problem is with the specificity of the assay. More interesting still is that there are infected cases that develop only IgM and no IgG, which has not yet been reported. The explanation by the authors that they were not the ones tested a second time is not satifactory. It is understandable that even though not everyone tested first round were retested a second time, but 100 out of them were retested a second time. It is very surprising that all seven IgM positive cases were not retested. The failure to retest the cases with IgM reflects a major weakness in the study design, and a scientific flaw.

3. The main point of the paper is to promote mass serological screening of the population, this recommendation should not be made given the concerns on the serology test itself and the detection of IgM only data.

Reviewer #2: Changes introduced into the manuscript fulfill with my comments and suggestions, for which the paper is now acceptable on my end

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Jan 2021

Dear Editor,

We thank you and the reviewers for the comments and suggestions. Please find our point-by-point responses below:

Editor: Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised during the review process.

Response: Thank you for your time and consideration. The manuscript was revised to meet PLOS ONE’s publication criteria. The Introduction section was updated with more recent references. The concerned weakness was additionally discussed in detail in the Discussion section. The main point of the paper had been clarified and specified in the Discussion section.

Reviewer #1: 1. The information in the introduction is outdated and needs to be updated, for example lines 61-64.

Response: Thank you for your time and crucial comment. The information in the Introduction section was updated with more recent evidence published in late 2020 and Jan 2021 including original articles and systematic reviews. Some key information during early pandemics were preserved while unnecessary outdated information was deleted.

Reviewer #1: 2. The weakness of the paper is the failing to resolve the cases with IgM and no IgG, with a second Ab test to resolve whether the test is missing these cases during very early phases of infection before the development of IgG or the problem is with the specificity of the assay. More interesting still is that there are infected cases that develop only IgM and no IgG, which has not yet been reported. The explanation by the authors that they were not the ones tested a second time is not satifactory. It is understandable that even though not everyone tested first round were retested a second time, but 100 out of them were retested a second time. It is very surprising that all seven IgM positive cases were not retested. The failure to retest the cases with IgM reflects a major weakness in the study design, and a scientific flaw.

Response: The comments were well received; however, a seroprevalence study is usually cross-sectional. Several seroprevalence studies on COVID-19 performed only one antibody test in each asymptomatic individual (Xu 2020, Steensels 2020, Kammon 2020, Chen 2020, Nakamura 2020, Takita 2020, Nopsopon 2020, Chau 2020, Psichogiou 2020, Mughal 2020, Insúa 2020, Costa 2020, Olayanju 2020). For other types of study, multiple serological tests were performed in COVID-19 confirmed cases to assess the antibody dynamics (Long 2020, Post 2020, and Lee 2020). As our study attempted to get two consecutive antibody tests in some randomly selected asymptomatic individuals, along with the RT-PCR confirmation in the IgM positive participants that was permitted by the government, we believe that our study contributed relatively more information than a typical seroprevalence study and provided a real-life picture of the early COVID-19 pandemic.

Nonetheless, please allow us to clarify the design of our study as the description of testing sequences might have been confusing. The study randomly selected 100 individuals to get tested in April and then conducted hospital-wide antibody testing in May. According to the protocol approved by the institutional review board, a participant with positive IgM would get the nasopharyngeal swab for RT-PCR, which was gold standard during the study period, to confirm the diagnosis at the hospital expense whereas second antibody test for IgM positive patients was not in the approved protocol. All seven IgM positive participants were found in May, thus did not get their second antibody test. Given the limited test availability during the study period (early COVID-19 pandemic), the distribution and use of test kits were not totally under our control. Despite the importance of the second antibody test, we attempted to request to conduct the repeat surveillance but were not granted by the ethical committee due to the predilection of PCR testing according to the national policy mentioned above. Even if we could get the second antibody test among them, the sample size would still be too small to conclude in this regard. The manuscript was revised to clarify the concerning issue in the Result and Discussion sections.

Reviewer #1: 3. The main point of the paper is to promote mass serological screening of the population, this recommendation should not be made given the concerns on the serology test itself and the detection of IgM only data.

Response: Thank you for your comment. The message delivered in the Discussion section was revised to down tone the main point as advised.

Reviewer #2: Changes introduced into the manuscript fulfill with my comments and suggestions, for which the paper is now acceptable on my end

Response: Thank you for your time and great comments. The manuscript was drastically improved in quality through your precious advice.

We hope that our responses are satisfactory. Should there be anything that might improve our work, please kindly inform us. Thank you very much for your kind consideration.

Best Regards,

Assist. Prof. Dr. Krit Pongpirul, MD, MPH, PhD.

On behalf of the authors

22 Feb 2021

PONE-D-20-25321R2

Seroprevalence of Hospital Staff in a Province with Zero COVID-19 Cases

PLOS ONE

Dear Dr. Krit Pongpirul,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by March 30. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Daniela Flavia Hozbor

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors has addressed the concerns as best as they could without performing the requested additional testing results, which this reviewer still think is not satisfactory. Perhaps adding the rationale and explanation for having IgM and no IgG response in the tested case will provide additional scientific interest for the readers.

Reviewer #3: 1) Although I agree with the flaws/limitations of the study as summarized by Reviewer 1, the study does add informative descriptive data for this region in Thailand despite the limitations.

2) The statement that females have higher IgM % than males in Abstract and Results sections are not appropriate. Just provide the percentages and confidence intervals (CIs). As provided in the Results section, the CIs are nearly overlapping so it cannot be concluded that they are different. Please revise accordingly the statements below.

Abs: Females seemed to have higher IgM seropositive than male staff (1.0% vs. 0.5%).

Results: Female staff seemed to have higher rate of positive IgM (1.0%, 95% CI: 0.5–2.1%) than male (0.5%, 95% CI: 0.1–2.6%).

3) In the conclusion: Consider revising FROM:

“Antibody testing should be promoted for mass screening, particularly

in asymptomatic healthcare workers.”

TO:

“Accurate antibody testing is a valuable screening tool, particularly in

asymptomatic healthcare workers.”

4) Typo:

Page 7: Only 1% of participants had previous negative PCR results while the rest never get tested (Table 1).

… should be ‘got’ tested.

Please read the paper again carefully and correct any grammatical errors.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

26 Feb 2021

Dear Editor,

We thank you and the reviewers for the comments and suggestions. Please find our point-by-point responses below:

Editor: Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised during the review process.

Response: Thank you for your time and consideration. The manuscript was revised to meet PLOS ONE’s publication criteria. The concerned limitation was discussed in detail in the Discussion section. The result interpretation, wording choices, and typos were revised throughout the manuscript.

Reviewer #1: The authors has addressed the concerns as best as they could without performing the requested additional testing results, which this reviewer still think is not satisfactory. Perhaps adding the rationale and explanation for having IgM and no IgG response in the tested case will provide additional scientific interest for the readers.

Response: Thank you for your suggestion. The rationale and explanation for having IgM and no IgG response in the tested case were added in the Discussion section.

Reviewer #3: 1) Although I agree with the flaws/limitations of the study as summarized by Reviewer 1, the study does add informative descriptive data for this region in Thailand despite the limitations.

Response: Thank you for your supportive comments. We believe that this study provides a useful piece of evidence for COVID-19 pandemic control from the region with a unique situation.

Reviewer #3: 2) The statement that females have higher IgM % than males in the Abstract and Results sections is not appropriate. Just provide the percentages and confidence intervals (CIs). As provided in the Results section, the CIs are nearly overlapping so it cannot be concluded that they are different. Please revise accordingly the statements below.

Abs: Females seemed to have higher IgM seropositive than male staff (1.0% vs. 0.5%).

Results: Female staff seemed to have higher rate of positive IgM (1.0%, 95% CI: 0.5–2.1%) than male (0.5%, 95% CI: 0.1–2.6%).

Response: Both statements were revised accordingly by providing only the percentages and confidence intervals (CIs) of each gender.

Reviewer #3: 3) In the conclusion: Consider revising FROM:

“Antibody testing should be promoted for mass screening, particularly in asymptomatic healthcare workers.”

TO:

“Accurate antibody testing is a valuable screening tool, particularly in asymptomatic healthcare workers.”

Response: The statement in the conclusion was revised accordingly.

Reviewer #3: 4) Typo:

Page 7: Only 1% of participants had previous negative PCR results while the rest never get tested (Table 1). … should be ‘got’ tested. Please read the paper again carefully and correct any grammatical errors.

Response: The typo on Page 7 was corrected. The manuscript was perused by the authors for any typo or grammatical error which was corrected accordingly.

We hope that our responses are satisfactory. Should there be anything that might improve our work, please kindly inform us. Thank you very much for your kind consideration.

Best Regards,

Assoc. Prof. Dr. Krit Pongpirul, MD, MPH, PhD.

On behalf of the authors

Submitted filename: Response to Reviewer Ranong 2.11 TN KP clean.docx

Click here for additional data file.

5 Mar 2021

Seroprevalence of Hospital Staff in a Province with Zero COVID-19 Cases

PONE-D-20-25321R3

Dear Dr. Krit Pongpirul,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Daniela Flavia Hozbor

Academic Editor

PLOS ONE

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Reviewer #3: All comments have been addressed

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: The authors have responded adequately to all the points raised in my previous review. Thank you for responding.

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Reviewer #3: No

19 Mar 2021

PONE-D-20-25321R3

Seroprevalence of hospital staff in a province with zero COVID-19 cases

Dear Dr. Pongpirul:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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