1 Seroprevalence of Hospital Staff in Province with Zero COVID-19 Cases 2 Tanawin Nopsopon, M.D

medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20151944; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

1 Seroprevalence of Hospital Staff in Province with Zero COVID-19 Cases 2 Tanawin Nopsopon, M.D. 3 Lecturer, Department of Preventive and Social Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, 4 Thailand 5 [email protected] 6 7 Krit Pongpirul, M.D., M.P.H., Ph.D. (Corresponding Author) 8 Assistant Professor, Department of Preventive and Social Medicine, Faculty of Medicine, Chulalongkorn 9 University, Bangkok, Thailand; Department of International Health, Johns Hopkins Bloomberg School of Public 10 Health, Baltimore, MD, USA; Bumrungrad International Hospital, Bangkok, Thailand 11 [email protected] 12 13 Korn Chotirosniramit 14 Medical Student, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 15 [email protected] 16 17 Wutichai Jakaew, M.D. 18 Chairman of Infectious Control Board, Ranong Hospital, Ranong, Thailand 19 [email protected] 20 21 Chuenkhwan Kaewwijit, B.Sc. 22 Head of Medical Laboratory Department, Ranong Hospital, Ranong, Thailand. 23 [email protected] 24 25 Sawan Kanchana, M.D., MFRCPT. 26 Hospital Director, Ranong Hospital, Ranong, Thailand 27 [email protected] 28 29 Narin Hiransuthikul, M.D., Ph.D. 30 Professor, Department of Preventive and Social Medicine, Faculty of Medicine, Chulalongkorn University, 31 Bangkok, Thailand 32 [email protected] 33 Words: 1,939 words (Text), 260 words (Abstract), 1 figure, 2 tables

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20151944; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

35 ABSTRACT:

36 BACKGROUND. COVID-19 seroprevalence data has been scarce, especially in less developed countries with a

37 relatively low infection rate.

38 METHODS. A locally developed rapid immunoglobulin M (IgM) / immunoglobulin G (IgG) test kit was used for

39 screening hospital staff in Ranong hospital which located in a province with zero COVID-19 prevalence in Thailand

40 from April 17 to May 17, 2020. A total of 844 participants were tested; 82 of which were tested twice with one

41 month apart. (Thai Clinical Trials Registry: TCTR20200426002)

42 RESULTS. Overall, 0.8% of the participants (7 of 844) had positive IgM, none had positive IgG. Female staff

43 seemed to have higher IgM seropositive than male staff (1.0% vs. 0.5%). None of the participants with a history of

44 travel to the high-risk area or a history of close contact with PCR-confirmed COVID-19 case had developed

45 antibodies against SARS-CoV-2. Among 844 staff, 811 had no symptom and six of them developed IgM

46 seropositive (0.7%) while 33 had minor symptoms and only one of them developed IgM seropositive (3.0%). No

47 association between IgM antibody against SARS-CoV-2 status and gender, history of travel to a high-risk area,

48 history of close contact with PCR-confirmed COVID-19 case, history of close contact with suspected COVID-19

49 case, presence of symptoms within 14 days, or previous PCR status was found. None of the hospital staff developed

50 IgG against SARS-CoV-2.

51 CONCLUSION. COVID-19 antibody test could detect a substantial number of hospital staffs who could be

52 potential silent spreaders in a province with zero COVID-19 cases. Antibody testing should be encouraged for mass

53 screening, especially in asymptomatic healthcare workers.

54 TRIAL REGISTRATION. This study was approved by the Institutional Review Board of Chulalongkorn

55 University (IRB No.236/63) and the Institutional Review Board of Ranong Hospital. (Thai Clinical Trials Registry:

56 TCTR20200426002)

57 FUNDING. None.

60 Introduction

61 Seroprevalence data has been scarce in Asian countries besides China. Along with the gold-standard polymerase

62 chain reaction (PCR) testing, antibody testing is beneficial for epidemiological investigation and epidemic control of

63 infectious diseases including the present coronavirus disease 2019 (COVID-19). In Singapore, serological evidence

64 was used to trace and identify missing spreader for three clusters (1). Asymptomatic silent spreaders have been of

65 major concern as suggested by a systematic review—could be as low as 1.6% of COVID-19 confirmed cases in

66 China or as high as 51.7% of confirmed cases in Diamond Princess cruise (2).

67 Early COVID-19 prevalence studies were based only on PCR for the diagnosis of severe acute respiratory

68 syndrome coronavirus 2 (SARS-CoV-2) infection in individuals. However, recent studies tended to report PCR

69 along with serological test results. Our rapid systematic review of peer-reviewed evidence up to May 1, 2020,

70 reported a range of seroprevalence among healthcare workers from 2.0% in Lebanon and Claremont, the USA to

71 4.5% in Padova, Italy (3). China reported an overall seroprevalence of 2.5% in a hospital setting, in which 1.8% and

72 3.5% were among healthcare workers and asymptomatic patients, respectively (4). Additionally, China studied the

73 development of antibodies against SARS-CoV-2 in symptomatic confirmed COVID-19 cases and found that

74 immunoglobulin M (IgM) reached its peak 20–22 days after onset while immunoglobulin G (IgG) reached its peak

75 17–19 days after onset (5). Some works emphasized on antibody testing for hospital workforce and policy issues.

76 Another seroprevalence study in Belgium conducted on healthcare personnel who worked in a tertiary hospital

77 found 6.4% seroprevalence and identified some risk factors for developing antibodies against SARS-CoV-2 (6).

78 More recent studies in the medRxiv preprint repository reported a range of seroprevalence in healthcare

79 personnel from 0% (7) to 45.3% (8) with intra-regional variations. In China, for example, seroprevalence among

80 hospital staff ranged from 1.8% (4) to 17.1% (9) whereas seroprevalence of healthcare workers in Asia besides

81 China ranged from 3.3% (10) to 5.4% (11). In Southeast Asia, there was only a study from Thailand reported a 3.7%

82 seroprevalence (12). Seroprevalences in healthcare personnel varied from 1.1% (13) to 45.3% in Europe (8) and

83 from 2% (3) to 7.6% (14) in North America. Only one African study reported zero seroprevalences in hospital staff

84 in Libya (7) whereas no seroprevalence studies were from South America, Antarctica, or Australia.

85 Ideally, both PCR and antibody testing provided complementing information to shape the picture of the

86 situation in a specific hospital, area, or country. However, most low- to middle-income countries could not afford

87 the cost of laboratory tests and had to develop criteria-based policies for resource-use optimization. In Thailand, for

88 instance, the PCR is reserved for individuals who meet the national criteria for COVID-19 polymerase chain

89 reaction testing.

90 Ranong is one of 77 provinces in Thailand with zero cumulative confirmed COVID-19 case from April 17

91 to May 17, 2020, and still had no case as of July 4, 2020 (Figure 1). Hospital is one of the highest risk areas for

92 receiving and spreading pathogens—healthcare workers developed a higher chance of getting infected by co-

93 workers or patients and vice versa. This study aims to estimate the hospital-wide seroprevalence in healthcare

94 workers who worked in the largest public hospital in Ranong to develop the strategies to slow down the pandemic

95 and ensure the safety of healthcare workers who come to work and patients who visit the hospital.

96 Results

97 Healthcare workers' demographics. All 844 Thai hospital staff were invited to participate in the study and tested for

98 IgM and IgG antibodies against SARS-CoV-2 (100% participation rate). Their median age was 42 years

99 (interquartile range 32–50). Most of them (71.7%) were female and 96.1% had no symptoms. The 33 symptomatic

100 participants (3.9%) reported cough (1.7%), rhinitis (1.5%), sore throat (1.4%), dyspnea (0.5%), and fever (0.2%).

101 History of travel to the high-risk area was 2.5%, history of close contact PCR confirmed COVID-19 case was 2.0%,

102 history of close contact suspected case was 38.1%. Only 1% of participants had previous negative PCR results while

103 the rest never get tested (Table 1).

104 ( insert Table 1 about here )

105 Serological results of healthcare workers in Ranong hospital. Overall, seven hospital staff tested positive for

106 COVID-19 IgM (0.8%, 95% CI: 0.4%, 1.7%) while none of the participants developed IgG. Female staff seemed to

107 have higher rate of positive IgM (1.0%, 95% CI: 0.5%, 2.1%) than male (0.5%, 95% CI: 0.1%, 2.6%). None of the

108 participants with a history of travel to the high-risk area or a history of close contact with PCR-confirmed COVID-

109 19 case developed antibodies against SARS-CoV-2. There was no statistical difference in IgM seroprevalence

110 between staff with and without a history of close contact with suspected COVID-19 case. Among 844 staff, 811 had

111 no symptom and six of them developed IgM seropositive (0.7%, 95% CI: 0.3%, 1.6%) while 33 had minor

112 symptoms and only one of them developed immunoglobulin M (3.0%, 95% CI: 0.5%, 15.3%). Of 12 staff with sore

113 throat, one had positive IgM (8.3%, 95%CI: 1.5%, 35.4%). There was zero IgM seroprevalence in staff with fever,

114 cough, rhinitis, or dyspnea. Of 844 participants, eight had previous negative PCR result and none has developed the

115 antibody for SARS-CoV-2 (Table 1). No association between IgM antibody against SARS-CoV-2 status and gender,

116 history of travel to a high-risk area, history of close contact with PCR-confirmed COVID-19 case, history of close

117 contact with suspected COVID-19 case, presence of symptoms within 14 days, or previous PCR status was found.

118 None of the hospital staff developed IgG against SARS-CoV-2.

119 Characteristics of seropositive participants. Seven participants developed IgM antibody in May. Their age ranged

120 from 20 to 49 years. Six of them were female while only one staff was male. All of them were Thai, had no history

121 of travel to a high-risk area, no history of contact with PCR-confirmed COVID-19 case, and no previous PCR status.

122 Three had a history of contact with suspected COVID-19 case while the other three did not. Participant 7 was only

123 one with IgM positive who had a symptom (i.e. sore throat) within 14 days before antibody testing. Nasopharyngeal

124 swabs of all seven staff with a positive antibody tested negative for SARS-CoV-2 on PCR (Table 2).

125 ( insert Table 2 about here )

126 Repeating antibody tests in random sampling participants. Of 844 participants, 100 were randomly selected to get

127 repeating antibody testing in April and May. None of them had any immunoglobulin developed in April whereas 82

128 also participated in antibody testing in May which showed no antibody against COVID-19.

129 Discussion

130 In a small seaside province, Ranong, which had 193,370 inhabitants, located in the south of Thailand closed to the

131 Andaman Sea and had border close to Myanmar, there was zero officially PCR-confirmed COVID-19 case

132 compared to the current situation in Thailand (0.048 per thousand). This zero prevalence could be either from good

133 compliance to public health recommendations or a low number of individuals who get PCR testing due to the

134 stringent national criteria.

135 In this study, we reported a 0.8% IgM seroprevalence in Ranong hospital staff while their PCR tests were

136 negative. While critics might argue that the antibody test had a high false-positive rate, especially in a population

137 with high pre-test probability such as hospital staff, the PCR test could have a high false-negative rate, especially

138 without a highly sensitive diagnostic test (15).

139 Of seven seropositive hospital staff, only one was symptomatic and none of them had a history of travel to

140 high-risk area or history of close contact with PCR-confirmed COVID-19 case. The current national criteria and

141 policies for PCR testing mostly rely on risk histories and symptoms so asymptomatic individuals or those with

142 minor symptoms were left out, resulting in an underestimation of the actual prevalence of COVID-19.

143 Our study reported a lower seroprevalence in healthcare workers than hospitals in China (0.8% vs. 1.8%)

144 (4), a tertiary hospital in Belgium (0.8% vs. 6.4%) (6), and a rural hospital located in low COVID-19 prevalence

145 county of Germany (0.8% vs 2.7%) (16). Unlike China, Belgium, and Germany where the seroprevalences were

146 mostly from positive IgG, our study revealed mostly positive IgM. Comparison with Belgium and Germany hospital

147 should be interpreted with caution due to the unknown PCR status of subjects of the two studies.

148 COVID-19-confirmed patients developed peak IgM at 20–22 days and peak IgG at 17–19 days after

149 symptom onset (5). For patients with no or minor symptoms, the onset might not be possible to determine. In this

150 study, we used a qualitative antibody test kit so the relatively lower antibody level in early infected persons might

151 not be detectable. Repeating PCR or quantitative antibody tests for suspected individuals was practiced in many

152 countries. However, the cost would be doubled or more and might not be possible for low- or middle-income

153 countries. We attempted to repeat the antibody test in randomly selected participants; however, only 82 of them

154 could participate in the follow-up test and none of them developed antibodies against SARS-CoV-2 in both first and

155 second tests. Rapid antibody tests should be more affordable to permit multiple tests among high-risk asymptomatic

156 individuals who could be silent spreaders.

157 Serological testing provides some crucial epidemiological information and would have been more effective

158 when combined with other diagnostic tests such as PCR. With immunoglobulin status and PCR results, we can

159 shape the situation more precisely for both individual and regional views. Hopefully, with this and other vigorous

160 and dedicated studies on antibody status around the globe, antibody testing would provide useful information for

161 pandemic control.

162 In conclusion, the COVID-19 antibody test could detect a substantial number of hospital staffs who could

163 be potential silent spreaders in areas with zero COVID-19 case. Antibody testing should be encouraged for mass

164 screening, especially in asymptomatic healthcare workers.

165 Methods

166 Participants

167 All 844 healthcare workers including the physician, nurse, medical assistance, medical technician, and non-medical

168 officers of the largest public hospital in Ranong province that reported zero cumulative PCR-confirmed COVID-19

169 cases during the entire study period (April 17 to May 17, 2020), were invited to participate in this study. All of them

170 accepted to participate with written informed consent. Participants with active symptoms suiting national criteria for

171 polymerase chain reaction testing were quarantined and excluded. Participants were asked to answer a survey about

172 risk history for COVID-19, recent symptoms in the past two weeks, and previous PCR tests if available.

173 Antibody testing

174 Locally developed Baiya Rapid IgG/IgM test kit (Baiya Phytopharm, Thailand) which reported the presence of IgM

175 and IgG qualitatively, was used for antibody testing in this study. The internal validation of the test kit using the

176 serum of 51 PCR confirmed COVID-19 cases and 150 controls showed sensitivity 94.1% (48 of 51) and specificity

177 98.0% (147 of 150) of IgM or IgG antibody. Participants with positive IgM were encouraged to have PCR test if

178 available.

179 Study procedures

180 On April 17, 2020, of 844 participants, 100 were randomly selected to have their first antibody testing. On May 17,

181 2020, all the remaining participants were tested for serological immunity whereas the 100 healthcare workers who

182 tested in April had their second antibody testing.

183 Statistics

184 Categorical data were presented with counts and percentages while continuous data were reported with median and

185 interquartile range. Association between categorical variables and the status of immunoglobulin was analyzed using

186 Fisher’s exact test. The 95% confidence interval (CI) of the seroprevalence was calculated by Wilson’s method

187 using binomial probabilities. Missing data were excluded. All data were analyzed using Stata 16.1 (College Station,

188 TX).

189

190 Study approval

191 This study was approved by the Institutional Review Board of Chulalongkorn University (IRB No.236/63) and the

192 Institutional Review Board of Ranong Hospital. (Thai Clinical Trials Registry: TCTR20200426002)

193 Author Contributions

194 TN, KP, and NH conceptualized the study, designed methodology, and administrated the project. TN, KC, KP, WJ,

195 CK and SK contributed to data curation and clinical investigation. TN, KC, and KP validated the data. TN and KP

196 performed formal analysis and provided software. TN visualized the data. KP, WJ, SK, NH provided resources for

197 the study. KP, SK, and NH supervised the study. TN and KP wrote the original draft of the manuscript. All authors

198 reviewed and edited the final manuscript.

199 Acknowledgements

200 We thank Baiya Phytopharm, Thailand for supporting the Baiya Rapid COVID-19 IgG/IgM test kit. The company

201 did not involve in the data analysis, interpretation, or manuscript preparation.

202

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10 medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20151944; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Confirmed COVID-19

Ranong

Figure 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.