High Number of RNA Copies in Asymptomatic Individuals Infected with SARS-CoV-2 in an Area of the Colombian Caribbean

doi:10.21203/rs.3.rs-32302/v1

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High Number of RNA Copies in Asymptomatic Individuals Infected with SARS-CoV-2 in an Area of the Colombian Caribbean

https://doi.org/10.21203/rs.3.rs-32302/v1

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Background: Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is an emerging pandemical virus that has caused millions of reported cases and hundreds of thousands of deaths in less than six months. South America has suffered the pandemic because it lacks the hospital and economic capacities needed to contain the pandemic's advance. Public health implications of transmission, while asymptomatic is a critical concern at the current pandemic.

Objective: Describe the socio-demographic, clinical, and viral kinetics features of a cohort of SARS-CoV-2 infected individuals from the Colombian Caribbean.

Methods: Six hundred eighty-six clinical samples from several hospital centers in the province were received between April 9th and May 16th, 2020. RNA was extracted using lysis buffers and spin columns. The samples were tested for SARS-CoV-2 by RT-qPCR (Reverse transcription real-time polymerase chain reaction) using commercially available multiplex real-time PCR assay for simultaneous detection of 3 target genes of SARS-CoV-2 (AllplexTM, 2019-nCoV assay, Korea). Viral copies quantification was done using a standard curve constructed from seriated dilutions of a SARS-CoV-2 positive control.

Results: Thirty-five nasopharyngeal samples were positive for SARS-CoV-2 infection; the average age was 43 (range, 1-95 years). Seventeen of 35 (49%) of the patients showed symptoms. Most of them had cough, fever, and odynophagia, 3 of the patients reported having arthralgia. Only two patients required hospitalization. None of the patients had known co-morbidities. RT-qPCR results show that two of the symptomatic patients had significantly higher RNA copies than the rest of the patients. Eighteen of 35 (51%) individuals were asymptomatic, the average age was 30 (range, 6-61 years. Four individuals showed a higher copy than some symptomatic patients. Nonetheless, the average of RNA copies 8.26 x10+10 was lower than the symptomatic.

Conclusions: the population studied was young with an average of 43 years in symptomatic and 30 years of asymptomatic; this is important because of the high impact in the economy, and probably it is the cause of the reduced lethality observed in the department. Because a large proportion of infections probably result from transmission from asymptomatic or pre-symptomatic persons, the usefulness of public health interventions in Colombian provinces should be based in the molecular screening in a vast conglomerate's population and to quantify the viral load.

Virology

Asymptomatic infections

COVID-19

Coronavirus infections

pre symptomatic disease

environment and public health

Communicable Disease Control

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is an emerging pandemical virus that has caused millions of reported cases and hundreds of thousands of deaths in less than six months. South America has suffered the pandemic because it lacks the hospital and economic capacities needed to contain the pandemic's advance. South America has 433,497 infected people. The top five countries' distribution is as follows: Brazil has 241,080 cases, Peru 92,273, Chile 43,781, and Ecuador 33,182, and Colombia 15,574 cases [1]. The mortality per million people in Ecuador, Peru, Brazil, Chile, Bolivia, and Colombia are 155, 80, 74, 24, 14, and 11, respectively, with a total of 23,117 people who died in the continent [1]. Meanwhile, Colombia's first case was on March 6th, 2020, and has been shocked by the number of cases with an infection rate of 294/million [2].

In the department of Cordoba, Colombia Caribbean area, the first case was reported on March 26th. Fortunately, only 10% of 78 cases have not required hospitalization; as of today, the mortality rate reaches 0.22/100,000 population [2].

Public health implications of transmission, while asymptomatic is a critical concern at the current pandemic [3, 4]. This study aims to describe the socio-demographic, clinical, and viral kinetics features of a cohort of SARS-CoV-2 infected individuals from the Colombian Caribbean.

Type of study, geographic area and sample collection

The present work is a prospective, descriptive study carried out in the department of Cordoba, located in the northwest of Colombia, and is a part of the Caribbean savanna (Fig. 1A). The mean annual temperature is 28 °C, occurring a dry and a rainy season. The department's population is 1,828,947 inhabitants, and Monteria, Cordoba's capital city, has a population of 505,334. This study was conducted in the laboratory of Universidad de Cordoba, which is licensed by Colombia's National Health Institute for the molecular diagnostic of SARS-CoV-2 human infection. Six hundred eighty-six clinical samples from several hospital centers in the province were received and processed by RT-qPCR between April 9th and May 16th, 2020, with 35 positive results for SARS-CoV-2 infection. The ethical standards of the Ministry of Health of Colombia Resolution No. 8430 of 1993 were followed. The data of the present study correspond to patients coded under strict anonymity with an internal laboratory number.

RNA extraction and SARS-CoV-2 detection

Briefly, RNA was extracted using lysis buffers and spin columns. After RNA extraction, the samples were tested for SARS-CoV-2 by RT-qPCR using commercially available multiplex real-time PCR assay for simultaneous detection of 3 target genes of SARS-CoV-2 (Allplex™, 2019-nCoV assay, Korea). The assay is designed to detect RdRP (RNA dependent RNA polymerase) and N genes specific for SARS-CoV-2, and E gene for all of Sarbecovirus, including SARS-CoV-2. Viral copies quantification was done using a standard curve constructed from seriated dilutions of a SARS-CoV-2 positive control. This control includes synthetic RNA target sequences for the three genes (RdRP, S, and N). Samples tested with a Ct value < 40 were considered positive. If a tested sample had a Ct between 40, and 45 the test was repeated, and the sample was considered positive if the curve showed a trend to arise, and the individual had an epidemiological link with an infected patient.

Thirty-five nasopharyngeal samples were positive for SARS-CoV-2 infection. Seventeen of 35 (49%) of the patients showed symptoms, 9/17 (53%) of them were female, eight patients were from the urban area of Monteria (505,334 inhabitants), eight from Sahagun (107,636 inhabitants), and one from Cerete (105,815 inhabitants). Most of them had cough, fever, and odynophagia, 3 of the patients reported having arthralgia (Table 1). Only two patients required hospitalization. So far, all of the symptomatic patients are alive and have recovered from the infection. The average age was 43 (range, 1–95 years). Both pediatric patients were symptomatic (Table 1). None of the patients had known co-morbidities. Only four symptomatic patients reported not having expositional contact with an infected person. RT-qPCR results show that two of the symptomatic patients (codes 505, 568) had significantly higher RNA copies than the rest of the patients. The average RNA copies were very high 4 × 9^+ 11 (Table 1).

Table 1

Epidemiological, clinical, demographic information, and RNA virus copies.
Anonymous code	Date of sample	Symptomatic (onset date) / Asymptomatic	Municipality	Age/Gender /Health staff	Contact	Ct
a 505	09/05/2020	Symptomatic (30-apr) Fe, Od	Sahagun	65/M/No	No	24,00089021
556	11/05/2020	Symptomatic (9-May) Co, Ar, Od	Sahagun	32/F/Yes	Yes	36,9420150638368
557	12/05/2020	Symptomatic (10-May) Fe, Ar, Od	Sahagun	95/M/No	No	39,2656400322052
a 568	12/05/2020	Symptomatic (9-May) Od	Sahagun	52/M/No	Yes	24,0295340854369
593	13/05/2020	Symptomatic (6-May) Co, Od	Sahagun	28/F/Yes	Yes	38,6431519592528
594	13/05/2020	Symptomatic (11-May) Co, Od	Sahagun	31/F/Yes	Yes	37,4174202687062
595	13/05/2020	Symptomatic (11-May) Co	Sahagun	56/M/Yes	Yes	38,0353148721936
615	13/05/2020	Symptomatic (10-May) Fe	Sahagun	24/F/No	Yes	36,9054554946246
b	09/05/2020	Symptomatic (2-May) Co, Od	Monteria	26/M/No	No	37,120751845905
602	12/05/2020	Symptomatic (10-May) Co, Od	Monteria	41/F/Yes	Yes	40,3710395393544
b 605	13/05/2020	Symptomatic (4-May) Co	Monteria	23/F/No	Yes	34,7068841210098
b 606	13/05/2020	Symptomatic (10-May) Co	Monteria	60/M/No	Yes	38,1288150448141
b 608	13/05/2020	Symptomatic (11-May) Co	Monteria	43/F/No	Yes	37,6696185948042
b 609	13/05/2020	Symptomatic (10-May) Od	Monteria	1/M/No	Yes	37,2655603597797
b 610	13/05/2020	Symptomatic (12-May) Fe	Monteria	6/M/No	Yes	41,1847268770343
684	15/05/2020	Symptomatic (1-May) Ar	Monteria	72/F/No	Yes	37,7517373406786
559	11/05/2020	Symptomatic (7-May) Od	Cerete	74/F/No	No	36,6196282425225
a 558	11/05/2020	Asymptomatic	Sahagun	46/F/No	Yes	36,6196282425225
583	13/05/2020	Asymptomatic	Sahagun	32/F/Yes	Yes	37,6280851364822
585	13/05/2020	Asymptomatic	Sahagun	30/F/Yes	Yes	24,8419460495114
592	13/05/2020	Asymptomatic	Sahagun	21/M/Yes	Yes	38,9647343140328
c 666	15/05/2020	Asymptomatic	Sahagun	34/F/No	Yes	42,9440997130024
c 674	15/05/2020	Asymptomatic	Sahagun	27/F/Yes	Yes	39,7340514680728
c 677	15/05/2020	Asymptomatic	Sahagun	34/F/Yes	Yes	44,77372237901
c 678	14/05/2020	Asymptomatic	Sahagun	30/F/Yes	Yes	36,6311907471569
c 682	15/05/2020	Asymptomatic	Sahagun	50/M/Yes	Yes	36,8407657818134
d 574	12/05/2020	Asymptomatic	Monteria	26/M/No	Yes	38,5063617495545
d 575	12/05/2020	Asymptomatic	Monteria	17/F/No	Yes	37,1084176398085
599	13/05/2020	Asymptomatic	Monteria	35/M/No	Yes	39,3886493111317
600	12/05/2020	Asymptomatic	Monteria	26/M/Yes	Yes	33,6632756072637
601	12/05/2020	Asymptomatic	Monteria	25/M/Yes	Yes	39,0785098708386
604	12/05/2020	Asymptomatic	Monteria	61/M/Yes	Yes	36,0769730910986
b 607	13/05/2020	Asymptomatic	Monteria	17/M/No	Yes	38,2068660785118
d 612	13/05/2020	Asymptomatic	Monteria	24/F/No	Yes	37,6687265771356
d 613	13/05/2020	Asymptomatic	Monteria	6/F/No	Yes	35,5706197133347
Fe: fever, Co: cough, Ar: arthralgia, Od: odynophagia, M: male, F: female.

Individual codes 585, 600, 613, and 612 showed a higher copy than some symptomatic patients (Table 1, Fig. 1C). Nonetheless, the average of RNA copies 8.26 × 10 + 10 was lower than the symptomatic. The Wilcoxon test for independent samples showed that there is no significant difference (p > 0.05) between the viral RNA copy number of symptomatic and asymptomatic patients (Fig. 1B). Nine of 18 individuals were from Monteria, the remaining nine from Sahagun. All asymptomatic individuals had a known infected contact (Table 1); the figure shows the number of infected contacts (Fig. 1C). We do not know if the asymptomatic individuals of the present study develop a COVID-19 disease after taken their nasopharyngeal sample, but we believe that they are pre-symptomatic or asymptomatic. 43% of the health staff was infected, an important issue because they are in the front line facing the pandemic (Table 1).

Eighteen of 35 (51%) individuals were asymptomatic, 10/18 (56%) were female, the average age was 30 (range, 6–61 years. Most studies report that male is more affected by coronaviruses than female; however, in the present study, women were more affected than men (53% symptomatic and 56% asymptomatic). This trend, 52,56%, continues throughout the department of Cordoba and is opposing to national behavior (44,25%). The present study strengthens the concern about public health implications of pre-symptomatic or asymptomatic SARS-CoV-2 infection [3, 5]. Our results show a 51%) of asymptomatic infected individuals, of which 78% (14/18) presented a significative viral copy, some of them higher than several of symptomatic patients (Fig. 1C). Moreover, RT-PCR Ct lower than 34 of some of them presumes the possibility to isolate infectious SARS-CoV-2 to demonstrate viral viability [3, 5, 6].

If a considerable percentage of infections are asymptomatic, increased testing approaches may be needed to detect these persons [6, 7]. Colombia has tested 3,741 per million people in a country with 50 million people, Brazil, with 330 million people have tested 3,462 subjects, Peru and Chile are the countries with more tested people in South America, exceeding the 25,000 tests. Because a large proportion of infections probably result from transmission from asymptomatic or pre-symptomatic persons, the usefulness of public health interventions in Colombian provinces should be based in two steps, first the molecular screening in a vast conglomerate's population and second to quantify the viral load. Finally, a remarkable issue is that the population studied is very young with an average of 43 years in symptomatic and 30 years of asymptomatic; this data is essential because produce a high impact in the economy and probably it is the cause of the reduced lethality observed in the country and the department.

SARS-CoV-2: Severe acute respiratory syndrome Coronavirus 2; RNA: ribonucleic acid; RT-qPCR: Reverse transcription real-time polymerase chain reaction; RdRP: RNA dependent RNA polymerase; Ct: threshold cycle; COVID-19: Coronavirus diseases 2019.

Ethical approval and consent to participate.

The study follows the ethical standards of the Ministry of Health of Colombia Resolution No. 8430 of 1993. The data of the present study correspond to patients coded under strict anonymity with an internal laboratory number. This study is the results of a research project that was approved by the Comité de ética del Instituto de Investigaciones Biológicas del Trópico de la Universidad de Cordoba, with the number N° 0410- 2020.

Consent to publication.

Not applicable

Availability of data and material.

Our results are preliminary, and we do not wish to share this preliminary data for the moment.

Competing interests.

The authors declare no competing interests.

Funding.

This study was supported by the University of Cordoba.

Authors' contributions.

All authors participated for equal in the conception of the manuscript, design of the study, collection, analysis, and interpretation of the data. Besides, all authors did review the manuscript and approved it before submit it.

Acknowledgments.

To the department of Cordoba, Secretary of Health. To the Rectory of the University of Cordoba for their determined commitment to face coronavirus's pandemic and financial support. To Paola Diaz for the elaboration of the Figure, and Instituto Nacional de Salud of Colombia.

Worldometers, 2020; https://www.worldometers.info/coronavirus/#countries. Accessed 17 May 2020.
Instituto Nacional de Salud de Colombia. https://www.ins.gov.co/Noticias/Paginas/Coronavirus.aspx. Accessed 19 May 2020.
Dabiao Ch, Wenxiong X, Ziying L, Zhanlian H, Jing L, Zhiliang G, et al. Recurrence of positive SARS-CoV-2 RNA in COVID-19: A case report. International Journal of Infectious Diseases 2020; 93: 297–299.
Furukawa NW, Brooks JT, Sobel J. Evidence supporting transmission of severe acute respiratory syndrome coronavirus 2 while pre symptomatic or asymptomatic. Emerg Infect Dis. 2020; https://doi.org/10.3201/eid2607.201595
Liu Y, Yan LM, Wan L, Xiang TX, Le A, Liu JM, et al. Viral dynamics in mild and severe cases of COVID-19. Lancet Infect Dis. 2020 Jun;20(6):656-657. doi: 10.1016/S1473-3099(20)30232-2.
Sun J, Xiao J, Sun R, Tang X, Liang C, Lin H, et al. Prolonged persistence of SARS-CoV-2 RNA in body fluids. Emerg Infect Dis. 2020; http://dx.doi.org/10.3201/eid2608.201097
Wajnberg A, Mansour M, Leven E, Bouvier NM, Patel G, Firpo A, et al. Humoral immune response and prolonged PCR positivity in a cohort of 1343 SARS-CoV 2 patients in the New York City region. 2020 doi:https://doi.org/10.1101/2020.04.30.20085613. Accessed 20 May 2020.

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High Number of RNA Copies in Asymptomatic Individuals Infected with SARS-CoV-2 in an Area of the Colombian Caribbean

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Abstract

Figures

Background

Methods

Type of study, geographic area and sample collection

RNA extraction and SARS-CoV-2 detection

Results And Discussion

Conclusions

Abbreviations

Declarations

Ethical approval and consent to participate.

Consent to publication.

Availability of data and material.

Competing interests.

Funding.

Authors' contributions.

Acknowledgments.

References

Supplementary Files

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