Asymptomatic SARS-CoV-2 infection of healthcare workers (HCWs) has been reported as a key player in the nosocomial spreading of COVID-19. Early detection of infected HCWs can prevent spreading of the virus in hospitals among HCWs and patients. We conducted a cross-sectional study to determine the asymptomatic infection of HCWs in a private clinic in the city of Santiago, Chile. Our study was conducted during a period of 5 weeks at the peak of transmission of SARS-CoV-2 in Chile. Nasopharyngeal samples were obtained from 413 HCWs and tested for the presence of SARS-CoV-2 using RT-qPCR. We found that a 3.14% of HCWs were positive for the presence of SARS-CoV-2 (14/413). Out of these, 7/14 were completely asymptomatic and did not develop symptoms within 3 weeks of testing. Sequencing of viral genomes showed the predominance of the GR clade; however, sequence comparison demonstrated numerous genetic differences among them suggesting community infection as the main focus of transmission among HCWs. Our study demonstrates that the protocols applied to protect HCWs and patients have been effective as no infection clusters due to asymptomatic carriers were found in the clinic. Together, these data suggest that infection with SARS-CoV-2 among HCWs of this health center is not nosocomial.
All sequencing files will be available from the NCBI database (accession number PRJNA665485).
SARS-CoV-2 originated in Wuhan, China, presumably in a food market. Since then it has spread widely around the world, leaving more than 53.5 million positive cases and 1.3 million deaths (WHO) as of November 2020. The first case of COVID-19 in Chile was reported in March 3rd, 2020 and from that point most of the country has had cases of SARS-CoV-2 infection leaving more than 450000 cases and 15000 deaths (Chilean Government official data).
COVID-19 presents in patients with fever, cough and chest discomfort that frequently evolves into pneumonia [
Healthcare workers are a high-risk population for viral transmission, particularly because social distancing is not always possible while caring for patients. This is despite all the efforts to provide adequate personal protection elements (PPE) and for establishing protocols to limit SARS-CoV-2 transmission in the workplaces [
Here we investigate asymptomatic infection of HCWs in a private clinic in the city of Santiago, Chile. Our study was conducted during the peak of transmission of SARS-CoV-2 in Chile and aims to assess whether the correct use of PPE and the infection control protocols established at most health institutions are sufficient to control nosocomial transmission of SARS-CoV-2.
A total number of 413 asymptomatic HCWs were enrolled into the final study. Only 14 tested positive for the presence of SARS-CoV-2 by RT-qPCR, representing a 3.4% of positivity in asymptomatic HCWs. Importantly, our results show that there is no relationship between the SARS-CoV-2 PCR Ct values or viral load of an individual and the subsequent development of any symptoms related to COVID-19. Moreover, sequencing of the viral genomes confirms that there are at least 5 different infection sources among the infected individuals, suggesting that these HCWs became infected at their individual communities outside of the clinic.
All HCWs from Clinica Indisa that had not been infected by SARS-CoV-2 previously and did not present any symptoms at the time of enrollment were able to join the study. Public announcements in the clinic were posted to recruit volunteers. Enrollment to our study was completely voluntary. We were able to reach ~30% of the total HCWs in the clinic. (413/1379). Before sampling, an online questionnaire was applied to all asymptomatic subjects to record: Sex, age, profession, residence commune, comorbidities (chronic respiratory, cardiovascular, rheumatic diseases, others), and other variables related to their occupation as a service in which they work, and exposition. Also, written consent of every enrolled HCW was obtained. This study was approved by the Medical Director of INDISA Clinic and the Scientific Ethical Committee of Universidad Andres Bello.
Nasopharyngeal swabs were obtained from enrolled subjects and kept in viral transport media at 4°C until processing, following the protocols established by the World Health Organization and the Chilean Public Health Institute (ISP). Samples were collected in the morning and results were informed in the following 8–10 hours. This rapid processing of samples allowed efficient detection of new asymptomatic cases, thereby drastically reducing any potential nosocomial transmission by the subjects. HCWs did not return to work until the result from their qPCR test was informed and was negative for the presence of SARS-CoV-2.
For isolation of viral genomes, we made use of a Kingfisher Duo Prime automated system using the MagMAX Viral/pathogen nucleic acid isolation kit (Thermofisher, USA), following manufacturer’s instructions. Briefly, 200 μL were taken from nasopharyngeal swabs and mixed with 5 μL of proteinase K solution and 275 μL of binding solution/magnetic beads mix. Samples were incubated at room temperature for 15 minutes, inside a laminar flow hood, to inactivate any viral particles. After incubation, samples were transfer to the Kingfisher Duo Prime system and the protocol was run for 25 minutes, which included 3 washes and then the RNA was eluted in 50μL with elution buffer. To quantify the viral genomes in the samples, the LigthMIX SARS-COV-2 RdRP Roche kit was used using a Roche Lightcycler 480 II PCR machine, following manufacturer’s instructions. Briefly, 5 μL of eluted samples (2-10ng/μL) were used as template for RT-PCR reactions together with a master mix and a set of primers targeting de RdRP gene. An incubation for 5 min at 55°C was performed for RT reaction to proceed. 45 PCR cycles were carried out to amplify SARS-CoV2 genomes. SARS-CoV-2 PCR Ct values <39 were considered positive for the presence of SARS-CoV-2 as indicated by the manufacturer. All HCWs positive in our study were re-tested by an official government licensed laboratory.
We performed the reverse transcription and PCR based on information provided by the Artic Network initiative (
We checked the quality of the raw reads using Fastqc v0.11.8, and then, the reads were filtered and trimmed using Trim_galore v0.5.0. We discarded the reads with a Phred score less than 30 and a minimum read length of less than 50bp. We assembled the SARS‐CoV‐2 genomes by IRMA v0.9.3 using as reference NCBI sequence ID NC_045512.2, and we performed the sequence alignment using MAFFT [
We aimed to screen the HCW population that were in direct contact with COVID-19 patients at Clinica Indisa in Santiago, Chile. Our study was carried out between May 1st and July 1st 2020, which was concomitant with the peak of SARS-Cov-2 transmission in the city of Santiago. At the start of the pandemic in March, Clinica Indisa had 79 high complexity intensive care unit (ICU) beds. In response to the drastic increase in the transmission of SARS-CoV-2 and in the admission of COVID-19 patients to ICU, the clinic increased its capacity to 172 beds in June (
(A) Total number and (B) occupancy percentage of ICU beds between March and July 2020; the peak transmission times of SARS-CoV-2 in Santiago, Chile.
Subjects were enrolled voluntarily from all services in the clinic. If subjects had previously been infected with SARS-CoV2 they were not able to enroll in the study. Subjects were asked to fill a questionnaire including personal information where they had to detail whether they had presented any COVID-19 symptoms, the presence of any comorbidities and if they had been in direct contact with SARS-CoV-2 infected people. Only volunteers that had not shown any symptoms related to COVDI19 at the moment of sample collection were enrolled in the study. At the same time, an informed consent document was signed. A nasopharyngeal swab was then taken from each individual to pursue the analysis. Our study was conducted to target most of the HCW that had been in contact with COVID-19 patients. Therefore, a total number of 413 asymptomatic HCWs were finally enrolled into the study. Ninety-five point four percent of these HCWs declared that their work had required close physical interaction with patients that may have had COVID-19, that is a distance shorter than 2 meters away. Out of the total individuals enrolled, 75.5% of them were women, with a median age for entire group of 33 years old ([IQR] = 29–40 years) and where only 27.1% claimed to have comorbidities. The study group congregated different professionals in the clinic with a higher representation in the sample of nursing technician (NT) (38.7%) and professional nurses (32.9%) (
Subjects were separated by the complete absence of symptoms or if they developed symptoms within 3 weeks of sample collection. A Ct Value <39 was considered as a positive sample for the presence of SARS-CoV-2.
Characteristics of HCWs (n = 413) | SARS-CoV-2 PCR | p value | ||
---|---|---|---|---|
Negative | Positive | |||
0.368 | ||||
Male | 99 (24.8) | 2 (14.3) | ||
Female | 300 (75.2) | 12 (85.7) | ||
Total | 399 (100) | 14 (100) | ||
0.810 | ||||
Male | 34 (29–42) | 37 (30–44) | ||
Female | 33 (28–39) | 32 (29–38) | ||
Total | 33 (29–40) | 32 (30–38) | ||
0.634 | ||||
Nurse | 130 (32.5) | 6 (42.9) | ||
Physiotherapist | 19 (4.8) | 0 (0) | ||
Medical Doctor | 11 (2.8) | 1 (7.1) | ||
Other | 71 (17.8) | 1 (7.1) | ||
AS | 14 (3.5) | 0 (0) | ||
NT | 154 (38.6) | 6 (42.9) | ||
Total | 399 (100) | 14 (100) | ||
0.007* | ||||
BC | 7 (1.7) | 1 (7.1) | ||
ES | 39 (9.8) | 5 (35.7) | ||
HW | 61 (15.3) | 1 (7.1) | ||
ICU | 292 (73.2) | 7 (50.0) | ||
Total | 399 (100) | 14 (100) | ||
0,462 | ||||
No | 292 (73.2) | 9 (643) | ||
Yes | 107 (26.8) | 5 (35.7) | ||
Total | 399 (100) | 14 (100) |
AS: Administrative Staff; NT: Higher Level Nursing Technicians; BC: Bronchopulmonary Consultations; ES: Emergency Service; HW: Hospital Wards; ICU: Intensive Care Units (*) = Statistically significant.
Positive samples from HCWs with Ct value <30, were used for subsequent sequencing of the viral genome. All the samples clustered at the GR GISAID clade (equivalent to clade 20B), as all the genomes presented the polymorphisms C241T, C3037T, A23403G, G28882, S-D614G, and N-G204R (
We constructed a maximum-likelihood tree using 169 full-sequence genomes. Additionally, we included the genome reference NCBI sequence ID NC_045512.2 on the tree. The letters in the tree indicate the lineage to which each clade belongs based on the GISAID classification (L, V, G, GH, and GR;(Han et al., 2019)). The purple circles indicate the bootstrap value in percent for 1,000 iterations. (A) the dendrogram of the 169 full-sequence genomes from Chilean samples (161 full-sequence genomes of SARS-CoV-2 isolated from Chilean patients, which are available in GISAID and eight full-sequence genomes of SARS-CoV-2 isolated from asymptomatic HCWs samples). (B) GR lineage where are located the 8 full-sequence genomes obtained from asymptomatic HCWs samples.
ID | Asymptomatic | Developed Symptoms | Ct | Sequence | ID seq NCBI | Coverage (X) | Linage |
---|---|---|---|---|---|---|---|
yes | yes | 26.72 | yes | SAMN16255338 | 100 | GR | |
yes | yes | 25.89 | yes | SAMN16255337 | 304 | GR | |
CoV 121 | yes | yes | 35.9 | no | - | - | |
CoV 143 | yes | no | 34.74 | no | - | - | |
CoV 147 | yes | yes | 35.20 | no | - | - | |
yes | no | 28.23 | yes | SAMN16255336 | 163 | GR | |
CoV 186 | yes | no | 37.24 | no | - | - | |
CoV 193 | yes | no | 37.47 | no | - | - | |
yes | no | 25.54 | yes | SAMN16255335 | 161 | GR | |
yes | yes | 25.63 | yes | SAMN16255334 | 183 | GR | |
yes | no | 18.11 | yes | SAMN16255333 | 235 | GR | |
yes | no | 25.88 | yes | SAMN16255332 | 182 | GR | |
yes | yes | 29.1 | yes | SAMN16255331 | 183 | GR | |
CoV 332 | yes | yes | 38.4 | no | - | - |
Samples used for sequencing are highlighted in bold letters.
In summary, these results indicate that only a minor fraction (3.45%) of the HCWs tested from Clinica Indisa were positive for the presence of SARS-CoV-2. Moreover, sequencing of the viral genomes shows that there are at least 5 different infection sources among the infected individuals, supporting the idea that these HCWs became infected at their individual communities outside of the clinic.
Since the beginning of the SARS-CoV-2 pandemic, countries have taken different approaches to stop spreading of the virus. They all coincide at this point that testing and tracing SARS-CoV-2 infected subjects is primordial for slowing down the spread of the virus. However, limited resources and the dependency of diagnostic reagents produced overseas, have diminished the ability of each country to adequately respond to this challenge, especially in Latin America.
It is now known that an elevated proportion of infected people is asymptomatic. Some studies have shown that these individuals infected with SARS-CoV-2 may represent up to 45% of the cases [
Our study focused on the identification of asymptomatic HCWs infected with SARS-CoV-2 during the peak of infection rates in Chile. It was found that only 14/413 (3,4%) of HCWs tested positive for SARS-CoV-2 in the absence of symptoms, a significantly small proportion compared to a 9.1% of asymptomatic infections in the country during the time our study was conducted (
Our study has some limitations, which include the number of enrolled subjects. This was a voluntary study open to all 1379 workers in the clinic, but it was limited by the time of sample collection and HCWs shifts due to law requirements of a mandatory quarantine after a RT-PCR test was run and results were informed. Nevertheless, we were able to reach approximately 30% of the HCWs in the clinic. Our study was conducted in a private clinic in Santiago, Chile. Thus, our results do not necessarily reflect the public hospital system where the use and availability of PPE was limited at the time. At the moment the study was conducted, no testing was performed routinely at either private or public hospitals to detect symptomatic or asymptomatic SARS-CoV-2 HCWs infections. Nowadays in hospitals HCWs are routinely being screened for SARS-CoV-2 infection as part of their protocols.
The higher percentage of positive cases in Bronchopulmonary ward (14.3%) and Emergency Services (12.8%) suggests that these 2 areas are at higher risk of infection than the ICU or general wards. This could be due to the constant changes of PPE that HCWs are required to do between patients, thus at least for these two areas we cannot completely discard nosocomial infection.
Interestingly, the comparison of the SARS-CoV-2 PCR Ct values obtained from asymptomatic individuals and those from individuals that finally developed symptoms, did not show any significant differences. This result suggests that there is no direct relationship between viral load at the moment of testing and the emergence of symptoms associated with COVID-19. As asymptomatic subjects may exhibit elevated viral titers they can be cataloged as “super-spreaders”, capable of silently transmitting the virus among the population to a large number of people [
Previous studies have shown high nosocomial transmission rates of SARS-CoV-2 between HCWs and/or patients [
We thank Gonzalo Fernández, Washington Valverde, and José Vega for their help collecting samples.