Estimating the prevalence of persistent symptoms after SARS-CoV-2 infection (post-COVID-19 syndrome): a regional cross-sectional study protocol

Abstract

Introduction

The COVID-19 pandemic, driven by the SARS-CoV-2 virus, has had a significant global impact, with over 775 million cases reported and more than 7 million deaths as of July 2024. In Chile, approximately 5.4 million people have been infected, with a substantial proportion experiencing persistent symptoms known as post-COVID-19 syndrome. This study aims to estimate the prevalence of post-COVID-19 syndrome in Punta Arenas, Chile, and to explore the associated symptoms, mainly focusing on psychological, physical and molecular impacts on the affected population.

Methods and analysis

This cross-sectional study will use stratified random sampling to select a representative sample of 282 adults from Punta Arenas. Participants eligible for the study are those who had tested positive for SARS-CoV-2 by reverse transcription-quantitative PCR between July 2022 and July 2023. Data collection will include comprehensive clinical assessments, psychological evaluations and laboratory analyses of inflammatory biomarkers. Standardised instruments will be used to ensure consistency and reliability in measuring persistent symptoms. Statistical analyses will include descriptive statistics, regression models and subgroup analyses to identify risk factors and the prevalence of post-COVID-19 syndrome.

Ethics and dissemination

The Human Research Ethics Committee of the Clinical Hospital of the University of Chile approved the study protocol (Memorandum No 007/2023). We will present the results in peer-reviewed publications and national and international professional and academic meetings.

Trial registration number

NCT05855382.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• The study location is an isolated region with low population mobility, enabling a precise prevalence assessment.

• Recruitment from the Chilean Ministry of Health Epidemiological Surveillance System database ensures a representative sample.

• A limitation of the study is the challenge of distinguishing whether self-reported symptoms are solely due to persistent COVID-19 or are influenced by other underlying illnesses or conditions.

• The study relies on participants’ recall of symptoms and medical history; there is a potential for recall bias.

Introduction

The SARS-CoV-2 virus pandemic has left a profound global footprint, affecting over 775 million individuals and tragically claiming more than 7 million lives as of July 2024.^{1 2} In Chile, the infection has been documented in approximately 5.4 million people with a prevalence that could reach over 28% of the population.³,⁵

COVID-19 is characterised by multisystemic impact, affecting the respiratory, gastrointestinal, cardiovascular and neurological systems.⁶,⁹ While many individuals develop mild symptoms, with 36% of infections reported as asymptomatic infections in a seroprevalence study conducted in Chile,³ approximately 15% of cases can progress to severe forms requiring hospitalisation and around 5% can suffer life-threatening complications.¹⁰ A subset of infected individuals can develop a postinfection syndrome characterised by persistent and fluctuating symptoms. This syndrome has been known by various terms, including ‘persistent COVID-19’, ‘post-COVID-19 syndrome’, ‘chronic COVID-19 syndrome’, or ‘post-acute sequelae of SARS-CoV-2 infection’.¹¹,¹³ The term post-COVID-19 will be used in the remainder of this study. Post-COVID-19 constitutes a syndrome linked to a spectrum of previous asymptomatic, moderate or severe infections.^{6 12 14} The WHO officially defined post-COVID-19 syndrome as the persistent manifestation of symptoms up to 3 months following a history of probable or confirmed SARS-CoV-2 infection lasting at least 2 months.^{11 15 16} These symptoms can arise during or after the initial disease and include physiological, psychological and cognitive impairments, significantly impacting individuals’ daily life.¹⁷,²¹

While associated risk factors are still under investigation, current evidence suggests an elevated likelihood of developing post-COVID-19 syndrome among individuals aged 35–70 years, predominantly women, with a history of underlying conditions such as type 2 diabetes mellitus, obesity, previous Epstein-Barr virus infection, presence of autoantibodies and various social health determinants.²⁰²²,²⁶ The post-COVID-19 syndrome is a diagnosis ‘by exclusion’ characterised by the persistence of symptoms for over 3 months following the initial infection and requiring the ruling out of alternative conditions.^{11 27 28} Worldwide, the prevalence of post-COVID-19 syndrome is estimated to be between 6% and 7% in the adult population and 1% in the paediatric population. However, reported prevalence ranges from 2% to 45%.^{313 29},³¹ The reasons for this widespread variation across different populations are as yet unclear.³²,³⁶ Several factors have been proposed, including individual as well as the environment, in particular the study design and location.^{3 27 37 38}

Subantarctic Chile was one of the most affected regions in the world at the beginning of the COVID-19 pandemic.³⁹ On the one hand, the low mobility rates of this territory, because of its unique geographical isolation and extreme latitude, enable high traceability of cases, facilitating recruitment and reducing variability induced by migratory flows.^{20 39} This context highlights the need for comprehensive prevalence estimates across the country. Despite this significant burden, a gap remains in understanding the long-term impact of COVID-19 on this population. Additionally, the estimation of post-COVID-19 syndrome prevalence is made more difficult due to different methodological approaches, hindering cross-study comparisons. Therefore, adherence to standardised strategies is essential.

The aim of this study is to estimate the prevalence and linked symptoms of post-COVID-19 syndrome in the city of Punta Arenas, Chile. The design is based on collecting a representative sample using age-based stratification and includes a comprehensive psychological assessment. Data collection includes clinical, epidemiological, psychological, respiratory, physical and molecular variables. This strategy will help identify the most prevalent persistent symptoms, ascertain their duration and gauge their impact on affected individuals’ quality of life. In addition, the study will assess inflammatory biomarkers in blood, including cytokines and B-cell immune repertoire, to explore their potential association with post-COVID-19 syndrome and disease severity.¹⁴⁴⁰,⁴⁶ The results could contribute to inform decision-making for post-pandemic management.

Methods and analysis

The methods of this study were designed in concordance with the WHO definition of post-COVID-19 syndrome⁴⁷ and are reported following the Strengthening the Reporting of Observational Studies in Epidemiology statement for cross-sectional studies.⁴⁸

Study aims and design

The post-COVID-19 syndrome study is a cross-sectional observational study with a population-based approach and stratified random sampling. The primary aim is to estimate the prevalence of post-COVID-19 syndrome following SARS-CoV-2 infection in adult patients who tested positive for COVID-19 by reverse transcription-quantitative PCR (RT-qPCR). The secondary aims are exploratory and include: (1) describing and establishing the frequency of physical and psychological signs and symptoms, (2) identifying individuals who meet the WHO case definition of post-COVID-19 syndrome in Chile, (3) exploring risk factors associated with post-COVID-19 syndrome and (4) exploring inflammatory and molecular biomarkers associated with post-COVID-19 syndrome. The study is carried out by the Centro Asistencial Docente e Investigación de la Universidad de Magallanes (CADI-UMAG) in Punta Arenas, Chile. Participant recruitment began in May 2023 and is ongoing, with an expected completion date of December 2024. Participants are being contacted at three separate points in time.

Population and eligibility

To be eligible for participation in this transversal study, an individual must satisfy the following criteria: (1) be 18 years or older, (2) have a history of a confirmed or positive diagnosis of SARS-CoV-2 infection by RT-qPCR between July 2022 and July 2023 and reside in Punta Arenas, (3) be registered in the Local Health Authority-Secretaría Regional Ministerial de Salud (SEREMI) epidemiological surveillance system database and (4) be able to understand and provide written informed consent. The study will also include participants who report persistent symptoms associated with positive SARS-CoV-2 infection test results, categorised as self-reported post-COVID-19 syndrome condition cases.

Sample size

A sample size determination was carried out to ascertain the number of participants required to achieve the desired precision in prevalence estimates, ensuring a 95% CI, as per:

$$n=\frac{Z^{2}P\left(1-P\right)}{d^2}$$

In equation (1), p represents the expected prevalence rate, while d denotes the precision level. The post-COVID-19 syndrome prevalence rates reported in the literature were used as inputs to calculate the required sample sizes. Based on official WHO guidelines,⁴⁷ the expected prevalence of post-COVID-19 syndrome in the general population was estimated at 20%, and this estimate was used as the basis for the sample size calculation at the time the study was designed. It is anticipated that 20% of individuals may experience persistent symptoms (eg, fatigue, pain, concentration problems and others) for at least 3 months. This estimation allows for the calculation of a sample size that considers the entire population of the area, along with a current inability to precisely determine prior exposure to the virus. A margin of error of ±5% in estimating the prevalence with a 95% CI, and a potential 12.5% sample loss was also considered. This potential anticipated sample loss could be due to participants declining some or all specimen collections post consent or it becoming impossible to re-contact patients during follow-up visits at CADI. This sample size estimation was calculated using the Scalex SP calculator.⁴⁹

Based on the above estimates and calculations, it was determined that the study would have to enrol a total of 282 participants. This sample size represents approximately 0.30% of the regional population aged 18 years and older. The sample was stratified into three age groups: youth (18–29 years, 14.6%), adulthood (30–64 years, 48%) and older adulthood (≥65 years, 11.4%), based on census data from the Chilean Institute of Statistics.⁵⁰ Recruitment will continue beyond the initial target to ensure robust and accurate estimates of post-COVID-19 syndrome.

Recruitment of participants

Figure 1 shows the flow diagram of participant recruitment in the post-COVID-19 syndrome study. Participants will be recruited through the following two ways:

Figure 1. Recruitment and assessment process in the post-COVID-19 syndrome study. The diagram illustrates the recruitment, eligibility assessment and clinical visits of the participants. Participants complete three stages of clinical visits: baseline assessments, kinesiological and respiratory evaluations and musculoskeletal analysis. The final step will be a comprehensive diagnosis, outcome analysis and estimation of the prevalence of post-COVID-19 syndrome. CADI, Centro Asistencial Docente e Investigación; RT-qPCR, reverse transcription-quantitative PCR; PEM, post-exertional malaise; SEREMI, Local Health Authority-Secretaría Regional Ministerial de Salud.

Via community health services message

Individuals who tested positive for SARS-CoV-2 and were registered with the Ministry of Health Services in Punta Arenas will be invited to participate in the post-COVID-19 syndrome study. The initial call for participants will be made via e-mail and will include a general adherence survey sent to a sample of 4000 people from the Local Health Authority-SEREMI database in Punta Arenas. From those who respond, 282 SARS-CoV-2 positive individuals, aged 18 years and above, will be selected in the order of response. Registration to participate will be conducted through the post-COVID-19 syndrome study website or via email.

Self-registered participants

Individuals interested in participating in the post-COVID-19 syndrome study will be able to self-enrol. However, only those with a positive SARS-CoV-2 test recorded in the Local Health Authority-SEREMI database will be eligible for inclusion through this method. The study population will be frequency-matched to the proportional stratified sampling distribution according to the age ranges defined in this study, and cases will be randomly selected.

Self-registration will be one strategy to minimise selection bias if enrolment rates are low. If many eligible participants refuse to enrol, the resulting sample may differ substantially from the target population, potentially introducing bias into the results. To minimise this, we will work closely with healthcare workers at each site to facilitate enrolment and will engage local patient associations and the community to raise awareness and encourage participation.

Data collection

The study data will be collected and managed using the Research Electronic Data Capture (REDCap) system, hosted on a secure server at the University of Magallanes. REDCap is a secure, web-based software platform designed to support data acquisition for research studies using devices and REDCap software.⁵¹ Continuous automated and manual data validation measures will be implemented throughout the survey fieldwork to optimise data integrity. Before the initiation of fieldwork, the survey team will undergo training on data collection protocols and data security measures. All collected data, including demographic information such as age, gender, history of current or past COVID-19 diagnosis and treatment, history of household COVID-19 contact, symptoms and laboratory results and source documentation, will be entered into an electronic case report file within REDCap. Additional data will be collected from individuals reporting symptoms suggestive of post-COVID-19 syndrome to assess health-seeking behaviour and barriers to care related to their symptoms. These fields are detailed in the online supplemental material. The principal investigators will ensure that the data entered into REDCap are accurate, complete and reliable, with range checks applied to data values.

Data deidentification

Each participant will be assigned a unique subject ID code number (post-COVID-19 XXX) consecutively on inclusion. This study number will be entered into the data collection form. Data collectors will generate a separate list containing the study number and the participant’s name. This list will be used to identify potential participants for the second component of the study (the survey). The list with identifying information will remain at the participating organisation and will not be disclosed to the research team. ID numbers linked to sensitive patient data (including name, ID number and date of enrolment) will be stored in a secured file, separate from other data, according to Chilean law requirements on data protection and research involving human subjects (Act No 20584, Act No 19628 and Act No 20120). Deidentified data will be incorporated into the source documentation, which will be stored in locked cabinets and retained for a maximum of 5 years in the confidential storage room 1010 at CADI-UMAG.

Data sources and measurements

Table 1 outlines the primary study domains and response variables to be assessed through standardised questionnaires, physical assessments and laboratory analyses. Participants will attend three visits at CADI-UMAG, each lasting up to 90 min. A multidisciplinary team will assist during the initial visit to ensure thorough and accurate data collection.

Table 1. Summary of study visits, domains and assessment instruments^*.

Visit	Step	Domain	Instrument	Purpose	Timing
V1	S1	Sociodemographic	Survey form	Baseline characterisation	Baseline
		Medical history	Medical history form	Identify risk/modifying factors
		Quality of life	QoL instrument	Assess functional impact
		Health behaviours	Behavioural questionnaire	Lifestyle-related recovery factors
	S2	Blood biomarkers	Clinical lab panel	General physiological status
		Immune profiling	Research lab panel	Immune system evaluation
	S3	Cognitive function	WAIS-IV	Cognitive sequelae detection
		Mental health	Standardised scales	Psychological impact
		Sleep quality	Sleep Quality Index	Evaluate sleep-related symptoms
V2	S1	Physical performance	FAS, InBody, dynamic handgrip.	Functional impact assessment	Follow-up
		PEM	DePaul PEM section	Evaluate PEM response
		Respiratory function	Pulmonary tools	Pulmonary sequelae
V3	S1	Gait and posture	3D photogrammetry	Motor system evaluation
	S2	Integrated assessment	All instruments used	Diagnostic confirmation of post-COVID	Final visit

^*See online supplemental table 1 for full variable details and measurement methods.

FAS, Fatigue Assessment Scale; InBody, body composition analyser; PEM, post-exertional malaise; QoL, quality of life; WAIS-IV, Wechsler Adult Intelligence Scale (fourth edition).

Clinic visit 1: initial patient assessment

Informed consent will be obtained from all participants before their assessment during the initial clinic visit. Baseline data collection will include demographic variables. A structured questionnaire, divided into five sections of categorised, multiple-choice clinical questions, will be used. Comprehensive laboratory evaluations will be conducted, including a complete blood count, inflammatory biomarkers in blood and an analysis of B and T cell immune repertoires. Additionally, standardised questionnaires will assess psychological and cognitive functions .⁵²,⁵⁸

Informed consent

In compliance with Chilean legal requirements, this study is classified as low-risk research involving human subjects, necessitating written informed consent from all participants. Data collectors will explain the study, providing participants with an explanatory statement and a consent form. Given the study’s focus on prevalence, an open call will be made to the general population. To accommodate participants with varying health conditions and potential impairments, different consent options will be provided:

• Participants capable of providing consent: the study will be clearly explained, and written consent will be obtained by the investigating physicians after allowing time for reflection.

• Participants unable to provide written consent: consent will be sought from a trusted individual or, if unavailable, a family member. The participant’s refusal will be respected, and written consent will be obtained from this representative.

Written consent will be secured whenever possible on the day of study admission to facilitate prompt procedures, such as blood tests. Participants will be informed of their rights to access, modify, oppose or cancel their participation. A revocation consent form will be provided, and on withdrawal, the participant will be excluded from further study procedures, although previously collected data will still be analysed. The research team will promptly communicate significant findings to participants.

Clinic visit 2: kinesiological and respiratory assessment

During the second visit, participants will undergo kinesiological assessments, including musculoskeletal capacity tests such as fatigue evaluation, body composition analysis and handgrip strength measurement using a dynamometer (administered by a healthcare professional). Additionally, participants will complete a standardised questionnaire assessing post-exertional malaise (PEM), characterised by the delayed or prolonged exacerbation of symptoms before and after physical activity to evaluate baseline status and immediate postactivity changes.⁵⁹,⁶¹

This evaluation complements the fatigue and physical function assessments ^{62 63} . Anthropometric measurements will include height, measured with a stadiometer, and weight and body composition, assessed through bioelectrical impedance analysis (using the InBody 770 device) .⁶⁴,⁶⁶ Furthermore, respiratory assessments will be conducted, including pulmonary function tests and spirometry, covering seven categorised respiratory parameters. ⁶⁷,⁷⁰

Clinic visit 3: functional movement and clinical assessment

Musculoskeletal function will be assessed using the 6-minute walk test (measuring distance covered), heart rate variability, stability evaluation, postural control, electroencephalography and electromyography, covering six categorised items. Any abnormalities detected during these assessments, including previously undiagnosed mental health issues, will be reported by the medical staff. Participants experiencing significant fatigue or functional decline after the test may be re-evaluated for PEM using the same standardised questionnaire from clinic visit 2 to monitor potential symptom exacerbation ^{71 72} .

Following these assessments, a medical evaluation will be conducted to diagnose the presence or absence of post-COVID-19 syndrome. Diagnosis will be based on clinical criteria following WHO guidelines.^{2 47} Participants not diagnosed with post-COVID-19 syndrome will either be discharged or referred to a healthcare centre for further evaluation or specialised care as needed.

Outcome measures

Primary outcome

The primary outcome measure will be to determine the prevalence of persistent symptoms in individuals who tested positive for SARS-CoV-2 in Punta Arenas. The severity of symptoms—including fatigue, pain, dyspnoea and cognitive impairment—will be assessed using standardised questionnaires with population-based norm cut-off scores for clinically significant severity. These assessments will be based on the 10 symptoms identified by the WHO, according to the Delphi consensus, which is associated with long covid (post-COVID-19 syndrome).^{2 47}

Secondary outcomes

• Assess the impact of persistent COVID-19 symptoms on daily life and well-being, including psychological, physical and respiratory aspects.

• Explore inflammatory and molecular biomarkers in blood samples to investigate their association with the persistence and severity of COVID-19 symptoms.

Biobank storage and accessory research

After laboratory analysis, blood residues (serum and plasma, at least 1 mL each) will be stored in the Molecular Medicine Laboratory—CADI-UMAG Biobank at −80°C and labelled with the subject ID number, thus deidentifying the samples. If future use of these samples is required, approval will be sought from the corresponding ethics committee, and the participant will be recontacted to approve the use of their samples.

Statistical analysis

Data will be processed and analysed using the R statistical programming language. Descriptive analyses will be conducted, where continuous variables will be summarised using either the mean and SD or the median and IQR, depending on the data distribution. For categorical variables, proportions will be calculated. Depending on the data distribution, we will apply one-sample or two-sample t-tests or, alternatively, Wilcoxon and Mann-Whitney tests. In addition, univariable and multivariable regression models, along with χ² and Fisher’s exact tests, will be employed. The study will also use a general linear mixed model, with subject IDs as the random effect. For questionnaire assessments, rank correlation methods will be applied. Statistical significance will be set at a threshold of p<0.05. If sufficient data are available, we will create subgroups based on post-COVID-19 syndrome status within the substudies to evaluate within-group associations across all parameters.

Patient and public involvement

Primary care clinics will be involved from the planning phase, collaborating on the development of educational materials to ensure message clarity and relevance. Patients and the community are considered key stakeholders in the project.

Community dissemination will begin prior to recruitment through a targeted communication campaign (local radio and press) led by the local health authority and the research team. The campaign will outline the study’s objectives, the research questions and the relevance of the findings to encourage participation. Information will be provided via town hall meetings and leaflets distributed in pharmacies and consultation offices. On study completion, results will be presented to the community and local authorities, emphasising public health implications related to post-COVID-19 syndrome. All communications will be simplified for public understanding.

Protocol amendments and dissemination policy

This protocol is the first version, completed on 10 May 2023. The trial has been registered at ClinicalTrials.gov (NCT05855382). Amendments will be published under this registration number. If required, the online version at ClinicalTrials.gov will be updated. The study will be performed in accordance with the Declaration of Helsinki and the principles of local legal and regulatory requirements. The trial status is recruiting; recruitment began in May 2023. The expected date of completion is December 2024.

Ethics and dissemination

The Human Research Ethics Committee of the Clinical Hospital of the University of Chile approved the study protocol, as stated in Memorandum No 007/2023. The observational design entails minimal risk to participants. No direct benefits to participants are anticipated, and neither incentives for participation will be provided nor reimbursement for expenses. Personal data will be handled according to Chilean laws and regulations (Act No. 19 628), and data will be anonymised as described above. At enrolment, potential participants will be fully informed on all aspects of the study and will be prompted to share their full contact details for the next follow-up clinic visit. It is possible that post-COVID-19 syndrome screening may elicit some anxiety among participants. To mitigate this risk, experienced clinicians within the study teams will be available to educate and support participants. Screening results will be made available to participants rapidly to minimise their wait time, and medical professionals will present these results.

We will present the results of peer-reviewed publications and national and international professional and academic meetings. We will organise seminars with relevant stakeholders (health policymakers and authorities, experts and academics) and hold town hall meetings with the local community. We will coordinate with the local health authority for press releases and radio broadcasts to disseminate the study’s purpose, introduce the research team and share milestones.