prospective case control study traduccion

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"case-control study" en español.

• volume_up estudio de casos y controles
• volume_up estudio de control de casos

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Traducciones similares para case-control study en español.

• investigación
• case probably
• case report
• case the joint
• case the place
• case worker
• case-by-case analysis
• case-by-case approach
• case-by-case basis
• case-by-case decision
• case-control study
• case-harden
• case-hardened
• case-sensitive
• casehardened
• casein protein
• caseload reduction

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Prospective vs. Retrospective Studies

Prospective

A prospective study watches for outcomes, such as the development of a disease, during the study period and relates this to other factors such as suspected risk or protection factor(s). The study usually involves taking a cohort of subjects and watching them over a long period. The outcome of interest should be common; otherwise, the number of outcomes observed will be too small to be statistically meaningful (indistinguishable from those that may have arisen by chance). All efforts should be made to avoid sources of bias such as the loss of individuals to follow up during the study. Prospective studies usually have fewer potential sources of bias and confounding than retrospective studies.

Retrospective

A retrospective study looks backwards and examines exposures to suspected risk or protection factors in relation to an outcome that is established at the start of the study. Many valuable case-control studies, such as Lane and Claypon's 1926 investigation of risk factors for breast cancer, were retrospective investigations. Most sources of error due to confounding and bias are more common in retrospective studies than in prospective studies. For this reason, retrospective investigations are often criticised. If the outcome of interest is uncommon, however, the size of prospective investigation required to estimate relative risk is often too large to be feasible. In retrospective studies the odds ratio provides an estimate of relative risk. You should take special care to avoid sources of bias and confounding in retrospective studies.

Prospective investigation is required to make precise estimates of either the incidence of an outcome or the relative risk of an outcome based on exposure.

Case-Control studies

Case-Control studies are usually but not exclusively retrospective, the opposite is true for cohort studies. The following notes relate case-control to cohort studies:

• outcome is measured before exposure
• controls are selected on the basis of not having the outcome
• good for rare outcomes
• relatively inexpensive
• smaller numbers required
• quicker to complete
• prone to selection bias
• prone to recall/retrospective bias
• related methods are risk (retrospective) , chi-square 2 by 2 test , Fisher's exact test , exact confidence interval for odds ratio , odds ratio meta-analysis and conditional logistic regression .

Cohort studies

Cohort studies are usually but not exclusively prospective, the opposite is true for case-control studies. The following notes relate cohort to case-control studies:

• outcome is measured after exposure
• yields true incidence rates and relative risks
• may uncover unanticipated associations with outcome
• best for common outcomes
• requires large numbers
• takes a long time to complete
• prone to attrition bias (compensate by using person-time methods)
• prone to the bias of change in methods over time
• related methods are risk (prospective) , relative risk meta-analysis , risk difference meta-analysis and proportions

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Case Control Studies

Affiliations.

• 1 University of Nebraska Medical Center
• 2 Spectrum Health/Michigan State University College of Human Medicine
• PMID: 28846237
• Bookshelf ID: NBK448143

A case-control study is a type of observational study commonly used to look at factors associated with diseases or outcomes. The case-control study starts with a group of cases, which are the individuals who have the outcome of interest. The researcher then tries to construct a second group of individuals called the controls, who are similar to the case individuals but do not have the outcome of interest. The researcher then looks at historical factors to identify if some exposure(s) is/are found more commonly in the cases than the controls. If the exposure is found more commonly in the cases than in the controls, the researcher can hypothesize that the exposure may be linked to the outcome of interest.

For example, a researcher may want to look at the rare cancer Kaposi's sarcoma. The researcher would find a group of individuals with Kaposi's sarcoma (the cases) and compare them to a group of patients who are similar to the cases in most ways but do not have Kaposi's sarcoma (controls). The researcher could then ask about various exposures to see if any exposure is more common in those with Kaposi's sarcoma (the cases) than those without Kaposi's sarcoma (the controls). The researcher might find that those with Kaposi's sarcoma are more likely to have HIV, and thus conclude that HIV may be a risk factor for the development of Kaposi's sarcoma.

There are many advantages to case-control studies. First, the case-control approach allows for the study of rare diseases. If a disease occurs very infrequently, one would have to follow a large group of people for a long period of time to accrue enough incident cases to study. Such use of resources may be impractical, so a case-control study can be useful for identifying current cases and evaluating historical associated factors. For example, if a disease developed in 1 in 1000 people per year (0.001/year) then in ten years one would expect about 10 cases of a disease to exist in a group of 1000 people. If the disease is much rarer, say 1 in 1,000,0000 per year (0.0000001/year) this would require either having to follow 1,000,0000 people for ten years or 1000 people for 1000 years to accrue ten total cases. As it may be impractical to follow 1,000,000 for ten years or to wait 1000 years for recruitment, a case-control study allows for a more feasible approach.

Second, the case-control study design makes it possible to look at multiple risk factors at once. In the example above about Kaposi's sarcoma, the researcher could ask both the cases and controls about exposures to HIV, asbestos, smoking, lead, sunburns, aniline dye, alcohol, herpes, human papillomavirus, or any number of possible exposures to identify those most likely associated with Kaposi's sarcoma.

Case-control studies can also be very helpful when disease outbreaks occur, and potential links and exposures need to be identified. This study mechanism can be commonly seen in food-related disease outbreaks associated with contaminated products, or when rare diseases start to increase in frequency, as has been seen with measles in recent years.

Because of these advantages, case-control studies are commonly used as one of the first studies to build evidence of an association between exposure and an event or disease.

In a case-control study, the investigator can include unequal numbers of cases with controls such as 2:1 or 4:1 to increase the power of the study.

Disadvantages and Limitations

The most commonly cited disadvantage in case-control studies is the potential for recall bias. Recall bias in a case-control study is the increased likelihood that those with the outcome will recall and report exposures compared to those without the outcome. In other words, even if both groups had exactly the same exposures, the participants in the cases group may report the exposure more often than the controls do. Recall bias may lead to concluding that there are associations between exposure and disease that do not, in fact, exist. It is due to subjects' imperfect memories of past exposures. If people with Kaposi's sarcoma are asked about exposure and history (e.g., HIV, asbestos, smoking, lead, sunburn, aniline dye, alcohol, herpes, human papillomavirus), the individuals with the disease are more likely to think harder about these exposures and recall having some of the exposures that the healthy controls.

Case-control studies, due to their typically retrospective nature, can be used to establish a correlation between exposures and outcomes, but cannot establish causation . These studies simply attempt to find correlations between past events and the current state.

When designing a case-control study, the researcher must find an appropriate control group. Ideally, the case group (those with the outcome) and the control group (those without the outcome) will have almost the same characteristics, such as age, gender, overall health status, and other factors. The two groups should have similar histories and live in similar environments. If, for example, our cases of Kaposi's sarcoma came from across the country but our controls were only chosen from a small community in northern latitudes where people rarely go outside or get sunburns, asking about sunburn may not be a valid exposure to investigate. Similarly, if all of the cases of Kaposi's sarcoma were found to come from a small community outside a battery factory with high levels of lead in the environment, then controls from across the country with minimal lead exposure would not provide an appropriate control group. The investigator must put a great deal of effort into creating a proper control group to bolster the strength of the case-control study as well as enhance their ability to find true and valid potential correlations between exposures and disease states.

Similarly, the researcher must recognize the potential for failing to identify confounding variables or exposures, introducing the possibility of confounding bias, which occurs when a variable that is not being accounted for that has a relationship with both the exposure and outcome. This can cause us to accidentally be studying something we are not accounting for but that may be systematically different between the groups.

Copyright © 2023, StatPearls Publishing LLC.

• Introduction
• Issues of Concern
• Clinical Significance
• Enhancing Healthcare Team Outcomes
• Review Questions

Publication types

• Study Guide

• Study protocol
• Open access
• Published: 30 January 2023

A prospective, longitudinal, case–control study to evaluate the neurodevelopment of children from birth to adolescence exposed to COVID-19 in utero

• Rachel A. Hill   ORCID: orcid.org/0000-0001-6111-355X 1 , 2 ,
• Atul Malhotra 3 ,
• Vathana Sackett 3 ,
• Katrina Williams 3 , 4 , 5 ,
• Michael Fahey 3 ,
• Kirsten R. Palmer 6 , 7 ,
• Rod W. Hunt 3 , 8 ,
• Hayley Darke 1 ,
• Izaak Lim 1 , 9 ,
• Vesna Newman-Morris 1 , 9 ,
• Jeanie L. Y. Cheong 5 , 8 , 10 ,
• Clare Whitehead 5 , 11 ,
• Joanne Said 5 , 12 ,
• Paulo Bignardi 13 ,
• Evelin Muraguchi 13 ,
• Luiz Carlos C. Fernandes Jr 13 ,
• Carlos Oliveira 13 &
• Suresh Sundram 1 , 14  

BMC Pediatrics volume  23 , Article number:  48 ( 2023 ) Cite this article

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The Coronavirus disease (COVID-19) pandemic has created unprecedented acute global health challenges. However, it also presents a set of unquantified and poorly understood risks in the medium to long term, specifically, risks to children whose mothers were infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy. Infections during pregnancy can increase the risk of atypical neurodevelopment in the offspring, but the long-term neurodevelopmental impact of in utero COVID-19 exposure is unknown. Prospective, longitudinal studies are needed to evaluate children exposed in utero to SARS-CoV2 to define this risk.

We have designed a prospective, case-controlled study to investigate the long-term impacts of SARS-CoV2 exposure on children exposed in utero. Women infected with SARS-CoV-2 during pregnancy will be recruited from Monash Health, the Royal Women’s Hospital and Western Health (Melbourne, Australia) and Londrina Municipal Maternity Hospital Lucilla Ballalai and PUCPR Medical Clinical (Londrina, Brazil). A control group in a 2:1 ratio (2 non-exposed: 1 exposed mother infant dyad) comprising women who gave birth in the same month of delivery, are of similar age but did not contract SARS-CoV-2 during their pregnancy will also be recruited. We aim to recruit 170 exposed and 340 non-exposed mother-infant dyads. Clinical and socio-demographic data will be collected directly from the mother and medical records. Biospecimens and clinical and epidemiological data will be collected from the mothers and offspring at multiple time points from birth through to 15 years of age using standardised sample collection, and neurological and behavioural measures.

The mapped neurodevelopmental trajectories and comparisons between SARS-CoV-2 exposed and control children will indicate the potential for an increase in atypical neurodevelopment. This has significant implications for strategic planning in the mental health and paediatrics sectors and long-term monitoring of children globally.

Peer Review reports

Historically, it is well documented that infections during pregnancy increase the risk for atypical neurodevelopment in offspring such as intellectual disability, cerebral palsy, autism and schizophrenia [ 1 ]. This has been noted in large epidemiological studies following influenza and measles epidemics, with varying degrees of severity depending on the pathogen and the gestation at the time of exposure to the infection [ 2 ]. A plausible but unknown prospect are severe long-term neurodevelopmental impacts following in utero exposure to SARS-CoV-2. This highly concerning prospect must be tested to establish the absolute risk and enable early intervention.

Transplacental or vertical transmission of SARS-CoV-2 has been reported [ 3 ]. Several case reports have confirmed the presence of SARS-CoV-2 in the amniotic fluid and umbilical cord blood [ 3 , 4 , 5 ], although this appears to be rare. Limited case studies also report elevated anti-SARS-CoV-2 Immunoglobulin M (IgM) and IgG antibodies and positive nasopharyngeal swab tests in neonates born to SARS-CoV-2 infected mothers [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. However, while vertical transmission is rare, of considerable concern is the maternal immune response to SARS-CoV-2 and the so called ‘cytokine storm’ that is a common occurrence following infection. Concern over this immune response is borne from previous ecological studies, birth cohort studies and animal models that have established key links between the activation of pro-inflammatory pathways in the mother with adverse neurodevelopment outcomes in the infant [ 2 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ].

To this end, we have established a large-scale, multi-site international initiative to monitor the long-term neurodevelopmental outcomes of infants exposed to SARS-CoV-2 in utero. The aim of the study is to assess the neurodevelopmental outcomes for children exposed to SARS-CoV-2 in utero. We hypothesise that children of mothers who contracted SARS-CoV-2 infection during pregnancy will show a heightened risk for future neurodevelopmental disorders.

We describe here a prospective longitudinal protocol to assess children exposed to SARS-CoV-2 in utero at multiple key neurodevelopmental time points from birth to 15 years of age. This protocol was established at Monash University, Melbourne, Australia and adapted at the School of Medicine, Pontificia Universidade Catolica do Parana, Londrina, Brazil. We encourage international uptake of this protocol for standardised global monitoring of neurodevelopmental outcomes.

Methods / design

Aim and study setting.

The study is a case-controlled investigational assessment of the long-term impacts of SARS-CoV-2 in utero exposure on children from birth to 15 years old. Ethics approval has been obtained through Monash Health Human Research Ethics Committee RES-20–0000-801A (protocol #6, 17/03/2022) and the National Council of Research Ethics (CONEP, acronym in Portuguese) with protocol number 5.234.055. The study aligns with the SPIRIT guidelines. Women infected with SARS-CoV-2 during pregnancy are being recruited from Monash Health, the Royal Women’s Hospital and Sunshine Hospital (Melbourne sites), and Londrina Municipal Maternity Hospital Lucilla Ballalai and PUCPR Medical Clinic (Londrina, Brazil). A putative control group in a 2:1 ratio is also being recruited of women who gave birth in the same month of delivery, and are of similar age (within a 5-year age bracket) but who did not contract SARS-CoV-2 during their pregnancy. It is important to note here that there are currently no sufficiently specific or sensitive tests to differentiate past vaccination from past infection of COVID-19, and with vaccination rates over 95% in Australia we cannot definitively test if a mother has had a COVID-19 infection during their pregnancy. Therefore, this group is a putative control group based on whether the mother has reported infection with COVID-19 during the pregnancy or not. Demographic information is collected from the mother at the first visit. See Table 1 for the complete list of demographic data collected. Exclusion criteria are loss of pregnancy. Multiple births (twins) are included and matched to non-exposed multiple births. Assessments are planned at birth, 3 months, 1, 2, 3, 4, 5, 10 and 15 years (see Fig.  1 Timeline diagram).

Timeline of assessments

Maternal study specific data

For women who tested positive for COVID-19: timing of the illness (weeks of pregnancy), highest temperature recorded during illness, duration of illness and symptom severity (World Health Organisation (WHO), seven-point ordinal scale) [ 19 ], disease modifying treatments received are recorded. COVID-19 vaccination status at the first appointment is also recorded. If the participant is vaccinated, the date of each vaccination and brand of vaccine is recorded.

All mothers will complete the Edinburgh Postnatal Depression Scale (EPDS). The EPDS is a questionnaire designed to screen women for symptoms of emotional distress during pregnancy and the postnatal period [ 20 ]. This is a 10-question survey which takes approximately 5–10 min to complete. This test will be completed at the initial birth assessment as well as the 3 and 12-month follow up assessments and will be administered by the study coordinator.

All mothers will complete the Maternal Postnatal Attachment Scale (MPAS). The MPAS is 19-item self-report questionnaire to measure a mother’s subjective feelings of attachment to her infant [ 21 ]. The MPAS will be completed at birth, 3 month and 12-month time points and should take approximately 10–15 min to complete, and will be administered by the study coordinator.

Parent-completed questionnaires about the child

All parent-completed questionnaires are administered by the clinical trials coordinator through either Q Global web-based platform for test administration (Pearson Clinical Assessment, Sydney, NSW, Australia), ACER, Melbourne, Australia or Psychological Assessments Australia, NSW, Australia. A trained Allied health professional (psychologist / neuropsychologist) will score and interpret the data.

At birth, 3 and 12 months the following questionnaires will be administered:

1. The Vineland adaptive behaviour scale—Third Edition (VABS-3) is an assessment of the child’s adaptive functioning [ 22 ]. It assesses 4 domains: communication, daily living, socialisation and motor skills. The VABS takes approximately 15–20 min to complete and will be administered through Q Global web-based platform for test administration (Pearson Clinical Assessment, Sydney, NSW, Australia).

2. Sensory Profile-2 (SP-2 questionnaire) [ 23 ]: an assessment of the child’s sensory processing patterns to understand how they may be impacting their participation in home, school and community-based activities. It takes approximately 5–20 min to complete and will again be administered through the Q global web-based platform.

At 2 and 3 years of age the VABS-3 and SP-2 will be administered as well as the Child Behaviour Checklist (CBCL) and the Repetitive Behavior Scale-Revised (RBS-R).

The CBCL: Preschool Version assesses specific kinds of behavioural, emotional and social difficulties that can be experienced by pre-school and school-aged children [ 24 ]. The questionnaire is completed by parents and takes approximately 10–20 min to complete (ACER, Melbourne, Australia).

The RBS-R is a 43 item questionnaire that assesses presence and severity of stereotyped behaviour, self-injurious behaviour, compulsive behaviour, routine behaviour, sameness behaviour, and restricted behaviours, which are associated with autism [ 25 ]. The questionnaire takes approximately 5–15 min to complete and will be administered through the Q Global web-based platform.

At 4 years of age, the VABS-3, SP2, CBCL and RBS-R will be administered as well as the Children’s Communication Checklist – Second Edition (CCC-2). The CCC-2 screens children who are likely to have communication difficulties and pragmatic language impairments [ 26 ]. The questionnaire takes approximately 5–15 min to complete and will be administered through the Q global web-based platform.

At 5 years of age, the VABS-3, SP2, CBCL, RBS-R and CCC-2 will be administered as well as the Behaviour Rating Inventory of Executive Functioning (BRIEF) (child version). The BRIEF assesses aspects of executive functioning as observed in the home environment.

[ 27 ] (Psychological Assessments Australia, NSW, Australia).

At 10 and 15 years of age, the VABS-3, SP2, CBCL, RBS-R, CCC-2 and BRIEF (child version) will be administered as well as the Connors 3 rd Edition-Parent assessment of Attention Deficit / Hyperactivity disorder [ 28 ]. The Connors assessment is commonly used to assess for ADHD and its common comorbidities in children aged 6 to 18 years.

Study-specific data collected from the infant

Birth time point.

The following information will be collected at birth (or within gestational ages 40–44 weeks):

1. Anthropometry: weight, length, and head circumference.

2. Hammersmith Neonatal Neurological Examination (HNNE). The HNNE is a 34-item examination assessing tone, motor patterns, observation of spontaneous movements, reflexes, visual and auditory attention and behaviour [ 29 ]. This assessment will be scored by a health professional trained in the administration of the HNNE who is blinded of the maternal COVID-19 status, and takes approximately 10–15 min.

3. General movements assessment (GMA). The GMA is used to identify normal writhing, or abnormal cramped synchronised, poor repertoire or chaotic movements [ 30 ]. The assessment is scored from a 3–5 min video of the infant while they are lying on their back in a calm but alert state. This assessment will be scored by a health professional trained in the administration of GM’s who is blinded of the maternal COVID-19 status.

3 months (corrected age) time point

At 3 months of age (corrected for prematurity) anthropometry (weight, length, head circumference) will be recorded as well as the GMA and the Hammersmith Infant Neurological Examination (HINE). At 3 months of age the GMA is used to assess normal fidgety or absent or abnormal movement. The HINE is a neurological assessment for infants aged between 2 and 24 months. The assessment includes a neurological examination which is scored, developmental milestones and behaviour (which are not scored) [ 31 ]. The neurological examination consists of 26 items from 5 domains, including cranial nerve function, posture, quality and quantity of movements, muscle tone, and reflexes and reactions. The GMA and HINE at 3 months will be scored by a health professional trained to administer these assessments and who is blinded of the maternal COVID-19 status.

12 months (corrected age) time point

At 12 months of age, anthropometric data are collected. In addition, the following scales are administered by a trained allied health professional:

The Bayley’s Scale of Infant and Toddler Development Fourth Edition (BSID IV), which is a test of development quotient [ 32 ].

The Ages and Stages Questionnaire (ASQ-3) as well as the ASQ: social and emotional 2. This questionnaire is a developmental screening tool for children aged between one month to 5 1/2 years [ 33 ].

24 months (corrected age) time point

At 24 months of age anthropometric data will be collected and a medical examination for vision, hearing and cerebral palsy is conducted. In addition, the following scales are administered by trained health professionals (psychologist and speech pathologist) who are blinded of the maternal COVID-19 status:

1. Bayley’s Scale of Infant and Toddler Development Fourth Edition (BSID IV).

2. The Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) [ 34 ]

3. Preschool Language Scales-Fourth Edition (PLS-4) [ 35 ], a test for communication skills.

3-year time point

At 3 years of age, the BSID IV, ADOS-2 and PLS-4 will be administered (as above at the 2-year time point).

4-year time point

At 4 years of age, the ADOS-2 and PLS-4 as well as the Stanford-Binet Intelligence Scale (SBIS) [ 36 ] – intelligence quotient, will be administered by trained health professionals.

5-year time point

At 5 years of age, the ADOS-2 and SBIS as well as the Clinical Evaluation of Language Fundamentals- Fourth Edition (CELF-4) [ 37 ] will be administered by trained health professionals. The CELF tests for communication and language skills for children 5 years and older.

10 and 15-year time point

At 10 years and at 15 years of age, the ADOS-2, SBIS and CELF-4 will be administered by trained health professionals.

Optional biospecimen collection

Maternal biospecimen collection.

For mothers who consent to biospecimen sample collection we will access their bio-banked samples collected during their infectious period. Blood samples and nasal mucosa will be assessed for viral load and inflammatory and cytokine marker analysis. If the mother has recovered prior to study participation biospecimens, including blood, saliva and buccal swabs, will be collected upon first visit. Blood samples will be collected by a health professional and assessed for levels of inflammatory markers [ 22 ]. Saliva will be collected to assess levels of cortisol [ 23 ]. Saliva samples are collected by the participant as soon as they wake, on the morning of their first assessment, in order to capture the waking cortisol response. Buccal swabs will be collected by a health professional and DNA will be extracted for epigenetic analysis.

Infant biospecimen collection

Parents may consent to provide a buccal swab sample from the infant. Biospecimens will be collected by a health professional at birth (or near the expected due date if born preterm). DNA will be extracted from buccal swabs for epigenetic analysis. At the time of birth, mothers who have a caesarean birth will also be given the option to consent to the collection of the umbilical cord blood and placental tissue. In cord blood and placental tissue, we will assess viral load (if infection was close to the time of birth), inflammatory and cytokine markers, mitochondrial function, and indices of mitochondrial structure and function. Additionally, placental morphology will be assessed using routine histopathological methods. Umbilical cord blood and stem and progenitor cell composition will be determined using flow cytometry.

Statistical analysis and power calculations

The data collected at each assessment will be compared between SARS-CoV-2 exposed and control groups longitudinally using a separate linear mixed effects analysis for each outcome measure. Given the number and frequency of measures there are likely to be missing datapoints, thus a mixed modelling approach will avoid the need for listwise deletion of incomplete data. Sociodemographic and clinical characteristics will be compared between groups using t-tests, Mann–Whitney U tests, or Chi-square tests as appropriate. If these confounders are statistically significant between the groups they will be included as covariates within the mixed modelling analysis. Machine learning approaches will be used to determine risk profiles based on demographic and biological data. Separate analysis will be done to split the COVID-19 group into those that scored higher than a 2 for illness severity (WHO ordinal scale) and those scoring under 2 (2 COVID-19 groups and 1 control group). Another analysis will split the COVID-19 group by those infected early in pregnancy (< 20 weeks) or late (> 20 weeks).

Power analysis using G*Power for an Analysis of Variance (ANOVA) repeated measure, between factor approach with 3 groups and 7 measures (7 assessment time points) suggests a sample size of 147 is required to have 95% power to detect a medium effect size of Cohen’s f = 0.25. To allow for potential dropouts we aim to recruit 170 mother-infant dyad cases and 340 mother-infant dyad controls to detect a medium effect.

Data management plan

Biospecimens will be stored and analysed in the laboratories at Monash Health, Monash Medical Centre, Monash University Clayton. Samples collected at Sunshine Hospital or at the Royal Women’s hospital will be stored short-term at these facilities before being transferred as a cohort to Monash Health (Behavioural neuroscience laboratory, Monash University). Samples collected will be de-identified at the time of collection and allocated a study code. This means that any information which could identify the participant, such as name, address, date of birth and hospital record number will be removed before the specimen is sent to the laboratory for analysis. We expect that all the blood, saliva and buccal swabs that we collect will be used for laboratory analysis. However, after the laboratory work has been completed, if there is any sample left over, it will either be stored at Monash Medical Centre (MMC) or discarded depending on the consent completed by the participant. Here we will give the participant the option (tick box) to either consent to immediate use, then any left over to be discarded, or to long-term storage of the samples for future unspecified use related to the study.

Maternal demographics and questionnaires will be stored in a password protected file, or in a locked cabinet held at MMC. Demographic and questionnaire data will be de-identified at the time of collection and allocated a study code. This means that any information which could identify the participant, such as name, address, date of birth and hospital record number will be removed prior to analysis. Data may only be accessed by researchers listed on the proposal.

Child developmental outcomes will be stored in a password protected file, or in a locked cabinet held at Monash Medical Centre. All assessment data will be de-identified at the time of collection and allocated a study code. This means that any information which could identify the participant, such as name, address, date of birth and hospital record number will be removed prior to analysis. Data may only be accessed by researchers listed on the proposal.

Plans for return of results of research to participants

We will generate a short summary report in lay terms following each assessment displayed as a ‘strengths and difficulties’ framework. Scores will not be shared with the parents/caregivers as assessment scores may be misinterpreted. We will ask for parent/caregivers’ permission to share data with professionals on an ‘as requested’ basis – as required for health, disability and/or education purposes.

The study is currently approved at Monash Health and Londrina participant health services. As Melbourne, Australia is currently experiencing a high prevalence of COVID-19 cases, thought to be attributed to the highly contagious Omicron strain, practical and operational issues to consider include hospital restrictions that discourage face to face participant involvement. Here, telehealth options have been explored, particularly for the 12-month assessments, which do not require neurological assessments, such as the Hammersmith neurological scales that must be done in person. For the birth and 3-month assessments, extra precautions have been planned, including personal protective equipment and social distancing compliance.

Another operational issue to consider is that with the high vaccination rates now in Victoria (~ 95% people aged 15 and over double vaccinated) and Brazil (~ 93% of the population have received 2 doses as at 09/03/2022), there is likely to be variation in that data, with some women having received 1, 2 or 3 doses and some being unvaccinated. This has been included in the study design as a question in the demographics; ‘Are you vaccinated? If so when? and How many doses?’. However, depending on the numbers this will need to be considered as a variable when analysing the data. We would anticipate that women who have been vaccinated will have a less severe course of illness, which will be reported through the WHO 7-point ordinal scale. These data will allow us to assess this anticipated hypothesis.

Our study design is such that biospecimen samples are collected at birth (or as close to birth as possible), then we will map our biomarker findings onto the neurodevelopmental trajectory of the child. For some women, the collection of biospecimens will be only shortly after they have been infected with COVID-19, while for others, they may have been infected early in their pregnancy. This variation in the time since infection is a limitation of the study. However, we have also linked this project to a COVID-19 biobank established through Monash Health, which collects mucosal swabs and serum samples at the time of infection. While not all participants will consent to both studies, these data will provide us with a unique opportunity to map biomarkers during infection to the neurodevelopmental trajectory of the child.

Overall, this established protocol will allow longitudinal, prospective analysis of the neurodevelopment of children exposed in utero to SARS-CoV-2 to determine the risk that COVID-19 infection during pregnancy poses to the infant. A secondary set of outcomes will be the biological findings from our biospecimen collections and consequent mapping of biological changes on the child’s neurodevelopmental trajectory. These data may provide valuable new knowledge on biomarkers or risk pathways of neurodevelopmental disturbances. The scales used in this study have been validated across multiple cultures, ensuring global uptake feasibility. With collaborations established in Londrina, Brazil, we call for international uptake of this protocol to inform health care professionals globally of the risk of COVID-19 infection during pregnancy to the neurodevelopment of the infant.

Availability of data and materials

The datasets generated during and/or analysed during the current study are not publicly available yet due to the majority of the data not collected yet but are available from the corresponding author on reasonable request.

Abbreviations

Severe acute respiratory syndrome coronavirus 2

Immunoglobulin M

Immunoglobulin G

Coronavirus disease of 2019

Edinburgh postnatal depression scale

Maternal postnatal attachment scale

Vineland adaptive behavior scale – 3 rd edition

Sensory profile-2

Child behavior checklist

Repetitive Behavior Scale-Revised

Children’s communication checklist – 2 nd edition

Behaviour Rating Inventory of Executive Functioning

Hammersmith neonatal neurological examination

Hammersmith infant neurological examination

General movement assessment

Bayley’s Scale of Infant and Toddler Development Fourth Edition

Ages and Stages Questionnaire 3 rd edition

Autism Diagnostic Observation Schedule-Second Edition

Preschool Language Scales-Fourth Edition

Stanford-Binet Intelligence Scale

Clinical Evaluation of Language Fundamentals- Fourth Edition

World health organisation

Analysis of variance

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Acknowledgements

We would like to thank the Clinical Trials Facility at Monash Health Translation Precinct for facilitating biospecimen collections.

This research is supported on a research grant awarded by the philanthropic organisation, One in Five, as well as internal departmental research funds. The external funding body has not played a role in the design, collection, analysis or interpretation of data or the writing of this manuscript or the decision to submit this manuscript.

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All authors have read and approved the manuscript. RH conceptualised and designed the study, wrote the study design and ethics submission, obtained philanthropic funding and will lead the data collection. AM assisted in the study design, ethics submission and the collection of neonatal data, and manuscript editing. VS assisted in the study design and the collection of neonatal data. KW assisted with study design and manuscript editing. MF assisted with study design and manuscript editing. KP assisted with study design and recruitment and manuscript editing. RH assisted with study design and manuscript editing. HD assisted with study design, ethics submission and manuscript editing. IL assisted in study design and manuscript editing. VNM assisted in study design and manuscript editing. JC assisted in study design, ethics submission and manuscript editing. CW assisted in study design, ethics submission and manuscript editing. JS assisted in study design, ethics submission and manuscript editing. PB adapted the protocol and coordinated the ethics submission for the Brazilian site, manuscript editing. EM adapted the protocol and assisted with the ethics submission for the Brazilian site. LF adapted the protocol and assisted with the ethics submission for the Brazilian site. CO adapted the protocol and assisted with the ethics submission for the Brazilian site. SS co-conceptualised the study and assisted in the study design, ethics submission and manuscript editing.

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Ethics has been approved through Monash Health Human Research Ethics Committee RES-20–0000-801A. All participants sign a consent form prior to participation in the study. Upon signing the consent form, they consent to the collection of the above described information (methods/ design) for themselves and their child. Individual tick boxes have been included for all biospecimen collections (blood, saliva and buccal swab) as these are optional. Consent for long-term storage of biospecimen samples is also included as a tick box.

Ethics for Brazilian site: Ethics has been approved through the National Council of Research Ethics (CONEP, acronym in Portuguese) under protocol no. 5.234.055. All participants sign a consent form prior to participation in the study. Upon signing the consent form, they consent to the collection of the above-described information (methods/ design) for themselves and their child. Individual tick boxes have been included for all biospecimen collections (blood, saliva and buccal swab) as these are optional.

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Hill, R.A., Malhotra, A., Sackett, V. et al. A prospective, longitudinal, case–control study to evaluate the neurodevelopment of children from birth to adolescence exposed to COVID-19 in utero. BMC Pediatr 23 , 48 (2023). https://doi.org/10.1186/s12887-023-03858-w

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• Published: 28 April 2021

A prospective case–control pilot study to evaluate bone microarchitecture in children and teenagers on long-term parenteral nutrition using HR-pQCT

• Typhaine Louazon 1 , 2 ,
• Pierre Poinsot 1 ,
• Lioara Restier 1 ,
• Abdelouahed Belmalih 1 ,
• Irène Loras-Duclaux 1 ,
• Stéphanie Marotte 1 ,
• Sophie Heissat 1 ,
• Didier Barnoud 3 ,
• Cécile Chambrier 3 ,
• Cyrille B. Confavreux 4 , 5 ,
• Alain Lachaux 1 , 2 , 7 ,
• Justine Bacchetta 2 , 5 , 6 &
• Noel Peretti 1 , 2 , 7  

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Long-term parenteral nutrition (PN) may induce bone complications. Tridimensional bone imaging techniques such as high-resolution peripheral quantitative computed tomography (HR-pQCT) allow the assessment of both compartmental volumetric densities and microarchitecture. Our aim was to evaluate these parameters in children and teenagers receiving long-term PN. This cross-sectional, case–control study included children older than 9 years undergoing PN for at least 2 years. They were age-, gender- and puberty-matched with healthy controls (1:2). Evaluation included biological assessment of bone metabolism (serum calcium, phosphate, and albumin; urinary calcium and creatinine; 25-OH vitamin D, osteocalcin and PTH), dual X-ray absorptiometry (DXA) and HR-pQCT at the ultradistal tibia and radius. Results are presented as median [range]. Eleven patients (3 girls) with a median age of 16 [9–19] years were included. Bone parameters assessed by HR-pQCT at the ultradistal radius and tibia were similar in patients and controls. Parathyroid hormone (PTH) levels were higher (14 [7–115] vs 16 [12–27]) and osteocalcin levels were lower (44 [15–65] vs 65 [38–142]) in patients than in controls, although within the normal range. Conclusions: there were no differences for compartmental bone densities and microarchitecture in patients undergoing chronic PN. Further longitudinal studies are required to confirm these quite reassuring preliminary results.

Introduction

Long-term parenteral nutrition (PN) is the most efficient way to prevent malnutrition in patients with severe intestinal failure (IF). However, PN may induce complications such as bone impairment, also called PN-associated metabolic bone disease (PN-MBD) 1 , 2 , 3 , 4 , 5 , 6 . PN-MBD was first described in 1980 5 , 7 . It is associated with osteoporosis and impaired mineralization 8 . Very few data are available on its exact prevalence, but it is estimated to range from 40 to 90% in adults and from 25 to 80% in children and teenagers 9 , 10 , 11 , 12 .

PN-MBD may be associated with metabolic abnormalities such as hypercalcemia, hypercalciuria, acidosis and hypophosphatemia. Even though the pathophysiology of PN-MBD remains to be fully determined, different parameters are nevertheless known to increase the risk of PN-MBD, namely inadequate PN composition (calcium, phosphate, and lactate), the presence of an underlying inflammatory disease, and treatment with corticosteroids.

Teenagers are particularly at risk for PN-MBD because of growth and acquisition of 25% of bone mass during puberty (and 90% over the first two decades of life). However, there are very limited data on long-term bone impairment in children and teenagers undergoing long-term PN.

Dual X ray Absorptiometry (DXA) is the most used technique to assess bone quantity; it is recommended to assess the longitudinal bone health for children at risk 13 . Total bone mineral content (TBMC) must be used for bone assessment according to these recommendations, expressing z scores with reference data based at least on age and gender. However, DXA has some technical limitations, especially in growing children and teenagers 13 , 14 , 15 : (i) DXA measures areal density rather than volumetric density that can be modified by growth; (ii) it is unable to evaluate separately trabecular and cortical bone that are sometimes impaired independently; and (iii) DXA cannot determine bone microarchitecture, which is a major determinant of the risk of bone fracture 16 .

Therefore, innovative tridimensional and non-invasive bone imaging techniques have been developed in the early 2000s, such as high-resolution peripheral quantitative computed tomography (HR-pQCT), allowing the assessment of both compartmental (i.e. total, cortical and trabecular) volumetric densities and trabecular microarchitecture in vivo 14 , 17 , 18 , 19 , 20 . This improved bone assessment, especially in children and teenagers whose bones continually grow in mass, size, and shape. To our knowledge, HR-pQCT has never been studied in pediatric patients receiving long-term PN.

We hypothesized that children and teenagers receiving chronic PN may display significant bone microarchitecture differences compared with controls matched for age, gender and pubertal status.

The objectives of this pilot study were (1) to determine the density and bone microarchitecture in children and teenagers receiving long-term PN compared to healthy controls, using HR-pQCT; (2) to compare HR-pQCT results to DXA measurements; and (3) to evaluate the biological markers of bone metabolism.

Patients and methods

This cross-sectional, comparative case-controlled pilot study was performed in the home parenteral nutrition pediatric unit of a university pediatric hospital between March 2014 and June 2015.

Inclusion criteria were children receiving home PN for more than 2 years for intestinal failure, which seems a sufficiently long period to evaluate the impact of PN on bone metabolism; older than 9 years of age due to technical limitations of HR-pQCT (need to have a perfectly still child), and with regular follow-up in a university HPN center. Exclusion criteria were congenital bone diseases (2 patients with McKusick syndrome) and corticosteroid use for more than one month during the 6 previous months (1 patients with rheumatologic inflammatory disease).

For each assessment, the patient’s anthropometric data were collected: birth term, birth height and weight, gender, weight, height, BMI, and Tanner status. For the same patient, all anthropometric measurements were performed by the same operator during follow-up appointments in a standardized manner: for height, with a wall height chart with increments of 0.5 cm; for weight, with a calibrated scale with increments of the nearest 0.1 kg. The Z-scores were calculated for each value according to national data references 21 . Tibia length was measured, the knee flexed at 90%, from the proximal margin of the medial malleolus to the proximal border of the medial tibial condyle (manually located) with a tape measure by the same trained operator. For each patient, IF characteristics and bone complication histories were obtained from medical files: IF etiology and intestinal residual length with or without ileocecal valve, fractures and bone pain.

PN was formulated according to the child’s individual needs.

The study was approved by a local independent ethics committee ( CPP Lyon Sud-Est II, ID-RCB2013-A01245-40 ), parental informed consent was obtained for each pediatric patient, and all methods were performed in accordance with the relevant guidelines and regulations . The study was registered in Clinical Trial (NCT02368496; 23 February 2015).

Healthy volunteers

Each patient undergoing PN participating in this study was matched by gender, age and pubertal status (Tanner stage) to two controls recruited concomitantly and locally in the VITADOS cohort of healthy children (NCT 01832623; 16 April 2013), as previously published 22 .

Biological data

All biological analyses were performed in the same laboratory, immediately after blood sample, in the morning with fasted patients. Usually, in children or adolescent with long-term PN, the frequency of standard biological follow-up is each 3 month when patients are stable. Serum calcium, phosphate, albumin, urinary calcium and creatinine were measured by routine laboratory methods. Intact parathyroid hormone was measured with a second generation assay (Roche Elecsys, Roche Diagnosis, Mannheim, Germany), 25-OH vitamin D with a radio-immunological technique (DiaSorin Assay, DiaSorin Diagnosis, Saluggia, USA), and serum NMID osteocalcin (N-terminal mid fragment of osteocalcin) with immune-chemiluminescence (LIAISON XL, DiaSorin Diagnosis, Saluggia, USA). Glomerular filtration was calculated with the new equation published before 23 .

HR-pQCT measurements were performed at the non-dominant limb unless there was a history of fracture, as previously reported by our team in children 24 . Volumetric bone mineral density (vBMD) and bone microstructure were measured at the distal radius and tibia using a HR-pQCT device (XtremeCT, SCANCO Medical AG, Brüttisellen, Switzerland) that acquires a stack of 110 parallel CT slices with an isotropic voxel size of 82 µm. A scout-view (dorsal–palmar radiography) allowed positioning of a reference line at the endplate. The first slice of the ROI was set at 22.5 mm of the reference line at the ultra-distal tibia and 9.5 mm at the ultra-distal radius, which extended proximally on 110 slices, i.e., 9.02 mm in the axial direction, as previously described. The following imaging settings were used: effective energy = 60 kVp, X-ray tube current = 900µA, integration time = 100 ms. The 126 mm field of view was reconstructed on a 1536 × 1536 matrix, yielding 82 µm isotropic voxels. The total scan time was 2.8 min with an equivalent dose of approximately 3 μSv. Attenuation data were converted to equivalent hydroxyapatite (HA) densities. A phantom was scanned daily for quality control.

For the standard analysis, a trained operator generates semi-automatic contours around the periosteal surface in scans without motion artifact; the entire volume of interest is thereafter automatically separated into a cortical and trabecular region. The outcome variables included total area (Tt.Ar, mm 2 ), volumetric bone density (mg HA/cm 3 ) for total (Tt.BMD), trabecular (Tb.BMD), and cortical (Ct.BMD) compartments; cortical thickness (Ct.Th, µm); and trabecular number (Tb.N, mm -1 ), thickness (Tb.Th, µm), separation (Tb.Sp, µm), and intra-individual distribution of separation (Tb.Sp.SD, µm). In clinical practice, the higher the trabecular number and thickness, the better the trabecular status; the lower the trabecular separation and distribution, the better the trabecular status.

DXA: dual X-ray absorptiometry

Whole body DXA and spine DXA were performed with a fan beam (Hologic Discovery W, Hologic, Inc., Bedford, MA) in the array using standard positioning techniques. We performed the standard pediatric protocol of total body less head. The Lumbar spine measurements included L1-L4. The DXA whole body and spine scans were analyzed to generate measures of whole body projected bone area (cm 2 ), bone mineral content (BMC) (g) and bone mineral density (BMD) (g/cm 2 ). Lean mass (kg) and fat mass (FM) (kg) were obtained from the whole body DXA scan excluding the skull.

Statistical analysis

Statistical analyses were performed using the SPSS Software. All eligible patients were included. Comparisons between patients and controls were performed using the nonparametric Wilcoxon signed rank test since the number of patients was low. Correlations between density, microarchitectural and biological parameters were computed with the Spearman bivariate analysis. All statistical tests were performed at the two-sided 0.05 level of significance. Data are presented as median, minimum and maximum: med [min–max].

Subjects characteristics

We included 11 patients and 22 controls. Anthropometric and clinical data for patients and controls are summarized in Table 1 .

The median age of initiating PN was 17 (0–150) months, and this nutritional support was given for a median of 10.3 (6.4–18.3) years at the time of this study.

All patients receiving long-term PN had intestinal failure; etiologies are indicated in Table 1 .

Two patients at the time of examination had withdrawn PN since 4 months and 5 months respectively. Calories in parenteral nutrition represented a median intake of 77 (40–190) % of recommended dietary allowances. All patients also had oral supplementation: 100 000 UI of vitamin D 3 each 3 months. Median vitamin D3 intake in PN was 220 (0–250) UI/day. Table 2 summarizes the main PN parameters applied to this cohort.

Three patients had also enteral nutrition during PN or during nights without PN.

Four patients and one control had history of long bone fracture, none had a vertebral fracture and none had more than two fractures. All were traumatic fractures.

Biological parameters

Biological parameters for patients and controls are also summarized in Table 1 . Nutritional biological parameters (albumin, prealbumin, urea, creatinin and eGFR according to the recent equation) were normal in the 2 groups; however compared to controls, PN patients displayed lower median albumin levels (84% of controls; p  = 0.015) but higher prealbumin (127% of controls; p  = 0.035) and urea levels (114% of controls; p  = 0.048). Parameters of calcium/phosphate and bone metabolism (calcium, phosphate, urinary calcium to creatinine ratio, 25 hydroxy-vitamin D and total alkaline phosphatase) were similar between the two groups. However, PN patients displayed higher but normal PTH (256% of controls; p  = 0.003) and lower but normal osteocalcin plasmatic levels (67% of controls; p  = 0.005), as compared to controls.

At the radius, no significant differences were observed between controls and PN patients, as illustrated in Table 3 . However at the tibia, PN children displayed a lower trabecular area compared to controls (470 [124–675] vs 606.5 [369–897] µm 2 , p  = 0.003).

DXA and body composition

Body composition data are summarized in Tables 3 and 4 . BMC was similar to controls; only the BMD of the whole body was significantly lower in patients (0.81 [0.54–0.96] vs 0.92 [0.66–1.35], p  = 0.039). In terms of body composition, the global distribution was similar between patients and healthy patients. However patients differed according the gynoid lean and total mass distribution. The android-gynoid ratios and the BMI were similar to the controls.

This pilot prospective study is the first to evaluate bone microarchitecture with HR-pQCT in pediatric patients undergoing long-term PN in comparison to healthy controls. The main results are: (1) reassuring results for bone quality and body composition evaluated by both HR-pQCT (with similar densities and microarchitecture between PN patients and controls) and DXA (with BMC similar to controls); and (2) higher but normal PTH levels despite correct vitamin D levels, with lower but normal osteocalcin levels in PN patients as compared to controls.

PN-associated metabolic bone disease

The main previous studies that have described bone diseases in patients receiving home PN were adult studies 5 , 8 , 11 . The first pediatric study was published in 2010 25 . Of note, 83% of these patients had bone mineral deficiency and 17% had fractures. Different studies evaluated longitudinal evolution and predictive factors, including intestinal failure-related factors and parenteral nutrition-related factors 15 , 25 , 26 , 27 . Risk factors identified were: (1) the etiology of intestinal failure: patients with a small bowel syndrome were more at risk for bone disease; (2) the duration of PN was correlated to lower lumbar BMD; (3) others factors such as the small bowel length and the presence of the ileocecal valve did not predict BMD.

Compared to previous pediatric studies, our cohort had less PN-MBD (only 2 (18%) patients had a whole body BMC ≤ − 2 DS). Several reasons may explain this discrepancy: the etiologies are different since we excluded patients with inflammatory bowel disease and patients having received steroid therapy for more than 1 month during the 6 previous months. These etiologies differ from other studies describing lower bone mineral density in patients on long-term PN which may include inflammatory bowel disease 4 , 8 , 9 , 11 . We can also hypothesize that the treatment and patient care were effective in preventing bone deterioration.

A significant fracture history is defined by either one or more vertebral crush fractures, two or more long bone fractures by 10 years of age, or three or more fractures by 19 years of age 12 . The clinical bone history of our patients indicates that four patients had a past of bone fracture, and only one control subject. However, as noted below, radiological exams (HR-pQCT and DXA) were not significantly different between the two groups. It seems unlikely that all radiological measurements are ineffective to identify the risk of bone fracture, and we face here one of the main limitations of this cohort namely its limited size.

Determination of the density and bone microarchitecture in children and teenagers with long-term PN.

Compared to DXA, the main objective of HR-pQCT is to evaluate microarchitectural composition. Low trabecular area and high trabecular separation are associated with an increased risk of osteoporosis. This profile associated with bone fragility is described only at the ultra-distal tibia of our patients, and only with a small difference. However, there are no significant differences in cortical parameters that are involved more in bone strength than trabecular ones. Many studies have shown an increased cortical vBMD with age without modifications of trabecular parameter 18 , 28 , 29 . Cortical vBMD measured by HR-pQCT increased with puberty in both genders 28 . Therefore, this small difference reported only at the ultra-distal tibia of patients should have only a minor effect if any on bone solidity.

Comparison of HR-pQCT results to DXA measurements

Our study seems to indicate very mild differences of mineral status with both radiological techniques when compared patient to healthy controls. On one hand, HR-pQCT indicates no difference in BMD but only minor trabecular changes at the tibia; on the other hand DXA indicates no alteration of the main parameters (namely spine BMD, BMC or whole body BMC that correspond to the recommended measure sites in pediatrics) 30 .

BMD had divergent results with the two radiological techniques: HR-pQCT measured similar total volumetric BMD in patients and controls. In contrast, DXA showed a significant lower whole body subtotal BMD in patients. This may suggest that (1) inherent differences between the radiological techniques (HR-pQCT versus DEXA); (2) BMD of the ultra-distal radius and tibia do not reflect whole body BMD, but previous studies have found DXA spine and hip scans do correlate with HR-pQCT radius and tibia scans; (3) HR-pQCT and DXA evaluate bone composition differently; (4) our patients were smaller than controls but had a similar BMI. That could explain a lower BMD with DXA. DXA relies on areal density rather than volumetric density that can be modified by growth. Therefore, the risk of underestimating BMD in small children and overestimating BMD in tall children is well known 13 , 31 .

Evaluation of the biological markers of bone metabolism

Very few pediatric studies accurately assessed the biological markers of bone metabolism in PN. We did not find biological abnormalities in this study for the markers of bone metabolism; however, as compared to controls, our patients displayed different levels of PTH and osteocalcin that however both remained within the normal range.

Indeed, patients displayed a relative hyperparathyroidism: PTH levels were within the normal range, but significantly higher in patients compared to controls. Hyperparathyroidism in patients on long-term PN has been previously described 26 , 32 . A relationship between hyperparathyroidism and bone remodeling was also previously described in adults 33 , 34 . In our patients, this higher level of PTH is not explained by a lower vitamin D level, which is similar to healthy controls. Based on international recommendations, a partial deficiency could be considered in patients and controls since vitamin D levels should be higher than 75 nmol/L 35 . The recommendation for parenteral vitamin D intakes are 400 UI/day 36 . Our patients received only a median intake of 268 ± 0.87 UI/day in perfusion, but also an oral supplementation of vitamin D every 3 months. Therefore, intestinal malabsorption could explain their low plasmatic level of vitamin D; this result should encourage increasing oral doses and closely monitoring plasmatic vitamin D.

Low calcemia and high phosphoremia could explain a higher PTH. This hormone increases calcium levels and decreases phosphate levels, through a stimulation of 1–25 OH2 vitamin D synthesis and further activation of intestinal calcium absorption, through a stimulation of tubular calcium reabsorption and through an inhibition of the apical expression of the sodium/phosphate transporters Npt2 thus inducing phosphaturia. Last, PTH also has biphasic effects on bone, depending on the levels of PTH and its pulsatility. In our patients, calcium and phosphate parenteral intakes are within the range of the recommendations Calcium 0.35 for 0.25–0.4 mmol/kg/day and phosphate 0.6 for 0.2 to 0.7 mmol/kg/day, whereas the rates of calcemia and phosphatemia were similar in the two groups 37 . Unfortunately, we do not have urinary calcium and phosphate levels for all patients to evaluate tubular reabsorption. However, the Ca/creat urinary ratio was normal and not different between patients and controls.

Finally, an acid–base imbalance could also increase PTH levels. Indeed, metabolic acidosis rapidly inhibits the CaSR that causes PTH release and relative hyperparathyroidism. Acid–base balance was not specifically studied in this cohort, even if PN patients displayed significant lower plasma bicarbonate concentrations in comparison to controls. However, the values are normal and the difference does not seem clinically relevant.

Osteocalcin

Patients had a significantly lower osteocalcin levels than controls. Osteocalcin is produced by differentiated osteoblasts. Furthermore, non-carboxylated osteocalcin regulates glucose homeostasis by increasing insulin secretion and decreasing insulin resistance.

Osteocalcin level is influenced by different parameters: (1) gender: higher in women; (2) age with a decrease with age in both men and women, and especially during puberty 38 ; (3) physical activity: higher after a run. As our patients are gender- and age-matched with controls, variations of osteocalcin could indicate an impaired glucose metabolism 38 , 39 , 40 . Unfortunately, we do not have the parameters to evaluate this hypothesis such as, for example, the Homeostasis Model Assessment (HOMA) scale of insulin resistance (insulin (mUI/L) × glucose (mmol/L)/22.5) 41 , 42 , 43 . It would be interesting for a future study to collect insulin and glucose levels in these patients.

Several limitations of this study may be highlighted. It is a monocentric study with a small cohort. However, this study was designed as a pilot study, and children receiving long-term PN are rare. Another limitation in the interpretation of results is the significant difference observed in height and body weight between patients and controls, likely explaining the lower trabecular area observed at the weight-bearing tibia in PN patients. Body size differences alone could account for any discrepancies between the clinical and healthy groups, an adjustment for height between patients and controls would ideally be useful. Even though we selected the smallest subjects among the controls (after matching for gender, age and puberty), it was impossible to adjust for height given the difference of height between controls and patients. However, odd differences in height between groups is little, and limb length is similar between the 2 groups; therefore we believe that these two limitations are not crucial in view of the observed results, namely the absence of significant differences in bone status between controls and PN patients 28 , 44 , 45 .

Conclusion and perspectives

This first study evaluating microarchitecture in children receiving long-term PN provides quite reassuring results: the innovative imaging technique HR-pQCT did not find any deterioration of bone outcomes in our cohort after more than 10 years of PN. However, the small sample size requires caution in interpretation. Further longitudinal studies, with larger cohorts, are required to confirm these data and to determine if such techniques could help physicians improve the therapeutic management of children receiving long-term PN.

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An institutional funding for the VITADOS cohort was provided by the Programme Hospitalier de Recherche Clinique Inter-régional (PHRCi) (J Bacchetta, 2011). An institutional grant was provided by ALLP for this study.

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Louazon, T., Poinsot, P., Restier, L. et al. A prospective case–control pilot study to evaluate bone microarchitecture in children and teenagers on long-term parenteral nutrition using HR-pQCT. Sci Rep 11 , 9151 (2021). https://doi.org/10.1038/s41598-021-88366-6

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• What Is a Case-Control Study? | Definition & Examples

What Is a Case-Control Study? | Definition & Examples

Published on February 4, 2023 by Tegan George . Revised on June 22, 2023.

A case-control study is an experimental design that compares a group of participants possessing a condition of interest to a very similar group lacking that condition. Here, the participants possessing the attribute of study, such as a disease, are called the “case,” and those without it are the “control.”

It’s important to remember that the case group is chosen because they already possess the attribute of interest. The point of the control group is to facilitate investigation, e.g., studying whether the case group systematically exhibits that attribute more than the control group does.

Table of contents

When to use a case-control study, examples of case-control studies, advantages and disadvantages of case-control studies, other interesting articles, frequently asked questions.

Case-control studies are a type of observational study often used in fields like medical research, environmental health, or epidemiology. While most observational studies are qualitative in nature, case-control studies can also be quantitative , and they often are in healthcare settings. Case-control studies can be used for both exploratory and explanatory research , and they are a good choice for studying research topics like disease exposure and health outcomes.

A case-control study may be a good fit for your research if it meets the following criteria.

• Data on exposure (e.g., to a chemical or a pesticide) are difficult to obtain or expensive.
• The disease associated with the exposure you’re studying has a long incubation period or is rare or under-studied (e.g., AIDS in the early 1980s).
• The population you are studying is difficult to contact for follow-up questions (e.g., asylum seekers).

Retrospective cohort studies use existing secondary research data, such as medical records or databases, to identify a group of people with a common exposure or risk factor and to observe their outcomes over time. Case-control studies conduct primary research , comparing a group of participants possessing a condition of interest to a very similar group lacking that condition in real time.

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Case-control studies are common in fields like epidemiology, healthcare, and psychology.

You would then collect data on your participants’ exposure to contaminated drinking water, focusing on variables such as the source of said water and the duration of exposure, for both groups. You could then compare the two to determine if there is a relationship between drinking water contamination and the risk of developing a gastrointestinal illness. Example: Healthcare case-control study You are interested in the relationship between the dietary intake of a particular vitamin (e.g., vitamin D) and the risk of developing osteoporosis later in life. Here, the case group would be individuals who have been diagnosed with osteoporosis, while the control group would be individuals without osteoporosis.

You would then collect information on dietary intake of vitamin D for both the cases and controls and compare the two groups to determine if there is a relationship between vitamin D intake and the risk of developing osteoporosis. Example: Psychology case-control study You are studying the relationship between early-childhood stress and the likelihood of later developing post-traumatic stress disorder (PTSD). Here, the case group would be individuals who have been diagnosed with PTSD, while the control group would be individuals without PTSD.

Case-control studies are a solid research method choice, but they come with distinct advantages and disadvantages.

Advantages of case-control studies

• Case-control studies are a great choice if you have any ethical considerations about your participants that could preclude you from using a traditional experimental design .
• Case-control studies are time efficient and fairly inexpensive to conduct because they require fewer subjects than other research methods .
• If there were multiple exposures leading to a single outcome, case-control studies can incorporate that. As such, they truly shine when used to study rare outcomes or outbreaks of a particular disease .

Disadvantages of case-control studies

• Case-control studies, similarly to observational studies, run a high risk of research biases . They are particularly susceptible to observer bias , recall bias , and interviewer bias.
• In the case of very rare exposures of the outcome studied, attempting to conduct a case-control study can be very time consuming and inefficient .
• Case-control studies in general have low internal validity  and are not always credible.

Case-control studies by design focus on one singular outcome. This makes them very rigid and not generalizable , as no extrapolation can be made about other outcomes like risk recurrence or future exposure threat. This leads to less satisfying results than other methodological choices.

If you want to know more about statistics , methodology , or research bias , make sure to check out some of our other articles with explanations and examples.

• Student’s  t -distribution
• Normal distribution
• Null and Alternative Hypotheses
• Chi square tests
• Confidence interval
• Quartiles & Quantiles
• Cluster sampling
• Stratified sampling
• Data cleansing
• Reproducibility vs Replicability
• Peer review
• Prospective cohort study

Research bias

• Implicit bias
• Cognitive bias
• Placebo effect
• Hawthorne effect
• Hindsight bias
• Affect heuristic
• Social desirability bias

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A case-control study differs from a cohort study because cohort studies are more longitudinal in nature and do not necessarily require a control group .

While one may be added if the investigator so chooses, members of the cohort are primarily selected because of a shared characteristic among them. In particular, retrospective cohort studies are designed to follow a group of people with a common exposure or risk factor over time and observe their outcomes.

Case-control studies, in contrast, require both a case group and a control group, as suggested by their name, and usually are used to identify risk factors for a disease by comparing cases and controls.

A case-control study differs from a cross-sectional study because case-control studies are naturally retrospective in nature, looking backward in time to identify exposures that may have occurred before the development of the disease.

On the other hand, cross-sectional studies collect data on a population at a single point in time. The goal here is to describe the characteristics of the population, such as their age, gender identity, or health status, and understand the distribution and relationships of these characteristics.

Cases and controls are selected for a case-control study based on their inherent characteristics. Participants already possessing the condition of interest form the “case,” while those without form the “control.”

Keep in mind that by definition the case group is chosen because they already possess the attribute of interest. The point of the control group is to facilitate investigation, e.g., studying whether the case group systematically exhibits that attribute more than the control group does.

The strength of the association between an exposure and a disease in a case-control study can be measured using a few different statistical measures , such as odds ratios (ORs) and relative risk (RR).

No, case-control studies cannot establish causality as a standalone measure.

As observational studies , they can suggest associations between an exposure and a disease, but they cannot prove without a doubt that the exposure causes the disease. In particular, issues arising from timing, research biases like recall bias , and the selection of variables lead to low internal validity and the inability to determine causality.

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Schlesselman, J. J. (1982). Case-Control Studies: Design, Conduct, Analysis (Monographs in Epidemiology and Biostatistics, 2) (Illustrated). Oxford University Press.

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Research Article

Reduced dengue incidence following city-wide w Mel Wolbachia mosquito releases throughout three Colombian cities: Interrupted time series analysis and a prospective case-control study

Roles Conceptualization, Investigation, Project administration, Resources, Supervision, Writing – original draft

Affiliation World Mosquito Program, Universidad de Antioquia, Medellín, Colombia

Roles Formal analysis, Investigation, Methodology, Visualization, Writing – original draft

Affiliation World Mosquito Program, Monash University, Melbourne, Australia

Roles Investigation

Roles Investigation, Project administration

Roles Data curation

Roles Project administration

Affiliation Secretariat of Health, Medellín, Colombia

Affiliation Secretariat of Health, Bello, Colombia

Roles Conceptualization, Funding acquisition

Roles Conceptualization, Investigation

Roles Investigation, Visualization, Writing – review & editing

Roles Methodology

Affiliation Department of Medical Statistics, London School of Hygiene and Tropical Medicine, London, United Kingdom

Roles Formal analysis, Investigation, Methodology

Affiliation Division of Biostatistics, Department of Epidemiology and Biostatistics, University of California, San Francisco, California, United States of America

Roles Conceptualization, Methodology, Writing – review & editing

•  [ ... ],

Roles Conceptualization, Formal analysis, Methodology, Project administration, Supervision, Visualization, Writing – original draft

* E-mail: [email protected]

• [ view all ]
• [ view less ]
• Ivan Dario Velez, 
• Stephanie K. Tanamas, 
• Maria Patricia Arbelaez, 
• Simon C. Kutcher, 
• Sandra L. Duque, 
• Alexander Uribe, 
• Lina Zuluaga, 
• Luis Martínez, 
• Ana Cristina Patiño, 

• Published: November 30, 2023
• https://doi.org/10.1371/journal.pntd.0011713
• Reader Comments

The introduction of Wolbachia ( w Mel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Randomised and non-randomised studies in multiple countries have shown significant reductions in dengue incidence following field releases of w Mel-infected Ae . aegypti . We report the public health outcomes from phased, large-scale releases of w Mel- Ae . aegypti mosquitoes throughout three contiguous cities in the Aburrá Valley, Colombia.

Methodology/Principal findings

Following pilot releases in 2015–2016, staged city-wide w Mel- Ae . aegypti deployments were undertaken in the cities of Bello, Medellín and Itagüí (3.3 million people) between October 2016 and April 2022. The impact of the Wolbachia intervention on dengue incidence was evaluated in two parallel studies. A quasi-experimental study using interrupted time series analysis showed notified dengue case incidence was reduced by 95% in Bello and Medellín and 97% in Itagüí, following establishment of w Mel at ≥60% prevalence, compared to the pre-intervention period and after adjusting for seasonal trends. A concurrent clinic-based case-control study with a test-negative design was unable to attain the target sample size of 63 enrolled virologically-confirmed dengue (VCD) cases between May 2019 and December 2021, consistent with low dengue incidence throughout the Aburrá Valley following w Mel deployments. Nevertheless, VCD incidence was 45% lower (OR 0.55 [95% CI 0.25, 1.17]) and combined VCD/presumptive dengue incidence was 47% lower (OR 0.53 [95% CI 0.30, 0.93]) among participants resident in w Mel-treated versus untreated neighbourhoods.

Conclusions/Significance

Stable introduction of w Mel into local Ae . aegypti populations was associated with a significant and sustained reduction in dengue incidence across three Colombian cities. These results from the largest contiguous Wolbachia releases to-date demonstrate the real-world effectiveness of the method across large urban populations and, alongside previously published results, support the reproducibility of this effectiveness across different ecological settings.

Trial registration

NCT03631719 .

Author summary

Dengue fever is a viral disease transmitted by Aedes aegypti mosquitoes and is an increasing public health concern globally. A novel evidence-based tool for dengue control involves the release of Ae . aegypti mosquitoes infected with a naturally-occurring insect bacteria called Wolbachia ( w Mel strain). w Mel significantly reduces the mosquito’s ability to transmit dengue and other viruses and can establish long-term in local Ae . aegypti populations following short-term releases. Previous studies have shown that establishment of w Mel in local mosquito populations significantly reduces local dengue incidence. In Colombia, w Mel-infected Ae . aegypti were released throughout the cities of Bello, Medellín, and Itagüí in the Aburrá Valley between 2015 and 2022, covering a population of 3.3 million people. The rate of dengue case notifications in the three cities declined by 95–97% after Wolbachia releases, compared to the prior decade, and dengue case numbers since 2020 have been the lowest in twenty years. A case-control study in Medellin in 2019–2021 showed that laboratory-confirmed dengue incidence was significantly lower in neighbourhoods with Wolbachia releases compared to untreated neighbourhoods. These results confirm the real-world effectiveness of the Wolbachia method, and show that it can be implemented at a city-wide scale to protect communities against dengue.

Citation: Velez ID, Tanamas SK, Arbelaez MP, Kutcher SC, Duque SL, Uribe A, et al. (2023) Reduced dengue incidence following city-wide w Mel Wolbachia mosquito releases throughout three Colombian cities: Interrupted time series analysis and a prospective case-control study. PLoS Negl Trop Dis 17(11): e0011713. https://doi.org/10.1371/journal.pntd.0011713

Editor: Elvina Viennet, Australian Red Cross Lifelood, AUSTRALIA

Received: May 16, 2023; Accepted: October 10, 2023; Published: November 30, 2023

Copyright: © 2023 Velez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data underlying the analyses reported here are available at the following links: Aburra Valley notified dengue cases and wMel Wolbachia prevalence by comuna and month: https://doi.org/10.6084/m9.figshare.22724663 Medellin test-negative design case control study analysis dataset: https://doi.org/10.6084/m9.figshare.24086139 Line listed data for dengue and severe dengue notified cases in Bello, Medellin and Itagui, 2008-2017: https://doi.org/10.6084/m9.figshare.24086160 Annual dengue case notifications in 57 dengue endemic Colombian cities, 2008-2023: https://doi.org/10.6084/m9.figshare.24086148 .

Funding: This work was supported by grants from the Bill & Melinda Gates Foundation (Grant OPP1159497 to SLO), the Wellcome Trust in partnership with the UK Department for International Development (Grant 102591/Z/13/A to SLO), and the US Agency for International Development (Grant AID-OAA-A-16-00081 to SLO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Dengue is a growing global health challenge, with climate change and urbanisation driving an increase in the population vulnerable to dengue epidemics [ 1 , 2 ]. Latin America has seen the greatest relative increase in dengue disease burden over the past two decades [ 3 ]. The primary vector for dengue, the Aedes aegypti mosquito, also transmits the chikungunya and Zika viruses, both of which have been circulating in Colombia since their first detection in 2014 and 2015 respectively [ 4 , 5 ].

The introduction of the insect bacterium Wolbachia ( w Mel strain) into Ae . aegypti mosquitoes reduces their ability to transmit human pathogens including dengue, Zika, chikungunya, and yellow fever [ 6 – 8 ]. Wolbachia is maternally inherited through successive generations and manipulates insect reproduction to favour its own population dissemination through a process of cytoplasmic incompatibility [ 8 , 9 ]. These characteristics facilitate its application as a public health tool, delivered as short-term releases of w Mel-infected Ae . aegypti into residential areas which drives introgression of w Mel into local Ae . aegypti populations [ 10 ], resulting in a mosquito population that is refractory to local dengue virus transmission. The feasibility, acceptability, effectiveness and durability of the Wolbachia method has been demonstrated in numerous field trials in Asia-Pacific and Latin American countries [ 11 – 19 ]. A cluster randomised efficacy trial in Yogyakarta, Indonesia, demonstrated a 77% reduction in the incidence of virologically-confirmed dengue and an 86% reduction in dengue hospitalisations in Wolbachia -treated versus untreated areas [ 14 ]. These results, together with consistent findings from non-randomised w Mel deployments in multiple countries [ 11 – 13 , 15 – 17 ], led the Vector Control Advisory Group of the World Health Organisation to endorse the evidence for Wolbachia as an effective method of dengue control [ 20 ]. To date, w Mel-infected Ae . aegypti have been deployed in communities in 11 countries, reaching an estimated 11 million people.

In Colombia, pilot Wolbachia deployments were undertaken in several neighbourhoods in the municipality of Bello in 2015–2016. The declaration of Zika as a public health emergency by the WHO in early 2016 [ 21 ] accelerated the planned expansion of pilot releases to city-scale, with the aim of optimising methods for scaled deployment under operational conditions while also evaluating the epidemiological effectiveness against Aedes- borne viruses [ 22 ]. We report here the public health outcomes of city-wide deployments of w Mel-infected Ae . aegypti mosquitoes throughout the adjacent municipalities of Bello, Medellín and Itagüí in the Aburrá Valley in Colombia, which represent the largest contiguous implementation of the Wolbachia method to date. The entomological outcomes of the deployments are reported in a concurrent publication [ 23 ]. The impact of the Wolbachia intervention on dengue incidence was evaluated in two parallel studies. A quasi-experimental study in all three municipalities used interrupted time series analysis to quantify the reduction in the incidence of dengue cases notified to the routine disease surveillance system. In parallel, a prospective clinic-based case-control study was conducted in one quadrant of Medellín to evaluate the impact of the w Mel deployment on the incidence of virologically-confirmed dengue.

Ethics statement

The protocol for the case-control study was approved by the human research ethics committees of Universidad de Antioquia and Monash University, and has been published previously [ 22 ]; ClinicalTrials.gov identifier NCT03631719]. Written informed consent was obtained from all participants, or from their guardian where a participant was under 18 years of age.

Study setting

The Aburrá Valley is located in the Department of Antioquia ( Fig 1 ), in the northwest of Colombia, and is among the most populous areas in Colombia. Medellín, Bello and Itagüí are the main urban centres of the valley with populations in 2021 of 2.53 million, 545 thousand and 270 thousand, respectively (3.3 million combined population in an area of 135 km 2 ) [ 24 ]. The three cities accounted for 91% of the dengue case burden in Antioquia in the ten years 2008–2017 [ 25 ].

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Deployment of w Mel across Medellín, Bello and Itagüí —combining pragmatic staged (Bello and Medellín) and municipality-wide (Itagüí) deployments (dark blue, blue and yellow) with a case-control study with test-negative design in a focused study area of intervention (orange) and untreated (green) areas (produced in QGIS version 3.28.3 using administrative boundaries freely available from the municipal governments of Bello ( https://www.datos.gov.co/Ordenamiento-Territorial/Divisi-n-Pol-tico-Administrativa-Barrios-Bello-Ant/pnhh-ccwd ), Medellín ( https://www.medellin.gov.co/geomedellin/datosAbiertos/1043 ), and Itagüí ( https://www.datos.gov.co/Ordenamiento-Territorial/Localizaci-n-Geogr-fica-de-los-Barrios-del-Municip/didi-drqa )).

https://doi.org/10.1371/journal.pntd.0011713.g001

Summary of wMel implementation

A detailed description of the implementation of the w Mel method within the Aburrá Valley is reported in a concurrent publication [ 23 ]. Briefly, after initial pilot releases in the París comuna of Bello between June 2015 and August 2016, staged city-wide deployments of w Mel-infected Ae . aegypti throughout all 10 comunas of Bello commenced in October 2016 and concluded in April 2019 ( S1 Fig ). Two parallel streams of w Mel deployments were undertaken in Medellín: deployments into the intervention arm of a case-control study in one quadrant of Medellín (4 comunas divided into 3 intervention and 3 control areas; August 2017—May 2019) and staged deployments throughout the rest of Medellín (12 comunas; October 2017—October 2019). Subsequent to the completion of participant enrolment for the case-control study in December 2021 (see below), between January and April 2022 w Mel-infected mosquitoes were released into the remaining three areas that had served as control areas for the duration of the case-control study. w Mel was deployed simultaneously across the whole of Itagüí between August 2019 and December 2020.

w Mel introgression into Ae . aegypti populations throughout release areas was determined through regular monitoring [ 23 ] and is summarised in S1 Fig . Mosquitoes were captured through a network of BG-Sentinel traps (BioGents) deployed throughout release areas or via aspirators operated by study personnel. Mosquito w Mel infection status was determined by screening with polymerase chain reaction (PCR)-based assays as described elsewhere [ 23 ], and w Mel prevalence was calculated as the percentage of Ae . aegypti screened that were w Mel-positive, aggregate by comuna and month. The COVID-19 pandemic and associated restrictions disrupted both w Mel monitoring and deployment between March 2020 and March 2021, resulting in incomplete monitoring data for most release areas as well as delays in w Mel deployment into some areas.

Quasi experimental study

Routine disease surveillance data..

The monthly count of notified dengue cases by comuna was obtained from the Health Secretariats of Medellín and Bello, and municipality-level data for all Colombian cities was obtained from the National Health Institute’s National Public Health Surveillance System ( Instituto Nacional de Salud [INS] SIVIGILA [ 25 ]). Comuna-level data was available from 2009 for Medellín and 2010 for Bello, up to June 2023. Municipality-level data was available from 2008 for all Colombian cities, up to 27 May 2023 (epidemiological week 21). The INS weekly epidemiological bulletins in published in 2023 refer to there being 57 dengue-endemic cities in Colombia with population greater than 100,000 inhabitants [ 26 ]. A list of these 57 cities (including Medellín, Bello and Itagüí) was compiled ( S1 Table ) based on information from the INS weekly epidemiological bulletins [ 26 ], census population data [ 24 ], and municipality-level dengue notifications data [ 25 ].

Nationally, the case definition used for dengue surveillance and reporting includes all laboratory-confirmed dengue cases and any clinically suspected dengue cases who were not laboratory-tested [ 27 ]. Suspected dengue cases who test negative for dengue IgM antibody are excluded from national reporting, although these cases are retained in the datasets maintained by the Medellín and Bello Health Secretariats. For consistency with the national datasets, we employed the same case definition and excluded test-negative suspected dengue cases from the comuna-level datasets used in interrupted time series (ITS) analysis for determining the impact of w Mel deployments on the incidence of notified dengue. For the ITS analysis we collated dengue case counts aggregated by month and by patients’ comuna of residence (Medellín and Bello) or municipality of residence (Itagüí) to align with the geographical areas used for w Mel releases and monitoring. For descriptive analyses of the baseline dengue epidemiology prior to large-scale Wolbachia deployments, we used the INS SIVIGILA datasets for all three cities.

Data on notified cases of Zika and chikungunya were also available from the Health Secretariats of Medellín and Bello from 2014–2022, and were analysed as a secondary endpoint.

Population data stratified by age and sex for each municipality was obtained from the Colombian National Administrative Department of Statistics 2018 census [ 24 ], and population data by comuna for Bello and Medellín was obtained from the municipal governments.

Statistical analysis.

Per capital annual dengue incidence (notified cases per 100,000 inhabitants) was calculated for each of the 57 dengue-endemic cities in Colombia, 2008 to 2023. For 2023, data was only available to epidemiological week 21 (27 May 2023), so the calculation of 2023 per capita incidence was annualised by adjusting by a factor of 52/21 = 2.476. For each year 2008–2023, the median and interquartile range of dengue incidence among the 57 dengue-endemic cities was calculated.

The analysis of the public health outcomes of w Mel deployments included dengue notification data from January 2008 (Itagüí), January 2009 (Medellín) or January 2010 (Bello) until June 2023. The comuna of París in Bello was excluded from analysis because the pilot releases in 2015 introduced w Mel in only one neighbourhood of the comuna, whereas the dengue data was reported for the whole of París comuna and cannot be disaggregated by neighbourhood to consider the pilot release areas separately from the rest of the comuna.

w Mel prevalence in months where no w Mel monitoring was conducted was interpolated using a regression line between the last w Mel monitoring event and the next. The final measured w Mel prevalence was carried forward through subsequent months until June 2023. The w Mel exposure status of each comuna in each month was defined as untreated (prior to w Mel releases), partially treated ( w Mel releases ongoing or completed but not stably at ≥60% w Mel), or fully treated (releases completed and stably at ≥60% w Mel). The ‘interruption’ in the ITS analysis is thus represented by the time point at which an individual comuna becomes partially- or fully-treated. An alternative w Mel exposure definition used categorised levels of comuna-level (city-level for Itagüí) monthly w Mel prevalence as a predictor of dengue incidence (<20%; 20% to <40%; 40% to <60%; ≥60%). Here, comunas can move across w Mel levels from month to month and thus each w Mel category level may represent a different set of comunas each month.

In Bello and Medellín where data was disaggregated by comuna, the ITS analysis was implemented using mixed-effect negative binomial regression to model the monthly count of dengue case notifications in each comuna as a function of w Mel treatment status (fully, partially or untreated in the primary analysis, and by level of w Mel prevalence in the secondary analysis), with an offset for population size, calendar month as a fixed-effect covariate, and comuna modelled as a random effect. In Itagüí where data was aggregated at the level of the municipality, a fixed effect negative binomial model was used with an offset for population size and calendar month as a covariate. The models estimate the w Mel intervention effect as the dengue incidence rate ratio (IRR) in fully- or partially-treated vs untreated periods (or by stratum of w Mel prevalence compared with the lowest stratum), adjusted for seasonality. Robust standard errors were used in all analyses by specifying the vce(cluster comuna ) option in Stata to account for non-independence of observations within comunas [ 27 ].

Case control study

Study design..

This clinic-based prospective case-control study used a test-negative design and was designed to measure experimentally the degree to which dengue incidence was reduced in three neighbourhoods where w Mel Wolbachia had been released (population 323,000 in 6.9 km 2 ), compared to three adjacent untreated neighbourhoods (population 331,000 in 8.3 km 2 ) ( Table 1 ). Randomised allocation was not feasible due to the small number of clusters, which was driven by the imperative at the time of project conception to prioritise rapid phased deployment throughout the rest of Medellín and Bello, to address an urgent need for novel scalable strategies to address the threat of Zika [ 22 ]. Instead, the allocation of the six areas into two arms was done in a way that maximised balance between the arms with respect to measured factors that may be associated with baseline dengue risk.

https://doi.org/10.1371/journal.pntd.0011713.t001

Participant recruitment and data collection.

To measure the epidemiological endpoint, participants were recruited from a network of 11 clinics across the study area. Febrile patients were invited to participate in the study if they met the following inclusion criteria: fever with a date of onset between 1–4 days prior to the day of presentation to the health care facility; aged ≥3 years old; and lived in the case-control study area for the 10 days preceding illness onset. Participants were not eligible if localizing features suggestive of a specific diagnosis were identified or if they had been enrolled in the previous four weeks. After obtaining written informed consent, basic demographic details, eligibility against the inclusion criteria, illness onset date, and a retrospective travel history encompassing days 3–10 prior to illness onset were recorded in a standardised electronic data collection form. A single 6 ml venous blood sample was collected from all consenting participants on the day of enrolment. Only participants enrolled from the 16th of May 2019, following completion of w Mel releases in the intervention area, were included in the analysis dataset. Enrolment was paused between 1 April 2020 and 31 January 2021 due to COVID-19 pandemic restrictions, and concluded on the 31st of December 2021.

Case and control classification.

The ZDC Multiplex RT-qPCR (Bio-Rad) was used to detect DENV, CHIK and Zika viruses in plasma samples from all enrolled participants. Samples were tested for the presence of dengue NS1 antigen by ELISA (Panbio Dengue Early ELISA [n = 720 samples] or BioRad Platelia [n = 92 samples]), according to manufacturers’ instructions. Samples which tested negative by DENV/CHIK/Zika RT-qPCR and DENV NS1 were tested for dengue IgM antibodies by IgM capture ELISA (Panbio [n = 763 samples] or InBios [n = 46 samples]). Samples positive for DENV in the triplex RT-qPCR were tested in a serotype-specific RT-qPCR to determine the infecting serotype, as described previously [ 28 ].

Virologically-confirmed dengue (VCD) cases (primary endpoint) were defined as participants with a positive result in DENV RT-qPCR or NS1 ELISA ( S2 Fig ). Presumptive dengue cases (secondary endpoint) were defined as participants who tested negative for dengue by DENV RT-qPCR and NS1 ELISA but who had a positive DENV IgM test. Controls were defined as participants meeting the clinical criteria for enrolment, but with negative test results for DENV RT-qPCR, DENV NS1 ELISA, DENV IgM ELISA, CHIK RT-qPCR, and Zika RT-qPCR. Equivocal test results were re-tested and participants with two equivocal test results were excluded from analysis.

Sample size calculation.

Initial sample size estimates calculated that 88 test-positive cases plus four times as many controls would be sufficient to detect a 50% reduction in dengue incidence with 80% power, based on standard formulae for calculating sample size/power in a case control study ( http://www.openepi.com/SampleSize/SSCC.htm ). This aligned with the proposed approach to estimating the intervention effect, which compares the exposure odds among test-positive cases versus test-negative controls, with the null hypothesis that the odds of residence in the Wolbachia intervention arm is the same among test-positive cases as test-negative controls, and did not account for clustering of participants in the 3 treated and 3 untreated zones. A re-evaluation of sample size requirements was conducted in April 2021 following 14 months of participant enrolment (May 2019 –March 2020 and February–April 2021), to consider the minimum effect size that would be detectable for a smaller sample size than originally estimated, given the 77% efficacy that had since been reported from a cluster randomised trial of w Mel in Yogyakarta [ 14 ] and the low incidence of dengue in Medellín since case-control enrolment began. This found that 42 test-positive dengue cases (and four times as many controls) would be sufficient to detect a 65% reduction in dengue. Clustering was not explicitly accounted for in the sample size calculation, rather an inflation factor of 1.5 was applied, to give a revised target sample size of 63 virologically-confirmed dengue cases and at least 252 test-negative controls.

The statistical analysis plan was published [ 29 ] and is available in the supporting information ( S1 Appendix ). The dataset for analysis included all enrolled virologically-confirmed dengue cases and presumptive dengue cases, and all test-negative controls that were matched to a case by calendar quarter of enrolment ( S3 Fig ). The intention-to-treat analysis considered w Mel exposure as a binary classification based on residence in the intervention or untreated area. The intervention effect was estimated from an aggregate odds ratio (OR) comparing the exposure odds (residence in the intervention area) among test-positive cases versus test-negative controls (for data aggregated across all three release areas within each study arm) with cluster-robust variance estimates. Area-level averages for age and sex were included as covariates in the model. The OR provides an unbiased estimate of the relative risk of dengue in Wolbachia -treated versus untreated areas, providing that the key assumptions underlying a test-negative design are upheld, namely that test-negative controls are allowed to include participants who may test positive for dengue at any other time during the study period, and the distribution of non-dengue febrile illness is not associated with the intervention status [ 30 , 31 ]. Efficacy of the intervention was calculated as 100*(1-aggregate OR).

The per-protocol analysis considered w Mel exposure as a quantitative index based on measured w Mel prevalence in local Ae . aegypti mosquitoes in the participant’s area of residence, and in locations visited by the participant during the period 3–10 days prior to illness onset. A weighted Wolbachia exposure index (WEI) was defined for each participant, as follows: the aggregate w Mel prevalence for each of the six areas was calculated each month as the number of w Mel-positive Ae . aegypti mosquitoes in that area divided by the total number of Ae . aegypti that were screened for w Mel in that area. The WEI for each participant was then calculated by multiplying the area-level w Mel prevalence (in the calendar month of participant enrolment) at each of the locations visited, by the proportion of time spent at each location, and summing across locations to give a value on a continuous scale from 0 to 1. An additional per-protocol analysis was conducted in which the WEI was calculated using only the area-level w Mel prevalence in the participant’s area of residence (in the calendar month of participant enrolment), ignoring the participant’s recent travel history. Cases and controls were classified by strata of their WEI: 0–0.2; 0.2–0.4; 0.4–0.6; 0.6–0.8; and 0.8–1. A mixed-effects logistic regression was used to model the relationship between WEI and dengue incidence, with age and sex as covariates, and area as a random intercept term. The WEI strata were modelled as an unordered covariate to calculate stratum-specific ORs (relative to the baseline 0–0.2 stratum). Efficacy was calculated as 100*(1-OR).

Baseline dengue epidemiology

In the ten years from January 2008 to December 2017 prior to city-wide w Mel releases in Bello, Medellín, and Itagüí, there were 60,896 dengue cases notified to the national surveillance system from the three cities (Bello: 5569 cases corresponding to 378 cases per 1000 population; Medellín: 47,212 cases, 20 per 1000; Itagüí: 8115 cases, 777 per 1000). Forty-five percent (n = 27,166) were classified as either laboratory-confirmed or epidemiologically-linked cases, and the remainder as probable cases. Less than one percent of notified cases were classified as severe dengue (n = 539) and 21.3% of cases (n = 12,959) were hospitalised. The median age of dengue cases was 28 years (interquartile range 16–44 years), and was similar among cities, between severe and non-severe cases and between hospitalised and non-hospitalised cases. Overall, 51.4% of dengue cases were female but after accounting for the demographic structure of the underlying population, dengue incidence was slightly higher in males (20.1 cases per 1000 males during 10 years vs 19.0 cases per 1000 females). S4 Fig shows the age and sex distribution of dengue cases notified from the three cities between 2008 and 2017.

Public health outcomes of wMel deployments: Quasi experimental study

Among 10 comunas in Bello, w Mel prevalence was stably ≥60% (i.e. fully treated) immediately following the end of releases in two comunas, and within 4–21 months (median 9.5 months) post-release in the remaining eight. Of the 18 release areas in Medellín, w Mel prevalence was stably ≥60% within 2 months of the end of releases in five areas, and within 4–17 months (median 8 months) in another five areas. Eight release areas remained partially treated (not stably at ≥60% w Mel) at the time of the last entomological monitoring between July 2021 and January 2022, 21–34 months after the end of releases. In Itagüí, w Mel prevalence was stably ≥60% (i.e. fully-treated) immediately following the end of releases.

The incidence of notified dengue in Bello, Medellín and Itagüí before, during and after w Mel deployments is shown in Fig 2 . Stable introgression of w Mel into local Ae . aegypti populations was associated with a significant reduction in dengue incidence in each municipality. In Bello, there were 110 dengue cases (6.4 per 100,000 person-years) notified in the fully treated period and 215 cases (19.9 per 100,000 person-years) notified in the partially treated period, compared to 4,109 cases (144.7 per 100,000 person-years) in the untreated period ( Fig 3 ). In the interrupted time series analysis, this was equivalent to a 95% reduction in dengue incidence (incidence rate ratio 0.047 [95% CI 0.037, 0.060]) in the fully treated period and an 85% reduction (IRR 0.147 [95% CI 0.095, 0.227]) in the partially treated period compared to the untreated period. In Medellín, there were 309 cases (9.0 per 100,000 person-years) notified in the fully treated period and 1,918 (25.2 per 100,000 person-years) in the partially treated period, compared to 43,130 cases (180.5 per 100,000 person-years) in the untreated period ( Fig 3 ), equating to a 95% (IRR 0.051 [95% CI 0.038, 0.069]) and an 85% (IRR 0.152 [95% CI 0.123, 0.189]) reduction in dengue, respectively. In Itagüí, there were 47 cases (7.0 per 100,000 person-years), 55 cases (15.7 per 100,000 person-years) and 8,199 cases (299.8 per 100,000 person-years) notified in the fully treated, partially treated and untreated periods respectively, equating to a 97% (IRR 0.032 [95% CI 0.020, 0.053]) and a 93% (IRR 0.070 [95% CI 0.045, 0.110]) reduction in dengue.

Dark blue line is the monthly incidence of dengue case notifications per 100,000 population (left-hand Y axis; note different scale among graphs) from January 2008 (Itagüí)/2009 (Medellín)/2010 (Bello) to June 2023. Light blue shading indicates the w Mel area coverage (km 2 ) in partially w Mel-treated areas ( w Mel releases were ongoing or completed but not stably at ≥60% w Mel), and darker blue shading indicates the w Mel area coverage (km 2 ) in fully w Mel-treated areas (releases were completed and stably at ≥60% w Mel) (right-hand Y axis).

https://doi.org/10.1371/journal.pntd.0011713.g002

Point estimates (symbols) and 95% confidence intervals (horizontal bars) from interrupted time series analysis of monthly dengue case notifications to the routine surveillance system. Efficacy is expressed as 100x(1-IRR). w Mel exposure was defined as ‘untreated’ prior to w Mel releases, ‘partially treated’ if w Mel releases were ongoing or completed but not stably at ≥60% w Mel, and ‘fully treated’ if releases were completed and stably at ≥60% w Mel.

https://doi.org/10.1371/journal.pntd.0011713.g003

Significant reductions in dengue incidence were observed for all category levels of w Mel prevalence, with no apparent dose response relationship ( Fig 4 ). In Itagüí, reductions in dengue of similar magnitudes ranging from 93–98% were seen across all w Mel levels compared to the lowest category level. In Bello and Medellín, dengue incidence was 95% lower, when comparing the highest w Mel level with the lowest, and significant reductions in dengue incidence (range 85–90%) were also observed at intermediate levels of w Mel.

Point estimates (symbols) and 95% confidence intervals (horizontal bars) from interrupted time series analysis of monthly dengue case notifications to the routine surveillance system. Efficacy is expressed as 100x(1-IRR).

https://doi.org/10.1371/journal.pntd.0011713.g004

In Bello, there were 106 chikungunya cases notified between 2014–2019, only one of which (in December 2019) was resident in a fully-treated area ( w Mel prevalence ≥60%), and 52 Zika cases notified between 2015–2017, all from untreated areas. There have been no chikungunya or Zika cases notified from Bello since December 2019 and June 2017, respectively. In Medellín there were 794 chikungunya cases notified between 2014–2020 and 494 Zika cases notified between 2015–2022, none of which were resident in a fully-treated area.

Notified dengue incidence in wMel-treated versus untreated Colombian cities, pre- and post-intervention

Among the 57 cities in Colombia with population >100,000 inhabitants and classified as dengue-endemic ( S1 Table ), there was substantial heterogeneity in per capita dengue incidence both within and between years, 2008–2023 ( Fig 5 ). All three w Mel-treated cities in the Aburrá Valley have ranked among the 10 lowest-incidence cities every year since 2019, whereas this was not the case in any year 2010–2017, prior to Wolbachia deployments.

Coloured filled circles indicate annual dengue incidence in the three cities in the Aburrá Valley where staged roll-out of the w Mel intervention was implemented between 2017 and 2021: Bello (orange), Medellín (blue) and Itagüí (green). Open grey circles represent annual dengue incidence in the remaining 54 cities (note the log10 scale on the Y-axis). The horizontal lines indicate the median and interquartile range of dengue incidence among the 57 cities in each year. The vertical dashed line roughly demarcates the pre-intervention and post-intervention periods.

https://doi.org/10.1371/journal.pntd.0011713.g005

Public health outcomes of wMel deployments: Case-control study

Participant characteristics..

Among 25,304 febrile patients presenting to the participating 11 primary care clinics between 10 November 2017 and 31 December 2021 and screened for eligibility, 1,665 were found to be eligible and consented to be enrolled. A total of 725 participants (351 from the intervention area and 374 from the untreated area) met the criteria for inclusion in the analysis dataset ( S3 Fig ). The most common reason for exclusion from analysis was enrolment prior to w Mel establishment (88% of excluded participants). Within the analysis dataset, the median (interquartile range) age was 23.8 years (9.2–35.9 years) among participants from the intervention area and 24.5 years (11.0–39.2 years) among participants from the untreated area, and 49% and 53% of participants were female, respectively.

Twenty-three participants were classified as VCD on the basis of either a positive DENV PCR result (n = 20) or a positive NS1 antigen result with negative PCR (n = 3). The infecting serotype was DENV1 in 17 participants, DENV3 in two participants, and could not be determined for the remaining four VCDs. No DENV2 or DENV4 cases were detected in this study, nor any Zika or chikungunya cases. An additional 15 participants were negative in DENV PCR and NS1 antigen testing but were DENV IgM-positive and were classified as presumptive dengue cases, giving a total of 38 participants meeting the secondary endpoint of virologically-confirmed or presumptive dengue (i.e. any dengue).

Dengue incidence in the intervention vs untreated area (intention-to-treat analysis).

The incidence of VCDs was 45% lower in the intervention area compared to the untreated area, though this was not statistically significant (8/23 VCDs and 338/687 test-negatives resident in intervention and 15/23 VCDs and 349/687 test-negatives in untreated areas; OR 0.55 [95% CI 0.25, 1.17]) ( Fig 6 ). A similar but statistically significant reduction was observed for the endpoint of any dengue, including both VCDs and presumptive dengue cases (13/38 dengue cases in intervention and 25/38 in untreated areas; OR 0.53 [95% CI 0.30, 0.93]). Serotype-specific analysis could only be performed for DENV1 cases, which showed a non-significant 57% reduction in DENV1 cases in the intervention compared to the untreated area (OR 0.43 [95% CI 0.09, 2.04]).

Shown is the protective efficacy (expressed as 100×(1−OR)) of w Mel-infected Aedes aegypti deployments against virologically-confirmed dengue of any serotype (VCD), serotype 1 dengue (DENV1), and VCD and presumptive dengue (any dengue).

https://doi.org/10.1371/journal.pntd.0011713.g006

Dengue incidence across Wolbachia Exposure Index strata (per-protocol analysis).

After tabulation of VCDs by WEI stratum, the decision was made to combine the top 2 strata to boost sample size. The incidence of VCD was 73% lower (95% CI 4%, 92%) among participants in the top WEI stratum (≥0.6) compared to the lowest stratum (0–0.2) for WEI calculated based on duration-weighted w Mel frequencies in the cluster of residence only, and 10% and 51% lower among those with WEI 0.4–0.6 and 0.2–0.4, though these differences were not statistically significant ( Fig 7A ). Similar results were found for WEI calculated based on duration-weighted w Mel frequencies in the cluster of residence and other visited locations, though none of these estimates reached statistical significance. There was no indication of a threshold effect nor a dose response relationship with increasing WEI, though we acknowledge that the number of cases in each WEI stratum above 0.2 was very small (range 1–4). The incidence of any dengue was 69% (95% CI 16, 88%) and 67% (95% CI 19, 87%) lower in participants with WEI ≥0.6 compared to the lowest stratum, for WEI calculated based on residence and visited locations and WEI calculated based on residence alone, respectively ( Fig 7B ).

Markers show stratum-specific efficacy (and 95% confidence intervals) against dengue by categorised level of Wolbachia exposure index, with WEI based on duration-weighted w Mel frequencies in the cluster of residence and other visited locations (open circles) or w Mel frequency in cluster of residence only (closed circles).

https://doi.org/10.1371/journal.pntd.0011713.g007

Since the large-scale roll-out of Wolbachia -infected Ae . aegypti mosquitoes across a continuous population of 3.3 million people in Bello, Medellín and Itagüí, Colombia, the incidence of notified dengue cases has been 95–97% lower than during the decade prior to Wolbachia introduction, after adjustment for seasonal trends. A causal association between the Wolbachia deployments and reduced dengue incidence is supported by the results of a parallel prospective case-control study, which found that the incidence of virologically-confirmed and presumptive dengue cases detected prospectively in outpatient clinics was significantly lower in three Wolbachia -treated neighbourhoods in northeast Medellín than in three well-matched untreated neighbourhoods. The infecting serotype was DENV1 in three-quarters of cases detected in the case-control study.

The reduction in notified dengue incidence following w Mel Wolbachia establishment in Bello, Medellín and Itagüí is consistent with results from Australia [ 11 , 12 ], Indonesia [ 13 , 14 ], and Brazil [ 17 , 19 ], and with model predictions of a collapse in dengue virus transmission following introduction of Wolbachia into local Ae . aegypti populations [ 32 , 33 ]. The Aburrá Valley, where Bello, Medellín and Itagüí are located, experienced large dengue epidemics in 2010 and 2016, but has seen very little dengue since. The end of the 2016/2017 epidemic coincided with the start of the phased w Mel deployments across Medellín and most of Bello, and it is plausible that the post-intervention period has coincided with a natural trough in dengue incidence. However, the average annual incidence since city-wide w Mel releases commenced in 2017 has been lower than any other period previously recorded, and the sustained suppression of dengue through seven typical transmission seasons from January 2017 to June 2023 gives increasing confidence in a Wolbachia -mediated effect.

Due to the infrequency of dengue epidemics in the Aburrá Valley in the baseline period, and because secular trends and other parallel control measures can confound the demonstration of a Wolbachia intervention effect in a before-and-after analysis, we designed a priori a prospective case-control study to evaluate experimentally the public health impact of Wolbachia releases in one area of Medellín, alongside the time series analysis of routine disease surveillance data for all three cities [ 22 ]. Randomised allocation of the Wolbachia intervention was not feasible, given the small number of neighbourhoods within the case-control area and the necessarily pragmatic approach to deployments in Medellín in order to achieve large-scale coverage within a short time frame. However, the allocation of the six neighbourhoods into two arms of the case-control study was done in a way to maximise balance between the arm with respect to measured factors that may be associated with baseline dengue risk, in order to minimise risk of bias in the estimation of the Wolbachia intervention effect. Although the sustained suppression of dengue throughout Medellín, Bello and Itagüí since enrolment began in 2019 meant that the case-control study was unable to reach its target sample size of 63 enrolled dengue cases in 2.5 years, two-thirds of the 23 virologically-confirmed dengue cases (and an additional 15 presumptive dengue cases) enrolled were resident in the untreated neighbourhoods. The results showed a 47% reduction in the incidence of any dengue (virologically-confirmed or presumptive) among residents of Wolbachia -treated neighbourhoods compared to untreated neighbourhoods. Human movement between neighbourhoods, incomplete Wolbachia establishment in treated areas, and Wolbachia contamination into untreated areas are likely to have limited the measurement of Wolbachia ’s true effectiveness in the primary analysis of the case-control study. A per-protocol analysis which attempted to account for this exposure misclassification using the measured Wolbachia prevalence in the cluster of residence and at other locations visited during the week prior to illness demonstrated a 69% reduction in dengue incidence among participants with highest Wolbachia coverage levels, compared to those with the lowest.

The rate of Wolbachia establishment in local Ae . aegypti populations was variable among the Aburrá Valley release areas, with stable w Mel prevalence above 60% achieved immediately post-release in Itagüí; in a median of 8 months (range 1–21 months) post-release in Bello; and in a median of 16 months in Medellín (minimum 1 month, with stable establishment not yet verified in 7/18 areas at last monitoring). Sustained suppression of dengue incidence was observed throughout all three cities in the period since Wolbachia releases despite this heterogeneity, consistent with the previously published results of field trials in the Brazilian cities of Niteroi and Rio, which showed significant reductions in dengue and chikungunya incidence even with intermediate w Mel levels [ 17 , 19 ]. The complexities in achieving homogeneous Wolbachia introgression in these large complex urban environments are likely to be common to many other tropical urban centres—including challenges in accessing high-rise areas, gated communities and informal or insecure settlements, diversity in Ae . aegypti ecology across the city, and spatial heterogeneity in mosquito abundance. Long-term entomological and public health monitoring in the Aburrá Valley will be valuable for ascertaining the degree to which Wolbachia prevalence continues to increase without further intervention, as well as for demonstrating the durability of Wolbachia once established in the mosquito population and the long-term impact of Wolbachia on Aedes-borne disease incidence. Recent evidence from the earliest w Mel Wolbachia release sites in northern Queensland, Australia, shows Wolbachia self-sustaining at a high prevalence 7–10 years post-release with few genomic changes [ 34 , 35 ], and local dengue transmission has been effectively eliminated following area-wide releases [ 11 , 12 ]. In the earliest Colombian release area in París, Bello, w Mel prevalence was >90% when last monitored in September 2021, more than five years after the completion of releases [ 23 ], supportive of an expectation that the area-wide establishment of Wolbachia in Ae . aegypti mosquito populations throughout the Aburrá Valley will be effective for many years in controlling dengue and other Aedes-borne diseases.

A limitation of relying on routine disease surveillance data to evaluate the long-term public health impact of Wolbachia is that case notification in Colombia is based on a clinical diagnosis of dengue, without requiring confirmatory laboratory tests such as NS1 antigen detection or nucleic acid tests for viral RNA. This means that a subset of notified cases will be febrile illness of another aetiology, and these ‘false positive’ dengue cases will continue to be reported even in the absence of true local transmission. Furthermore, the case notification data does not reliably distinguish between autochthonous dengue cases and ‘imported’ cases with a recent travel history outside of the city of residence, so increased dengue activity elsewhere in Colombia could plausibly lead to an uptick in cases resident in the Aburrá Valley release areas but who acquired their infection elsewhere. Nonetheless, routine surveillance data provides a readily available and pragmatic signal for monitoring the long-term effectiveness of Wolbachia .

Around half of Colombia’s population live in dengue endemic areas, and the frequency and magnitude of dengue outbreaks is increasing: in six of the ten years 2010–2019, the annual reported dengue cases in Colombia exceeded any of the previous thirty years [ 36 ]. Central Latin American countries—including Colombia—are among the regions predicted to see the largest increases in populations affected by increased temperature suitability for dengue transmission in a warming climate [ 2 ]. Conventional approaches to dengue control based on chemical control of adult and immature mosquitoes and environmental management to reduce breeding sites, together with effective clinical management, are the mainstay of dengue control programs in endemic countries but have been unable to curtail the spread of dengue. The urgent need for coordinated and sustainable strategies for dengue control is reflected in the World Health Organization’s launch in March 2022 of the Global Arbovirus Initiative [ 37 ], one pillar of which is focused on the scale-up and integration of innovative evidence-based interventions for Aedes-borne disease control. In September 2020, Wolbachia implementation began in the city of Cali in southwest Colombia, with phased deployments reaching more than 700,000 Cali residents by September 2022 [ 38 ]. A recent economic analysis indicates that the initial financial investment required to implement Wolbachia in high-burden Colombian cities would generate sustained savings in the long term from the significant reduction in dengue cases and consequent offsets in healthcare and vector control costs [ 39 ], consistent with previous findings from Indonesia that Wolbachia releases would be highly cost-effective—and even cost-saving—in high-density urban areas [ 40 ]. The results presented here demonstrate the feasibility, acceptability and real-world effectiveness of implementing Wolbachia across large urban populations and, alongside previously published results, support the reproducibility of this effectiveness across different ecological settings.

Supporting information

S1 table. list of 57 dengue-endemic municipalities in colombia with population >100,000 inhabitants, based on information from colombia national institute of health (ins) weekly epidemiological bulletins, in 2023 [ 26 ], municipality-level dengue notifications data [ 25 ], and national population data [ 24 ]..

https://doi.org/10.1371/journal.pntd.0011713.s001

S1 Fig. w Mel introgression by commune in Bello and Medellín, and in Itagüí, July 2016 –July 2022.

Points indicate the wM el infection prevalence in local Aedes aegypti mosquito populations categorised into levels. Light blue shading indicates the period during which the area is considered ‘partially treated’, commencing from the beginning of w Mel releases. Dark blue shading indicates the period during which the area is considered ‘fully treated’, defined as w Mel releases completed and w Mel prevalence stably at ≥60%. Absence of shading indicates no w Mel releases in that area. Itagüí was not disaggregated by commune as w Mel was released simultaneously across the whole city.

https://doi.org/10.1371/journal.pntd.0011713.s002

S2 Fig. Flowchart of data and sample collection procedures and diagnostic algorithm.

Blue boxes indicate participant recruitment and enrolment activities undertaken at health clinics, including screening against inclusion/exclusion criteria, obtaining written informed consent, and collection of demographic and travel history data and a blood sample. Yellow boxes indicate the laboratory diagnostic testing performed at the project laboratory, the results of which (white boxes) will be used to classify participants as virologically confirmed dengue, presumptive dengue, Zika or chikungunya cases, or arbovirus-negative controls (grey boxes) according to the algorithm shown.

https://doi.org/10.1371/journal.pntd.0011713.s003

S3 Fig. Participant enrolment and inclusion in the analysis dataset.

The commonest reasons for exclusion from the analysis dataset were enrolment before the predefined time point of w Mel establishment (16 May 2019) and enrolment in a calendar quarter without any VCD and presumptive dengue cases (‘unmatched controls’; July—September 2021).

https://doi.org/10.1371/journal.pntd.0011713.s004

S4 Fig. Dengue cases notified to the Colombian National Public Health Surveillance System (SIVIGILA) from Bello, Medellin and Itagui between January 2008 and December 2017, by age and sex.

Bars show dengue case numbers and lines show per capita incidence in each five-year age band for males (blue) and females (green), aggregated across the three cities and ten years 2008–2017. Data sources: dengue case data (Instituto Nacional de Salud: http://portalsivigila.ins.gov.co/ ); age- and sex-specifc population by municipality from 2018 census (DANE: https://sitios.dane.gov.co/cnpv/ ).

https://doi.org/10.1371/journal.pntd.0011713.s005

S1 Appendix. Statistical analysis plan.

Published 31 March 2022 at https://www.clinicaltrials.gov/study/NCT03631719 .

https://doi.org/10.1371/journal.pntd.0011713.s006

S1 STROBE Checklist. STROBE checklist.

Completed checklist of items that should be included in reports of case-control studies.

https://doi.org/10.1371/journal.pntd.0011713.s007

Acknowledgments

We are grateful to all the World Mosquito Program staff in Colombia and in WMP Global who contributed to this work. We acknowledge the collaboration of the Health Secretariats of Medellín, Bello and Itagüí in supporting this work.

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• Study Protocol
• Open access
• Published: 21 November 2023

Neurophysiological explorations across the spectrum of psychosis, autism, and depression, during wakefulness and sleep: protocol of a prospective case–control transdiagnostic multimodal study (DEMETER)

• Valeria Lucarini 1 , 2 ,
• Anaëlle Alouit 3 ,
• Delphine Yeh 4 ,
• Jeanne Le Coq 2 ,
• Romane Savatte 2 ,
• Mylène Charre 2 ,
• Cécile Louveau 2 ,
• Meryem Benlaifa Houamri 2 ,
• Sylvain Penaud 4 ,
• Alexandre Gaston-Bellegarde 4 ,
• Stéphane Rio 5 ,
• Laurent Drouet 5 ,
• Maxime Elbaz 5 ,
• Jean Becchio 6 ,
• Sylvain Pourchet 6 ,
• Estelle Pruvost-Robieux 3 , 7 ,
• Angela Marchi 8 ,
• Mylène Moyal 1 , 2 ,
• Aline Lefebvre 9 ,
• Boris Chaumette 1 , 2 ,
• Martine Grice 10 ,
• Påvel G. Lindberg 3 ,
• Lucile Dupin 11 ,
• Pascale Piolino 4 ,
• Cédric Lemogne 12 ,
• Damien Léger 5 , 13 ,
• Martine Gavaret 3 , 7 ,
• Marie-Odile Krebs 1 , 2 &
• Anton Iftimovici 1 , 2  

BMC Psychiatry volume  23 , Article number:  860 ( 2023 ) Cite this article

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Quantitative electroencephalography (EEG) analysis offers the opportunity to study high-level cognitive processes across psychiatric disorders. In particular, EEG microstates translate the temporal dynamics of neuronal networks throughout the brain. Their alteration may reflect transdiagnostic anomalies in neurophysiological functions that are impaired in mood, psychosis, and autism spectrum disorders, such as sensorimotor integration, speech, sleep, and sense of self. The main questions this study aims to answer are as follows: 1) Are EEG microstate anomalies associated with clinical and functional prognosis, both in resting conditions and during sleep, across psychiatric disorders? 2) Are EEG microstate anomalies associated with differences in sensorimotor integration, speech, sense of self, and sleep? 3) Can the dynamic of EEG microstates be modulated by a non-drug intervention such as light hypnosis?

This prospective cohort will include a population of adolescents and young adults, aged 15 to 30 years old, with ultra-high-risk of psychosis (UHR), first-episode psychosis (FEP), schizophrenia (SCZ), autism spectrum disorder (ASD), and major depressive disorder (MDD), as well as healthy controls (CTRL) ( N  = 21 × 6), who will be assessed at baseline and after one year of follow-up. Participants will undergo deep phenotyping based on psychopathology, neuropsychological assessments, 64-channel EEG recordings, and biological sampling at the two timepoints. At baseline, the EEG recording will also be coupled to a sensorimotor task and a recording of the characteristics of their speech (prosody and turn-taking), a one-night polysomnography, a self-reference effect task in virtual reality (only in UHR, FEP, and CTRL). An interventional ancillary study will involve only healthy controls, in order to assess whether light hypnosis can modify the EEG microstate architecture in a direction opposite to what is seen in disease.

This transdiagnostic longitudinal case–control study will provide a multimodal neurophysiological assessment of clinical dimensions (sensorimotor integration, speech, sleep, and sense of self) that are disrupted across mood, psychosis, and autism spectrum disorders. It will further test the relevance of EEG microstates as dimensional functional biomarkers.

Trial registration

ClinicalTrials.gov Identifier NCT06045897.

In light of the genetic and neuroanatomical continuum among psychiatric illnesses [ 1 , 2 ], transdiagnostic neurophysiological approaches have demonstrated shared neurofunctional abnormalities between schizophrenia, mood, and autism spectrum disorders [ 3 , 4 ]. High-level cognitive processes have long been shown to rely on synchronized neuronal oscillations, resulting from a balance between excitatory and inhibitory populations of neurons, which can be directly measured by electroencephalography (EEG). This balance is maintained by a network of GABAergic interneurons that regulate the activity of superficial pyramidal cells [ 5 ]. The topographically precise inhibitory activity of interneurons also allows for spatial sensory coding, which is key to a variety of memory processes [ 6 ]. Disruption in these systems may therefore explain a range of cognitive symptoms seen across the spectrum of psychiatric disorders. Moreover, the timing of the disruption may explain the neurodevelopmental continuum between autism spectrum on the one hand, and psychotic and mood disorders on the other. For instance, glutamatergic NMDA receptors, which regulate interneuron activity, can be affected by genetic mutations disrupting the function of subunits expressed either in early development or later on, leading respectively to neurodevelopmental phenotypes, such as ASD, or to schizophrenia spectrum-disorders [ 7 ]. In addition, interneurons are crucial to prefrontal maturation during adolescence and early adulthood [ 8 ], a timeframe when most psychiatric disorders occur [ 9 ]. EEG quantitative approaches therefore appear as accessible and promising tools to investigate the pathophysiology of psychiatric disorders, from autism to schizophrenia spectrum disorders.

Beyond frequential or oscillatory activities, EEG analyses now allow to study the temporal dynamics of neuronal networks throughout the brain [ 10 ]. At rest, brain activities alternate very rapidly, every 80 ms or so, between states of unstable equilibrium, called microstates, and characterized by a particular polarization of the entire cerebral electrical potential field. EEG coupled with functional MRI has suggested that these microstates may correspond to particular modes of spatial organization of information processing [ 11 ]. For instance, microstate classes have been associated with various functioning profiles: verbal (class A), visual (class B), self-oriented/self-referential processing (class C), cognition (class D), and interoception and sensorimotor processing (class E) [ 12 ]. Moreover the same microstate structures have been described from waking rest to deep sleep, confirming that they may reflect a robust large-scale resting-state network architecture, similar to the resting-state connectivity seen in fMRI that is also preserved in sleep [ 13 ]. Disruption in these microstate systems has been described across the spectrum of psychiatric disorders, in schizophrenia [ 14 ], autism spectrum disorder [ 15 ], or depression [ 16 ], but also in neurological disorders such as epilepsy [ 17 ]. Thus, preliminary results from our group suggested that a certain pattern of microstates could be associated with specific stages of disease progression in psychosis, but also translated a shared dimension on the schizophrenia-autism continuum [ 18 ]. They may therefore contribute to understanding the range of transdiagnostic endophenotypes shared between neuropsychiatric diseases, such as anomalies in sleep, sensorimotor integration, speech, and sense of self. Moreover, since microstates can be modulated under hypnotic conditions [ 19 ], and medical hypnosis has been associated with improved attentional and executive control over self-referential processes [ 20 ], EEG microstates may also provide a proxy for psychotherapeutic response. Thus, the effectiveness of hypnosis has been suggested in psychiatric disorders associated with overactivation of the default mode, such as depression [ 21 ].

Sleep disturbances are strongly linked to the pathophysiology of most neuropsychiatric disorders and may explain many of the cardiovascular, pneumologic, and neurologic comorbidities of psychiatric disorders [ 22 ]. Various mechanistic models have been described in bipolar disorder and depression, including circadian rhythm anomalies, internal desynchronization, or anomalies of sleep architecture [ 23 , 24 ]. Sleep dysregulation is also highly prevalent in autism spectrum disorders, leading to severe distress and impact on quality of life [ 25 ], while in the early stages of psychosis, there is a high prevalence of insomnia, nightmare disorder, sleep-related hallucinations, excessive sleepiness disorders or restless leg syndromes [ 26 ]. Moreover, having a sleep disorder exacerbates psychotic and mood symptoms among patients with psychosis [ 26 ]. From a neurophysiological perspective, the most replicated macroscopic EEG anomaly across the spectrum of psychosis, mood disorders, and autism, is the decrease in density of sleep spindles [ 27 , 28 , 29 ], which are determinant for cognitive processes such as memory consolidation [ 30 ]. However, more quantitative EEG analyses remain to be done to further explore brain connectivity during sleep.

• Sensorimotor integration

Sensorimotor abnormalities are a cross-cutting neuropsychiatric dimension [ 31 , 32 , 33 ], which can be robustly analyzed with quantitative EEG [ 34 ]. Motor deficits linked to alterations in cortical excitability/inhibition modulation of motor areas have been identified in various neurodevelopmental pathologies such as schizophrenia or autism [ 35 , 36 ], and in particular during adaptation to a probabilistic context [ 37 ].

Specifically, autism spectrum disorders have been shown to exhibit anormal context-sensitive processing mechanisms, sensorimotor gating deficits, as well as repetitive motor movements and atypical integration of sensory stimuli [ 38 , 39 , 40 ]. Recent behavioral and imaging studies investigating tactile processing in autism, suggested no difference in light touch detection and texture, but increased sensitivity in vibration [ 41 , 42 , 43 ]. Moreover, decreased connectivity in finger somatosensory areas and slower perceptual processing speed were shown [ 44 , 45 ]. Although sensory perturbations are well known, literature on sensory integration prior to motor movement is lacking. In schizophrenia, and more generally in psychotic disorders, it is still unclear how sensorimotor mechanisms are impaired. It has been hypothesized that a general disruption may cause a functional disintegration between sensory and cognitive processes [ 32 , 46 ], yet, further investigations are needed in order to shed light on precise sensorimotor integration. Only a handful of studies showed tactile perception accuracy deficits [ 47 , 48 , 49 ], and abnormal sensory predictions in a self- and non-self-elicited sensation discrimination task [ 50 ]. This indicates a failure of normal inhibitory regulation of sensory, motor, and attentional mechanisms, common in several neurodevelopmental disorders.

Another accessible neurophysiological function that reflects thought processing and also results from a complex integration of sensorimotor signals is represented by speech, considered in its quantitative dimensions, which has also been correlated with EEG microstate patterns [ 51 ]. Given that communication difficulties are key features of autistic and psychotic disorders [ 52 ], computational methods have recently been introduced to objectively quantify linguistic anomalies in the psychosis spectrum and to identify subtle and early linguistic peculiarities in UHR individuals [ 53 ]. Recent studies have shown that analyses in the semantic and syntactic areas could predict psychotic transition [ 54 ], but the predictive role of other linguistic domains, such as phonetics, has so far been poorly investigated. A main aspect of phonetic research is prosody, the tone of voice with which words are pronounced, crucial for communication [ 55 ]. Researchers from both the phonetic and psychiatric fields have invested significant effort into trying to precisely characterize the prosodic profile of patients with schizophrenia, generally finding reduced pitch variability and increased pause duration [ 56 ]. However, among the limitations of the existing research, are a weak generalizability of the results to languages other than English, a lack of comparisons with other clinical groups and scant attention devoted to voice quality [ 56 ]. Besides, prosodic cues have scarcely been explored in individuals with high risk of psychosis and more research is needed to clarify the potential predictive role of these features [ 57 , 58 ].

Alongside traditional approaches investigating communicative behavior in psychosis focusing only on the voice of the patient, it is also necessary to investigate what happens at the interactional level [ 59 ]. Turn-taking analysis specifically explores dialogical interactional behaviors. Turn-taking is the organization of the conversation into alternating speaking turns between different interlocutors and its main goal is to assure that no more than one person is speaking at any time. Another goal is to avoid long silent gaps between the end of one speaking turn and the beginning of the next one [ 60 ]. Turn-taking analysis has rarely been applied to individuals with psychosis and at-risk mental states so far [ 61 , 62 , 63 ].

It appears that in this group turn-taking patterns involving increased mutual silence are prevalent. Interestingly, voice atypicalities have also been quantified in individuals with autism spectrum disorders, both in childhood and adulthood [ 64 ]. Moreover, recent studies have found an increased number of silent gaps as compared to controls in the early stages of dialogues [ 65 , 66 ]. Of note, there is evidence suggesting that there are shared social cognition deficits between autism and schizophrenia spectrum disorders [ 67 ]. From this perspective, there is additional motivation for comparing prosodic and turn-taking patterns in individuals with ASD and along the psychosis spectrum.

Crucially, the possible link between prosodic and turn-taking variables and their neurophysiological substrate in microstates has never been studied in patients with these profiles.

Self-reference effect and disorders of the self

Neurophysiological measures may also shed light on the individual’s phenomenological experience, such as self-consciousness [ 68 ], and its alteration in patients with psychotic disorders [ 69 ]. The sense of self is multifaceted and can be examined through two main prisms: firstly, as knowledge about “Me”, object of a reflexive construct of the self-concept, stored in long-term memory (narrative self) [ 70 ], and secondly as an “I” subject of the pre-reflexive and embodied subjective experience in the here and now (minimal self) [ 71 ]. Self-disorders constitute a core feature of the schizophrenia spectrum, markers of vulnerability to psychosis and predictors of psychotic conversion in patients at ultra-high risk or who had a first episode of psychosis. One of the possible prisms for studying these self-disorders is based on the evaluation of the self-reference effect on memory, according to which processing information closely related to the self is the most effective strategy for remembering new material [ 72 ]. Indeed, the self is intimately linked to memory and acts as a processing bias that determines how and what information is encoded and retrieved [ 73 , 74 ], particularly in episodic memory, which refers to the memory of the past experiences of the self and contributes to one’s feeling of identity and temporal continuity. However, minimal or narrative self-disorders appear associated to an altered or even lack of self-reference effect on memory [ 75 , 76 ]. Studying the self-reference effect in early psychosis could therefore contribute to characterizing the extent and course of self-disorders in prodromal (ultra-high risk) and early (first episode of psychosis) stages of schizophrenia. An innovative task has been designed using immersive virtual reality to evaluate the self-reference effect on episodic memory via a naturalistic approach, relying on the encoding of multisensory daily life events rather than simplistic lists of words or objects.

Objectives of the DEMETER study

Building on our preliminary results, the DEMETER project (“Détermination Des Microétats EEG associés Aux Troubles Psychiques Dans Les États à Risque”—EEG Microstates Across At-Risk Mental States) is a prospective observational study that aims to characterize the EEG microstate signature with regard to underlying neurophysiological functions, including sensorimotor integration, speech, sleep, and sense of self, across a population of adolescents and young adults, with ultra-high-risk of psychosis (UHR), first-episode psychosis (FEP), schizophrenia (SCZ), autism spectrum disorder (ASD), and major depressive disorder (MDD), compared with healthy controls (CTRL) ( N  = 21 × 6), between two timepoints one year apart.

Participants will undergo deep phenotyping based on psychopathology and neuropsychological assessments at baseline and after one year of follow-up, high-resolution EEG (64 electrodes) with a resting period and a sensorimotor task, a recording of the characteristics of their speech (prosody and turn-taking), a one-night polysomnography, and biological sampling for multi-omic analyses, and a self-reference effect task in virtual reality (the latter only in UHR, FEP, and CTRL).

The main questions it aims to answer are as follows. 1) Are EEG microstate anomalies associated with specific disorders, and clinical and functional prognosis, both in resting conditions and during sleep ? 2) Are EEG microstate anomalies associated with differences in sensorimotor integration, speech, and sense of self ? 3) An interventional ancillary study will involve only healthy controls, in order to assess whether light hypnosis conditions can modify the EEG microstate architecture in a direction opposite to what is seen in disease.

Participant recruitment

All participants will be included at the Clinical Research Centre (CRC), University Hospital Group Paris Psychiatry and Neurosciences (GHU). Inclusion criteria are: an age between 15 and 30 years old; French as the maternal language or spoken in the context of bilingualism; a DSM-5 diagnosis of schizophrenia or major depressive disorder or autism spectrum disorder; a diagnosis of ultra-high-risk of psychosis or first-episode psychosis based on the Comprehensive Assessment of at risk mental state (CAARMS) translated in its French version [ 77 ]; and healthy control subjects recruited from the general population. Exclusion criteria are: suicidal risk; severe or non-stabilized somatic and neurological disorders; epilepsy; head trauma; IQ below 70; presence of other psychiatric disorders (bipolar disorder, obsessive–compulsive disorder, or substance use disorders, except for tobacco or cannabis, tolerated up to 5 joints/day); for healthy control subjects, a family history of psychosis is an exclusion criterion. Pregnant or breast-feeding women will not be included.

Participants will be screened among the population of patients seen at an early psychosis outpatient clinic (Centre d’évaluation des jeunes adultes et adolescents—CJAAD, GHU). Healthy controls will be reached through the healthy volunteers database of the CRC. Participant assessment will be as follows (Fig.  1 , Table 1 ). 1) During the pre-inclusion visit, participants will be informed of all the details of the protocol, orally and in writing, and eligibility criteria will be verified. Then a two-week reflection period will be observed, before the signature of a written consent at the baseline inclusion. 2) The baseline visit will consist of a first visit of three half-day sessions including medical, psychopathological and neuropsychological assessments, biological sampling (described below), and speech recording. In the following two months, participants will undergo one night of polysomnography (everyone) and two half-day sessions for the sensorimotor task (everyone) followed by light hypnosis (only controls), and the self-reference effect task in virtual reality (only controls, UHR, and FEP). 3) The follow-up visit will consist of a second psychopathological and neuropsychological assessment, biological sampling, and shorter EEG recording (5–10 min).

Protocol design. MDD: major depressive disorder. UHR: ultra-high-risk of psychosis. FEP: first-episode psychosis. SCZ: schizophrenia. ASD: autism spectrum disorder. CTRL: healthy controls

Healthy controls will receive a compensation of 120€, and participants with a psychiatric disorder will receive a financial compensation of 60€, as they will benefit to an access to more personalized care. This protocol has been approved by the ethics committee (Comité de protection des personnes) Ouest II (approval number: 2021-A01919-32).

Clinical, psychopathological and neuropsychological assessment

A clinical assessment will include anamnestic data collection (socio-demographic characteristics, treatment history, medical history, psychiatric history); a clinical examination with a physical examination, a psychiatric examination, the CAARMS, the positive and negative syndrome scale (PANSS), the Montgomery–Åsberg Depression Rating Scale (MADRS), Social and Occupational Functioning Assessment Scale (SOFAS), and neurological soft signs [ 78 ]. The neuropsychological assessment will include intellectual functioning (WAIS-IV), executive functioning (fluences, attentional capacities, episodic and working memory), and social cognition.

Speech assessment

Each participant will undergo a semi-structured interview with an experimenter. Interviews will focus on interests and passions, to elicit as free and spontaneous dialogues as possible. Topics related to the participants’ clinical symptomatology will not be approached, unless participants explicitly wish to do so. The recordings will not have a fixed duration, but an attempt will be made to obtain at least 15–20 min of dialogue. Both speakers will wear head-set AKG-C544L microphones, connected via AKG MPA VL phantom adaptors to a Zoom H4n Pro Handy recorder. Speech will be recorded at a sampling rating of 44,000 Hz (16-bit). The distance between the mouth and the microphone will be kept at 2 cm to ensure consistent levels of vocal loudness. Moreover, the two speakers will be placed as far as possible, to prevent crosstalk (i.e. speech of one interactant caught by the other interactant’s microphone). Finally, the recordings will be carried out in a quiet room to limit environmental noise. This is consistent with previous analyses on acoustic patterns in psychiatry [ 79 ]. The.wav files obtained will be annotated using Praat software and subsequently analyzed with Praat [ 80 ] and R. Prosodic features will be extracted using the Prosogram tool (a set of Praat scripts, open-source) [ 81 ] and voice quality features will be computed with a modified version of scripts from the Prosogram tool [ 82 ]. Turn-taking variables will be quantified with combined Praat and R scripts [ 62 , 66 , 83 ]. This task has been designed and will be supervised by an expert in Phonetics and by a psychiatrist trained in linguistic data extraction and analysis (MGr and VL).

Sensorimotor integration task

Sensorimotor integration is investigated using a visuo-tactile task. On each trial, the participant, seated in front of a screen, has a visual instruction: a point to the right or left of the screen. The task consists of pressing one of the two buttons positioned on each side of the body with the index finger of the corresponding hand according to the visual instruction. A vibrotactile stimulator (small bone conduction speakers wired to an Arduino electronic card modulated by an amplifier) is applied to the first dorsal interosseous muscle of both hands. 400 msec before the visual instruction, one of the two hands receives a tactile cue (vibration) on one hand for 100 msec. The tactile cue can be congruent or incongruent with the visual cue, both indicating or not the same hand. Depending on the block, the tactile cue can be more or less reliably coupled with the visual stimulus. In reliable blocks, 90% of the trials present the vibration and visual instruction congruently (indicating the same hand). In non-reliable blocks, only 50% of the trials are congruent, and in this case, the tactile cue is not reliable. Two blocks with 70% congruent cases are carried out intermediately. Finally, a baseline block which does not contain any tactile cues is presented at the beginning and the end of the task. The order of the 90% and 50% blocks is randomized. The tactile and visual stimuli are generated with a MATLAB script. Each block consists of 100 trials, in total 500 trials. EEG data is recorded throughout the task, using a 64-channel EEG cap (from Biosemi). The setup is coupled to an eye tracker in order to control that the participant is fixing the cross at the center of the screen during each block. At the end of the task, a five minute eyes closed resting-state EEG will also be recorded. In order to examine attentional modulation, measurement of alpha power band (in Hz) is computed. Cortical excitability and inhibition are analyzed with mu and theta bands (in Hz), and integration of sensory information as somatosensory evoked potentials (SEPs), where amplitudes (in µV) and latencies (in msec) are extracted. The adaptation of the reaction time (in msec) to the button press according to the probabilistic context of congruency is examined. Analysis is conducted with Python scripts with dedicated libraries such as MNE-Python [ 84 ]. This task has been designed and will be supervised by researchers trained in neurophysiology recording and analysis (AA, LD).

Hypnosis task

After the sensorimotor integration task, healthy controls will undergo a light hypnosis task coupled with two control tasks, in addition to the eyes-closed resting-state already recorded. First, participants will be asked to listen to a neutral text (a refrigerator manual) read by the investigator, with the instruction to listen attentively in order to be able to answer specific questions regarding the content of text. Second, participants will do a mental calculation test. Third, participants will undergo the light hypnosis task. Light hypnosis is based on Ericksonian hypnosis without inducing a trance state, and has been developed by the Collège International des Techniques par Activation de la Conscience (CITAC; Jean Becchio, Sylvain Pourchet) as part of the Paris-Saclay university training in clinical hypnosis. Participants will be asked to focus on any type of preoccupation they may have, and then to picture the first step towards resolving this preoccupation. They will then be asked to provide resources or qualities they have. The light hypnosis session will then start by asking the participant to assume a comfortable and at the same time tonic position, sitting straight, the back lifted from the chair. They will be asked to close their eyes, while being informed that they can open them at any time if needed. They will then be asked to picture their objective, and the first step toward its solution. Then, they will be asked to picture themselves in a situation where they learnt to do something. Proprioceptive, sensory (each of the five senses), and metaphorical suggestions based on their resources will be provided in order to guide the participant in this exercise. This task has been designed and will be performed by two psychiatrists trained in clinical hypnosis (AI, CLo).

Self-reference effect task in virtual reality

Virtual reality immersion will be achieved using the HTC VIVE Pro Eye (Taoyuan City, Taiwan: HTC corporation) virtual reality headset. The self-reference effect task will consist in a walk through a virtual city, where participants will encounter a total of 32 multisensory daily life events that aim to be incidentally encoded in episodic memory. Participants will embody a virtual avatar and navigate twice through two distinct parts of the city. Prior to each navigation, avatar embodiment will be induced using a visuomotor stimulation in front of a virtual mirror, asking participants to move their different body parts while looking at them directly or in the mirror. To manipulate the minimal self-reference, one navigation will be associated with a synchronous avatar to induce a high sense of embodiment, and therefore a stronger sense of minimal self. The other navigation will be associated with an asynchronous avatar with a 700 ms-delay between participants’ real performed movements and the seen movements of the avatar, to induce a low sense of embodiment, and thus a weaker sense of minimal self. To manipulate the narrative self-reference, in each navigation path, half of the events will be associated to the participants themselves (Self), and the other to someone else (Other). The association will be induced by asking participants to take a picture of each event and rate its personal significance for either Self or Other. All conditions will be counterbalanced.

Following each navigation, participants will be submitted to self-reported questionnaires assessing their sense of embodiment (Embodiment Questionnaire) [ 85 ], sense of presence (Igroup Presence Questionnaire) [ 86 ], cybersickness (Simulator Sickness Questionnaire) [ 87 ], and current emotional state (Mood Visual Analogue Scale) [ 88 ].

Finally, participants will undergo two episodic memory tests: a free recall task and a recognition task. The free recall will be based on a verbal interview of 20 min, during which participants will be asked to recall all the events that they remember encountering in the virtual city. The recognition test will be programmed using the Python module Neuropsydia [ 89 ] and consists of displaying on a computer screen all 32 encountered events mixed with 16 lures which were not encountered in a random order, and asking participants whether they encountered this event in the virtual city. For both memory tests, participants will be asked to provide systematically and the most precisely possible, for each recalled event: description of the event, spatiotemporal situation, source, referent for the personal significance rating, perceptive and phenomenological details, degree of reliving or familiarity of the event. This task has been designed by Delphine Yeh under the supervision of Pascale Piolino, and derived from Sylvain Penaud’s protocol for the procedures linked to the minimal self-reference [ 90 ]. The virtual environment has been developed by Alexandre Gaston-Bellegarde using Unity.

Polysomnography

An overnight polysomnography with 19 EEG channels and ventilatory polygraphy will be recorded for all participants within the first two months of inclusion, at the sleep medicine department of Hôtel-Dieu hospital, in Paris (Centre du Sommeil et de la Vigilance). Trained sleep technicians will set-up the head-sets in the evening and supervise the recording during the night. The recordings will be analyzed by trained sleep specialists (SR, LD).

Microstates analysis

Microstate analysis will be performed on eyes-closed resting-state, during the sensorimotor integration task, and during sleep. A minimal preprocessing will be done with the MNE EEG software on Python, which includes a bandpass filter between 0.5 and 40 Hz, rereferencing to the mean, and visual and automatic correction for artifacts using independent component analysis (ICA). Each recording will be visually reanalyzed by clinical neurophysiologists to check for any residual artifact. Microstate analysis will be done using the Pycrostates package [ 91 ]. Global field power (GFP) will be determined for each participant. Only EEG topographies at GFP peaks will be retained to determine microstates’ topographies, through a modified K-means clustering. For each subject the same number of GFP peaks will be extracted and concatenated into a single data set for clustering. A combined score will be used to compute the optimal number of clusters. The resulting clusters will be backfitted to each individual maps. Temporal smoothing will be used to ensure that periods of inter-peak noise, of low GFP, did not interrupt the sequences of quasi-stable segments. For each subject, three parameters will be computed for each microstate class: frequency of occurrence (“occurrence”), temporal coverage (“coverage”) and mean duration. Occurrence is the average number of times a given microstate occurs per second. Coverage (in %) is the percentage of total analysis time spent in a given microstate. Mean duration (in ms) is the average time during which a given microstate was present in an uninterrupted manner (after temporal smoothing).

Biological sampling

Peripheral blood samples will be collected for genetic, epigenetic, proteomic, and metabolomic studies.

Statistical power estimates

We considered a minimum expected effect size around 0.5, based on pairwise comparisons of EEG microstate parameters (mean duration, time coverage, occurrence) between chronic schizophrenia and relatives of subjects with schizophrenia [ 14 ]. Accepting an alpha risk of 0.05 and 95% power, we estimate the necessary number of subjects to be included in each of the 6 groups at 15 (calculated in R with the pwr.anova.test function) (Fig.  2 ). Given the risk of overestimating the minimum effect size associated with publications based on small cohorts, we estimate that a number of 21 subjects significantly increases the chances of obtaining a power greater than 95%. This represents a total of 126 subjects.

Power calculation

Statistical analysis design

For all neurophysiological variables, the investigators will apply a repeated measures ANOVA, and use the following contrasts:

“UHR, FEP, SCZ, ASD, MDD” vs. “Healthy subjects”, in order to test the variables as markers of general psychopathology;

“UHR, FEP, SCZ” vs. “ASD, MDD”, in order to test the variables as specific markers of psychosis; equivalently, their specificity in MDD and ASD will be tested);

“UHR” vs. “FEP" vs. “SCZ”, in order to test the variables as markers of state;

Finally, in a dimensional approach, the functional correlates of microstates will be studied across wakefulness and sleep, and during speech, regardless of diagnosis.

The Research Domain Criteria (RDoC) strategy has led to a paradigmatic change in psychiatric research, promoting the integration of dimensional constructs beyond current nosographic boundaries. In this context, sensorimotor integration, speech, sleep–wake rhythms, and sense of self appear as relevant phenotypes to understand transdiagnostic functional impairments that lead to a high burden at the individual level [ 92 , 93 ]. In a multimodal approach, the use of neurophysiological tools such as high-density EEG, polysomnography, or audio recorders offer an accessible means to study these dimensions at a very good temporal resolution. Hypnosis or virtual reality tools further give the opportunity to non-invasively modulate and test perceptions in relation with these neurophysiological assessments. Moreover, applying quantitative EEG analyses in this framework, such as microstates, may shed light on the connectivity networks underlying thought processing and provide clinically-relevant biomarkers of state that could be easily implemented in daily practice.

This protocol describes a transdiagnostic longitudinal case–control study that includes a multimodal neurophysiological assessment of sensorimotor integration, speech, sleep, and sense of self, in patients with major depressive disorder, ultra-high-risk of psychosis, first-episode psychosis, schizophrenia, and autism spectrum disorders, compared to healthy controls, in a population of adolescents and young adults aged 15 to 30 years old. Preliminary retrospective analyses from our group, with routine clinical low-resolution EEG recordings, have suggested that a variation in EEG microstates class D may be a marker of stage across psychotic disorders, as it decreases from UHR to FEP and schizophrenia. However, these changes were not specific to psychosis, and they appeared to reflect a shared dimension on the schizophrenia-autism spectrum. We also suggested that a microstate ratio imbalance between class C and class D may perhaps be more specific to schizophrenia, although it did not appear that EEG microstates were sufficient to differentiate between different groups of diseases [ 18 ]. Building on this preliminary data, we propose this prospective study with higher resolution EEG recordings to test whether anomalies in the EEG microstate architecture may be associated with diagnosis, clinical and functional prognosis, both in resting conditions and during sleep, across psychiatric disorders. We postulate that we may find EEG microstate anomalies associated with differences in sensorimotor integration, speech, sense of self, and sleep, and that the dynamic of EEG microstates may be modulated by a non-drug intervention such as light hypnosis, as a proof-of-concept of potential usefulness in psychotherapeutic approaches.

We further hypothesize that the attentional component of somatosensory integration in preparation for a motor response is modulated through visuo-tactile stimuli in healthy subjects, and is altered in patients with psychotic disorders, probably with abnormal inhibition mechanism responses. Specifically, we expect that primary sensory cortex activity, measured as alpha and beta oscillations, influences motor cortex excitability and would be desynchronized in psychosis. We also anticipate an impaired connectivity among the primary sensorimotor network, as well as altered synchrony states in an attentional context. Finally, at rest, we expect these anomalies to be associated with a C/D microstate imbalance and with microstate class E anomalies (postulated to be correlated with interoception and sensorimotor processing).

We also expect to find differences in prosodic and turn-taking patterns between patients and healthy controls. In particular, we predict all patients to display reduced pitch variability, reduced speech output and increased pause duration. Moreover, we expect patients’ voices to overlap more with the interlocutor’s. We also hypothesize that linguistic cues could be markers of the stage, with increasing levels of atypicality from UHR to SCZ. Finally, we speculate that patients with ASD and participants along the schizophrenia spectrum will present similar prosodic and turn-taking patterns.

FInally, we postulate a reduced self-reference effect on memory performance in UHR and FEP individuals, due to patients’ self disturbances. Specifically, we expect a preserved narrative self-reference effect but no minimal self-reference effect in UHR and FEP individuals, since minimal self disturbances are already present in early stages of psychosis whereas narrative disturbances of the self are less marked, as opposed to controls who are expected to exhibit both minimal and narrative self-reference effects.

This study will address several methodological challenges. Its transdiagnostic design will allow us to test the specificity of any relevant observed association. At the data collection level, the pipeline that integrates the sensorimotor task with the high-density EEG recording will follow stringent quality checks so that the EEG recordings are interpretable with regard to the underlying task. The one-night polysomnography recordings will require to anticipate all the risks associated with prolonged recordings, such as electrodes that come off during sleep due to participant movement. This implies time-consuming regular check-ups during the night from trained technicians. In order to allow reproducible results, the ancillary light hypnosis protocol will require the use of a simple standardized strategy from the two clinicians trained in hypnosis. Regarding the linguistic data collection, high-quality double-channel audio recordings are crucial to allow precise and reliable analyses with the Praat software. Regarding the self-reference effect, the innovative task using immersive virtual reality will enable to study this effect in a naturalistic and standardized context, which will capture the richness of episodic memory and its links with the self in everyday life better than the simplistic lists of words or objects that are traditionally used in self-reference effect studies. Moreover, the task will integrate both minimal and narrative self-reference, which will enable to examine under the same design the respective but also joint contributions of both facets of the self to the self-reference effect in patients with self-disorders.

In conclusion, this multimodal, transdiagnostic neurophysiological approach will help pave the way for personalized medicine through in-depth endophenotyping of sleep, speech, sensorimotor integration and self-perception, four dimensions that overlap in the spectrum of psychiatric disorders.

Availability of data and materials

Anonymized data will be stored at GHU Paris Psychiatrie et Neurosciences, and will be made available upon reasonable request to the authors.

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Acknowledgements

We thank the team of the Délégation à la Recherche Clinique et à l’Innovation (DRCI) of GHU Paris Psychiatrie et Neurosciences for providing administrative and legal support to build this protocol, with special thanks to Bernadette Lemercier, Thujin Yoharajah, Khaoussou Sylla, Didier André, and Kahina Belkhir Hadid. We thank the Centre de Recherche Clinique (CRC) of GHU Paris Psychiatrie et Neurosciences for providing the material support of subject inclusions. Figures were done with Biorender ( https://www.biorender.com/ ).

This study is funded by the DEMETER Starting Grant, GHU Paris Psychiatrie et Neurosciences (principal investigator: Anton Iftimovici). Valeria Lucarini was supported by the doctoral grant ‘Young Talents in Psychiatry 2021’ from the Fondation FondaMental—Fondation Bettencourt-Schueller. Anaëlle Alouit was also financed by the French government’s “Investissements d’Avenir” programme (ANR-18-RHUS-0014 PsyCARE).

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Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Team “Pathophysiology of psychiatric disorders”, GDR 3557-Institut de Psychiatrie, 102-108 Rue de la Santé, Paris, 75014, France

Valeria Lucarini, Mylène Moyal, Boris Chaumette, Marie-Odile Krebs & Anton Iftimovici

GHU Paris Psychiatrie et Neurosciences, Pôle Hospitalo-Universitaire d’évaluation, Prévention, et Innovation Thérapeutique (PEPIT), Paris, France

Valeria Lucarini, Jeanne Le Coq, Romane Savatte, Mylène Charre, Cécile Louveau, Meryem Benlaifa Houamri, Mylène Moyal, Boris Chaumette, Marie-Odile Krebs & Anton Iftimovici

Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Team “Stroke: from prognostic determinants and translational research to personalized interventions”, Paris, 75014, France

Anaëlle Alouit, Estelle Pruvost-Robieux, Påvel G. Lindberg & Martine Gavaret

Laboratoire Mémoire, Cerveau et Cognition, UR7536, Université Paris Cité, Boulogne-Billancourt, F-92100, France

Delphine Yeh, Sylvain Penaud, Alexandre Gaston-Bellegarde & Pascale Piolino

Centre du Sommeil et de la Vigilance, AP-HP, Hôtel-Dieu, Paris, France

Stéphane Rio, Laurent Drouet, Maxime Elbaz & Damien Léger

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Jean Becchio & Sylvain Pourchet

Service de Neurophysiologie Clinique, GHU Paris Psychiatrie et Neurosciences, Paris, France

Estelle Pruvost-Robieux & Martine Gavaret

Epileptology and Cerebral Rhythmology, APHM, Timone Hospital, Marseille, France

Angela Marchi

Department of Child and Adolescent Psychiatry, Fondation Vallee, UNIACT Neurospin CEA - INSERM UMR 1129, Universite Paris Saclay, Gentilly, France

Aline Lefebvre

IfL-Phonetics, University of Cologne, Cologne, Germany

Martine Grice

INCC UMR 8002, CNRS, Université Paris Cité, Paris, F-75006, France

Lucile Dupin

Inserm, INRAE, Center for Research in Epidemiology and StatisticS (CRESS), Service de Psychiatrie de l’adulte, AP-HP, Hôpital Hôtel-Dieu, Université Paris Cité and Université Sorbonne Paris Nord, Paris, France

Cédric Lemogne

VIFASOM, ERC 7330, Université Paris Cité, Paris, France

Damien Léger

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Contributions

This project is the fruit of a collaborative effort, including: project funding and overall coordination (AI), speech study design (VL, MGr), sensorimotor integration study design (AA), self-reference effect in virtual reality study design (DY, PP, SP, AGB), sleep study design (AI), polysomnography set-up and analysis (SR, LD, ME, DL), hypnosis study design (AI, CLo, JB, SP), participant screening, inclusion and clinical assessment (AI, VL, MC, MBH, MOK), neuropsychological assessment (JLC and RS), EEG preprocessing and analysis protocols (AI, EPR, AM, MM, AL, MGa), biological assessment (BC). AI, VL, AA, and DY drafted the initial manuscript. All authors read and approved the final manuscript.

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Correspondence to Anton Iftimovici .

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This study was approved by the ethics committee “Comité de protection des personnes” Ouest II (Approval Number: 2021-A01919-32), and is registered on ClinicalTrials.gov (ID: NCT06045897).

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Lucarini, V., Alouit, A., Yeh, D. et al. Neurophysiological explorations across the spectrum of psychosis, autism, and depression, during wakefulness and sleep: protocol of a prospective case–control transdiagnostic multimodal study (DEMETER). BMC Psychiatry 23 , 860 (2023). https://doi.org/10.1186/s12888-023-05347-x

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Case control studies.

Steven Tenny ; Connor C. Kerndt ; Mary R. Hoffman .

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Last Update: March 27, 2023 .

• Introduction

A case-control study is a type of observational study commonly used to look at factors associated with diseases or outcomes. [1]   The case-control study starts with a group of cases, which are the individuals who have the outcome of interest. The researcher then tries to construct a second group of individuals called the controls, who are similar to the case individuals but do not have the outcome of interest. The researcher then looks at historical factors to identify if some exposure(s) is/are found more commonly in the cases than the controls. If the exposure is found more commonly in the cases than in the controls, the researcher can hypothesize that the exposure may be linked to the outcome of interest. 

For example, a researcher may want to look at the rare cancer Kaposi's sarcoma. The researcher would find a group of individuals with Kaposi's sarcoma (the cases) and compare them to a group of patients who are similar to the cases in most ways but do not have Kaposi's sarcoma (controls). The researcher could then ask about various exposures to see if any exposure is more common in those with Kaposi's sarcoma (the cases) than those without Kaposi's sarcoma (the controls). The researcher might find that those with Kaposi's sarcoma are more likely to have HIV, and thus conclude that HIV may be a risk factor for the development of Kaposi's sarcoma.

There are many advantages to case-control studies.  First, the case-control approach allows for the study of rare diseases.   If a disease occurs very infrequently, one would have to follow a large group of people for a long period of time to accrue enough incident cases to study. Such use of resources may be impractical, so a case-control study can be useful for identifying current cases and evaluating historical associated factors.  For example, if a disease developed in 1 in 1000 people per year (0.001/year) then in ten years one would expect about 10 cases of a disease to exist in a group of 1000 people. If the disease is much rarer, say 1 in 1,000,0000 per year (0.0000001/year) this would require either having to follow 1,000,0000 people for ten years or 1000 people for 1000 years to accrue ten total cases. As it may be impractical to follow 1,000,000 for ten years or to wait 1000 years for recruitment, a case-control study allows for a more feasible approach. 

Second, the case-control study design makes it possible to look at multiple risk factors at once. In the example above about Kaposi's sarcoma, the researcher could ask both the cases and controls about exposures to HIV, asbestos, smoking, lead, sunburns, aniline dye, alcohol, herpes, human papillomavirus, or any number of possible exposures to identify those most likely associated with Kaposi's sarcoma.

Case-control studies can also be very helpful when disease outbreaks occur, and potential links and exposures need to be identified.  This study mechanism can be commonly seen in food-related disease outbreaks associated with contaminated products, or when rare diseases start to increase in frequency, as has been seen with measles in recent years.

Because of these advantages, case-control studies are commonly used as one of the first studies to build evidence of an association between exposure and an event or disease.

In a case-control study, the investigator can include unequal numbers of cases with controls such as 2:1 or 4:1 to increase the power of the study.

Disadvantages and Limitations

The most commonly cited disadvantage in case-control studies is the potential for recall bias. [2]   Recall bias in a case-control study is the increased likelihood that those with the outcome will recall and report exposures compared to those without the outcome.  In other words, even if both groups had exactly the same exposures, the participants in the cases group may report the exposure more often than the controls do.  Recall bias may lead to concluding that there are associations between exposure and disease that do not, in fact, exist. It is due to subjects' imperfect memories of past exposures.  If people with Kaposi's sarcoma are asked about exposure and history (e.g., HIV, asbestos, smoking, lead, sunburn, aniline dye, alcohol, herpes, human papillomavirus), the individuals with the disease are more likely to think harder about these exposures and recall having some of the exposures that the healthy controls.

Case-control studies, due to their typically retrospective nature, can be used to establish a correlation  between exposures and outcomes, but cannot establish causation . These studies simply attempt to find correlations between past events and the current state. 

When designing a case-control study, the researcher must find an appropriate control group. Ideally, the case group (those with the outcome) and the control group (those without the outcome) will have almost the same characteristics, such as age, gender, overall health status, and other factors. The two groups should have similar histories and live in similar environments. If, for example, our cases of Kaposi's sarcoma came from across the country but our controls were only chosen from a small community in northern latitudes where people rarely go outside or get sunburns, asking about sunburn may not be a valid exposure to investigate.  Similarly, if all of the cases of Kaposi's sarcoma were found to come from a small community outside a battery factory with high levels of lead in the environment, then controls from across the country with minimal lead exposure would not provide an appropriate control group.  The investigator must put a great deal of effort into creating a proper control group to bolster the strength of the case-control study as well as enhance their ability to find true and valid potential correlations between exposures and disease states.

Similarly, the researcher must recognize the potential for failing to identify confounding variables or exposures, introducing the possibility of confounding bias, which occurs when a variable that is not being accounted for that has a relationship with both the exposure and outcome.  This can cause us to accidentally be studying something we are not accounting for but that may be systematically different between the groups. 

The major method for analyzing results in case-control studies is the odds ratio (OR). The odds ratio is the odds of having a disease (or outcome) with the exposure versus the odds of having the disease without the exposure. The most straightforward way to calculate the odds ratio is with a 2 by 2 table divided by exposure and disease status (see below). Mathematically we can write the odds ratio as follows.

Odds ratio = [(Number exposed with disease)/(Number exposed without disease) ]/[(Number not exposed to disease)/(Number not exposed without disease) ]

This can be rewritten as:

Odds ratio = [ (Number exposed with disease) x (Number not exposed without disease) ] / [ (Number exposed without disease ) x (Number not exposed with disease) ] 

The odds ratio tells us how strongly the exposure is related to the disease state. An odds ratio of greater than one implies the disease is more likely with exposure. An odds ratio of less than one implies the disease is less likely with exposure and thus the exposure may be protective.  For example, a patient with a prior heart attack taking a daily aspirin has a decreased odds of having another heart attack (odds ratio less than one). An odds ratio of one implies there is no relation between the exposure and the disease process.

Odds ratios are often confused with Relative Risk (RR), which is a measure of the probability of the disease or outcome in the exposed vs unexposed groups.  For very rare conditions, the OR and RR may be very similar, but they are measuring different aspects of the association between outcome and exposure.  The OR is used in case-control studies because RR cannot be estimated; whereas in randomized clinical trials, a direct measurement of the development of events in the exposed and unexposed groups can be seen. RR is also used to compare risk in other prospective study designs.

• Issues of Concern

The main issues of concern with a case-control study are recall bias, its retrospective nature, the need for a careful collection of measured variables, and the selection of an appropriate control group. [3]  These are discussed above in the disadvantages section.

• Clinical Significance

A case-control study is a good tool for exploring risk factors for rare diseases or when other study types are not feasible.  Many times an investigator will hypothesize a list of possible risk factors for a disease process and will then use a case-control study to see if there are any possible associations between the risk factors and the disease process. The investigator can then use the data from the case-control study to focus on a few of the most likely causative factors and develop additional hypotheses or questions.  Then through further exploration, often using other study types (such as cohort studies or randomized clinical studies) the researcher may be able to develop further support for the evidence of the possible association between the exposure and the outcome.

• Enhancing Healthcare Team Outcomes

Case-control studies are prevalent in all fields of medicine from nursing and pharmacy to use in public health and surgical patients.  Case-control studies are important for each member of the health care team to not only understand their common occurrence in research but because each part of the health care team has parts to contribute to such studies.  One of the most important things each party provides is helping identify correct controls for the cases.  Matching the controls across a spectrum of factors outside of the elements of interest take input from nurses, pharmacists, social workers, physicians, demographers, and more.  Failure for adequate selection of controls can lead to invalid study conclusions and invalidate the entire study.

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2x2 table with calculations for the odds ratio and 95% confidence interval for the odds ratio. Contributed by Steven Tenny MD, MPH, MBA

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Disclosure: Connor Kerndt declares no relevant financial relationships with ineligible companies.

Disclosure: Mary Hoffman declares no relevant financial relationships with ineligible companies.

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• Cite this Page Tenny S, Kerndt CC, Hoffman MR. Case Control Studies. [Updated 2023 Mar 27]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-.

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