Diarrhea hospitalization costs among children <5 years old in Madagascar.

BACKGROUND: Following a recommendation by the World Health Organization, Madagascar introduced rotavirus vaccine in 2014. Though national rotavirus vaccine coverage has remained <80%, rotavirus hospitalizations declined by 78%. Gavi, the Vaccine Alliance, has provided financial support for rotavirus vaccine, however the Malagasy government has increasing responsibility for the financial cost. METHODS: In this evaluation, we describe the direct medical, direct non-medical, and indirect cost of illness due to diarrhea among children <5 years old at a public pediatric referral hospital. A 3-part structured questionnaire was administered during and following the hospitalization and the child's hospital record was reviewed. RESULTS: In total, 96 children were included in this analysis. The median total cost of the illness was $156.00 (IQR: 104.00, 210.86) and the median direct medical cost was $107.22. Service delivery costs represented a median of 44% of the inpatient costs; medications and diagnostic tests represented a median of 28% and 20% of the total costs of the hospitalization, respectively. The median percentage of the total illness costs paid by the household was 67%. Among households with income of <$61/month, the median costs of the illness paid by the household were $78.55, representing a median of 168% of the household's monthly expenses. Among households earning >$303/month, the median costs paid by the household were $147.30, representing a median of 53% of the household's monthly expenses. Among all household income levels, caregivers commonly paid these bills from savings, borrowed money, and donations. CONCLUSIONS: Our findings will be useful in assessing the cost-effectiveness of rotavirus vaccine by decisionmakers. These results may also help hospital administrators and healthcare providers better understand the financial constraints of families.

Cost estimates of diarrhea hospitalizations among children <5 years old in Zimbabwe.

INTRODUCTION: Diarrhoea is a leading killer of children <5 years old, accounting for 480,000 deaths in 2017. Zimbabwe introduced Rotarix into its vaccination program in 2014. In this evaluation, we estimate direct medical, direct non-medical, and indirect costs attributable to a diarrhea hospitalization in Zimbabwe after rotavirus vaccine introduction. METHODS: Children <5 years old admitted to Harare Central Hospital from June 2018 to April 2019 with acute watery diarrhea were eligible for this evaluation. A 3-part structured questionnaire was used to collect data by interview from the child's family and by review of the medical record. A stool specimen was also collected and tested for rotavirus. Direct medical costs were the sum of medications, consumables, diagnostic tests, and service delivery costs. Direct non-medical costs were the sum of transportation, meals and lodging for caregivers. Indirect costs are the lost income for household members. RESULTS: A total of 202 children were enrolled with a median age of 12 months (IQR: 7-21) and 48 (24%) had malnutrition. Children were sick for a median of 2 days and most had received outpatient medical care prior to admission. The median monthly household income was higher for well-nourished children compared to malnourished children (p < 0.001). The median total cost of a diarrhea illness resulting in hospitalization was $293.74 (IQR: 188.42, 427.89). Direct medical costs, with a median of $251.74 (IQR: 155.42, 390.96), comprised the majority of the total cost. Among children who tested positive for rotavirus, the median total illness cost was $243.78 (IQR: 160.92, 323.84). The median direct medical costs were higher for malnourished than well-nourished children (p < 0.001). CONCLUSION: Direct medical costs are the primary determinant of diarrhea illness costs in Zimbabwe. The descriptive findings from this evaluation are an important first step in calculating the cost effectiveness of rotavirus vaccine.

Evidence for Household Transmission of Rotavirus in the United States, 2011-2016.

BACKGROUND: Rotavirus is a leading cause of acute gastroenteritis (AGE) in children and is highly transmissible. In this study, we assessed the presence of AGE in household contacts (HHCs) of pediatric patients with laboratory-confirmed rotavirus. METHODS: Between December 2011 and June 2016, children aged 14 days to 11 years with AGE were enrolled at 1 of 7 hospitals or emergency departments as part of the New Vaccine Surveillance Network. Parental interviews, medical and vaccination records, and stool specimens were collected at enrollment. Stool was tested for rotavirus by an enzyme immunoassay and confirmed by real-time or conventional reverse transcription-polymerase chain reaction assay or repeated enzyme immunoassay. Follow-up telephone interviews were conducted to assess AGE in HHCs the week after the enrolled child's illness. A mixed-effects multivariate model was used to calculate odds ratios. RESULTS: Overall, 829 rotavirus-positive subjects and 8858 rotavirus-negative subjects were enrolled. Households of rotavirus-positive subjects were more likely to report AGE illness in ≥1 HHC than were rotavirus-negative households (35% vs 20%, respectively; P < .0001). A total of 466 (16%) HHCs of rotavirus-positive subjects reported AGE illness. Of the 466 ill HHCs, 107 (23%) sought healthcare; 6 (6%) of these encounters resulted in hospitalization. HHCs who were <5 years old (odds ratio, 2.2 [P = .004]) were more likely to report AGE illness than those in other age groups. In addition, 144 households reported out-of-pocket expenses (median, $20; range, $2-$640) necessary to care for an ill HHC. CONCLUSIONS: Rotavirus-associated AGE in children can lead to significant disease burden in HHCs, especially in children aged <5 years. Prevention of pediatric rotavirus illness, notably through vaccination, can prevent additional illnesses in HHCs.

Effect of propensity of seeking medical care on the bias of the estimated effectiveness of rotavirus vaccines from studies using a test-negative case-control design.

BACKGROUND: Rotavirus is the leading cause of severe diarrhea among children worldwide, and vaccines can reduce morbidity and mortality by 50-98%. The test-negative control (TNC) study design is increasingly used for evaluating the effectiveness of vaccines against rotavirus and other vaccine-preventable diseases. In this study design, symptomatic patients who seek medical care are tested for the pathogen of interest. Those who test positive (negative) are classified as cases (controls). METHODS: We use a probability model to evaluate the bias of estimates of rotavirus vaccine effectiveness (VE) against rotavirus diarrhea resulting in hospitalization in the presence of possible confounding and selection biases due to differences in the propensity of seeking medical care (PSMC) between vaccinated and unvaccinated children. RESULTS: The TNC-based VE estimate corrects for confounding bias when the confounder's effects on the probabilities of rotavirus and non-rotavirus related hospitalizations are equal. If this condition is not met, then the estimated VE may be substantially biased. The bias is more severe in low-income countries, where VE is known to be lower. Under our model, differences in PSMC between vaccinated and unvaccinated children do not result in selection bias when the TNC study design is used. CONCLUSIONS: In practice, one can expect the association of PSMC (or other potential confounders) with the probabilities of rotavirus and non-rotavirus related hospitalization to be similar, in which case the confounding effects will only result in small bias in the VE estimate from TNC studies. The results of this work, along with those of our previous paper, confirm the TNC design can be expected to provide reliable estimates of rotavirus VE in both high- and low-income countries.

Epidemiologic challenges in norovirus vaccine development.

Norovirus is the leading cause of acute gastroenteritis (AGE) worldwide. In the United States norovirus is estimated to cause 19-21 million illnesses, 1.7-1.9 million outpatient visits, 56,000-71,000 hospitalizations, and 570-800 deaths annually. Through direct costs and loss of productivity, norovirus disease cost the US economy more than $5.5 billion annually. Due to the lack of available therapies to treat norovirus infections and their highly infectious nature, preventing norovirus illness through vaccination is an appealing strategy. Currently, several norovirus vaccines are in development, including five vaccines in preclinical trials, an oral monovalent vaccine (Vaxart, Inc.) that recently completed a phase IB clinical trial, and a bivalent intramuscular vaccine (Takeda Pharmaceutical Company Limited) in a phase IIB clinical trial. However, no norovirus vaccines are currently available on the market. In this commentary we aim to describe some of the barriers faced in norovirus vaccine development, particularly focusing on vaccine effectiveness and defining the target population.

Preparing for safety monitoring after rotavirus vaccine introduction - Assessment of baseline epidemiology of intussusception among children <2 years of age in four Asian countries.

Intussusception is the invagination of one segment of the bowel into a distal segment, characterized by symptoms of bloody stool, vomiting, and abdominal pain. Previous studies have found regional differences in incidence but the etiology of most intussusception cases is unknown. Rotavirus vaccines were associated with a slightly of increased risk of intussusception in post-licensure evaluations in high- and middle-income countries, but not in low income African countries. To describe the baseline epidemiology of intussusception in young children prior to rotavirus vaccine implementation, active sentinel hospital surveillance for intussusception in children < 2 years of age was conducted in 4 low income Asian countries (Bangladesh, Nepal, Pakistan and Vietnam). Over a 24-month period, 15 sites enrolled 1,415 intussusception cases, of which 70% were enrolled in Vietnam. Overall, 61% of cases were male and 1% (n = 16) died, ranging from 8% in Pakistan to 0% in Vietnam. The median age of cases enrolled ranged from 6 months in Bangladesh and Pakistan to 12 months in Vietnam. The proportion of cases receiving surgical management was 100% in Bangladesh, 88% in Pakistan, 61% in Nepal, and 1% in Vietnam. The high proportion of males and median age of cases around 6 months of age found in this regional surveillance network are consistent with previous descriptions of the epidemiology of intussusception in these countries and elsewhere. Differences in management and the fatality rate of cases between the countries likely reflect differences in access to healthcare and availability of diagnostic modalities. These baseline data will be useful for post-rotavirus vaccine introduction safety monitoring.

Using surveillance and economic data to make informed decisions about rotavirus vaccine introduction.

While rotavirus vaccines are available, safe, and effective, many countries are not yet widely using these vaccines. Surveillance for rotavirus disease and potential vaccine adverse events is critical for country decision making about rotavirus vaccine. This special issue shares rotavirus and intussusception disease surveillance data and rotavirus vaccine cost-effectiveness analyses from countries that have yet to introduce rotavirus vaccines into their routine infant immunization programs. The studies highlight the substantial burden of rotavirus disease and the cost-effectiveness of rotavirus vaccine in a broad set of countries without rotavirus vaccine in their routine immunization programs.

Impact of rotavirus vaccines in Sub-Saharan African countries.

By the end of 2017, 32 (68%) of 47 countries in the World Health Organization's African Region had introduced rotavirus vaccine into their national immunization programs, including 27 countries that received financial support from the Gavi, the Vaccine Alliance. Several early introducing African countries previously evaluated the impact, vaccine effectiveness, and/or cost effectiveness of their routine rotavirus vaccination programs and found that rotavirus vaccine was effective and resulted in substantial declines in hospitalizations due to rotavirus. This Special Issue of Vaccine provides additional rotavirus vaccine effectiveness and impact data from a broader range of African countries, describes the longer term impact and potential indirect benefits of rotavirus vaccination programs, describes trends in circulating genotypes in the pre- and post-vaccine introduction eras, and evaluates the cost-effectiveness of a rotavirus vaccination program in a post-introduction setting. As countries begin transitioning from Gavi support, the findings of these studies provide evidence of the impact and effectiveness of rotavirus vaccination programs under conditions of routine use.

Burden of Severe Norovirus Disease in Taiwan, 2003-2013.

Background: Despite the increasingly recognized role of norovirus in global acute gastroenteritis (AGE), specific estimates of the associated disease burden remain sparse, primarily due to limited availability of sensitive norovirus diagnostics in the clinical setting. We sought to estimate the incidence of norovirus-associated hospitalizations by age group in Taiwan using a previously developed indirect regression method. Methods: AGE-related hospitalizations in Taiwan were identified using International Classification of Diseases, Ninth Revision, Clinical Modification codes abstracted from a national database; population data were provided from the Department of Household Registration Affairs. Population and hospitalizations were aggregated by month and year (July 2003-June 2013) and grouped by age: <5 years, 5-19 years, 20-64 years, and ≥65 years. Monthly counts of cause-unspecified AGE hospitalizations were modeled as a function of counts of known causes, and the residuals were then analyzed to estimate norovirus-associated hospitalizations. Results: Over the study period, an annual mean of 101400 gastroenteritis-associated hospitalizations occurred in Taiwan (44 per 10000 person-years), most of which (83%) had no specified cause. The overall estimated rate of norovirus-associated hospitalizations was 6.7 per 10000 person-years, with the highest rates in children aged <5 years (63.7/10000 person-years). Predicted norovirus peaked in 2006-2007 and 2012-2013. Conclusions: Our study is one of the first to generate a population-based estimate of severe norovirus disease incidence in Asia, and highlights the large burden of norovirus in Taiwan, particularly in children. Predicted peak norovirus seasons coincided with the emergence of new strains and resulting pandemics, supporting the validity of the estimates.

Rotavirus Vaccination Is Associated With Reduced Seizure Hospitalization Risk Among Commercially Insured US Children.

Rotavirus commonly causes diarrhea but can also cause seizures. Analysis of insurance claims for 1773295 US children with 2950 recorded seizures found that, compared to rotavirus-unvaccinated children, seizure hospitalization risk was reduced by 24% (95% confidence interval [CI], 13%-33%) and 14% (95% CI, 0%-26%) among fully and partially rotavirus-vaccinated children, respectively.

Global rotavirus vaccine introductions and coverage: 2006 - 2016.

An estimated 215,000 children died of rotavirus infections in 2013, accounting for 37% of diarrhea-related deaths worldwide, 92% of which occurred in low and lower-middle income countries. Since 2009 the World Health Organization (WHO) recommends the use of rotavirus vaccines in all national immunization programs. This review compares rotavirus vaccine (RV) introductions and vaccine coverage by region, country income status and Gavi-eligibility from 2006-2016. Gross National Income data from the World Bank and surviving infant population from United Nations Population Division was obtained for 2016. Data from WHO were collected on rotavirus vaccine coverage, national immunization schedules, and new vaccine introductions for 2016 while estimated rotavirus deaths were collected for 2013, the last year of available WHO data. As of December 2016, the majority of countries (57%, 110/194) had not introduced universal rotavirus vaccine despite WHO's 2009 recommendation to do so. Countries in the WHO African region had the greatest proportion of introductions (37%, 31/84) by December 2016 and a great majority of these (77%, 24/31) were supported by new vaccine introduction (NVI) grants from Gavi. Almost half (48%) of global introductions were in low and lower-middle income Gavi-eligible and Gavi-graduating countries. Conversely, countries in the Southeast Asia WHO region and those not eligible for Gavi NVI support have been slow to introduce rotavirus vaccine. High-income countries, on average, had poorer rotavirus vaccine coverage compared to low and lower-middle income countries. The over-representation of African countries within the Gavi subset and high estimated rotavirus deaths in these African countries, likely explains why introduction efforts have been focused in this region. While much progress has been made with the integration and implementation of rotavirus vaccine into national immunization programs, 110 countries representing 69% of the global birth cohort had yet to introduce the vaccine by December 2016.

Incidence and Clinical Profile of Norovirus Disease in Guatemala, 2008-2013.

Background: Acute gastroenteritis (AGE) is a leading infectious cause of morbidity worldwide, particularly among children in developing countries. With the decline of rotavirus disease rates following introduction of rotavirus vaccines, the relative importance of norovirus will likely increase. Our objectives in this study were to determine the incidence and clinical profile of norovirus disease in Guatemala. Methods: We analyzed data from a population-based surveillance study conducted in Guatemala from 2008 through 2013. Demographic information, clinical data, and stool samples were collected from patients who presented with AGE (≥3 liquid stools within 24 hours that initiated 7 days before presentation). Estimated incidence of hospitalized, outpatient, and total community norovirus disease was calculated using surveillance data and household surveys of healthcare use. Results: We included 999 AGE hospitalizations and 3189 AGE outpatient visits at facilities, of which 164 (16%) and 370 (12%), respectively, were positive for norovirus. Severity of norovirus was milder than of rotavirus. Community incidence of norovirus ranged from 2068 to 4954 per 100000 person-years (py) in children aged<5 years. Children aged <5 years also had higher incidence of norovirus-associated hospitalization (51-105 per 100000 py) compared with patients aged ≥5 years (0-1.6 per 100000 py and 49-80 per 100000 py, respectively). Conclusions: This study highlights the burden of norovirus disease in Guatemala, especially among young children. These data can help prioritize development of control strategies, including the potential use of vaccines, and provide a baseline to evaluate the impact of such interventions.

Epidemiology of childhood intussusception in Bangladesh: Findings from an active national hospital based surveillance system, 2012-2016.

INTRODUCTION: Rotavirus vaccines have significantly decreased the burden of diarrheal diseases in countries that have introduced them into their immunization programs. In some studies, there has been a small association between rotavirus vaccines and intussusception in post-marketing surveillance, highlighting the importance of tracking incidence before and after vaccine introduction. The objective of this study was to describe the epidemiology of intussusception among Bangladeshi children pre-vaccine introduction. METHODS: We conducted active, hospital-based surveillance for intussusception at 7 tertiary care hospitals with pediatric surgical facilities during July 2012 to September 2016. Hospitalized children under 2years of age were identified according to Brighton Collaboration level 1 criteria for intussusception. The frequency and proportion of intussusception among overall surgical admissions, as well as the demographic and clinical information of the cases is described. RESULTS: Overall 153 cases of intussusception among children <2years-old were identified at participating sites over the enrolment period, confirmed by Level 1 Brighton criteria. These cases represented 2% of all surgical admissions under 2years of age. One hundred twelve cases (73%) were male; the median age was 7months; and the median duration of hospitalization was 7days. One hundred forty-six (95%) children with intussusception required surgery, and 11 (7%) died. CONCLUSIONS: Confirmed cases of intussusception represented nearly 2% of pediatric surgical admissions at tertiary referral centers in Bangladesh during the study period and 7% of children with intussusception died. Given the high burden of rotavirus disease in Bangladesh, vaccine introduction is warranted, however, further studies after introduction of rotavirus vaccine are necessary to determine any association between vaccine and intussusception in this setting.

Preparing for rotavirus vaccine introduction - A retrospective assessment of the epidemiology of intussusception in children below 2 years of age in Nepal.

BACKGROUND: Rotavirus is the most common cause of severe diarrhea in Nepali children, accounting for 25-33% of childhood diarrhea hospitalizations. Two rotavirus vaccines recommended for inclusion in national immunization programs have been associated with a low risk of intussusception in post-marketing studies conducted in several countries. Data on the epidemiology of intussusception hospitalizations are lacking in Nepal. Thus, we aimed to describe the epidemiology of intussusception-associated hospitalizations among Nepali children in preparation for rotavirus vaccine introduction. METHODS: A retrospective review of intussusception hospitalizations for a three year period was conducted at two major pediatric hospitals in Kathmandu, Nepal. Possible intussusception cases were identified through admission, discharge, and operation theater logs and ultrasound registers. Cases with a diagnosis of possible intussusception were selected for medical record review and classified as confirmed if they met the Brighton Collaboration level 1 criteria of diagnostic certainty and the child was aged < 24 months. Data on demographics, clinical course, and outcome were abstracted and analyzed. RESULTS: Eight-five confirmed intussusception cases were identified; most (96%) were confirmed at surgery. The number of intussusception cases peaked between ages 4-7 months; no cases occurred in children 0-2 months. Fifty-nine (64%) case-patients were male. The median duration of symptoms before admission was 2 days (range: 0-14). Abdominal pain, bloody stool, and vomiting were the most common clinical features. All cases underwent surgical treatment; there was only one death. CONCLUSIONS: This is the first study to evaluate the epidemiology of intussusception hospitalizations among children aged < 24 months in Nepal. Because the public health impact of rotavirus vaccination could be substantial in Nepal, where childhood diarrhea-related morbidity and mortality are high, this baseline knowledge of intussusception prior to introduction of rotavirus vaccine in the national immunization schedule will provide useful information for post-vaccine introduction safety monitoring.

National Estimates of Reductions in Acute Gastroenteritis-Related Hospitalizations and Associated Costs in US Children After Implementation of Rotavirus Vaccines.

We compared acute gastroenteritis (AGE)-related hospitalization rates among children <5 years of age during the pre-rotavirus vaccine (2000-2006) and post-rotavirus vaccine (2008-2013) periods to estimate national reductions in AGE-related hospitalizations and associated costs. We estimate that between 2008 and 2013, AGE-related hospitalizations declined by 382000, and $1.228 billion in medical costs were averted.

Potential for a booster dose of rotavirus vaccine to further reduce diarrhea mortality.

Concern has grown that children vaccinated against rotavirus in developing countries may be vulnerable to rotavirus diarrhea in the second year of life due to waning immunity. Adding a booster dose of rotavirus vaccine at 9 or 12 months of age with measles vaccine has been suggested as a strategy to address this. We evaluated the hypothetical potential benefits of a booster dose on reduction of rotavirus mortality. The projected number of deaths averted were calculated using national level full series vaccination coverage, estimated national rotavirus deaths by week of age, and VE at <12 months of age and ≥12 months of age derived from the published literature. We assumed three functional forms of waning based on the VE estimates: stepwise, linear, and logarithmic. We modeled three potential boosting scenarios: (a) reduced VE waning in the second year of life by 50%, (b) reestablished second year of life VE to the levels in the first year of life, and (c) boosted first year VE by 50% of the difference between VE in the first and second years. To express uncertainty resulting from the parameters, each of the nine models were run 1000 times using a random sample of input values. Across all WHO regions, with the stepwise models we estimated a median of 9800 (95%CI: 9400, 10,200), 19,600 (95%CI: 18,800, 20,400), and 29,400 (95%CI: 28,200, 30,700) additional rotavirus deaths averted in the reduced VE waning, reestablished VE, and boosted VE scenarios. These estimates were highly sensitive to the assumed functional form of waning with approximately 65-80% fewer deaths averted if immunity waned in a linear or logarithmic fashion compared to the stepwise model. While these projections will benefit from improved input data points, our resultsinform consideration of booster doses of rotavirus vaccine.

Estimating the full public health value of vaccination.

There is an enhanced focus on considering the full public health value (FPHV) of vaccination when setting priorities, making regulatory decisions and establishing implementation policy for public health activities. Historically, a therapeutic paradigm has been applied to the evaluation of prophylactic vaccines and focuses on an individual benefit-risk assessment in prospective and individually-randomized phase III trials to assess safety and efficacy against etiologically-confirmed clinical outcomes. By contrast, a public health paradigm considers the population impact and encompasses measures of community benefits against a range of outcomes. For example, measurement of the FPHV of vaccination may incorporate health inequity, social and political disruption, disruption of household integrity, school absenteeism and work loss, health care utilization, long-term/on-going disability, the development of antibiotic resistance, and a range of non-etiologically and etiologically defined clinical outcomes. Following an initial conference at the Fondation Mérieux in mid-2015, a second conference (December 2016) was held to further describe the efficacy of using the FPHV of vaccination on a variety of prophylactic vaccines. The wider scope of vaccine benefits, improvement in risk assessment, and the need for partnership and coalition building across interventions has also been discussed during the 2014 and 2016 Global Vaccine and Immunization Research Forums and the 2016 Geneva Health Forum, as well as in numerous publications including a special issue of Health Affairs in February 2016. The December 2016 expert panel concluded that while progress has been made, additional efforts will be necessary to have a more fully formulated assessment of the FPHV of vaccines included into the evidence-base for the value proposition and analysis of unmet medical need to prioritize vaccine development, vaccine licensure, implementation policies and financing decisions. The desired outcomes of these efforts to establish an alternative framework for vaccine evaluation are a more robust vaccine pipeline, improved appreciation of vaccine value and hence of its relative affordability, and greater public access and acceptance of vaccines.

Use of Patients With Diarrhea Who Test Negative for Rotavirus as Controls to Estimate Rotavirus Vaccine Effectiveness Through Case-Control Studies.

BACKGROUND: Case-control studies are often performed to estimate postlicensure vaccine effectiveness (VE), but the enrollment of controls can be challenging, time-consuming, and costly. We evaluated whether children enrolled in the same hospital-based diarrheal surveillance used to identify rotavirus cases but who test negative for rotavirus (test-negative controls) can be considered a suitable alternative to nondiarrheal hospital or community-based control groups (traditional controls). METHODS: We compared calculated VE estimates as a function of varying values of true VE, attack rates of rotavirus and nonrotavirus diarrhea in the population, and sensitivity and specificity of the rotavirus enzyme immunoasssay. We also searched the literature to identify rotavirus VE studies that used traditional and test-negative control groups and compared VE estimates obtained using the different control groups. RESULTS: Assuming a 1% attack rate for severe rotavirus diarrhea, a 3% attack rate for severe nonrotavirus diarrhea in the population, a test sensitivity of 96%, and a specificity of 100%, the calculated VE estimates using both the traditional and test-negative control groups closely approximated the true VE for all values from 30% to 100%. As true VE decreased, the traditional case-control approach slightly overestimated the true VE and the test-negative case-control approach slightly underestimated this estimate, but the absolute difference was only ±0.2 percentage points. Field VE estimates from 10 evaluations that used both traditional and test-negative control groups were similar regardless of control group used. CONCLUSIONS: The use of rotavirus test-negative controls offers an efficient and cost-effective approach to estimating rotavirus VE through case-control studies.