Research priorities for investigation of the influence of vitamin-A supplementation on morbidity

A statement

by the Subcommittee on Vitamin A Deficiency Prevention and Control

The Subcommittee on Vitamin A Deficiency Prevention and Control of the Food and Nutrition Board's Committee on International Programs conducted workshop on Strategies and Priorities for Research on the Influence of Vitamin A Supplementation on Morbidity on 28 April 1988. The purpose of the workshop was to consider the influences of vitamin A on the immune system, the role of vitamin A in the differentiation and maintenance of epithelial tissue, the current research and recent findings on vitamin A and morbidity, and the ethical dimensions of such research.

This report was written by the subcommittee subsequent to the workshop and represents the consensus of its members. It does not give equal weight to all hypotheses considered at the workshop but emphasizes those relating vitamin A to infections within the context of field studies. In addition, the report includes a detailed discussion of methods of assessment of vitamin-A status. It does not discuss the ethical issues in any great detail because these were discussed very thoroughly in its previous report and in Dr. Porter's paper below (pp. 36-40).

The following hypotheses were addressed at the workshop:

• Marginal vitamin-A deficiency adversely affects the integrity of epithelial tissue.
• Marginal vitamin-A deficiency lowers resistance to infection by affecting the immune system.
• Marginal vitamin-A deficiency is a risk factor for the increased incidence and severity of diarrhoeal and respiratory infections.
• Vitamin-A supplementation to populations in which vitamin-A status is marginal increases immunocompetence and reduces the incidence and severity of diarrhoeal and respiratory infections.

The word marginal is used to describe the range of vitamin-A deficiency of interest to the subcommittee; it refers to the subclinical stage preceding the appearance of night blindness and the conjunctival and corneal changes characteristic of xerophthalmia. Although it is difficult to define marginal vitamin-A deficiency adequately, it is nonetheless clear that in countries where xerophthalmia is a significant public health problem a large proportion of children are afflicted with marginal deficiency. The World Health Organization considers prevalences of corneal xerosis and keratomalacia (X2 + X3A + X3B)* in excess of 0.01% to indicate a significant public health problem affecting large numbers of children [1]. In such areas, many more children will show less severe clinical signs (X1A, X1B) and night blindness (my), and even more will have low plasma vitamin A, low vitamin-A stores in the liver, and vitamin-A-deficient diets. Xerophthalmia is known to be associated with diminished host defences and increased risk of infection. Similarly, marginal vitamin-A deficiency may lower resistance to infection, a possibility that needs to be confirmed and quantified through adequately designed studies. Because so many children are likely to be affected by marginal vitamin-A deficiency, the study of this condition and its implications constitutes a significant research priority.

The subcommittee's first report considered effects of vitamin A on mortality and provided advice on the conduct of field studies designed to test the hypothesis that vitamin-A supplementation lowers child mortality rates [2]. Mortality studies require very large samples in order to achieve satisfactory statistical power.

The study of morbidity related to vitamin A is more feasible since infections occur more frequently and fewer subjects need be studied. Mortality and morbidity studies are complementary. The demonstration of an effect of vitamin-A supplementation on the incidence and severity of gastrointestinal and respiratory infections would strengthen the persuasiveness of findings on mortality because such a demonstration would provide a plausible pathway leading to the mortality effects. Similarly, the demonstration of effects of marginal vitamin-A deficiency on epithelialtissue differentiation and maintenance and on immunocompetence would further validate both morbidity and mortality findings.

Research gaps

The subcommittee has assessed progress to date in regard to morbidity effects and has determined that further research is required. Salient research gaps are as follows.

Immuncompetence

Vitamin-A deficiency both in man and in laboratory animals impairs immunological responses [3]. Although not all of the immune responses have been tested in man, the consensus of results in published studies indicates that the following parameters are altered. Reduced vitamin-A intake and low serum-rehnol concentrations are associated with decreased delayed hypersensitivity response, lower serum-antibody response to some antigens, decreased lymphocyte response to mitogens, decreased natural-killer-cell activity, delayed rejection of grafts, changes in complement level, and phagocyte dysfunction. Several published reviews are available [4-6]. In animals deficient in vitamin A, morbidity and mortality increased after infectious challenge 13]. The significance of these findings in humans remains to be established.

In general, changes in immunocompetence often precede obvious infection and can be viewed as a functional index of nutritional deficiency. Changes in immune responses may mediate heightened susceptibility to infection in populations deficient in vitamin A.

More information should be obtained through research in the following areas:

• epithelial-tissue integrity and barrier function in vitamin-A-deficient individuals, particularly in marginal stages of deficiency, including examination of the effect of vitamin-A supplementation;
• the pattern of recovery of immunocompetence following either small, frequent physiological amounts of vitamin A or massive pharmacological doses of vitamin A;
• secretory immunoglobulin and cell-mediated immune responses to immunization and the extent of protective immunity achieved in vitamin-A-deficient groups;
• investigation of the mechanism(s) by which vitamin-A deficiency alters immunocompetence;
• examination of the quantitative and temporal relationship between vitamin-A status and different indices of immune function.

Morbidity

The literature suggests that marginal vitamin-A deficiency is associated with increased incidence or severity of infections (or both). The evidence seems to be much stronger for respiratory than for gastrointestinal infections. The interpretation of the results to date is difficult because studies have failed to fully document vitamin-A status or to control for factors associated with both vitamin-A deficiency and the risk of infection.

An example is the need to control for environmental sanitation. Vitamin-A-deficient children may be from lower-income families with poorer housing and sanitary facilities. Children with adequate vitamin-A status, even when living in the same communities, may be from better-off homes. Thus, failure to control for environmental sanitation would overestimate the importance of vitamin A for morbidity. Similarly, vitamin-A deficiency generally coexists with other nutritional problems that are thought to affect the risk of morbidity. Studies of marginal vitamin-A deficiency and morbidity must at least control for protein-energy malnutrition and other deficiencies such as of zinc and iron.

Further research is recommended in the following areas:

• Identification of the extent to which the incidence and severity of infections are affected by marginal vitamin-A deficiency.
• The relation of marginal vitamin-A deficiency to specific types of morbidity: What types of morbidity are most affected by vitamin-A deficiency? What is the natural outcome of commonly occurring infections in the community and the role of immunocompetence in mediating the outcome? Are there different effects of vitamin-A supplementation or depletion on different infections - for example, on measles, rotavirus, and bacterial or viral pneumonias?
• Interaction of vitamin-A status with other deficiencies (for example, protein-energy, zinc, and iron) in regard to effects on morbidity.
• Interaction of vitamin-A status with factors related to socio-economic status, such as level of exposure to infection and access to health services.
• Effectiveness of massive doses of vitamin A versus lower doses given daily or weekly, adverse effects of massive doses, and mechanisms responsible for these different effects.

Indicators of vitamin-A status

In its first report [2], the subcommittee noted the importance of adequately characterizing the vitamin-A status of study populations. Such data will allow for stratification by vitamin-A status if required in the data analysis and will increase flexibility in extrapolating the results to other populations. The report also noted the problems in the measurement of vitamin-A status that require further methodological work:

• Biochemical indicators: How reliable and accurate are serum concentrations of vitamin A as indicators of overall vitamin-A status? How useful is the relative dose-response test?
• Cytological indicators: How useful is impression cytology as an index of vitamin-A status? Can buccal cytology be used instead of conjunctival cytology?
• Clinical history: How accurate are historical reports of night blindness?
• Dietary histories: How well do dietary histories for consumption of vitamin-A-rich foods actually correlate with vitamin-A intake and vitamin-A status?

Vitamin-A status is determined by total body stores and can be thought of in relative terms as deficient, depleted, adequate, excessive, or toxic. Deficient, excessive, and toxic concentrations are manifested by clinical signs and symptoms or by biochemical measures interpretable both for individuals and populations. Quantitative measurement of intermediate levels of vitamin A (depleted and adequate) is problematic and in need of research.

Vitamin-A status is also affected by absorption and other factors that increase metabolism, including infection and protein-energy malnutrition. Such factors should be considered in the design of studies to investigate the impact of vitamin A on morbidity.

Field-applicable indicators of marginal vitamin-A status currently recognized include serum concentrations, the relative dose-response test, and conjunctival-impression cytology. Each of these has limitations in feasibility and practicality in the field as well as in quantitative interpretation. Detailed discussions of methodologies for clinical histories and dietary histories are not specific to studies of vitamin A and morbidity and thus will not be discussed in detail here.

Serum concentrations

The interpretation of serum concentrations of vitamin A as reflectors of status on an individual and population basis have been discussed in a previous publication [2]. In the past, the interpretation of population distribution curves was largely based on comparisons with non-representative population surveys from developing countries from which cutoff points for deficient, low, and acceptable levels were derived [7]. Recently available are age- and sex-specific distribution curves for representative, relatively well nourished populations living in the United States [8; 9]. Although these reference curves appear to be independent of ethnic differences, they are influenced by environment [10; 11], and their usefulness as a universal reference needs to be validated. The most appropriate reference curve for serum levels for field studies of vitamin A and morbidity would be from a representative population known to have adequate body stores while living in an environment characteristic of an at-risk population. Studies are needed to establish appropriate reference curves for populations residing outside the United States and in high-risk areas. To obtain such references, distribution curves established before and repeated after large-dose supplements - for example, 30 days after a 200,000IU supplement - would be appropriate. These curves could then be compared with the US reference curves to determine whether there are differences.

Relative dose-response test

Whereas to demonstrate a rise in homeostatically controlled serum concentrations in response to increased intake or supplementation requires serially obtained samples with an intervening period for stabilization, the relative dose-response (RDR) test can detect depleted stores with only a five-hour interval between dosing and sampling [12; 13]. This test, modified to suit various conditions, has been successfully applied in both clinical and field-survey conditions. It has been validated by an intravenous procedure for dosing coupled with direct liver biopsy in a limited number of children with liver disease and adults [14;15]. The number of direct quantitative validation studies needs to be expanded to obtain greater assurance as to the range and confidence intervals of the association of a positive RDR test with vitamin-A concentrations in the liver. However, this can be accomplished only under specialized clinical circumstances, not in field studies in developing countries. Indirect validation with a before-and-after supplementation procedure also needs to be extended to population groups in which heavy intestinal parasitism, malabsorption, and protein-energy malnutrition are common. In addition, a proposed adaptation of the RDR method using didehydroretinol (DR, or vitamin A₂), a natural derivative of retinal (vitamin Al, the common form of vitamin A in foods and formed in the gut from carotenoids) [16], needs to be tested in human populations. If the DR adaptation proves reliable in human populations, only a single blood sample after five hours might be required, making the test much more feasible for field morbidity studies [17]. The RDR approach to assessment of depletion requires additional validity tests under field conditions to verify that mild to moderate protein-energy malnutrition, infections, or other conditions that may lower absolute serum concentration do not impede this short-term response and hence limit the applicability of the test for reliably indicating depletion of body stores.

Conjunctival-impression cytology

The histologically based conjunctival-impression cytology (CIC) test requires fewer logistical and technological resources than a direct biochemical determination of vitamin A. In theory, this increases its practicality for field studies in developing countries [18|. Validation against liver biopsies and the RDR test has been reported in a clinical setting among a very limited number of children with liver disease [19]. These studies suggest that the absence of goblet cells in the impressions corresponded to a liver concentration of <20 microg per gram, or a positive RDR, or both. Additional quantitative validation studies are needed but, as with the RDR, can be accomplished only under special clinical conditions. In a larger field study conducted in Guatemala, the CIC evaluated against the RDR was reported to lack sensitivity although it was specific [20; 21]. This field validation trial needs to be repeated. The effect of potential confounders such as concurrent eye infections, e.g. conjunctivitis and trachoma, should also be studied. The report of a strong linear correlation between abnormal CIC and relative plasma concentrations is not consistently reported by others under clinical or field conditions and needs confirmation [18]. Interpretation of the histological picture relative to degrees of vitamin-A depletion needs to be standardized.

Research needs in relation to vitamin-A assessment are as follows:

• Population reference curves of serum vitamin-A concentrations should be validated for developing countries.
• The relative dose-response test should be further validated under appropriate field conditions.
• Field trials to validate the conjunctival-impression cytology technique should be repeated.
• Standardization of the interpretation of histological findings for conjunctival-impression cytology relative to vitamin-A status is needed.

Indicators of nutritional status

The following issues related to indicators of nutritional status should be considered in designing studies of vitamin A and morbidity:

• What is the nature of the relation among nutritional status indicators and morbidity and immunocompetence? Is the relation linear, or are thresholds evident?
• While wasting is known to be related to increased risk of morbidity, the relation with stunting is less clear and should be explored further. In particular, studies should assess whether the process of stunting (linear growth retardation) is associated with diminished immunocompetence and increased morbidity.
• How should feeding histories be recorded, and which variables are most essential (i.e., duration of exclusive breast-feeding, type and timing of supplementary feeding, and duration of breastfeeding)?

Indicators of morbidity

• What are the validity and reliability of different methods of morbidity measurement?
• What is the definition of a "case" or episode?
• What are the diagnostic criteria for causal agents?

Measures of immunocompetence

The following measures of immunocompetence may be considered in the evaluation of vitamin-A-deficient individuals and populations before and after any proposed intervention. The rationale for choosing these four tests is based on the consistency of abnormalities observed in published studies and will be discussed further in the subcommittee's next report. The ultimate choice will be dictated by the nature of the study and practical considerations such as the availability of laboratory facilities and costs.

• delayed hypersensitivity,
• lymphocyte response to mitogens,
• salivary-immunoglobulin-A concentration,
• natural-killer-cell activity.

It would be important to role out the confounding effect of concurrent or recent infection on the basis of clinical findings and estimation of complement-reactive protein and endotoxins in the blood.

Strategies for research

The subcommittee recognizes that no single research design and set of procedures can or should be followed in a variety of settings in different countries with differing social contexts and resource limitations that affect feasibility. Nonetheless, the subcommittee wishes to present some guidelines that may prove useful in designing future studies.

Choice of population

It is desirable to test the major hypotheses in populations with various levels of vitamin-A nutriture.

Choice of deign

The subcommittee strongly recommends that double-blind randomized studies with placebo controls be used where feasible (as always, with due consideration for scientific and ethical issues). A controlled study is likely to yield information of greater scientific validity than a non-experimental study (e.g. a case-control study). Thus, experimental studies are preferable for unequivocally testing the hypotheses of interest.

In its previous report [2], the subcommittee reviewed theoretical and practical aspects of controlled trials. It noted, for example, that baseline comparability is ensured on average by random assignment of treatment and control interventions. The choice of the level of randomization - by individual, household, or community - is a matter of balancing considerations of study size with feasibility. If cluster sampling is used, appropriate statistical methods should be followed.

The likelihood of strict baseline comparability can be improved by randomization within strata on factors that relate to the outcome variables and that vary substantially among units to be randomized. Adequate stratification increases the precision of estimates, but must be weighed against the added field work that may result.

If strict comparability at baseline is not achieved in unstratified studies, adjustments are still possible during data analysis either by stratification or by regression analyses. This is possible, of course, only if the appropriate data have been collected at baseline.

The subcommittee recognizes the value of non-experimental studies and believes that they should be conducted where experiments are not feasible. Case-control studies, in particular, seem appropriate for studies of marginal vitamin-A status and risk of morbidity.

Case definition for such studies may present problems, however. If a case is defined as a child with diarrhoea at a given time and a control as a child without, the definition of a case will be weak as a result of the ephemeral nature of diarrhoea. Case definition could be improved by collecting histories of past infections; recall biases should be dealt with on the basis of the type of data. Another possible approach would be to select cases on the basis of marginal vitamin-A status, identify matched controls, and measure outcome through longitudinal follow-up. Such a design raises ethical issues related to the failure to treat those with identified clinical conditions. The subcommittee concurs with Feachem [22], who holds that such studies are unethical.

A major problem in case-control studies is the difficulty of satisfactorily matching for characteristics known to affect both the outcomes and vitamin-A status in the same direction. Case-control studies carried out to date have been equivocal because of poor matching. Variables to be matched should include age, sex, anthropometric and socio-economic status, nutritional status (anthropometric indicators of protein-energy malnutrition, indicators of iron and zinc deficiency, and other indicators where appropriate), environmental sanitation, and access to health care.

Content of the proposal

Below are listed issues which should be addressed in the design of proposals for future studies of vitamin-A supplementation and its relation to morbidity.

Specify the hypothesis

Research proposals should clearly state whether the hypothesis is to test the effect of marginal vitamin-A status on morbidity or to test the effect of vitamin-A supplementation on morbidity outcomes. If the hypothesis concerns testing the effect of marginal vitamin-A status, quantitative measures of depletion of vitamin A will be needed. The stated hypothesis should also indicate whether there is any intent to test the mechanisms by which vitamin-A deficiency or supplementation may lead to changes in morbidity - for example, by changes in immunocompetence. It should also state whether the intent is to study only morbidity per se or also severity of illness.

Specify sampling considerations and population description

• What type of population will be studied? What information is available that may be significant for vitamin-A studies?
• Is randomization to be done on the basis of the village or on an individual level?
• If randomization is on the level of village or cluster, how will cluster sampling affect estimates of sample size?

Specify expected effects

• What magnitude of effect on incidence or duration of morbidity is expected from vitamin-A supplementation? Is there any experimental basis for this estimate?
• How would different assumptions about the expected magnitude of the impact on morbidity affect sample size?

Specify methods

• Morbidity experience (for example, how will the frequency and duration of diarrhoea or respiratory disease be quantified?). Will attempts be made to identify causative agents?
• Data-collection plans.
• Indicators to be used for vitamin-A status (biochemical, clinical, dietary).
• Nutritional status (including feeding history, dietary history, and protein-energy, zinc, and iron nutritional status).
• Socio-economic status (for example, income, occupation, possessions, housing, education, exposure to infections, and access to services).
• Immunization status for immunizable diseases (especially measles).
• Intervening mechanisms such as immunocompetence.

Describe quality-control and data-management procedures

• What quality-control procedures will be implemented to ensure adequate coverage of the targeted population and satisfactory data quality?
• What data-management procedures will be used to ensure the availability of adequate information for analysis?

Specify the analysis plan

This should detail the specific methods to be used to test the study hypotheses and should, in addition, address the following issues:

• How will the analysis adjust for possible confounding factors such as nutritional status and socioeconomic status?
• What indicators will be used to quantify the key variables in the analysis (for example, vitamin-A status, nutritional status, socio-economic status, morbidity status)?
• Which indicators will be treated as dichotomous variables and which as continuous variables?
• If cluster sampling is used, how will adjustments be made for design effects when calculating confidence intervals?
• How will the analysis deal with multiple data points collected on the same individual? For example, if height and weight data are collected at several points, which measurements or combinations of measurements will be used to define nutritional status?
• What assumptions will be made in the statistical analysis, and what will be the effect of likely deviations from these assumptions?

This report is intended to provide guidance for developing programmes to assess the impact of vitamin A on child morbidity. It also gives an overview of the research priorities in this area, focusing on field studies of the impact of vitamin A on infectious diseases.

Subcommittee on Vitamin A Deficiency

Prevention and Control

Reynaldo Martorell (Chairman), Food Research Institute, Stanford University, Stanford, California

Abdelmonem A. Afifi, School of Public Health, University of California, Los Angeles, California

Guillermo Arroyave, School of Family and Consumer Sciences, San Diego State University, San Diego, California

Ranjit Kumar Chandra, Directory of Immunology, Memorial University of Newfoundland and Janeway Child Health Centre, St. John's, Newfoundland, Canada

Frank Chytil, Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee

Samuel Preston, Population Studies Center, University of Pennsylvania, Philadelphia, Pennsylvania

Mervyn W. Susser, Columbia University, New York, New York

Frederick Trowbridge, Division of Nutrition, Center for Health Promotion and Education, Centers for Disease Control, Atlanta, Georgia

Barbara A. Underwood, National Eye Institute, National Institutes of Health, Bethesda, Maryland

Virginia H. Laukaran, Staff Officer, Food and Nutrition Board

Susan Berkow, Staff Officer, Food and Nutrition Board

Frances Peter, Deputy Director, Food and Nutrition Board

Jean Shirhall, Editor

References

1. World Health Organization. Control of vitamin A deficiency and xerophthalmia. Report of a joint WHO/ UNICEF/USAID/Helen Keller International/IVACG meeting. WHO technical report series, no. 672. Geneva: World Health Organization, 1982.
2. National Research Council-National Academy of Sciences, Food and Nutrition Board. Vitamin A supplementation: methodologies for field trials. Report of the Subcommittee on Vitamin A Deficiency Prevention and Control. Washington, D.C.: National Academy of Sciences, 1987.
3. Beisel WR. Single nutrients and immunity. Am J Clin Nutr 1982;35:417-68.
4. Chandra RK, ed. Nutrition and immunology. New York: Alan R. Liss, 1988.
5. Keusch G. Nutrition and infection. In: Remington JS, Schwartz MN, eds. Current clinical topics in infectious diseases. New York: McGraw-Hill, 1984:106-23
6. Vyas D, Chandra RK. Vitamin A and immunocompetence. In: Watson RR, ed. Nutrition, disease resistance and immune function. New York: Marcel Dekker, 1984:325-44.
7. Arroyave G. Chichester CO, Flores H. et al. Biochemical methodology for the assessment of vitamin A status. Washington, D.C: Vitamin A Consultative Group, Nutrition Foundation, 1982.
8. Pilch SM, ed. Assessment of vitamin A nutritional status of the US population based on data collected in the Health and Nutrition Examination Surveys. Prepared for the Center for Food Safety and Applied Nutrition. US Food and Drug Administration Contract No. FAD 233-84-2059. Bethesda, Md, USA: Life Sciences Reseach Office, Federation of American Societies for Experimental Biology, 1985.
9. Pilch SM, ed. Analysis of vitamin A data from the Health and Nutrition Examination Surveys. J Nutr 1987;117:636-40.
10. Looker AC, Johnson CL, Woteki CE, Yetley EA, Underwood BA. Ethnic and racial differences in serum vitamin A levels of children aged 4-11 years. Am J Clin Nutr 1988;47:247-52.
11. Looker AC, Johnson CL, Underwood BA. Serum retinal levels of persons ages 4-74 years from three Hispanic groups. Am J Clin Nutr 1988;48: 1490-96.
12. Flores H. Campos F. Araujo CRC, Underwood BA. Assessment of marginal vitamin A deficiency in Brazilian children using the relative dose-response procedure. Am J Clin Nutr 1984;40:1281-89.
13. Loerch ID, Underwood BA, Lewis KC. Response of plasma levels of vitamin A to a dose of vitamin A as an indicator of hepatic vitamin A reserves in rats. J Nutr 1979;109:778-86,
14. Amédée-Manesme O. Mourey MS, Hanck A, Therasse J. Vitamin A relative dose response test: validation by intravenous injection in children with liver disease. Am J Clin Nutr 1987;46:286-89.
15. Amédée-Manesme O. Anderson D, Olson JA. Relation of the relative dose response to liver concentrations of vitamin A in generally well-nourished surgical patients. Am J Clin Nutr 1984;39:898-902.
16. Tanumihardjo SA, Olson JA. A modified relative dose-response assay employing 3,4-didehydroretinol (vitamin A2) in rats. J Nutr 1988;118:598-603.
17. Olson IA. Indicators of vitamin A status. Bull Xerophthalmia Club 1988;38:3.
18. Natadisastra G. Wittpenn JR, West KP, Muhilal, Sommer A. Impression cytology for detection of vitamin A deficiency. Arch Ophthalmol 1987;105:1224-28.
19. Amédée-Manesme O. Luzeau R. Wittpenn IR, Hanck A, Sommer A. Impression cytology detects subclinical vitamin A deficiency. Am J Clin Nutr 1988;47:875-78.
20. Gadomski AM, Kjolhede CL, Wittpenn J. Bulux 1, Rosas AR, Forman MR. Conjunctival impression cytology (CIC) to detect subclinical vitamin A deficiency: comparison of CIC with biochemical assessments. Am J Clin Nutr 1989;49:495-500.
21. Kjolhede CL, Gadomski AM, Wittpenn J. et al. Conjunctival impression cytology (CIC): feasibility of a field trial to detect subclinical vitamin A deficiency. Am J Clin Nutr 1989;49:490-94.
22. Feachem RG. Vitamin A deficiency and diarrhoea: a review of interrelationships and their implications for the control of xerophthalmia and diarrhoea. Trop Dis Bull 1987;84:RI-R16.