Serum vitamin A levels in respiratory syncytial virus infection

CLINICAL AND LABORATORY OBSERVATIONS

Serum vitamin A levels in respiratory syncytial virus infection K a t h l e e n M a l e t i c Neuzil, MD, William C. G r u b e r , MD, Frank Chytil, PhD, M i l d r e d T. S t a h l m a n , MD, B a r b a r a E n g e l h a r d t , MD, a n d B a r n e y S. G r a h a m , MD, PhD From the Divisionsof Infectious Diseases and Clinical Pharmacology, Department of Medicine; the Divisions of Infectious Diseases and Neonatology, Department of Pediatrics; the Department of Biochemistry; and the Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee

Respiratory syncytial virus causes worldwide e p i d e m i c s of respiratory disease. Of 23 children infected with respiratory syncytial virus, 65% had low serum concentrations of vitamin A during a c u t e illness; these low values were associated with more severe illness. Vitamin A supplementation m a y h a v e a role in the m a n a g e m e n t of infection with respiratory syncytial virus. (J PEDIATR1994;124:433-6)

Respiratory syncytial virus causes worldwide annual epidemics of respiratory disease; the highest morbidity and mortality rates occur in children younger than 12 months of age. 1 In most parts of the world there are no adequate therapeutic or supportive care options. Attention has recently focused on the role of vitamin A in viral infections because it is an essential micronutrient with important roles in immunity and maintenance of normal epithelial cell differentiation.2, 3 Vitamin A deficiency is associated with increased frequency of clinically significant respiratory and diarrheal illnesses and may be a determinant in overall childhood mortality rates. 4 Measles acutely depresses vitamin A (retinol) levels in serum, especially in children younger than 2 years of age. Morbidity and death caused by measles are associated with low vitamin A levels, and their incidence is diminished by vitamin A supplementation.5, 6 This has led the Committee on Infectious Diseases of the American Academy of Pediatrics to

recommend vitamin A supplementation for children 6 months to 2 years of age who are hospitalized with measles and its complications.7 Respiratory syncytial virus and measles virus are both paramyxoviruses that replicate within the respiratory tract and produce respiratory illness. Each can predispose a patient to secondary bacterial respiratory infection. The similarities between the measles virus and RSV led us to evaluate serum vitamin A and retinol-binding protein levels in children infected with respiratory syncytial virus. METHODS

Study population. Children _<12 months of age with RSV infection, respiratory symptoms for less than 4 days when first seen, and hospital stay -->72 hours were studied. The diagnosis of RSV infection was made by enzyme-linked immunosorbent assay of respiratory tract specimens and RBP RSV

Supported in part by U.S. Public Health Service grant No. HL-14214 and Medimmune, Gaithersburg, Md. Dr. Neuzil is supported by National Institutes of Health Training grant No. GM07569. Submitted for publication Aug. 12, 1993; accepted Oct. 14, 1993. Reprint requests: Barney S. Graham, MD, PhD, A-3310 Medical Center North, Divisionof Infectious Diseases, Vanderbilt University School of Medicine Nashville, TN 37232. Copyright © 1994 by Mosby-Year Book, Inc. 0022-3476/94 $3.00 + 0 9/22/52163

I

Retinol-binding protein Respiratory syncytial virus

[

I

confirmed with viral culture. Nineteen of these children were hospitalized between November 1991 and April 1993; they were followed prospectively from day 0 (day of admission) to day 56 after presentation. The remaining four children were clinic patients for whom serum samples were available before, during, and after the diagnosis of RSV infection, Children were classified as at high risk if they had bronchopulmonary dysplasia or other serious chronic lung

433

434

N e u z i l et al.

The Journal o f Pediatrics March 1994

-o

E

o

v

'It--

E

>

v

.-J

C=

-~

c~

B_ rn rr

>

E

E

2

2

09

CO

II

N

CD

Control

0

7

56

Day of Illness Fig. t. Bars depict mean (+_SE) serum vitamin A and RBP levels for control subjects and children with RSV infection at day 0 (presentation) and days 7 and 56 (after presentation). Insufficientsera were available to determine serum RBP levels for all samples; the number of samples for each time point is shown in parentheses. For retinol levels,p <0.0005 on day 0 compared with control subjects and p = 0.002 and p <0.0005 on day 0 compared with days 7 and 56, respectively. For RBP levels,p <0.0005 on day 0 compared with control and p = 0.001 on day 0 compared with day 7.

disease (oxygen supplementation needed for at least 30 days within 6 months of the start of the study), had congenital heart disease, or had been born prematurely (<32 weeks of gestational age) and were younger than 6 months of age at the onset of RSV infection. The control group consisted of 22 healthy age-matched control subjects who were seen for well-baby checkups or elective pediatric surgery. General procedures. Informed consent was obtained from a parent or guardian of each child with the approval of the Vanderbilt Institutional Review Board. Samples of 1 ml of blood were obtained from hospitalized children at presentation and at days 7 and 56 after presentation, and from outpatients between 2 and 3 months after illness. The samples were processed immediately, protected from light, and stored at - 2 0 ° C. Serum vitamin A concentration was determined in duplicate by fluorometry.8 Serum RBP concentration was determined in duplicate by quantitative radial immunodiffusion (LC-Partigen immunodiffusionplate RBP from Behring Diagnostics, Inc., Somerville, N.J.). Nasal wash specimens were obtained from hospitalized patients for quantitative plaque assays on HEp-2 cells 9 on the day 0, and each subsequent day of hospitalization until discharge, or until the virus was no longer detected by culture. Charts were reviewed to assess nutritional status (weight

for age and weight for length), cardiopulmonary status, daily maximum temperature, nutritional intake, and therapeutic interventions (use of antibiotics, bronchodilators, steroids, ribavirin, or immune globulin). The severity of illness was determined by the need for mechanical ventilation. Statistical methods. Statistical analysis was performed by means of a Macintosh StatWorks program. All p values were determined by a Student t test or Spearman rank correlation coefficient. RESULTS Mean age was 4.3 months (range, 0.5 to 12 months) in children with RSV infection and 4.4 months (range, 1 to 12 months) in healthy control subjects. Mean serum vitamin A and RBP levels were significantlylower in infants with RSV infection than in control subjects (p <0.0005 for both). All 23 study patients had vitamin A levels less than the mean control value; 15 (65%) had levels less than 20 #g/dl; and 7 (30%) had levels less than 10 #g/dl. Day 7 and day 56 values for vitamin A and RBP were statistically higher than day 0 values in the study population with RSV infection (Fig. 1). Day 56 levels in children with RSV infection did not differ significantly from control levels (p = 0.2). Low serum vitamin A levels at presentation were associ-

The Journal o f Pediatrics Volume 124, Number 3

N e u z i l et al.

435

60

50 J "13 ::k

40 > (D J r-

30

E

>

20

E 2

19

10

Pre-illness

During illness

Post-illness

Timing of Serum Sample Fig. 2. Each line represents one of the five patients with RSV infection for whom sera were available for testing before, during, and after illness. Four of the fivepatients had acute decreases in vitamin A levelsduring acute infection compared with their preillness control serum samples.

ated with more severe illness, as determined by the need for mechanical ventilation. Of seven children with day 0 vitamin A levels <10 ~g/dl, 6 required mechanical ventilation during their hospitalization, compared with 4 of 16 children with vitamin A levels >10 ~g/dl (p <0.02, Fisher Exact Test). Day 0 vitamin A levels did not correlate with age, temperature, peak viral titer, duration of viral shedding, risk group determined on the basis of cardiopulmonary status, or therapeutic intervention. Preillness sera were available for testing from four clinic patients with RSV infection and from one hospitalized patient. Mean preillness serum vitamin A levels were 31.5 ~g/dl compared with 21.9 #g/d1 during RSV infection (p = 0.1). By 8 to 12 weeks after infection, mean serum retinol levels returned to baseline (mean, 38.0 #g/d0 (Fig. 2). DISCUSSION The association between measles infection and acute hyporetinolemia has been established in both developing and developed countries, although the mechanism has not been well elucidated. 5, I0 Our preliminary study indicates that this phenomenon is not unique to measles infection. The majority of children _<12 months of age had low vitamin A

and RBP levels when they were acutely ill with RSV infection. Low serum vitamin A levels in children with RSV infection were associated with increased severity of illness. Healthy age-matched control children had normal vitamin A levels in accordance with previously published studies,ll as did our clinic patients before RSV infection. The low vitamin A levels in this study likely represent acute hyporetinolemia rather than true vitamin A deficiency. Vitamin A deficiency is rare in the United States. Vitamin A is a fat-soluble vitamin that is stored in the liver as retinyl esters. Because our study patients were sick for less than 96 hours before they were initially seen, poor intake as a result of their acute illness cannot solely account for the depressed levels. Retinol-binding protein transports vitamin A from the liver to peripheral target tissues, and its predominant form in serum is as a retinol-RBP complex.2 The low RBP levels in our study therefore do not reflect total body stores but serve as an indicator of available serum vitamin A. This is demonstrated in samples from clinic patients with RSV infection in whom vitamin A levels were normal before infection but dropped acutely during infection, which also indicates that general malnutrition was not the cause of acute hyporetinolemia in our study population. With mechanical ventilation used as an objective end

436

N e u z i l et al.

point, we found that hyporetinolemia was associated with increased severity of illness; 86% of children with very low vitamin A levels ( < 10 # g / d l ) required mechanical ventilation, compared with 25% of children with levels > 10 ~zg/dl. This association is similar to that for measles. Fever is thought to be a potential cause of reduced serum vitamin A levels during acute measles infection, 12 but in our study elevated temperature did not correlate with low vitamin A levels. The hyporetinolemia of measles infection, and its association with severity of illness, may be related to altered distribution or increased utilization of vitamin A. 6 We postulate that RSV-associated hyporetinolemia may have a similar pathogenesis. Vitamin A is essential in the maintenance of the tracheobronchial epithelium; both measles and R S V infection damage these epithelial surfaces. The body may require additional vitamin A for lung healing and repair. If acute viral infection impairs vitamin A release from the liver, rapid utilization of bioavailable vitamin A for repair of respiratory epithelium may result in the low serum vitamin A levels observed. Vitamin A supplementation after the onset of measles infection has improved both the course of the episode and the case mortality rate, regardless of preexisting vitamin A status. 5 The potential benefits of vitamin A supplementation in R S V infection include more rapid clearing of virus, more rapid healing of the epithelium with less chance for secondary infections, and a more durable immune response. W e conclude that the majority of children who are acutely ill with R S V lower respiratory tract infection have low serum vitamin A levels that are associated with increased severity of illness and spontaneously return toward normal on recovery. Additional studies are needed to determine the pathogenesis of these effects. Vitamin A supplementation would be an attractive therapy for R S V infection because of its low cost, ease of administration, low

The Journal o f Pediatrics March 1994

potential for toxic effects, and the current lack of a vaccine for RSV. We suggest that a randomized, controlled trial is needed. We thank Mark Hunt for technical assistance and Dr. Peter Wright for critical review of the manuscript. REFERENCES

1. Hall CB. RSV. In: Feigin RD, Cherry JD, eds. Textbook of pediatric infectious diseases. Philadelphia: WB Saunders, 1977:1653-75. 2. Chytil F. The lungs and vitamin A. Am J Physiol 1992;6:L51727. 3. Coutsoudis A, Keipiela P, Coovadia HM, Broughton M. Vitamin A supplementation enhances specific IgG antibody levels and total lymphocyte numbers while improving morbidity in measles. Pediatr Infect Dis J 1992;11:203-9. 4. Sommer A, Katz J, Tarwotjo I. Increased risk of respiratory disease and diarrhea in children with preexisting mild vitamin A deficiency. Am J Clin Nutr 1984;40:1090-5. 5. Hussey GD, Klein M. A randomized, controlled trial of vitamin A in children with severe measles. N Engl J Med 1990; 323:160-4. 6. Barclay AJG, Foster A, Sommer A. Vitamin A supplements and mortality related to measles: a randomised clinical trial. BMJ 1987;294:294-6. 7. American Academy of Pediatrics Committee on Infectious Diseases. Vitamin A treatment of measles. Pediatrics 1993; 91:1014-5. 8. Thompson JN, Erdody P, Brien R, Murray TK. Spectrofluorometric determination of vitamin A in serum. Biochem Med Metab Biol 1979;5:67-89. 9. Graham BS, Bunton LA, Wright PF, Karzon DT. Reinfection of mice with respiratory syncytial virus. J Med Virol 1991 ;34:713. 10. Butler JC, Havens PL, Sowell AL, et al. Measles severity and serum retinol (vitamin A) concentration among children in the United States. Pediatrics 1993;91:1176-81. 11. Arrieta AC, Zaleska M, Stutman HR, Marks MI. Vitamin A levels in children with measles in Long Beach, California. J PEDIATR 1992;121:75-8. 12. Rosales FJ, Kjolhede C. Low vitamin A during measles [Letter]. Am J Dis Child 1992;146:1133.