Journal of Acauied mmine Deficiency Sydrmes and Human Rervirolgy 30-515 61599 Lipinct Willan & iki, ne Pte Mortality Risk in Selenium-Deficient HIV-Positive Children *Adriana Campa, *Gail Shor-Posner, *Fernando Indacochea, *Guoyan Zhang, *Hong Lai, Deshratn Asthana, *Gwendolyn B. Scott, and *Marianna K. Baum *Center for Disease Prevention, Department of Psychiatry and Behavioral Sciences, }Department of Pediatrics, and ‘sDepartment of Medicine, University of Miami School of Medicine, Miami, Florida, US.A. Objective: To determine the independent contsibution of specific nutritional Factors ‘on disease progression and survival in HIV-1-infected children. Population: HIV-infected children (N = 24), who were perinatally exposed to the virus and symptomatic, were recruited between October and December of 1990 from the Jackson Memorial Pediatric Immunology Clinic, Miami, Florida, and observed for 5 years. ‘Methods: Immune status was measured by CD4 cell count; nutritional status was determined using serum albumin and plasma trace elements including iron, zinc, and selenium, Cox proportional hazards regression models were used to evaluate the te lationship of these parameters to survival. Use of antiretroviral treatment was consid: ered in the statistical model, and age at death was considered a parameter of disease progression. Results: Over the course of the study, 12 children died of HIV-related causes, The final Cox multivariate analysis indicated that, ofthe variables evaluated, only CD4 cell count below 200 (risk ratio [RR] = 7.05; 95% confidence interval {CI}, 1.87-26.5); p 004}, and low levels of plasma selenium (RR = 5.96; 95% Cl, 1.322681; p (02) were significantly and independently related to mortality. Among the children ‘who died, those with low selenium levels gesting more rapid disease progn Conclusions pendent predictor of mortal ‘gression. Key Words: Selenium —HIV—Survival—Progression ‘The World Health Organization and UNAIDS esti- mate that 1.1 million children are living with HIV/AIDS worldwide, >90% of them in developing countries. In 1997 alone, 590,000 children (age <15. years) were newly infected with the virus, and 460,000 died from AIDS or AIDS-related conditions. Approximately 2.7 million children have died since the beginning of the pandemic (1). In developing countries, aggravated by ‘maternal disability or death, widespread malnutrition, and lack of antiretroviral therapy, HIV infection often ‘Adkess correspondence and reprint requests to Adriana Campa Division of Metabolism and Disease Prevention, Department of Psy chiatry and Behavioral Sciences, 1400 NW 10th Avenue, 10th Floor, University of Miami, School of Medicine, Miami, FL 33136, US.A. “Manuscript received October 27, 1998; accepted January 5, 1999 In pediatric HIV-infectio 508. 5 g/L), died at a younger age, sug- low plasma level of selenium is an inde- and appears to be associated with faster disease pro- Pediatrics progresses quickly to AIDS in children. The U.S. Bureau Of the Census estimates that by the year 2010, if the HIV epidemic is not contained, it may increase infant mortal- ity by as much as 75%, and mortality in children under 5 by more than 100%, erasing the great strides made by many developing countries in infant and child survival (2), It has been suggested that early and aggressive nu- tritional intervention in HIV-infected children, especially in populations without access to retroviral therapy, may help prevent malnutrition and wasting, characteristic symptoms of this disease (3). Whereas adequate nutrition is important for healthy growth and development in ev- ery child, in pediatric HIV-infection, with its increased, nutrient needs, routine nutritional monitoring and the es- tablishment of individualized goals are essential for ef-MORTALITY RISK IN SELENIUM-DEFICIENT HIV-POSITI fective nutritional management (4). Research on the role of micronutrients in optimizing nutrition in pediatric HIV infection, although documented in adults, is limited in children. Considerable evidence indicates that specific nutri tional deficiencies are associated with HIV-1 disease progression (5-8) and increased HIV-1 related mortality in adults (9,10). Results from our recent study in HIV-1 seropositive drug-abusing men and women indicate that nutritional deficiencies are markers of disease pro- aression, and that selenium, a trace element, is strong and independent predictor of survival in HIV-1 infec- tion (11). Other findings indicate that nutrient abnormal ties, known to influence the immune system, are also present in HIV-infected children (3). These findings cor- roborate our previous results thus demonstrating a sig- nificant association between nutritional status and im- mune dysfunction in HIV-1-infected children (12). In light of evidence linking nutritional status to disease pro gression and mortality, our present investigation was conducted to determine the relationship between trace element status and HIV-related mortality in infected chil- dren MATERIALS AND METHODS The studies were conducted in acordance with the standards ofthe Universiy of Miami School of Medicine andthe National Institutes of Health. The study group was composed of 24 children (10 boys, 14 srl) Participants were recruited consecutively fom the peitrie HIV ‘outpatient clinic at Jackson Memorial Hospital in Miami, Florida, and ‘informed consent was obtained from the parents or guardians ofall individuals. Children recruited forthe study needed to he HIV positive by antibody testing, pernaally exposed tothe virus, an atthe symp. tomatic disease stage (P2) at the time of recruitment. Selection of participants by disease stage and ability « confiem HIV infection seat limited the size ofthe study group. Baseline immunologic pa rameters and notrtional measurements were obtained between October and December 1990, At the beginning of the study, 83% ofthe eildren were receiving antiretroviral treatment (e.g, zidovudine [ZDV)) Immunologic Determinations Immune assessment included measurements of lymphocyte sub Populations (CD4 counts). Flow eytomerty and monoclonal antibodies were employed to quantitate numbers of circulating T-helpefinducer (CDF) cell numbers. Considering pediatric differences in the levels of (CDA counts at which signs of severe immunosuppression are man fested, levels under 200 cell counts were selected as eu-off point, CD$ counts <200 cells/mm’ represent severe immunodeficiency in both in fans and school-aged children (13). Nutritional Status ‘enous blood samples were obtained from the left antecubital vein after approximately 4 hours of fasting. Blood was collected in hepa E CHILDREN 509 rinized trace element-fiee tubes. Evidence of malnutrition was eval ated using measurements of serum albumin determined by standard nephelometric procedures (Id), Albumin levels under 3.5 gd) were ‘considered t be inadequate, ‘Trace Elements Analyses were performed using atomic absorption spectrophotom- cry with graphite furnace attachment to determine levels of iron and Zine (15). All instrument readings were performed in concentration mode using appropriate standards, All assays were performed in du- plicate, and supplies and glassware regularly monitored for trace ee Tron Levels <40 15/100 mil were considered tobe deficient for this pop lation, Zine for ne was considered tobe 76 0 150 js/100 ml, With deficient status =75 p/100 a Selenium Selenium values were determined by a standardized fluorometric method, vit selenium reference, obtained from the National Institute of Standards and Technology (Standard Reference material 1988), us- ing bovine serum (16). Based on previous findings in the HIV. infected pediatric patients, selenium levels >85 g/L were considered adequate (3.12), Statistical Analysis (Cox proportional hazards regression models were used wo investigate the association of micronutrient deficiency on survival in HIV-I~ infected children. The endpoint was HIV-I-relaed death, Survival time was defined as time elapsed from date of recruitment to date of death, or date when the study was closed Gly 30, 1996) Kaplan Meier (product limit) estimator was used to estimate the survival time distributions, anda logank statistic was employed to test equality of| survival functions, To evaluate the relationship of nutrient deficiency, Immunologie parameters, and retroviral therapy t survival univariate analysis was used. Those factors that exhibited high RR in the in ‘model were jointly entered into a final model. The p values presente are two-sided. Neither nutritional plasma levels (albumin, icon, and ificant association inc), nor antiretoviral treatment showed a si ‘ith survival inthe initial model, and were excluded from the final mode ies The children included in the study were predominantly black (87.5%), with 8% Hispanic and 4% white non- Hispanic. OF the children, 58% were girls, and 42% boys. The mean age of the total group at the time of Journal of Acquired Immune Deficleney Syndromes and Haman Retroiology, Vol 20, No S. April 15, 1989510 A. CAMPA ET AL. TABI | Group characterises Sunived over Died during the course of the the 5.5 year Atrecrutment 55 year study sud =m 12) = 12) c ‘Age at recruitment (y) ‘Mean 42422 Median 44 Range 293) 19-53) Gender (%) Male 2% 43% 42% Female 58% 58% 58% Race (%) White L 8% 178% Block 87.50% on 83% Zidovudine Use (%) Yes 92% No. recruitment was 4.2 + 2.2 years, (age range, 1.2-9.3 years). Most participants were from families with in- ‘comes below poverty levels, who received benefits from, the welfare program, food stamps, Medicaid, Immigra- tion and Naturalization Services (INS), Health and Re~ habilitative Services (HRS), and private funds for AIDS. patients. As shown in Table 1, no significant differences in demographic characteristics and use of retroviral medication were found between the children who died, ‘and those who survived during the study period. Nutritional Status and Mortality At the end of the observational period, in July 1996, 12 of the original 24 children were still alive. Through reviews of clinical and hospital records by their attending physician, time and cause of death of the 12 children who had died were determined, and HIV-related deaths con- firmed. Cox proportional hazards models, which controlled for baseline CD4 count <200 cellsvmm’, a parameter of ad- vanced disease, were used to determine the relation bx tween specific micronutrient deficiency and mortality. ‘As shown in Table 2, neither levels of albumin, iron, zine, nor antiretroviral treatment was significantly asso- ciated with mortality. Selenium was significantly asso- ciated with mortality in the initial model (RR = 4.19; 95% Cl, 1.17-20.3; p = 03), and was included in the final model, in addition to CD4 count <200 celis/mm* (RR = 6.33; 95% Cl, 1.54-26.0; p = 01). In the final model, CD4 cell count (RR CI, 1.87-265; p = .004), and selenium deticien 5.96; 95% Cl, 1.32-26.81; p = .02) were signifi- cantly associated with mortality. As shown in Figure 1 survival time for the whole group, measured from date of Journal of Acquired Immane Deficiency Syndromes and Human Reroioly, Vol 20, No.5. April 1, 1998 recruitment, was significantly shorter in children who ‘were selenium deficient relative to those who were sele- nium adequate (p = .017, log-rank test). The 25th per- centile of survival time for the selenium-deficient chil dren was 356 days (95% Cl, 256-688) compared with 1020 days, (95% Cl, 474°) in the selenium-adequate group. ‘When only those children who had died (NV = 12) during the study period were considered, as shown in Figure 2, selenium-deficient children (=85 g/L) died at a younger age (median, 4.3 years; range, 2.6-6.7 years: p = 03), and were rapid progressors, compared with chil- dren (n = 6) whose selenium levels were adequate, and who died at a median age of 8 years. DISCUSSION Significant relationships between selenium status and immune function have previously been demonstrated in both HIV-infected children and adults (12,17). ‘The pre- sent study suggests that plasma level of selenium is a sensitive indicator of disease progression and mortality in pediatric patients, thus supporting an important role for nutrition in survival, OF the nutrients considered in this study, only selenium deficiency was found to be associated with a high risk of mortality [RR = 5.96, 95% Cl, 1.32-26.81), independent of CD4 cell count ‘The odds ratios moving away from 1, however, suggest some degree of interaction. In support ofthis interaction, several studies have found that the prevalence of seleni um deficiency increases as the HIV disease advances (from 2%—4% in asymptomatic patients, 33% in our P2 symptomatic cohort, to 75% in Stage IV ALDS) (18,19). In the present study, selenium deficiency was also asso- ciated with rapid HIV-disease progression, characterized TABLE 2, Relation between immune function, nutritional stats, ‘and survival Number of children in Variables thiscell RR 9S%CI_—_p Value Initial model CD4 count 200mm? 6 == 633. 154-2600 OL Zidovudine wse mM 09 00-935 93 ‘Albumin «3.5 g/l S27 059-1236 2 Jenium =85 jl 8 419 117-203 03 Iron <0 g/100m) M149 039463 Zinc =75 pg/ml ss .NG 023-574 86 Final model {CDA count <2onmm* — 7.05 187-265 004 Selenium =85 yg 8 59 132-2681 02 CC, confidence interval: RR, relative riskMORTALITY RISK IN SELENIUM-DEFICIED T HIV-POSITIVE CHILDRE SH. Pie alle 5 z g & 0.75 ° = 2 FIG. 1. The proportion of survivors 5 05 Se Adequate atthe close ofthe study was greater 8 tor eelonnradequale chieren (10 = . ‘of 16 children) than for selenium- oa ‘ deficient children (2 of 8 children). % 0.25 \ 3s E Se Deficient io 0 200 400 © 600 © 800» 1000 1200-1400 Days Survival Time After Recruitment us of a by death before 5 y 1¢ (P = .03), and closer to the point of assessment (p = .05), Although the small size of the group studied limits the capacity for generalizing our findings to the population, the present results are in accord with studies in HIV- infected adults, thus demonstrating that plasma level of selenium is an independent predictor of survival (11), and supporting suggestions that selenium may have a critical function in maintaining immune status (12,20), ‘The dramatic impact of selenium on HIV-1-related mor- tality (RR = 10.8; p <.002) shown in a previous study in adults (11), may be related 10 its role in antioxidant eo e 5 te ; a. = a a | hes 8 ig ‘Albumin Selenium FIG. 2. Of the tweive children who died during the study, sele- nium-adequate children (NV = 6) were significantly older at death (p <.05). Albumin status was not assocated with age of chidren at death, Journal. Acquired Immune Defi det 25). ‘Supplementation with selenium has been shown to promote glutathione peroxidase and superoxide dismu- tase activities and to increase glutathione (21), all essen- tial parts of the antioxidant defense against viral peroxi- snse and possible modulation of viral expression (21— dative damage. In addition, viral infection may generate ly destructive free-radicals by stimulating the forma- tion of mononuclear and polymorphonuclear leukocytes (26). In selenium deficiency, other antioxidants have demonstrated compensatory activity in preventing some symptoms of oxidative stress (27-29), In HIV infection, however, several antioxidant mechanisms may be com- promised by multiple nutrient deficiencies. Thus, supple ‘mentation with selenium in HIV infection may offer the potential to moderate oxidative stress and immunosup- pression (3,17,30), The relationship between selenium deficiency and early death may be explained not only by selenium’s role in preventing oxidative stress in intracellular and extracellular compartments, but also by its proposed role within an infected cell in maintaining viral latency (27) Selenoproteins appear to be important for gene regula- tion (23,24). In vitro studies have demonstrated that selenium inhibits viral cytotoxic effects and the reacti- vation of HIV-1 by hydrogen peroxide and. protects against activation of HIV-I by tumor necrosis factor 31). Ithas been proposed that selenium deficiency com- bined with oxidative stress could stimulate HIV replica- tion through formation of viral selenoproteins. Several Viral genomic features required for expression of these512 A. CAMPA ET AL. clenoproteins have been experimentally confirmed 2). ‘The opportunity to examine disease progression and mortality retrospectively in a perinatally infected pe- diatric group, although limited by the small size of the group, is exceptional. In contradistinetion to that in infected adults, the time of infection in these children is well determined, and age can be used as a parameter for length of infection, These factors facilitate the iden- tification of rapid progressors (children who died be- fore the age of 5 years). Another important charac- teristic of this pediatric cohort is that all partici- pants were at the same stage of disease progression (ie., P2) at the beginning of the study. In addition, the ethnic distribution of this cohort was not signifi- cantly different from that of the pediatric HIV-in- fected population in Florida at the close of the study (88.5% black, 7% Hispanic, and 4% white non-Hispanic) 3). 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