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International Health Institute, Brown University, Providence, Rhode Island
Research Institute of Tropical Medicine, Manila, Philippines
In a cross-sectional study of 641 Schistosoma japonicuminfected individuals in Leyte, Philippines, who were 730 years old, we determined the grade of hepatic fibrosis (HF) by ultrasound and used anthropometric measurements and hemoglobin levels to assess nutritional status. Serum levels of interleukin (IL)-1, IL-6, and IL-10; tumor-necrosis factor (TNF); soluble TNF- receptor I; and C-reactive protein (CRP) were measured to examine the association between these markers of inflammation and HF grade. HF was present in 8.9% of the cohort; the majority of cases were mild (grade I), and severe (grade II or grade III) cases occurred only in male individuals. Compared with individuals without HF, those with severe HFand, to a lesser degree, those with mild HFhad a significantly lower body-mass index (BMI) and BMI z-score, a higher prevalence of anemia, and a higher level of CRP and were more likely to produce IL-6; furthermore, those with severe HF had a significantly higher level of IL-1, compared with those either without HF or with mild HF. These findings suggest that even mild HF is associated with nutritional morbidity and underscore the importance of early recognition and treatment. In addition, our data are consistent with the hypothesis that, by systemically increasing the levels of the proinflammatory cytokines IL-1 and IL-6, HF causes undernutrition and anemia.
Periportal fibrosis of the liver, or periportal hepatic fibrosis (HF), is the most serious manifestation of chronic schistosomiasis infection, eventually leading to portal hypertension with subsequent hepatosplenomegaly and esophageal varices. Risk factors for schistosomiasis-associated periportal HF include older age, male sex, longer duration and intensity of infection, and genetic factors [14].
Several studies have shown, in children and adolescents, a causal relationship between chronic schistosomiasis japonica and undernutrition and anemia [5, 6]; however, the association between HF and these morbidities has not been examined. No ultrasound studies have been performed in patients with the clinical syndrome of schistosomal dwarfism, which is characterized by severe malnutrition and growth stunting, hepatosplenomegaly, anemia, and endocrinologic abnormalities [7]; thus, the association between HF and the aforementioned morbidities in this syndrome remains unclear.
Poor nutritional status associated with chronic schistosomiasis may be mediated by proinflammatory cytokinesspecifically, interleukin (IL)1, IL-6, and tumor-necrosis factor (TNF). This mechanism has been proposed as being involved in growth failure associated with chronic infections in general [8]. Furthermore, these proinflammatory cytokines are thought to play a crucial role in the development of cachexia-anorexia syndrome, which is associated with chronic parasitic infections [9]. Increased levels of IL-1, IL-6, and TNF- and decreased levels of IL-10 have been associated with HF [1014]. On the basis of these data, we hypothesized that a systemically increased level of proinflammatory cytokines in schistosomiasis-associated HF mediates undernutrition and anemia of inflammation. In addition, we speculated that IL-10 may be protective. These effects may be especially strong in children and adolescents, because these age groups experience periods of rapid growth and increased nutritional demands.
The aim of the present cross-sectional study was to evaluate nutritional status, hemoglobin (Hgb) levels, and hepatosplenomegaly in children, adolescents, and young adults with schistosomiasis-associated HF in Leyte, Philippines. In addition, we examined whether differences in morbidity existed between those without HF and those with only mild HF, because the latter group is commonly considered to be clinically insignificant. Finally, we addressed potential immunologic mechanisms underlying the relationship between HF and undernutrition. We hypothesized that elevated serum levels of IL-1, IL-6, and TNF- act as primary mediators of HF-associated morbidity.
SUBJECTS AND METHODS
Study site and population.
The present cross-sectional study was conducted in 3 Schistosoma japonicumendemic rice-farming villages in Leyte, Philippines. Treatment in this study area was provided by the Schistosomiasis Control Program, which conducts annual mass-treatment campaigns. Participation is reported to be low because of the absence of pretreatment screening for the presence of infection (personal communication, Dr. A. Ida, Director, Schistosomiasis Control Program, Leyte, Philippines). S. japonicuminfected individuals were recruited as part of a longitudinal study investigating resistance to reinfection. Approximately 90% of the inhabitants of the 3 villages were screened for the presence of S. japonicum infection. Individuals were eligible for enrollment in the study if they were infected with S. japonicum, lived primarily in 1 of the 3 study villages, were 730 years old, were not pregnant or lactating, and provided informed consent; use of these criteria resulted in the identification of 646 individuals. Participants were enrolled during 2 periods, October 2002 and April 2003.
Evaluation by ultrasound.
Participants were evaluated by ultrasound, by use of a Hitachi EUB-200 with a 3.5-Mhz probe (Hitachi Medical). Liver size (in centimeters) was measured as the size of the left liver lobe in the right parasternal line; spleen size (in centimeters) was measured in the left intercostal oblique view. Because reference measurements for liver size and spleen size in healthy Filipinos were not available, normal values in a healthy Chinese population were used [15]. Hepatomegaly and splenomegaly were defined as values that were >2 SD above the mean. HF grades IIII (GIGIII) were based on a modification of the grading system described by Doehring-Schwerdtfeger et al. [16]. Severe HF was defined as the presence of either GII HF or GIII HF.
Stool examination.
Parasite burden was determined by duplicate examination of 3 consecutive stool specimens obtained from each study participant. Each of these specimens was evaluated, by use of the Kato Katz method, for infection with S. japonicum, Ascaris lumbricoides, Trichuris trichuria, and hookworm [17]. Low, moderate, and high intensity of infection with S. japonicum was determined by use of World Health Organization (WHO) criteria [18].
Nutritional assessment.
Particpants were measured while wearing light clothing and no shoes [19]; weight, to the nearest 0.1 kg, was measured by use of a Seca digital scale (model 880); height was measured, to the nearest 0.1 cm, by use of a portable anthropometer. These measurements were used to determine the height-for-age z-score (HAZ), body-mass index (BMI) (calculated in terms of weight [in kilograms] divided by the square of height [in meters]), and BMI z-score (BMIZ). BMI is the index of choice for the assessment of recent undernutrition in adults and adolescents [20, 21]; HAZ represents long-term growth and nutritional status [20]. Recent (year 2000) Centers for Disease Control and Prevention reference curves were used to calculate z-scores [22], by use of EpiInfo 2000 software (version 2000). Growth stunting and wasting were defined, respectively, as an HAZ or a BMIZ that was >2 SD below the mean; because z-scores were calculable only for individuals <20 years old, wasting in the case of individuals 20 years old was additionally defined as a BMI that was <17. Severe wasting was defined as either a BMIZ that was >3 SD below the mean, for individuals 720 years old, or a BMI that was <16, for individuals 20 years old. Test-retest and interrater reliability assessments of anthropometric measurements were evaluated and were found to be very good to excellent (range, 0.860.99).
Blood collection and processing.
Venipuncture was performed, blood was collected into Vacutainer tubes (Becton Dickinson) [17], and this venous sample was used to obtain a complete hemogram, by use of a hematology analyzer (Serono-Baker Diagnostics). Anemia was defined according to the age- and sex-specific criteria described by WHO [18].
Serum was analyzed by use of a multiplex bead-based platform (BioRad) and custom assay kits. Albumin and bilirubin were determined by use of commercial kits (ThermoDMA). All pipetting and sample identification were performed by a barcode-enabled, high-speedpipetting robot (Tecan).
We developed a 12-plexsandwich capture assay and a 3-plex competitive-binding assay. Because they were measured concurrently, we present details of all analytes included in these assay kits, although several of these analytes were not used in the present study. For the 12-plex assay, 500 g of each detection antibody (IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon , and TNF- [BD Pharmingen]; IL-1, TNF receptor I (TNF-RI), TNF receptor II (TNF-RII), and leptin [R&D Systems]; erythropoietin [StemCell Technologies]; and ferritin [Maine Biotechnology Services]) was coupled to 6.25 × 107 microspheres from unique bead regions, according to the manufacturer's (Luminex) instructions. Standards (IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-, and TNF- [BD Pharmingen]; IL-1, TNF-RI, TNF-RII, EPO, and leptin [R&D Systems]; and ferritin [Fitzgerald Industries International]) were pooled at appropriate concentrations and as a single lot, were aliquoted into single-use tubes, and were stored at -80°C. Biotinylated detection antibodies were pooled into single-use aliquots and were stored at -80°C. Custom (all analytes) and commercial (ferritin, dehydroepiandrosterone sulfate , and EPO) controls were stored in single-use aliquots at -80°C.
The 3-plex competitive-assay kit for DHEAS, serum transferrin receptor (sTfR), and C-reactive protein (CRP) was developed in a similar fashion; however, instead of capture antibody, the analyte (either DHEAS , sTfR , or CRP [Scripps Laboratories]) was coupled to the beads. Detection antibodies and controls were prepared as described above.
On all plates, we included a custom, pooled control that contained all analytes, as well as commercial controls with expected values for ferritin, EPO, and DHEAS. The assay kits demonstrated <2% interanalyte interference, and the median interassay coefficient of variation, as assessed by use of 48 replicate controls on consecutive plates, was 15%.
Data management and statistical analyses.
Normality diagnostics were performed, and nonnormally distributed variables, including egg counts and levels of bilirubin, CRP, and all serum cytokines, were loge transformed (calculated in terms of ln[n + 1]). Because we were unable to achieve a normal distribution for IL-6, it was dichotomized into responders (detectable cytokine level) and nonresponders (undetectable cytokine level).
All final analyses were performed in SAS (version 8.02; SAS Institute). P < .05 was considered to be statistically significant. For all analyses, multilevel statistical analyses were used to adjust for clustering at the household level. Specifically, random-intercept models were constructed by use of Proc Mixed (with household as random effect and with a compound symmetry correlation matrix), for continuous outcomes, and generalized-estimating-equation models were constructed by use of Proc Genmod (with household as repeated effect and with a compound symmetry correlation matrix), for dichotomous outcomes. For all measures, the robust SEs are reported. The least-squares mean values presented represent the mean adjusted for confounders.
Ethical approval and informed consent.
The present study was approved by the institutional review boards of Brown University and the Philippines Research Institute of Tropical Medicine. All S. japonicuminfected individuals were treated both at the beginning (i.e., at baseline) and at the end of the longitudinal study. Written, informed consent was obtained from each adult participant or from the parents of assenting children.
RESULTS
Association between HF and undernutrition.
In bivariate analysis, the presence of HF was associated with a lower HAZ and a lower BMIZ, compared with the absence of HF (table 1). To obtain a nonconfounded estimate of the association between HF grade and z-scores, multivariate linear-regression models were constructed. Mean BMIZ decreased, in a dose-dependent fashion, in all 3 groupsthose without HF, those with GI HF, and those with GIIIII HF (figure 2). Although HAZ showed a trend of decreasing with increasing severity of HF, it was not statistically significant. Because these z-scores are calculable only up to age 20 years, and because 39% of individuals with HF were 20 years old, we also evaluated the entire, 641-member cohort, to determine whether there was an association between HF grade and BMI. Mean BMI was found to decrease significantly with increasing severity of HF, independent of age and sex: for those without HF, those with GI HF, and those with GIIIII HF, mean BMI was 17.7 (95% CI, 17.517.9), 17.2 (95% CI, 16.717.6), and 16.2 (95% CI, 15.317.1), respectively; for the differences between those without HF and those with GI HF, between those with GIIIII HF and those with GI HF, and between those with GIIIII HF and those without HF, P values were .034, .042, and .002, respectively (data not shown). We also examined, in the entire cohort, the relationship between HF grade and the presence of wasting. The odds of wasting being present were significantly increased in those with GIIIII HF, compared with either those with GI HF (odds ratio [OR], 53.8; 95% CI, 8.8328.3; P < .0001) or those without HF (OR, 19.6; 95% CI, 6.559.0; P < .0001), independent of age and sex (data not shown). In those without HF, those with GI HF, and those with GIIIII HF, the prevalence of severe wasting was 1.2%, 0%, and 30%, respectively.
Association between HF and levels of Hgb and anemia.
The association between HF grade and mean level of Hgb was evaluated by use of a linear-regression model, with adjustment for age, sex, hookworm egg count, and S. japonicum egg count. Mean Hgb decreased with increasing levels of HF (mean Hgb in those without HF, those with GI HF, and those with GIIIII HF was 12.3, 12.0, and 10.5 g/dL, respectively; data not shown), but none of the differences were statistically significant. However, when analyzed dichotomously, the prevalence of anemia was significantly higher in those with HF, compared with those without HF (table 1), and increased with increasing severity of HF; its prevalence in those without HF, those with GI HF, and those with GIIIII HF was 32.4% (n = 190), 48.1% (n = 23), and 58.4% (n = 6), respectively. When adjusted for age, sex, hookworm egg count, and S. japonicum egg count, the OR for anemia, in comparisons between those with GI HF and those without HF, between those with GIIIII HF and those with GI HF, and between those with GIIIII HF and those without HF was 2.2 (95% CI, 1.24.2; P = .015), 1.8 (95% CI, 0.56.6; P = .40), and 3.9 (95% CI, 1.212.3; P = .019), respectively.
Association between HF and hepatosplenomegaly.
In the entire cohort, 97 individuals (15.1%) had hepatomegaly, and, of these 97 individuals, 74 (76.3%) were male and 25 (25.8%) had HF. The OR for hepatomegaly, in comparisons between those with GI HF and those without HF, between those with GIIIII HF and those with GI HF, and between those with GIIIII HF and those without HF was 3.5 (95% CI, 1.96.7; P = .0001), 6.4 (95% CI, 1.234.9; P = .032), and 22.7 (95% CI, 4.4117.1; P = .0002), respectively. Splenomegaly was uncommon, with an overall prevalence of 0.9% (n = 6), and it occurred only in male individuals with concomitant hepatomegaly.
A striking, 2-fold-higher level of CRP was found in individuals with HF, compared with those without HF (table 1), and multivariate linear-regression analysis revealed significant differences between HF grades, independent of age, sex, and S. japonicum egg count (figure 5): mean levels of CRP in those without HF, those with GI HF, and those with GIIIII HF were 5.2 (95% CI, 4.75.6), 8.0 (95% CI, 5.411.50), and 16.6 (95% CI, 11.823.2) g/mL, respectively.
DISCUSSION
In the present cross-sectional study, we found that HF in individuals 730 years old who were infected with S. japonicum was significantly associated with several measures of undernutrition and inflammation, as well as with S. japonicum egg count and hepatomegaly. Furthermore, there was a clear dose-response relationship between these morbidities and increasing severity of HF.
As expected, age and sex were important demographic determinants of HF. The strikingly higher prevalence of HF in male individuals compared with female individuals, which has been described elsewhere [3, 4], could not be explained by differences in either age or S. japonicum egg count. However, differences in sex steroids may contribute to this phenomenon: estrogen has been associated with both suppression of the induction of HF [23] and increased production of IFN-, a cytokine with important antifibrogenic properties [24], whereas androgens have been found to reduce production of IFN- [25].
It is important to note that we found (1) that both BMIZ and BMI were significantly lowerand that wasting was significantly more prevalentin individuals with severe HF and (2) that HAZ showed a trend of decreasing with increasing severity of HF. Our inability to detect a significant association between HAZ and severity of HF may be related to the fact that completion of linear growth occurs at 20 years of age [26], whereas schistosomiasis-associated HF is a chronic condition more common in older individuals [3, 27]. In addition, the small sample size, particularly in the case of those with severe HF who were <20 old (n = 5), may have contributed to this null finding.
Chronic inflammatory conditions such as HF may lead to growth stunting in children and adolescents and to wasting in children, adolescents, and adults, through systemically increased levels of proinflammatory cytokines. In line with this hypothesis, we found that both the level of IL-1 and the percentage of IL-6 responders were significantly higher in individuals with HFparticularly in the case of those with severe HFthan they were in individuals without HF. Evidence exists that prolonged overproduction of IL-6 impairs linear growth, through a decrease in circulating insulin-like growth factor I (IGF-I) [28], a hormone that is produced primarily by the liver and that has growth-promoting effects both independent of growth hormone (GH) and through mediation of the peripheral effects of GH [29]; this IL-6mediated decrease probably results from accelerated IGF-I clearance [30]. In addition, both IL-1 and TNF- have been shown to diminish levels of circulating IGF-I, by decreasing its hepatic synthesis [31, 32]. HIV-infected children with growth impairment were found to have both significantly increased IL-6 activity and decreased IGF-I levels, compared with HIV-infected children with normal growth patterns [33]; in a study of children with both schistosomiasis-associated HF and growth stunting, serum levels of IGF-I were found to be decreased [34].
In addition to interfering with hormonal pathways that are involved in growth, proinflammatory cytokines also cause undernutrition by inducing anorexiaand hence reducing food intake. Involvement of IL-1, IL-6, and TNF- in the pathophysiology of this process has been demonstrated in several experimental animal models [3537]. In humans, increased levels of serum IL-6 and TNF- have been found to be associated with loss of appetite in adults undergoing hemodialysis [38]. The mechanisms involved are complex, including both peripheral and central cytokine production and actionand likely an imbalance between pro- and anti-inflammatory cytokines [9, 39].
Anemia was significantly more prevalent in individuals with HF, particularly in the case of those with severe HF; furthermore, individuals with severe HF had a mean Hgb level that was nearly 2 g/dL lower than that in individuals without HF. These associations were independent of age, sex, and both hookworm egg count and S. japonicum egg count. Two potential mechanisms through which HF may contribute to schistosomiasis-related anemia are (1) proinflammatory cytokinemediated dyserythropoiesis, as is seen in anemia of chronic disease/inflammation [40, 41], and (2) anorexia and subsequently decreased dietary intake of iron [38].
An important finding of the present study is that, compared with individuals without HF, even individuals with GI HF had significantly lower mean BMIZ and BMIand that, furthermore, this group had a significantly greater prevalence of anemia, hepatomegaly, and IL-6 responders, and a higher mean level of CRP. These results underscore the clinical significance of mild HF, challenge the widely held view that mild HF is clinically silent, and indicate that it may be clinically inappropriate, in epidemiologic studies, to group individuals with mild HF together with those without HF.
The significantly higher levels of IL-10 that we observed in individuals with severe HF may reflect a counterregulatory response to increased levels of proinflammatory cytokines, an important property of this immunoregulatory cytokine. Although a previous study has shown that IL-10 plays a central regulatory role in the pathogenesis of schistosomiasis [42], its relation to nutritional status has not been studied; however, in a study of Toxoplasma gondii infection, mice that showed partial weight recovery after initial weight loss had elevated levels of IL-10 production, compared with mice that continued to lose weight [35].
The cross-sectional design of the present study limits our ability to impute causal inferences. We have assumed that development of HF alters serum cytokine profiles and leads to morbidity. Alternatively, certain cytokine patterns inherent in certain individuals may predispose to the likelihood that HF, as well as undernutrition, will develop. Furthermore, chronic infection with schistosomiasis may cause undernutrition, which, in turn, may lead to a more maladaptive, profibrotic immune response. Longitudinal studies may help to provide insight into these issues. Another limitation of the present study is the absence of data on alcohol use and on the presence or absence of infection with hepatitis B and C, which may influence the extent of liver disease; however, in a study of children, adolescents, and adults in a schistosomiasis-endemic area in China, Wiest et al. did not find an association between HF and either alcohol use or the presence of infection with hepatitis B and C [27].
We conclude that, in children, adolescents, and young adults infected with S. japonicum, HF is associated with impaired nutritional status. Our findings underscore the importance of early recognition and treatment of HF, because compelling evidence indicates that undernutrition has detrimental effects on morbidity, mortality, cognitive development, reproduction, and the capacity for physical activity [43]. It is important to note that we found that even low-grade HF was associated with significantly worse nutritional status, thereby identifying a heretofore unrecognized but clinically important group. We hypothesize that the main mechanism through which HF leads to undernutrition and anemia is a systemic increase in the levels of proinflammatory cytokines, specifically IL-1 and IL-6and possibly (although this is not strongly supported by the results of the present study) TNF-.
Acknowledgments
We thank our field staffBlanca Jarilla, Mario Jiz, Archie Pablo, Raquel Pacheco, Patrick Sebial, Mary Paz Urbina, and Jemaima Yufor their diligence and energy, and we thank the study participants from Macanip, Buri, and Pitogo, in Leyte, Philippines.
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