Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Gastroenterology
Faecal Calprotectin in HIV-infected, HAART-naïve Ugandan Children
Hestvik, E.*; Olafsdottir, E.†; Tylleskar, T.*; Aksnes, L.¶; Kaddu-Mulindwa, D.§; Ndeezi, G.‡; Tumwine, J.K.‡; Grahnquist, L.||
*Centre for International Health, University of Bergen
†Department of Paediatrics, Haukeland University Hospital, Bergen, Norway
‡Department of Paediatrics and Child Health
§Department of Microbiology, Makerere University School of Medicine, College of Health Sciences, Kampala, Uganda
||Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
¶Department of Clinical Medicine, University of Bergen, Bergen, Norway.
Address correspondence and reprint requests to E. Hestvik, Centre for International Health, University of Bergen, Postboks 7804, N0-5020 Bergen, Bergen, Norway (e-mail: email@example.com).
Received 18 April, 2011
Accepted 7 November, 2011
The present study received funding from the University of Bergen and the GlobVac programme by the Research Council of Norway, grant no. 172226 Focus on Nutrition and Child Health: Intervention Studies in Low-income Countries.
The authors report no conflicts of interest.
Objectives: Calprotectin is a calcium- and zinc-binding protein and a marker in faeces of gastrointestinal inflammation. Reference values have been established in children older than 4 years. The aim of the present study was to determine the concentration of faecal calprotectin (FC) in human immunodeficiency virus (HIV)–infected, highly active antiretroviral therapy–naïve Ugandan children and compare it with the reference value.
Methods: We tested 193 HIV-infected children ages 0 to 12 years in a hospital-based survey for FC. A standardised interview with sociodemographic information and medical history was used to assess risk factors. A cluster of differentiation 4 (CD4) cell percentage was prevalent in all of the children.
Results: The median FC concentrations decreased with increasing age, as in healthy children. The median concentration was 208 mg/kg in infants 0 to 1 year, 171 mg/kg among toddlers 1 to 4 years, and 62 mg/kg for children 4 to 12 years. Children with advanced disease and a low CD4 cell percentage had significantly higher FC concentrations than those with a high CD4 cell percentage. Children older than 4 years with diarrhoea had significantly higher FC concentrations compared with those without diarrhoea.
Conclusions: HIV-infected children older than 4 years had a median FC concentration above the reference value, and gut inflammation in the children with elevated values is likely. Children with more advanced disease had increased FC concentrations regardless of age.
In 2008, approximately 120,000 children were living with human immunodeficiency virus (HIV) in Uganda (1). Sub-Saharan Africa accounts for 67% of all people living with HIV and carries the highest burden of the global HIV epidemic (2). The gastrointestinal (GI) tract is the largest immunological site of the body and HIV infection profoundly affects gut function (3–6). There are numerous GI problems in children in low-income countries, particularly in those who are HIV infected (7–9). In low-income countries, diarrhoea may occur in up to 80% of HIV-infected individuals (10).
From a clinical aspect, it is difficult in a low-income setting to predict when HIV-infected children need more attention due to GI failure or failure to thrive. We do know that children with a low cluster of differentiation 4 (CD4) cell percentage are at greater risk for acute and persistent diarrhoea (11). In a Rwandan study of HIV-infected children, common conditions such as chronic cough, persistent generalised lymphadenopathy, chronic diarrhoea, and failure to thrive occurred much more frequently, and were associated with a significantly higher mortality and morbidity in HIV-infected compared with noninfected children (12). Common conditions in HIV-infected children are associated with high mortality and morbidity and are linked to GI failure. Microbial translocation due to increased permeability in the gut caused by the HIV virus (13) has been suggested as one of the causes of the massive burden of disease in the gut in HIV-infected people (14).
Calprotectin is a calcium- and zinc-binding protein, abundant in neutrophils and found in monocytes/macrophages, but absent in platelets and lymphocytes (15). It is remarkably resistant to degradation in the presence of calcium, stable in faeces stored for 7 days at room temperature (16), and no changes over time have been found by storing the faeces at −20°C (17). Faecal calprotectin (FC) is a marker of GI inflammation. A reference value of 50 mg/mL has been established in adults and children older than 4 years (18,19). FC concentrations are elevated in both adults (16,20) and children (21,22) with inflammatory bowel disease (IBD), as well as in adults and children with various GI infections (23,24), but the concentrations are lower than in people with IBD. HIV-infected adults had a high variability in FC, and half of them (27/53) had FC higher than the reference value (50 mg/mL) (25). Neutrophils in vitro from HIV positive subjects have shown a diminished inhibitory response to calprotectin (26). FC concentrations are increased with bleeding into the GI tract (27). In young infants, high FC concentrations are normal (22,28). We have shown that FC among healthy urban Ugandan children are within the reference values and comparable with those in healthy children living in high-income countries (19). The aim of the present study was to determine the concentrations of FC in HIV-infected, highly active antiretroviral therapy (HAART)–naïve Ugandan children.
Study Design, Site, and Data Collection
The survey was conducted from February to October 2008 at the Department of Paediatrics and Child Health, Mulago National Referral Hospital, Kampala, a government-run hospital, in collaboration with the Centre for International Health, University of Bergen. A detailed description is provided elsewhere (29). Participants were included in the study if they were HIV-infected, HAART-naïve, ages 0 to 12 years, and had received informed consent from their caregiver. All of the participants were tested for HIV as a part of the medical service before inclusion in the study. A total of 263 HIV-infected, HAART-naïve children were eligible for the survey during the 9 months of encounter. Of the potential participants, 53 (22%) were not included in the final analysis due to samples lost in transport (26), no CD4 cell count being carried out (17), failure to provide a stool sample (9), and incompleteness of the questionnaire (1) (Fig. 1).
We classified CD4 cell percentage as high or low with limits defined by age: for children younger than 12 months, high if CD4 cell >25%; for 12 to 36 months, high if CD4 cell >20%; and for 36 months or older, high if CD4 cell >15%. The limits chosen were concurrent with those recommended for starting HAART according to the World Health Organization (WHO) guidelines available at the time of the study (30). All of the children were clinically categorised using the WHO staging system for HIV-infected children (31) because it is recommended for evaluating the need to start up HAART in children when a CD4 cell count is unavailable (30). Weight and length were taken according to WHO standardised techniques (32). Weight and height were missing in 2 children. The software programs WHOAnthro (33) (for children 0–5 years) and WHOAnthroPlus (34) (for children older than 5 years) were used to calculate weight-for-age z scores (WAZ), height-/length-for-age z scores (HAZ), and body mass index-for-age z score (BAZ). A detailed description of the CALPRO Calprotectin ELISA test has been published (19).
Data from the questionnaires, the results of the CALPRO Calprotectin ELISA test, and CD4 cell percentage were double entered, using EpiData version 3.1 (www.epidata.dk). Data quality was ensured through careful selection and training of research assistants, supervision, and field editing by use of the “check” module at data entry combined with double data entry and validation. The data were exported to SPSS version 17.0 (SPSS Inc, Chicago, IL) for statistical analysis. The concentration of FC was expected to have a skewed distribution, so the median was used. The confidence interval (CI) reported was set at 95%. All of the tests were 2-sided, P ≤ 0.05 considered significant. FC values in the different groups were compared by using the Mann-Whitney U test (for 2 different groups) and the Kruskal-Wallis H test (for ≥3 groups). WAZ, HAZ, and BAZ were analysed if over/under<−2 standard deviations (SD). WAZ measures were not available in children older than 10 years.
Ethical approval was obtained from Makerere University, Faculty of Medicine, research and ethics committee, and the Regional Committee for Medical and Health Research Ethics, West Norway (REK-VEST) in Norway. The data collectors were trained in ethical issues before the study. Oral and written information about the study was given to the caregivers in either English or the local language. Informed consent was obtained from all of the caregivers of the participants in the study. All of the children participating in the study were independently managed for their medical needs by the doctor in charge of the ward.
The mean age (±SD) of the participants completing the study was 3.1 (±2.9) years. Fifty-nine (30.6%) children were older than 4 years. For these children, the mean age (±SD) was 6.7 (2.3) years. Sex was equally represented in the study: 98 (50.8%) girls and 95 (49.2%) boys (Table 1).
The FC had a skewed distribution in the 193 HIV-infected children (Fig. 2). In the 3 age groups, the median FC concentrations were 208 mg/kg in infants 0 to 1 year, 171 mg/kg among toddlers 1 to 4 years, and 62 mg/kg for children 4 to 12 years. Of the 59 children older than 4 years, more than half of them, 34 (57.6%), had FC >50 mg/mL, 25 (42.4%) had FC >100 mg/mL, and 18 (30.5%) had concentrations >150 mg/kg. The clinical features of the latter 18 children are presented in Table 2. Children older than 4 years presented with a high grade of variability in the FC values. There was a statistically significant difference in the FC concentration in children ages 0 to 4 years compared with children older than 4 years. There was no statistically significant difference between the children ages 0 to 1 and 1 to 4 years (Table 1). In the youngest age groups, the concentration of FC was more widespread and had a larger range than in the oldest children, in whom the values were skewed towards the lower end of the scale (Fig. 2).
There was a statistically significant difference in FC concentrations between children with high and low CD4 cell percentages (Table 3). When grouping the children into older or younger than 4 years, the numbers were too small to generate a statistically significant difference.
Children with WAZ and BAZ <−2 SD had significantly higher median FC than those with WAZ and BAZ >−2 SD. For HAZ, there was no such difference (Table 4).
We performed a subgroup analysis of children ages 4 to 12 years in which FC has been proven to be the most useful and reference values are available. By performing the Mann-Whitney U and the Kruskal-Wallis H test, there was a statistically significant difference in the concentrations of FC, diarrhoea, and sex at examination. There were no significant differences in concentration of FC by WHO staging, fever at examination, being colonised by Helicobacter pylori, education of the female caregiver, sources of drinking water, or child's habit of using mosquito netting (Table 5).
In all of the children and in the subgroup of children older than 4 years, we performed a linear regression analysis to determine whether diarrhoea at enrollment was a factor on its own or a confounder for a low CD4 cell percentage (data not shown). There were no significant changes in median FC concentrations with 95% CI after adjusting for diarrhoea. We also adjusted for all of the factors shown in Table 3 by linear regression (data not shown), but there were no significant changes in median FC with 95% CI.
Only 3 participants older than 4 years had pathogens detected in stool either in culture or by direct microscopy. All 3 were boys: Escherichia coli with FC 44.2 mg/kg, age 4 years; hookworm with FC 21.5 mg/kg, age 5.4 years; and Giardia intestinalis with FC 203.4 mg/kg, age 4.5 years. Two children had experienced nosebleeds within the 2 weeks preceding the study: an 11.2-year-old girl with FC 45.3 mg/kg and an 8.2-year-old boy with FC 99.1 mg/kg.
This is the first survey of FC concentrations in an HIV-infected population in sub-Saharan Africa. Children older than 4 years had a higher median FC concentration than the reference values suggested in the literature. Children with more advanced disease had the highest FC concentrations. FC was detectable in all of the children regardless of age, clinical staging of HIV, and CD4 cell percentage. HIV-infected children had more variability in concentrations of FC compared with immunocompetent subjects.
A weakness of the present study is the limited number of children older than 4 years, the failure being due to the inclusion criteria of being HAART naïve with a natural history of acquired immunodeficiency syndrome (12). Using the diagnostic tools available in routine services we have identified a few pathogens in the stool by culture, microscopy, and an antigen test. We assume that this is due to recurrent antibiotic treatment for opportunistic infections. Studies have shown that invasive investigations, such as endoscopy with biopsies, are necessary to identify GI opportunistic infections at high rates (5). The finding of a few identified pathogens in the stools from HIV-infected people is comparable with other studies (6). Although we invited all of the children available at the paediatric department and had few refusals, it may be that our population is unrepresentative of HIV-infected, HAART-naïve children in Kampala. The children we studied were admitted to a tertiary hospital, and they may have been more seriously sick than the average HIV-infected, HAART-naïve child due to their caregivers seeking medical attention. The numbers of Ugandan children enrolled in HIV treatment programmes are increasing, and children are accessing HAART earlier (35).
To the best of our knowledge, FC concentrations in HIV-infected patients have been reported only in 1 Italian study of asymptomatic adults (25). FC was >50 mg/mL in 27 of 53 HIV-infected patients, and the authors argue that this is clearly indicative of GI inflammation and that it confirms a breakdown of the intestinal barrier. In our study, a similar proportion (34/59 HIV-infected children) had FC >50 mg/mL, and gut inflammation in these children is likely. Schwartz et al (26) have shown in vitro that neutrophils from HIV-infected people have a diminished inhibitory response to calprotectin. We have shown that children with low CD4 cell count and malnourished children produce calprotectin and present with significantly higher levels. Schwartz et al investigated “asymptomatic” HIV-infected people and we have studied hospitalised children at all of the stages of HIV, which may make comparisons difficult. We have shown that HIV-infected children have more variability in FC than healthy children, and so it is a good tool to follow gut involvement in HIV. The findings of more variability in FC values and an increased median value compared with healthy children, but not as high as seen in children with IBD, may be explained by the effect of HIV on the gut itself with increased permeability, which, again, is linked to microbial translocation (14). More investigations are needed to determine whether FC can be used to monitor the degree of microbial translocation in HIV-infected humans. Increased intestinal permeability in HIV-infected populations has been confirmed by several studies (36–38). Sharpstone et al (38) used a lactulose/L-rhamnose test and showed that all of the HIV-infected participants, except those defined as “well,” had a significantly increased intestinal permeability compared with healthy controls. In our study, we found that children with a low CD4 cell percentage had significantly higher concentrations of FC than those with a high CD4 cell percentage. We argue that participants in our study with a low CD4 cell percentage can be compared with those in the Sharpstone et al study defined as all other than those being “well with HIV,” and that those children with a low CD4 cell percentage had a more advanced enteropathy.
In our study, children who had diarrhoea, acute or persistent at examination, had significantly higher FC concentrations compared with those without diarrhoea. Comparable findings in non–HIV-infected populations have been described (24). This can be explained by increased permeability of the gut either by GI-opportunistic infection or by HIV enteropathy (10). Greenson et al (39) reported that adults with HIV with and without diarrhoea have significantly altered gut mucosa anatomy compared with normal controls. This supports our findings that HIV-infected children have involvement of the GI tract and increased gut inflammation. Knox et al (6) investigated 671 HIV-infected people, of whom 39% had diarrhoea, but stool pathogens were identified in only 12% of these patients. Despite this, 48% of all patients had an abnormal D-xylose test, 22.5% had borderline or low serum B12 levels, and 7.2% had depressed albumin levels. Increased malabsorption is often associated with increased permeability of the GI tract (38), which can be seen as increased concentrations of FC, as found in our HIV-infected child population of children.
Colonisation by H pylori was common in our population, but no difference in FC level was found in these colonised or uncolonised. This is comparable to findings in healthy children (19). In addition, there were no differences in median FC according to health behaviour, sources of drinking water, and education level of female caregiver. All of these are in agreement with our findings in apparently healthy Ugandan children (19) and show that those factors probably do not contribute to gut inflammation in HIV-infected children living in poor socioeconomic conditions.
FC is a marker for GI inflammation, frequently used in diagnosis and follow-up of patients with IBD (20–22), and a marker for disease activity in individuals (40). We hypothesise that FC in HIV-infected patients can be used, as in patients with IBD, to follow disease activity in individuals and to investigate gut engagement in HIV-infected children in low-income countries where other, more sophisticated methods are not available. We believe that inflammation in the gut measured by FC may be a good marker in combination with CD4 cell percentage to decide on further investigations and treatment with HAART in children living with HIV.
FC can be used as a marker of GI inflammation in HIV-infected people. HIV-infected children older than 4 years had a median FC concentration above the reference values given in the literature. Children with more advanced disease had increased FC concentrations regardless of age.
We thank all of the children, their caregivers, the data collectors, and the laboratory technicians who participated.
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