Emergent and re-emergent parasites in HIV-infected children : immunological and socio-environmental conditions that are involved in the transmission of Giardia spp . and Cryptosporidium spp .

Introduction: Emergent and re-emergent waterborne protozoans have become a worldwide public health problem, especially among vulnerable groups. Methods: This cross-sectional study evaluated 17 HIV-infected children and their families. Results: A high (76.5%) percentage of parasite-infected children was observed, even among children with CD4+ T-cell counts of >200 cells/mm3. Giardia spp., Cryptosporidium spp. and Cyclospora spp. were observed in 41.2% of these children. Low income, poor hygiene practices, and co-infection in domestic, peridomestic and scholastic environments were signifi cant sources of these intestinal infections. Conclusions: Early diagnosis, timely treatment, and socio-educational interventions may improve the health conditions of this vulnerable population.

Since the 1980s, waterborne protozoan parasites have emerged and re-emerged and became a worldwide public health problem, especially among vulnerable population groups (1) (2) .For example, children with a suppressed immune system are more susceptible to infections that are caused by opportunistic agents, especially when their CD4 + T-lymphocyte counts are <200 cells/mm 3 or 15% (1) (3) , and are especially vulnerable to clinical complications that are caused by intestinal parasites.Acute diarrhea and persistent vomiting can lead to severe dehydration and poor nutrient absorption, which can delay the child's growth and development, and can lead to hospitalization for an unstable clinical condition or death in severe cases (2) .
Different factors may contribute to intestinal parasite transmission, including children's hand-to-mouth habits, interpersonal contact in daycare facilities, sanitation status, poor personal and/or food hygiene, and contact with parasite-infected people and/or animals (2) (4) (5) .However, there is little systematic data regarding the parasitological, immunological, and socioenvironmental conditions of human immunodefi ciency virus (HIV)-infected children, or regarding these factors' relationships with have emerging and re-emerging intestinal protozoans, which exhibit high persistence and infectivity in vulnerable populations.Therefore, the present study aimed to assess the presence of intestinal parasites in HIV-infected children, with an emphasis on Giardia spp.and Cryptosporidium spp.These data may help identify the correlation between intestinal parasitic infections and the patient's immunological condition, verify the intra-family/ household co-infection status for these patients, and characterize the socio-environmental conditions that may be involved in the transmission of emergent/re-emergent intestinal parasites.This quantitative cross-sectional cohort study was performed during March-September 2013, in a specialized unit for HIVinfected adults and children at a public hospital in the northeast region of the State of São Paulo, Brazil.We included all HIV-1-infected patients who visited the outpatient clinic, were ≤13 years old, provided a stool sample, and were living in Ribeirão Preto, State of São Paulo, Brazil.We identifi ed 24 children, although only 17 children (3-12 years old; 9 girls and 8 boys) were followed for the full 6 months..
The children's family members were also asked to participate in order to evaluate their intra-family/household co-infection status.Twenty family members of 10 HIV-infected children agreed to participate, and were evaluated for intestinal parasites.These family members were 3-65 years old, and included 13 women and 8 men.
We collected 2-3 stool samples on alternating days and analyzed these samples at the Laboratory of Ecotoxicology and Environmental Parasitology (University of São Paulo, Ribeirão Preto College of Nursing).A total of 47 stool samples from HIV-infected children and 57 samples from family/household members were analyzed.The modifi ed Ziehl-Neelsen technique was used to detect and identify intestinal coccidian parasites, including Cryptosporidium spp., Cystoisospora spp., and Cyclospora spp. (6).The sedimentation/Coproplus® technique (NL Comércio Exterior Ltda, São Paulo, Brazil) was used to identify Giardia spp.and other noncoccidian parasites (7) .Immunoenzymatic assays [Giardia Stool Antigen Detection Microwell enzyme-linked immunosorbent assay (ELISA) and Cryptosporidium Stool Antigen Detection Microwell ELISA; IVD Research, USA] were used to confi rm Giardia spp.and Cryptosporidium spp (2) .Macroscopic analyses of all samples were also performed to verify their diarrheal characteristics, including a soft consistency, mucus, and liquefaction of the stools.
Data regarding the patients' CD4 + T-lymphocyte counts (cells/mm 3 and percentage), viral load (copies/mL and logadjusted value), adherence to antiretroviral treatment (ART), and previous parasitological conditions were collected from their clinical records.A structured questionnaire was validated and used to collect data regarding the patients' socio-environmental conditions: family's monthly income, sanitation status, personal and food hygiene habits, water-based recreational activities, and contact with animals and other children.
Descriptive analyses were performed for the parasitological, immunological, and socio-environmental data.Fisher's exact test was used to identify potential correlations between the immunological characteristics and parasitic infection status (via a p-value of <0.05), using Statistical Package for the Social Sciences (SPSS) software (version 22.0; SPSS Inc. Chicago, IL).Ethical approval was obtained from the University of São Paulo at Ribeirão Preto College of Nursing (referee report no.159.935).Participation was voluntary, and informed consent/ assent was obtained from participants and/or their guardian.
The socio-environmental conditions of the children are shown in Table 1.Highlights from these data include the following: 88.2% of the patients' families had a low monthly income (up to 3 times the Brazilian minimum wage (8) ); 100% of the families were supplied with safe public drinking water, sewage collection, and treatment systems; 47.1% of the children did not wash their hands before meals; and all the children had frequent contact with other children and pets in the domestic, peridomestic, and scholastic environments.
The identified parasites, CD4 + T-lymphocyte counts, and viral loads are presented in  The parasite infections from the patients' family members organized according to age and relationship are shown in Table 3.At least one parasite was identifi ed in 70% of the patients' family members.Giardia spp.and Entamoeba coli were the most frequent parasites (50% each), and were identifi ed as independent infections or co-infections with other parasites, including Cryptosporidium spp., Cyclospora spp., Strongyloides stercoralis, and Enterobius vermicularis.In 60% of the families, at least two individuals from the same household presented with the same parasite(s), independent of HIV-infection status.
Monitoring HIV-infected children who are undergoing continuous ART in specialized units can ensure that acute parasitic infections remain asymptomatic in these children (1) .Furthermore, continuous contact between these children and parasites and/or environmental exposure to asymptomatic parasite genotypes may cause asymptomatic infections (9) .However, when the immunological system is suppressed or ART is discontinued, these patients' clinical conditions can deteriorate, and they can experience acute diarrhea and dehydration that requires hospitalization (1) .
It is known that Giardia spp., Cryptosporidium spp., Cyclospora spp., and Entamoeba coli have human and animal enteric origins, and are transmitted directly or indirectly via the fecal-oral route (2) .Therefore, families who live in poor sanitary conditions and exhibit inadequate hygiene habits are believed to be undergoing constant infection and re-infection by those protozoans (5) .However, Ribeirão Preto's public water supply comes from the Guarani Aquifer, which is an underground source of high-quality water (10) .Furthermore, all the families in this study had access to sewage disposal, which indicates that sanitation status did not strongly affect their parasitic infections.
Despite the existence of an adequate sanitation system, poor hygiene practices among the children and their family members may have led to the infections during meal preparation and/or consumption, via contaminated foods.For example, researchers have found Giardia spp., Cryptosporidium spp., and Cyclospora spp. in leafy vegetables that were produced in the United States, Canada, and Mexico (11) .Furthermore, these protozoans are remarkably resistant to common disinfectants and may survive in raw foods (2) (11) .Although they were not frequent activities, swimming in pools or rivers may also have contributed to the infections via the accidental ingestion of contaminated water.Giardia spp., Cryptosporidium spp., and Cyclospora spp.have been found throughout the world in recreational water, including chlorinated water, because they are resistant to adverse environmental conditions (12) (13) .Moreover, the identifi ed parasites have a high zoonotic potential, with the capacity to develop in human and animal hosts (2) .Different Brazilian regional studies have also reported the presence of Giardia spp., Cryptosporidium spp., and Cyclospora spp. in dogs and cats (13) (14) .Giardia spp.and Cryptosporidium spp.cysts are in their infective form in pets' feces, and it is possible that children develop parasitic infections via this route.Therefore, frequent contact with pets and/or their feces may also be related to the high frequencies of parasitic infections among these children.Another socio-environmental factor that may have contributed to the parasitic infections was personal hygiene.For example, the patients did not wash their hands before meals (47.1%), after using the bathroom (35.3%), or after playing (41.2%).Therefore, it is essential to increase the awareness of caregivers and HIV-infected children regarding preventive measures that can control intestinal parasitic infections, and to promote activities that encourage the adoption of healthy personal and food hygiene habits.Health education efforts may be facilitated via the incorporation of various technologies, as well as inter-sector commitment and responsibility to cooperation within the healthcare system, especially among primary healthcare and educational services.
The high prevalence of Giardia spp.and Entamoeba coli in the patients may be explained by the relatively easy inter-personal transmission of these parasites, as 94.1% of the patients were attending school or daycare.Giardia is one of the most common intestinal parasites that affect Brazilian children, especially children who attend these institutions, with a prevalence of 8-50% (5) (7) .Similarly, Cryptosporidium is a signifi cant protozoa that is disseminated in schools and daycare facilities (2) , which explains its presence in 23.5% of the HIV-infected patients.This prevalence is higher than that of Cryptosporidium spp.infections among healthy children in other Brazilian cities (≤15.5%) (4).However, few studies have attempted to identify Cyclospora spp. in children who attend daycare facilities, which may be due to the low likelihood of inter-personal transmission (2) .
Contact with cohabitating family members may also infl uence intestinal parasitic infections among HIV-infected children.For example, contact with parasite-infected family members or cohabitants is an important risk among HIV-infected individuals (2) (3) .The similarities among the identifi ed parasites in the patients' family members reaffi rm the fact that interpersonal contact during daily living increases the risk of parasitic infection (15) .Therefore, cohabitants or family members should also be considered in the processes of diagnosis and treatment.
Despite the high frequency of intestinal parasitic infections among HIV-infected children in this study, the absence of symptoms hinders any investigation regarding potentially pathogenic emerging and re-emerging agents, due to neglect and underreporting.Therefore, the introduction of routine diagnostic techniques to detect emergent parasites in HIV-infected patients can help facilitate an early diagnosis.Furthermore, parasite control in asymptomatic individuals is relevant because the intermittent elimination of feces can lead to environmental contamination.
Treatment with parasite-specifi c medications should be started immediately after an HIV-infected child is diagnosed with a potentially pathogenic parasite infection, in order to avoid recurrent infection symptoms.However, there is currently no effective and specifi c treatment for Cryptosporidium spp., although the Food and Drug Administration has approved nitazoxanide for treating cryptosporidiosis in individuals with healthy immune systems.Unfortunately, the effi cacy of this medication in immunosuppressed individuals is not clearly established and requires further study (2) .Therefore, permanent monitoring of the immune response, with antiretroviral and antiparasitic treatment adherence, is important for preventing the clinical symptoms of cryptosporidiosis (2) .
In conclusion, this study demonstrates that it is necessary to strengthen the clinical management of individual patients, as well as collective and environmental management practices.Improvements in health education strategies have the potential to transform lifestyles, especially among vulnerable population segments, such as HIV-infected children and their families.Furthermore, future studies should analyze the genotypes of regional parasite strains and isolates that are found in HIVinfected children and their families.
The authors declare that there is no confl ict of interest.

Fregonesi
BM et al. -Emergent/re-emergent parasites in HIV-infected children

Fregonesi BM et al. -
Emergent/re-emergent parasites in HIV-infected children

Table 2
according to patient age and sex.The presence of parasites was more frequent