Study site and sampling: This research was carried out in children attended at the Associação Municipal de Proteção ao Menor, Presidente Bernardes county, located at the western region of São Paulo State (22º00'21'' S; 51º33'10'' W), with territorial area about 754 km² and population estimated in 15.5 thousand people, 70% of which living in the urban area (Fig. 1).

The selection criteria for children were age ranging from 0 to 6 years old and agreement from parents or legally responsible person in taking part in the research. In case of agreement, the following questionnaire was applied:

In order to find a possible relationship betweenG. duodenalis isolated from children, feces samples from their parents, brothers, and/or pets were also taken when the parasitological results forG. duodenalis was positive in the respective child. Feces samples were also taken from nine workers at the daycare center. Three samples were taken in a period of ten days in order to increase the probability of obtaining cysts ofG. duodenalis, which have an intermittent elimination²⁶. The samplings occurred from January to July, 2004.

The Human Subject Ethics Committee at Unoeste previously approved the research (Process No. 054).

Parasitological assessment: Feces samples were examined at the Laboratory for Clinical Analyses, Pharmacy College, Unoeste - Campus I, Presidente Prudente, SP. Three methods were employed in order to recover enteroparasites: Lutz⁶ method for eggs, Faustet al.¹ method to concentrate the protozoa cysts and helminths eggs, and Rugaiet al.²⁹ method for recovery of larval forms of helminths.

Purification of G. duodenalis cysts from human feces: This procedure was performed according to ROBERTS-THOMPSONet al.¹⁹ with minor adaptations. Feces samples were diluted in water (1:5), passed through surgical cotton gauze and centrifuged at 700 xg for five min. The supernatant was discharged, the sediment suspended in water and the process repeated four more times as before in order to remove debris. The resulting sediment was suspended in 3 mL water, added 3 mL of 1 M sucrose and centrifuged at 180 xg for 20 min. The cysts in suspension were transferred to another centrifuge tube, suspended in water and centrifuged again at 700 xg for 10 min. The resulting sediment was suspended in 3 mL of water, added 3 mL of sucrose 0.75 M and centrifuged at 250 xg for 10 min. The cysts in suspension were washed in water to remove the sucrose residues and finally suspended in 1 mL of water.

DNA extraction: DNA extraction and RAPD analysis were carried out at the Laboratory for Cytogenetic and Molecular Biology, Unoeste - Campus II.

The purified cysts,ca. 50 per sample, were centrifuged at 700 xg for five min., the supernatant discarded, and the pellet received 500 µL of TEN solution (100 µL of 1 M Tris-HCl; 200 µL of 0.5M EDTA, 200 µL of 5M NaCl; pH 8.0). The suspension was then transferred to liquid nitrogen for five min, followed by water-bath at 97 ºC for five min. This procedure was carried out three times. The suspension, after cooled in ice, received 5 µL of lysozime, mixed and incubated in water-bath at 37 ºC for one h. After that, 50 µL of 5% sodium dodecyl sulfate was added, mixed, followed by addition of 2.5 µL of K proteinase (20 mg/mL of H₂O) and again incubated in water-bath for 30 min. The suspension was centrifuged at 7500 xg for five min and the supernatant discarded. The pellet was resuspended in isopropylic alcohol and again centrifuged at 15000 xg for five min. The resulting pellet was washed in 70% alcohol and resuspended in 200 µL of TE solution (10 mM Tris; 1 mM EDTA) and the DNA concentration determined spectrophotometrically.

PCR with specific primers for G. duodenalis: A fragment with 292 bp of the 18S rDNA region flanked by primers RH11 (5´-CAT CCG GTC GAT CCT GCC-3') and RH4 (5'-AGT CGA ACC CTG ATT CTC CGC CAG G-3')¹¹ (Invitrogen^TM) was amplified by PCR. The reaction mix (25 µL) was composed by 2.5 µL of 10x PCR buffer pH 8.5, 0.75 µL of 50 mM MgCl₂, 0.5 µL of 200 µM dNTPs, 0.5 µL of each primer (0.1 nM), 1.25 µL of dimethyl sulfoxide (DMSO), 0.2 µL (0.1 U) of Taq DNA polymerase, 17.8 µL of ultrapure water, and 1 µL (40 ρg) of template DNA. The amplification reaction was: forty-three cycles at 94 ºC for 60 s, 53 ºC for 40 s, and extension at 72 ºC for two min. The amplification product was verified under transluminator in 1.5% agarose gel after electrophoresis and treatment with ethyl bromide.

PCR with arbitrary primers (RAPD): The RAPD (Random Amplified Polymorphic DNA) technique²³ was carried out in 25 µL of reaction mix containing 0.1 U of Taq DNA polymerase (Promega^TM), 0.2 mM of dNTPs, 1.5 mM of MgCl₂, 50 mM of KCl, 10 mM of Tris-HCl (pH 8.5), 1 ng of DNA template, and 1 µM of each primer: IMIA2 (5´ TGC CGA GCT G 3'), IMIA3 (5' AGT CAC GCA C 3'), IMIA5 (5' AGG GGT CTT G 3'), IMIA8 (5' GTG ACG TAG G 3'), and IMIA10 (5' GTG ATC GCA G 3'). The primers were selected randomly among the available at the Laboratory for Molecular Genetics. Forty-three cycles consisting of denaturation at 94 ºC for 60 s, annealing at 50 ºC for one min and extension at 72 ºC for five min, were performed for each random primer.

Agarose gel electrophoresis for visualization of amplified DNA: PCR products were loaded on 1.5% agarose gel in the presence of bromopenol blue in the loading buffer (TBE 1X) and submitted to electrophoresis (100 V, 30 min) at room temperature. The molecular masses of the bands were estimated by comparison with molecular mass standards (100 bp, Promega^TM). After electrophoresis, the gel was photodocumented on a transluminator and analyzed with the image analyzer Alpha-innotech.

RAPD products analysis: The RAPD products were analyzed at Department for Biological Sciences, College of Pharmaceutical Sciences, Araraquara (Universidade Estadual Paulista) with the software Gel Compar II (Applied Maths). A consensus dendrogram was generated based on Dices's coefficient of similarity and UPGM (Unweighed Pair Group Method with Arithmetic Mean) for grouping. The intensity of bands was not considered in the analysis.

RESULTS

Parasitological assessments: According to the selection criteria, 101 out of 131 children attended at the daycare center had their feces sampled. From these, 61 were between 0 and 3 years old and 40 were between 4 and 6, and 61% were girls. The most part of the families (80%) had familiar inputs between two and three salaries and all children lived in houses supplied with water from municipal pipes. Most of the children (83%) were asymptomatic, while diarrhea was the most common complaining in 16 cases. Most of the fecal samples were loose, with few exceptions that were diarrheic.

Sixty-two children had no enteroparasite, 15 presentedG. duodenalis, 23 presented other parasite thanG. duodenalis, while only one presented simultaneous occurrence ofG. duodenalis and other enteroparasite (E. vermicularis).Entamoeba coli occurred in nine cases, whileEndolimax nana occurred in three cases. Helminths, such asEnterobius vermicularis occurred in nine cases, one with eggs ofAscaris lumbricoides, and other with eggs ofTrichuris trichiura. In children with diarrhea, four presentedG. lamblia (one of them in association withE. vermicularis), one presentedE. vermicularis, two presentedE. coli, and nine had no enteroparasites.

WhenG. duodenalis was confirmed in a child, his/her family members (parents and brothers) and pets (five cases of children presentingG. duodenalis which had dogs at home) were also submitted to feces examination. In 35 fecal samples from family members, 16 containedG. duodenalis, comprising seven mothers and nine brothers. Fathers, pets or the nine daycare workers did not haveG. duodenalis in their fecal samples. Thus, a total of 31G. duodenalis isolates were considered in this study.

Genetic variability of G. duodenalis by RAPD: The banding profiles generated by each primer were able to detect genetic variability among theG. duodenalis isolates. As illustration, only profiles generated with the primers IMIA8 are shown (Fig. 2a and2b). Each primer produced from two to eight fragments, depending on theG. duodenalis isolate and the primer used in the reaction.

Greater polymorphism was obtained with the primer IMIA 8, indicating different banding pattern among all isolates. Except IMIA 5, all primers produced strong amplicons. For this reason, the weak amplicons obtained with IMIA 5 were not considered in the similarity analysis. The other four primers produced different genetic profiles that were analyzed simultaneously to establish the relationships between the isolates by means of a dendrogram of similarity (Fig. 3).

The grouping analysis resulted in three genetic groups with similarity about 45% one another. The first group was formed by eight isolates, seven of them obtained from relatives and only one obtained from a child attended at the daycare center, including his mother. The second group was formed by 15 isolates, 14 of them were children attended at the daycare center and only one was isolated from the mother of one of the child. The third group was formed by eight isolates obtained from relatives (mother or brothers) of the children attended at the daycare center. Among the children in the group 2, the greatest similarity was found between the isolates obtained from the children PBC1 and PBC11, with 88% of similarity.

DISCUSSION

Human enteroparasitoses are a public health concern and can be considered as indicators of social-economic conditions of a population. Due to the lower mobility and greater vulnerability, children under five years old can indicate the contamination level of a given region²⁵.

Recent reductions in prevalence of intestinal parasitic infections in some Brazilian regions have been related to improvement of familiar incomings, maternal scholarship, housing, sanitary conditions and health services⁷. In this work, 38.6% of the children under six years old attended at the daycare center in Presidente Bernardes-SP showed cysts or eggs of at least one parasite. This result is quite above the encountered in children up to six years old attended at a daycare center in Belo Horizonte, Minas Gerais State, Brazil (24.6%)¹⁴. The protozoanG. duodenalis and the helminthE. vermicularis were the most frequent in our work, with 16 and 9% of prevalence, respectively. Assessment carried out with children attending the primary school in Kenya revealed prevalence of 87% for ancylostomosis, 88% for trichuriosis and 31% for ascariasis⁵. In Holambra, São Paulo State, Brazil, the most frequent helminth wasAscaris lumbricoides⁹, while in this workE. vermicularis was the most frequent.E. vermicularis expels embryonic eggs that may be transmitted directly by fecal-oral ways, what can explain its high prevalence among children in this study. Similar levels of giardiasis prevalence among children have been reported¹⁷, but in that case the assessments were made in children that were attended in a hospital in Goiânia, Goiás State, Brazil, due to diarrhea.

Despite the higher proportion of girls among the children (61%), only five presented giardiasis, while 10 boys presented this intestinal parasite. Previous work has also showed higher prevalence of intestinal parasitic infections in males than females, without an apparent reason⁴.

Although giardiasis is related to age and social-economical level¹¹, other risk factors for infection have been identified, such as presence of pets at home, specially cats, number of children at home, food hygiene, daycare centers attendance, and living on rural areas¹⁷. The occurrence of children presentingG. duodenalis that had pets at home was low, and in no one case pets were suspect to be the source of contamination, given that they did not presentG. duodenalis cysts. Nevertheless, the zoonotic transmission between dogs and children may also occur⁶.

Besides the several risk factors presented before, giardiasis can be transmitted by fecal-oral ways among children that are attended in daycare centers⁵ or even among the family members at home. Facing the high frequency of giardiasis among the children in this study, it was hypothesized that the transmission way could occur among children during their daily contact at daycare center or by household contact at home. All children in this study lived in houses supplied with water from municipal pipelines, what reduce the chances that infection withG. duodenalis had correlation with drinking water²⁸, supposing adequate water treatment before distribution.

The occurrence ofGiardia duodenalis in children was confirmed by PCR using specific primers for this species. DifferentG. duodenalis isolates studied by different authors, in spite of morphological appearance, have showed different degrees of genetic diversity^2,3,8,12. Two hypotheses could be considered to explain such heterogeneity². Firstly, the differentG. duodenalis isolates form only one species with high degree of intraspecific variation. Second,G. duodenalis would be a complex formed by two or more species morphologically similar.

Both genetic heterogeneity and zoonotic potential ofG. duodenalis may differ according to geographic region¹³ and thus it is essential to typify the parasite in different regions. The heterogeneity ofG. duodenalis isolated from human and dogs was not the same in two Australian communities⁸. In an urban area, isolates from human and dogs had the same genotype, probably because the man was contaminated with the parasites from dogs orvice-versa. However, in an aborigine community, isolates from dogs and humans belonged to different genotypes.

RAPD has been widely used to study the genetic diversity amongG. duodenalis isolates. This technique employs random primers and detects polymorphisms in differentloci, allowing for a wider view of the genome, independently on the knowledge about the degree of ploidia of the microorganism to be studied. Previous work with RAPD showed genetic variability amongG. duodenalis isolates¹⁵. FourteenG. duodenalis isolates extracted from different animals and geographical region were characterized by RAPD and compared with previous data based on isoenzyme analysis of the same isolates. Although the authors had found close correlation between the two methods, some inconsistencies were observed. Seven out of fourteen isolates grouped differently when considering each technique, although the 14 isolates had been separated in 10 groups in both cases.

The characterization ofG. duodenalis isolates in this work by RAPD also revealed genetic variability among the isolates. In 31 isolates, 31 different genetic profiles were found, considering the set of random primers used in the reaction. The highest similarity among the isolates (88%) occurred between two isolates from children attended at the daycare center. Most of isolates from children showed genetic profile forming only one genetic group (group 2), with about 45% of similarity, except one isolate. The isolates from relatives (mothers and brothers) were divided between two other genetic groups with about 45% of similarity (Groups 1 and 3), in which only one isolate from children was included in the group 1. These observations allowed us to conclude that children are not bringing the parasite from their houses. In addition, no one worker at the daycare center presented giardiasis. These results exclude the drinkable water and food at the daycare center as possible way of dissemination, as the nine workers drink the same water and eat the same food. Thus, it is possible that the transmission among children occurred during their contact at the daycare center.

Just after obtaining results of feces examination, the director of daycare center and children parents were informed, and children with positive cases of parasitosis received free medical assistance. In addition, parents and daycare center employees listened an explanation about enteroparasitosis transmission and how to act to reduce or avoid infections.

Despite recent advances, these results show the need for improvement of sanitary conditions and synchronized programs of assistance aiming the sanitary education, continuous assessment of enteroparasitic infections and participation and checking the effectiveness of recommended treatments. The combination of such actions would enable for an improvement of children life conditions and the whole community as well.

In summary, the prevalence ofG. duodenalis (16%) in children attended at the daycare center (Associação Municipal de Proteção ao Menor em Presidente Bernardes, SP) is on the average found in Brazil. By means of RAPD technique, allG. duodenalis isolates were genetically dissimilar, forming three main groups, with about 45% of similarity one another. The most part of the isolates obtained from children at the daycare center (93%) grouped together, showing greater similarity among them. On the other hand, the isolates obtained from their relatives formed other groups. Such results allowed us to conclude that infections in children at the daycare center probably occurred person-person, as a result of their daily contact.

ACKNOWLEDGMENTS

To Universidade do Oeste Paulista (UNOESTE) for financial support. M.A.N is CNPq scholar.

Received: 19 March 2008

Accepted: 23 December 2008

References

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