Edvaldo Carlos Brito Loureiro; Cintya Oliveira Souza; Eveline Bezerra Sousa; Daniel Vasconcelos Santos; Daniela Cristiane da Cruz Rocha; Francisco Lúziode Paula Ramos; Mônica Cristina Moraes Silva
Instituto Evandro Chagas/SVS/MS, Ananindeua, Pará, Brasil
Original Title: Detecção de bactérias enteropatogênicas e enteroparasitas em pacientes com diarreia aguda em Juruti, Pará, Brasil. Translated by: American Journal Experts
With the aim of determining the frequency rate of enteropathogenic bacteria and enteroparasites in patients with acute diarrhea in the Municipality of Juruti, Pará State, which has been facing an intense migratory process due to a great mining project, we analyzed 263 cases of acute diarrhea. A total of 19% of those cases were related to enteropathogenic bacteria, particularly Shigella spp. that accounted for 13.7% of the cases. Salmonella spp., Aeromonas spp. and Plesiomonas shigeloide were also detected. The most frequent Shigella serotypes found were S. flexneri (61.1%) and S. sonnei (38.9%). Among the Salmonella, we could observe the following serotypes: S. Panama (2), S. Newport (2), S. Bredeney, S. Saintpaul and S. Gaminara. The infection caused by protozoa reached 85.3%, mostly due to Blastocystis hominis (37.3%), Entamoeba histolytica/E. dispar (22.8%), Endolimax nana (16.7%), Entamoeba coli (9.5%) and Giardia lamblia (8.4%). These results provide valuable data to epidemiological and environmental surveillance because it presents the unsatisfactory basic sanitary conditions of the local population.
Keywords: Diarrhea; Enterobacteriaceae; Intestinal Diseases, Parasitic; Protozoan Infections.
Annually, diarrheal diseases are responsible for the deaths of approximately 2.5 million children younger than 5 years of age, with developing countries bearing the majority of this burden12. Early weaning, low education levels and poor sanitation conditions are predisposing factors for diarrheal diseases33.
Viruses, bacteria and protozoa are among the infectious causes of diarrhea. The main etiological agents include the following: rotavirus, calicivirus, astrovirus, diarrheagenic Escherichia coli, Shigella spp., Salmonella spp., Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum and Cyclospora cayetanensis10,22,28,29,32.
In Brazil, intestinal infections deserve attention, especially those caused by diarrheagenic E. coli2,4, rotavirus15,19, Cryptosporidium and Entamoeba histolytica21,25.
The treatment of most bacteria-induced cases of diarrhea only requires correction of the symptoms and the reestablishment of the patient's electrolyte balance. However, it is important to note that a simple case of diarrhea may trigger epidemiological surveillance systems, given the possibility of the involvement of potentially epidemic agents such as V. cholerae O127. It is also important to further direct therapeutic measures to interrupt the chain of transmission for diseases such as cholera. Additionally, actions should be taken to reduce or avoid the risk of complications due to certain virulent bacteria, such as Shigella and S. typhi6,17.
Some intestinal parasites have great importance in the pathogenesis of diarrhea, and several studies have associated diarrhea with Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica21,25,30,3. Intestinal parasites are highly transmissible, particularly in developing countries where population growth is not matched with improvements in living conditions26, creating permanent public health problems.
According to the Municipal Health Secretariat (SEMSA) in the city of Juruti, Pará State, Brazil, diarrhea is one of the main illnesses of the population due to the lack of infrastructure, sanitation and clean drinking water23. However, little is known about the infectious causes of local diarrheal diseases due to the lack of qualified personnel and appropriate diagnostic methods. Thus, the aim of this study was to analyze the relationship between the bacterial and parasitic enteropathogens and cases of acute diarrheal disease in the city of Juruti, Pará State, Brazil.
MATERIAL AND METHODS
For this study, 332 patients were selected; of these, 286 (86%) were originally from the urban area of Juruti. The remaining patients were from the surrounding rural region. Of the total, 263 patients presented with acute diarrhea, defined as experiencing three or more stools per day, and 69 patients had experienced no diarrhea symptoms within the past 15 days and were considered the control group. The subjects were children and adults that visited one of three health centers in the city of Juruti. All patients or their guardians gave their written, informed consent to participate in this study. This study was performed under protocol 030/05, CAAE 0013.0.072.000-06 and was approved by the Research Ethics Committee of the Instituto Evandro Chagas (IEC).
Parasitological examinations were carried out using optical light microscopy for the analysis of the varied protozoa life cycle stages and helminths. Direct methods such as physiological saline, Lugol and spontaneous sedimentation were also utilized5.
Bacterial isolations were performed using fecal suspension; aliquots of these suspensions were plated onto selective media including MacConkey agar (MC), Salmonella-Shigella agar (SS) and thiosulfate citrate bile sucrose agar (TCBS). Samples were also seeded into selenite cystine and alkaline peptone water (APW), pH 8.5, a selective enrichment media. Selected colonies were submitted for triple sugar iron (TSI) and Kligler iron agar (KIA) screening. Biochemical characterizations were performed using conventional methods and the API-20E system (bioMerieux)14. Serum characterizations were performed according to Ewing8 and Kauffmann11.
The categories of pathogenic E. coli were as follows: EPEC (enteropathogenic E. coli), ETEC (enterotoxigenic E. coli), STEC (Shiga toxin-producing E. coli) and EIEC (enteroinvasive E. coli). These pathogens were identified by their virulence genes. Bacteria were screened for each virulence factor using gene-specific primers in polymerase chain reactions (PCR)7,9,18,20,31,34.
As shown in Table 1, bacterial enteropathogens were isolated in 50 (1 9%) out of the 263 cases of acute diarrhea. Of these, 36 (13.7%) cases were caused by Shigella spp., 7 (2.7%) by Salmonella spp., 6 (2.2%) by Aeromonas spp., and 1 (0.4%) by P shigelloides. Regarding parasitic infections, it is important to note that 88 patients (33.5%) were infected with two or more parasitic species, whereas 74 (28.2%) patients were infected with a single parasite. The most common parasites observed were B. hominis, E. histolytica/E. dispar, E. nana, hookworms and A. lumbricoides. In addition, parasitism by G. lamblia was found in 22 cases (8.4%). Among the enteropathogenic bacteria, an association was found only with S. flexneri and A. sobria. The most common serotype of Shigella was S. flexneri (61.1%) followed by S. sonnei (38.9%). For Salmonella infections, the identified serotypes included S. Panama (2), S. Newport (2), S. Bredeney, S. Saintpaul and S. Gaminara. A total of 35 patients exhibited infection with different categories of E. coli pathogens. Of these, eight patients were infected with diarrheagenic E. coli, seven with enterotoxigenic E. coli (ETEC) (LT +  and Sta ) and one with atypical enteropathogenic E. coli (EPEC).
The analysis comparing the results between the diarrheic patients and control group found that the frequency of bacterial agents was higher in the former, especially for Shigella spp. This difference was not observed for patients with parasitic infections; however, the frequency of E. histolytica/E. dispar and G. lamblia is noteworthy (Table 2).
DISCUSSION AND CONCLUSION
Overall, enteropathogenic bacteria accounted for 19% of the examined diarrhea cases. The most common bacteria belonged to the genus Shigella; in particular, S. flexneri and S. sonnei were frequently identified. These results are consistent with the fact that these species are the most frequent species detected in Brazil2. Among the so-called emerging bacterial pathogen16, Aeromonas and Plesiomonas were identified in patients with acute diarrhea. Although five serovars of Salmonella were identified among the diarrheal cases, no cases of S. Typhi or Vibrio were found. Nevertheless, it is important to emphasize the need to screen for S. Typhi because it is the causative agent of typhoid fever, which has caused epidemics in several municipalities of Para State24.
Several authors have shown the importance of 'diarrheagenic' E. coli in diarrheal cases28,2,19. Here we provided evidence of its occurrence in Para State, Brazil, suggesting a wide distribution and public health relevance of this pathogen.
It has been observed that parasites are important in the enteric processes of the population of Juruti. A great diversity of protozoa and helminths were observed among patients with diarrhea; E. histolytica/E. dispar and B. hominis were the most frequent. However, the pathogenicity of B. hominis has recently been discussed1. In Brazil, protozoa-associated acute diarrhea has been reported by several authors25,30. As expected, patients between 1 and 4 years of age had a higher frequency of parasite infection, followed by patients between 5 and 10 years of age; these finding are consistent with published data13.
These results provide important support to epidemiological and environmental surveillance, demonstrating that the population is at risk of developing diarrheal disease as a consequence of poor sanitation conditions.
Continuation of this study, with the molecular analysis of pathogenic categories of E. coli, the detection of rotavirus, and the characterization of G. lamblia, E. histolytica and Cryptosporidium spp. antigens could provide data demonstrating the participation of these pathogens in the etiology of diarrheal diseases in Juruti.
The marked presence of enteropathogens in the population of Juruti, with the appearance of emerging agents, and the high immigration movement caused by the deployment of a large-scale development project being implemented in the area reflect the need for the continual surveillance of the causative agents of diarrhea; in particular, bacterial pathogens should be monitored because of their high epidemic potential.
We would like to thank the staff of the Instituto Evandro Chagas: Bruno Lima, Claudia Nascimento, Dolores Santos, José Caetano Silva, José Goes dos Santos, Madalena Lobato, Maria da Paz Mesquita, Maria do Socorro Garcez, Maria Odete Arouche, Raimundo Nonato Araujo, Patricia Yoshida and Suely Pacheco for their technical and administrative support. We would also like to thank the staff at the Juruti Municipal Health Secretariat and Alcoa / Omnia SA for their support during our fieldwork. This work was funded by Alcoa / Omnia, CNPq-403323.2005.3 and IEC/SVS/MS.
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Recebido em / Received / Recibido en: 31/7/2009
Instituto Evandro Chagas / Secretaria de Vigilância em Saúde / Ministério da Saúde
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