Prevalence of Salmonella enterica in children aged less than 5 years with acute diarrhea and controls in Teresina-PI

1. Doutora em Microbiologia; professora adjunta da Universidade Federal do Piauí (UFPI). 2. Pós-doutor em Microbiologia; professor titular da Universidade Federal de Minas Gerais (UFMG). 3. Médico do Instituto Nacional de Seguro Social (INSS) do Piauí. 4. Doutora em Microbiologia; professora adjunta da UFMG. 5. Doutor em Pediatria; professor titular da UFMG. J Bras Patol Med Lab • v. 48 • n. 2 • p. 105-108 • abril 2012


Introduction
Enteric infections are a leading cause of disease and death in children, particularly in the developing world (1,2) .Estimates from World Health Organization (WHO) indicate that 1.6/2.1 million deaths occurred in children aged less than 5 years during 2000 (3,9) .Although mortality rates due to diarrhea have decreased, morbidity levels have not followed the same trend, especially in poor regions (8) .Furthermore, despite most diarrhea episodes are selflimited and dehydration can usually be controlled with oral rehydration, it would be desirable to prevent diarrhea or treat patients presenting with more severe disease (11) .The number of pathogens that are responsible for diarrheal disease is large and increasing but the estimation of diarrhea episodes due to each pathogen is unclear.We conducted this study to better understand the distribution of SE diarrhea episodes in children aged less than 5 years in Teresina-PI, one of the poorest regions in Brazil.We also investigated clinical and epidemiological data associated with Salmonella enterica (SE) infection and the antimicrobial susceptibility of the isolates.To our knowledge this is the first systematic study on SE intestinal infection performed in this geographic area.

Patients and methods
This investigation was part of a prospective study of children's diarrhea and was conducted in Teresina.Fecal samples were collected from 400 children up to 60 months of age (mean 13.9 months; 56.7% male), all of them outpatients, from the low socioeconomic stratum (mean annual income ~ US$4,700) seen at two public hospitals in Teresina from January 2004 to August 2007.Among them, 250 (mean age 14.6 months; 56% male) presented acute diarrhea and 150 (mean age 12.6 months; 58% male) had no diarrhea in the previous 15 days.No subject had history of hospitalization or antimicrobial therapy in the previous 15 days.Fecal specimens were obtained and individually transferred to sterile vials containing a sterile solution of equal parts of glycerol and 0.033 M phosphate buffer pH 7. Specimens were transported to the Laboratório de Microbiologia of Departamento de Parasitologia e Microbiologia of the Universidade Federal do Piauí (UFPI) within an hour and streaked onto MacConkey agar and Salmonella-Shigella (SS) agar.After incubation at 35ºC overnight around five lactose-positive, five lactose-negative and five H 2 S-positive colonies were picked up and further identified by microbiological methods.SE isolates were sent to a reference laboratory (Laboratório de Enterobactérias of Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) for serotyping.
Antimicrobial susceptibilities of one selected SE isolate to ampicillin (AMP), ceftriaxone (CFX), chloranfenicol (CHL), ciprofloxacin (CIP), nalidixic acid (NAL), and sulfamethoxazole-trimethoprim (SXT) were assayed by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) 2005 guidelines (4) .Data were analyzed using the χ 2 test with Yates' correction or Fisher's exact test as appropriate with the level of significance set at 5%.
The research protocol was approved by the Ethics Committee of the Universidade Federal de Minas Gerais (UFMG) and of the UFPI.The parents or guardians of all children gave written informed consent.

Results
SE was isolated from feces of five (5 out of 250; 2%) patients, all of them with diarrhea.Isolates were further identified as SE O-C2-C3-ND, SE serovar Enteritidis, SE serovar Newport (n = 2), and SE serovar Muenchen.The organism was not isolated from control patients.SE strains were evenly distributed in dry and rainy seasons.No association with sex, and presence of blood, pus and mucus in the feces was found.Increased fecal volume, three-10 bowel movements daily, vomits and fever (from 37.5 to 41ºC) were reported for all SE infected children.SE infection was more common (three out of five) in patients aged less than 6 months; SE serovar Newport was identified in two out of these three patients.Association among SE serovars and clinical and epidemiological data is shown in Table 1.
The antimicrobial susceptibility profile of SE isolates is depicted in Table 2.No isolate was resistant to all antimicrobials tested.SE serovar O-C2-C3-ND was resistant to CIP, CHL, NAL, and SXT and SE serovar Enteritidis was resistant to NAL.All other strains were pansensitive.

Discussion
Salmonellosis is a worldwide health problem being the second leading cause of bacterial foodborne illness in the United States (6) .However, the actual magnitude of the problem in Brazil is unknown since no recent data on human salmonellosis is available in specialized literature.We showed that SE infection is rare in children aged less than 5 years in Teresina, in the Brazilian Northeast, one of the poorest areas in the country.There are now over 2,500 identified serotypes of SE.Among them, a smaller number of serovars are significantly associated with human disease especially Typhimurium and Enteritidis (5)(6)(7) .In contrast to these data, serovar Newport was the most common detected in our population, a finding that demonstrates the uneven distribution of SE serotypes over the world.As expected by the fact that salmonellosis is a foodborne disease frequently associated with the consumption of naturally contaminated animal foods or derivatives, no seasonality was seen in the distribution of the disease.SE can be associated with a number of different disease syndromes, the most common being enteritis, frequently characterized by nausea, vomiting, diarrhea, and fever (6) .As expected, these clinical manifestations were reported by our patients.The disease is typically self-limited, but patients presenting with more severe manifestations should be treated aiming to eradicate the organism.Increasingly, SE isolates are being detected that demonstrate resistance to multiple antimicrobial agents, including third-generation cephalosporins, commonly recommended for treating severe infections (6) .Our results are similar to those reported by Vaz et al. (10) who found high resistance levels of SE Enteritidis to NAL and SXT, two antimicrobials frequently employed in our country for the treatment of patients supposed to have inflammatory diarrhea.Surprisingly, all SE we identified were susceptible to AMP, a drug largely employed in Brazil for treating patients with different kinds of bacterial infections.
In brief, SE infection is rare in children from the Brazilian Northeast.Our data support the need to establish monitoring programs to evaluate the magnitude of SE infections in different parts of the world and the emergence of potential resistance patterns in order to drive the empirically selected antimicrobial drugs for treating patients with more severe presentations of the disease.

Table 1
Association among Salmonella enterica serovar and clinical and epidemiologic data 1: in Celsius degrees; 2 : in months;