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Fernández, Heriberto M.Sc., Ph.D.; Neto, Ulysses Fagundes M.D., Ph.D; Ogatha, Silvio M.D.

The Pediatric Infectious Disease Journal: November 1997 - Volume 16 - Issue 11 - p 1098-1099

Institute of Clinical Microbiology; Universidad Austral de Chile; Valdivia, Chile (HF)

Department of Pediatrics; Universidade Federal de Sao Paulo; Sao Paulo, Brazil (UFN, SO)

Accepted for publication Aug. 13, 1997.

Address for reprints: Heriberto Fernández, Ph.D., Institute of Clinical Microbiology, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile. E-mail

To The Editors:

Campylobacter jejuni subsp. doylei is a Gram-negative, S-shaped rod, first isolated in Australia, in 1985, from children <5 years old with diarrheal illness.1 Although their characteristics have been described since 1988 by Steele and Owen,2 little information is available regarding their ecologic and geographic distributions, as well as their prevalence in human diarrhea. We report in this study the first case of acute diarrhea associated with C. jejuni subsp. doylei in Brazil.

A 3.5-year-old girl was assisted at the Pediatric Emergency Clinic of the Sao Paulo Hospital, after 4 days of watery diarrhea associated with vomiting. She had been well until the onset of frankly liquid stools, with no mucus or blood, with a purging rate of eight times daily. The child was alert, active, with no signs of dehydration and without fever (37.2°C). Her abdomen was soft, nontender, without organomegaly or masses but with hyperactive bowel sounds.

Oral fluid therapy without antibiotics and dietetic antidiarrheic regimen were prescribed.

During examination the patient had two bowel movements and a stool sample was obtained. A direct Gram stain was performed. The sample was also examined for ova and parasites and rotavirus, and cultured for enteropathogenic Enterobacteriaceae, Vibrio as well as for Campylobacter spp. Stool culture for the classic thermophilic enteropathogenic Campylobacter species was done by direct inoculation onto a Karmali's medium plate (Probac do Brasil). For the emerging Campylobacter species the membrane filter method of Trevor et al.3 was used. A 47-mm 0.45-μm membrane pore size filter (Millipore) was placed on a sheep blood agar plate (Probac do Brasil) and various drops (10 to 12)4 of 1/10 fecal suspension in physiologic saline solution were placed on the membrane filter. The membrane was removed after 30 min, by which time the fluid and Campylobacter cells will have passed through.

The Karmali's medium plate was incubated at 42°C for 48 h, and the sheep blood agar plate was incubated at 37°C for up to 5 days. Both types of media were incubated in a microaerophilic atmosphere obtained by means of the Microaerobac System® (Probac do Brasil).

The isolated strain was identified as belonging to the genus Campylobacter with Gram stain, oxidase, catalase and motility on wet mounts. Their phenotypical behavior was studied by a standardized API CAMPY® system (BioMérieux) according to the manufacturer's instructions. A laboratory strain of C. jejuni subsp. jejuni was used as reference.

Direct Gram stain from the stool sample revealed abundant small and thinly curved Gram-negative rods, resembling Campylobacter morphology.

The screening for enteric parasites and rotavirus and the standard cultures for conventional bacterial enteropathogens as well as for the classic thermophilic Campylobacter species were negative. Culture on sheep blood agar by the membrane filter method of Steele and McDermott allowed isolation of small, pinpoint, concave nonhemolytic colonies with smooth edges after 5 days of incubation. Subcultures incubated at 42°C or aerobically failed to grow. Oxidase and catalase performed with these colonies gave positive reactions. Gram stain showed curved, S-shaped and spiral Gram-negative rods that, when observed on wet mounts, were actively motile with darting movements. Their growth and morphologic characteristics as well as their biochemical properties determined by means of the API CAMPY® system allowed identification of this isolate as C. jejuni subsp. doylei.

In previous years it was reported that some Campylobacter species, other than C. jejuni subsp. jejuni and Campylobacter coli, can be involved as causative agents of gastroenteritis in humans.4 One of them is C. jejuni subsp. doylei which has been isolated from patients with diarrhea in Australia,1, 2 South Africa,5 Malaysia,6 Spain7 and Sweden.8

C. jejuni subsp. doylei does not grow at 42°C and it grows slowly at 37°C. The organism is characterized by a positive hippurate hydrolysis test result, an inability to reduce nitrate to nitrite and the nonproduction of gamma-glutamyltrans-ferase, and it is more susceptible than C. jejuni subsp. jejuni to cephalothin and polymyxin.2, 7 The strain described here was isolated at 37°C, only in a blood agar plate after a filtration procedure, failed to grow at 42°C in media containing cephalothin and polymyxin and their biochemical behavior was compatible with that described for C. jejuni subsp. doylei.

According to the available data, C. jejuni subsp. doylei is related to production of diarrhea most likely in children younger than 5 years old.2 In this paper we report a child who appeared to be in good overall condition and well-hydrated despite the high number of bowel movements per day and vomiting, probably because of the use of a rehydrating solution administered empirically by her mother. The illness resolved after 5 days of diarrhea.

Considering the presence of liquid stools without mucus, blood and leukocytes and the clinical features observed suggestive of a secretory diarrhea, it is possible that C. jejuni subsp. doylei produces an enterotoxigenic factor. However, their pathogenic attributes as well as their reservoirs and ecologic distribution and the role of C. jejuni subsp. doylei in causing human illness are not yet defined. Additional studies must be performed to confirm the relationship between the presence of the bacterium and the development of diarrhea, to demonstrate virulence factors and to define their reservoirs and ecologic distribution. On the other hand, because of their cultural characteristics, it is necessary that laboratories use isolation methods such as the filtration technique that permit the isolation of this and other Campylobacter species that could be underdiagnosed (misdiagnosed) when solely the traditional selective media for Campylobacter are used.

Acknowledgments. This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil); and Grants FONDECYT 1930353 and S-97-21 of the Research and Development Bureau-Universidad Austral de Chile.

Heriberto Fernández, M.Sc., Ph.D.

Ulysses Fagundes Neto, M.D., Ph.D

Silvio Ogatha, M.D.

Institute of Clinical Microbiology; Universidad Austral de Chile; Valdivia, Chile (HF)

Department of Pediatrics; Universidade Federal de Sao Paulo; Sao Paulo, Brazil (UFN, SO)

1. Steele TW, Sangster N, Lanser JA. DNA relatedness and biochemical features of Campylobacter spp. isolated in Central and South Australia. J Clin Microbiol 1986;22:71-4.
2. Steele TW, Owen RJ. Campylobacter jejuni subsp. doylei subsp. nov., a subspecies of nitrate-negative campylobacters isolated from human clinical samples. Int J Syst Bacteriol 1988;38:316-8.
3. Trevor W, Steele TW, Mc Dermott SN. Technical note: the use of membrane filters applied directly to the surface of agar plates for the isolation of Campylobacter jejuni from feces. Pathology 1984;16:263-5.
4. Allos BM, Blaser MJ. Campylobacter jejuni and the expanding spectrum of related infections. Clin Infect Dis 1995;20:1092-101.
5. Lastovica AJ, Kirby R, Ambrosio RE. Clinical isolates of thermophilic Campylobacter spp. with no or weak catalase activity. In: Pearson AD, Skirrow MB, Lior H, Rowe B, eds. Campylobacter III. London: Public Health Laboratory Service, 1985:201.
6. Tay ST, Puthucheary SD, Devi S, Kautner Y. Characterization of campylobacters from Malaysia. Singapore Med J 1991;36:282-4.
7. Jiménez A, Velázquez JB, Rodríguez J, Chomón B, Villa TG. Biotyping of Campylobacter jejuni and Campylobacter coli in Spain. J Infect 1994;29:305-10.
8. Lindblom GB, Sjörgen E, Hansson-Westerberg J, Kaijser B. Campylobacter upsaliensis, Campylobacter sputorum and Campylobacter concisus as common causes of diarrhoea in Swedish children. Scand J Infect Dis 1995;27:187-8.

Campylobacter jejuni subsp doylei; diarrhea; children

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