Atypical enteropathogenic Escherichia coli infection and prolonged diarrhea in children.
Some clinical isolates of enteropathogenic enteropathogenic
having pathogenicity for the intestine.
enteropathogenic Escherichia coli
strains of E. coli which cause enteritis by close association with enteric cells. Includes attaching and effacing E. coli. Escherichia coli Escherichia coli (ĕsh'ərĭk`ēə kō`lī), common bacterium that normally inhabits the intestinal tracts of humans and animals, but can cause infection in other parts of the body, especially the urinary tract. (EPEC EPEC
enteropathogenic Escherichia coli.
EPEC Enteropathic Escherichia coli, see there ) lack bundle-forming pili pili /pi·li/ (pi´li) [L.] plural of pilus.
plural of pilus.
pili torti and are termed atypical EPEC. The aim of this study was to determine if atypical EPEC are pathogens by comparing the clinical features of patients infected with atypical EPEC with those of children infected with other causative agents of diarrhea. Fecal samples obtained from children attending the Royal Children's Hospital The Royal Children's Hospital in Melbourne, Australia is the major specialist paediatric hospital for Victoria offering a full range of clinical services, tertiary care and health promotion and prevention programs for children and adolescents. in Melbourne for investigation of diarrhea were examined for adenovirus adenovirus
Any of a group of spheroidal viruses, made up of DNA wrapped in a protein coat, that cause sore throat and fever in humans, hepatitis in dogs, and several diseases in fowl, mice, cattle, pigs, and monkeys. , rotavirus rotavirus /ro·ta·vi·rus/ (ro´tah-vi?rus) any member of the genus Rotavirus. ro´taviral
Rotavirus /Ro·ta·vi·rus/ (ro´tah-vi?rus , Campylobacter Campylobacter
Genus of gram-negative spiral-shaped bacteria infecting mammals. Many species, especially C. fetus, cause miscarriage in sheep and cattle. C. jejuni is a common cause of food poisoning. Sources include meats (particularly chicken) and unpasteurized milk. spp., Salmonella spp., protozoa, and pathogenic E. coli E. coli: see Escherichia coli.
in full Escherichia coli
Species of bacterium that inhabits the stomach and intestines. E. coli can be transmitted by water, milk, food, or flies and other insects. . Clinical data were obtained by using a standardized pro forma As a matter of form or for the sake of form. Used to describe accounting, financial, and other statements or conclusions based upon assumed or anticipated facts.
The phrase pro forma and analyzed separately. Patients infected with atypical EPEC experienced mild, nondehydrating, and noninflammatory diarrhea that was not particularly associated with fever, vomiting, or abdominal pain Abdominal pain can be one of the symptoms associated with transient disorders or serious disease. Making a definitive diagnosis of the cause of abdominal pain can be difficult, because many diseases can result in this symptom. Abdominal pain is a common problem. . However, the duration of diarrhea in patients infected with atypical EPEC was significantly longer than that caused by the other species or where no pathogens were identified. Infection with atypical EPEC is associated with prolonged diarrhea.
The varieties of Escherichia coli that cause diarrhea are classified into pathogenic groups (pathotypes) according to according to
1. As stated or indicated by; on the authority of: according to historians.
2. In keeping with: according to instructions.
3. their virulence determinants (1,2). The specific nature of these virulence determinants imbues each pathotype with the capacity to cause clinical syndromes with distinctive epidemiologic and pathologic characteristics (2). For example, enterotoxigenic en·ter·o·tox·i·gen·ic
Of or being an organism containing or producing an enterotoxin.
Enterotoxigenic E. coli causes watery diarrhea in children in developing countries and in travelers to those countries, whereas enterohemorrhagic E. coli (EHEC EHEC
enterohemorrhagic Escherichia coli.
EHEC Enterohemorrhagic Escherichia coli, see there ) may cause hemorrhagic colitis hemorrhagic colitis
Abdominal cramps and bloody diarrhea, without fever, attributed to a self-limited infection by a strain of Escherichia coli. and the hemolytic uremic syndrome hemolytic uremic syndrome
A syndrome in which hemolytic anemia and thrombocytopenia occur with acute renal failure, marked in children by sudden gastrointestinal bleeding, urine that contains red blood cells and is scanty in volume, and because of the production of Shiga toxins. Enteropathogenic E. coli (EPEC) shares several key virulence determinants with the most common varieties of EHEC but does not produce Shiga toxins nor cause hemorrhagic colitis or hemolytic uremic syndrome. Instead, it causes nonspecific nonspecific /non·spe·cif·ic/ (non?spi-sif´ik)
1. not due to any single known cause.
2. not directed against a particular agent, but rather having a general effect.
1. gastroenteritis gastroenteritis: see enteritis.
Acute infectious syndrome of the stomach lining and intestines. Symptoms include diarrhea, vomiting, and abdominal cramps. , especially in children in developing countries (3,4). EPEC also differs from other pathotypes of E. coli in that it typically carries an EPEC adherence factor plasmid (pEAF). This plasmid encodes 1) bundle-forming pili (Bfp), which promote bacterial adherence to epithelial cells Epithelial cells
Cells that form a thin surface coating on the outside of a body structure.
Mentioned in: Corneal Transplantation and are an essential virulence determinant (5), and 2) a transcriptional activator, Per, that upregulates genes within a chromosomal pathogenicity island Pathogenicity islands (PAIs) are a distinct class of genomic islands which are acquired by horizontal transfer.
They are incorporated in the genome of pathogenic microorganisms but are usually absent from those non-pathogenic organisms of the same or closely related species. , termed the locus for enterocyte enterocyte
the predominant cells in the small intestinal mucosa. They are tall columnar cells and responsible for the final digestion and absorption of nutrients, electrolytes and water. effacement effacement /ef·face·ment/ (e-fas´ment) the obliteration of features; said of the cervix during labor when it is so changed that only the external os remains. (6,7). This pathogenicity island encodes a number of essential virulence proteins, including the surface protein intimin (the product of the eae gene), which is required to produce the attaching-effacing lesions that are a key feature of EPEC-induced pathology. A subset of EPEC, known as atypical EPEC, do not carry pEAF and hence do not produce Bfp or Per (4). Accordingly, their role in disease is controversial. Recently, we and others investigated the causes of community-acquired gastroenteritis in Melbourne (8, 9). Among the infectious agents that were sought in these studies was atypical EPEC, which emerged as the single most frequent pathogen in the study population (9).
To determine if atypical EPEC are also responsible for diarrhea in hospitalized children, we undertook a comprehensive microbiologic study of patients with diarrhea at the Royal Children's Hospital in Melbourne.
Patients and Methods
Patients were children with diarrhea attending the Royal Children's Hospital, Melbourne, between March 1 and August 31, 2003. They were considered for inclusion in the study when an obviously loose stool sample from a child <14 years of age was received at the Diagnostic Microbiology Laboratory for investigation. After their caregivers, attending physicians, and medical records had been consulted, patients were considered eligible for inclusion in the study if, during the current illness, they had passed [greater than or equal to] 3 loose stools within a day or had experienced loose stools plus vomiting, abdominal pain, or rectal bleeding Rectal bleeding can refer to:
or nontropical sprue
Digestive disorder in which people cannot tolerate gluten, a protein constituent of wheat, barley, malt, and rye flours. , were excluded, as were those with cystic fibrosis cystic fibrosis (sĭs`tĭk fībrō`sĭs), inherited disorder of the exocrine glands (see gland), affecting children and young people; median survival is 25 years in females and 30 years in males. , leukemia, and other immunosuppressive Immunosuppressive
Any agent that suppresses the immune response of an individual.
Mentioned in: Antirheumatic Drugs, Graft-vs.-Host Disease, Immunosuppressant Drugs
1. pertaining to or inducing immunosuppression.
2. disorders. Repeat samples and samples from children who had received antimicrobial agents Antimicrobial agents
Chemical compounds biosynthetically or synthetically produced which either destroy or usefully suppress the growth or metabolism of a variety of microscopic or submicroscopic forms of life. within the preceding week were also excluded.
Clinical data were obtained in accordance with a standardized pro forma and were analyzed before the results of the laboratory findings were known. Data collected included age; gender; date of onset of illness; symptoms and clinical signs, including characteristics of stools, abdominal pain, vomiting (number per day and duration), fever, abdominal tenderness, largest number of bowel movements in a 24-hour period preceding the sample collection, and extent of dehydration. Duration of diarrhea was estimated from the passage of the first loose stool to the patient's last appearance in the ward or 1 day after discharge. Patients with temperatures of [greater than or equal to] 38[degrees]C, taken by tympanic tympanic /tym·pan·ic/ (tim-pan´ik)
1. tympanal; of or pertaining to the tympanum.
2. bell-like; resonant.
1. thermometer, were considered febrile febrile /feb·rile/ (feb´ril) pertaining to or characterized by fever.
Of, relating to, or characterized by fever; feverish. . Severity of illness was estimated by using the 20-point scale developed by Ruuska and Vesikari (10).
All stool specimens were the first specimen obtained from a patient on a hospital visit, and specimens were investigated within 4 hours of collection. Specimens were examined macroscopically mac·ro·scop·ic also mac·ro·scop·i·cal
1. Large enough to be perceived or examined by the unaided eye.
2. Relating to observations made by the unaided eye. for color and consistency and by light microscopy for leukocytes, erythrocytes Erythrocytes
Red blood cells.
Mentioned in: Bartonellosis
n.pl red blood cells. , and parasitic forms (amebas, cysts, and ova ova (o´vah) plural of ovum.
Mentioned in: Stool O & P Test
plural of ovum. ) by using a saline-and-iodine wet preparation and a modified Ziehl-Neelsen stain Ziehl-Neelsen stain
a carbol-fuchsin stain most used for the detection of Mycobacterium spp. for oocysts of Cryptosporidium cryptosporidium (krĭp'tōspərĭd`ēəm), genus of protozoans having at least four species; they are waterborne parasites that cause the disease cryptosporidiosis. spp. (11). Samples were tested by enzyme immunoassay Immunoassay
An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus. for enteric adenoviruses and rotaviruses and cultured for E. coli, Salmonella, Shigella shigella
Any of the rod-shaped bacteria that make up the genus Shigella, which are normal inhabitants of the human intestinal tract and can cause dysentery, or shigellosis. Shigellae are gram-negative (see gram stain), non-spore-forming, stationary bacteria. S. , Yersinia Yersinia
A genus of bacteria in the Enterobacteriaceae family. The bacteria appear as gram-negative rods and share many physiological properties with related Escherichia coli. Of the 11 species of Yersinia, Y. pestis, Y. enterocolitica, and Y. , and Campylobacter spp. (12).
To reduce the cost of the investigation, diarrheogenic strains of E. coli were sought only during the first 11 weeks of the study, from March 1 to May 15. Bacteria were isolated from fecal samples by direct plating on MacConkey agar MacConkey (also McConkey) agar is a culture medium designed to grow Gram-negative bacteria and stain them for lactose fermentation. It contains bile salts, crystal violet dye (to inhibit Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose), (Oxoid Ltd., Basingstoke, UK). After overnight incubation at 37[degrees]C, a sterile cotton swab "Q-Tip" redirects here. For the rapper, see Q-Tip (rapper). For the band, see Q-Tips (band).
Cotton swabs (British English: cotton buds) are used in first aid, cosmetics application, and a variety of other uses. was used to transfer the entire growth from each plate into Luria broth containing 30% (vol/vol) glycerol glycerol, glycerin, glycerine, or 1,2,3-propanetriol (prō`pāntrī'ŏl), CH2OHCHOHCH2OH, colorless, odorless, sweet-tasting, syrupy liquid. , which was then frozen at -70[degrees]C until required. E. coli pathotypes were identified by polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) ) and confirmed by Southern hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.
2. molecular hybridization
3. (9). Briefly, template DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. for use in PCR was prepared from bacteria isolated from MacConkey agar plates and grown in 2.5 mL MacConkey broth with shaking at 37[degrees]C overnight. Bacteria from this culture were washed in phosphate-buffered saline, resuspended in sterile distilled water Noun 1. distilled water - water that has been purified by distillation
H2O, water - binary compound that occurs at room temperature as a clear colorless odorless tasteless liquid; freezes into ice below 0 degrees centigrade and boils above 100 degrees centigrade; , and heated for 10 min at 100[degrees]C. Samples were then placed on ice for 5 min and recentrifuged for 5 min at 16,000 x g. Aliquots of the supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material.
the liquid lying above a layer of precipitated insoluble material. were pipetted into sterile tubes, stored at -20[degrees]C for <1 week, and then diluted 1 in 10 in distilled water before being added to the PCR mix. PCR amplifications were performed in a GeneAmp PCR System 9700 thermal cycler The Thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus used for PCR. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. (Applied Biosystems Applied Biosystems, Inc. (formerly NASDAQ: ABIO) is the original name of a pioneer biotechnology company founded in 1981 in Foster City, California, among the Silicon Valley cities of the southern San Francisco Bay Area. , Foster City, CA, USA) with the PCR primers and conditions described previously (9). Genes identified by these primers and their association with each pathotype of diarrheogenic E. coli are listed in Table 1. PCR for the lacZ gene, which is present in almost all wildtype strains of E. coli, was included as a control to ensure that negative PCR results were not due to the absence of viable bacteria in the sample or the presence of inhibitors in the reaction mixture. Samples that were PCR negative for lacZ were excluded from further analysis. At the conclusion of the PCR, 10 [micro]L of the reaction mixture underwent electrophoresis on 2.5% 96-well format agarose agarose
more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments. gels (Electro-fast; Abgene, Epsom, UK). Gels were stained with ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic acid stain in molecular biology laboratories for techniques such as agarose gel electrophoresis. , visualized on a UV transilluminator, and photographed. A portion of the PCR product was retained for Southern blotting, which was performed by using capillary transfer of separated DNA fragments onto positively charged Adj. 1. positively charged - having a positive charge; "protons are positive"
charged - of a particle or body or system; having a net amount of positive or negative electric charge; "charged particles"; "a charged battery" nylon membranes (Roche Diagnostics Roche Diagnostics Division is a subsidiary of Hoffmann-La Roche which manufactures equipment and reagents for research and medical diagnostic applications. Internally, it is organized into six major business areas: Roche Applied Science, Roche Centralized Diagnostics, Roche Ltd., Lewes, UK). Digoxigenin-labeled DNA probes were prepared from the control strains of diarrheogenic E. coli listed in Table 1 and used as described (9). PCR- and probe-positive bacteria were assigned to a pathotype according to the criteria in Table 1. Equivocal or ambiguous assays were repeated, and if still unclear, were excluded from further analysis. Atypical EPEC strains were isolated in pure culture from the original sample and then serotyped by using hyperimmune hyperimmune /hy·per·im·mune/ (hi?per-i-mun´) possessing very large quantities of specific antibodies in the serum.
possessing very large quantities of specific antibodies in the serum. rabbit antisera to O-antigens O1 through O181 (18). These strains were also subjected to PCR to determine the intimin subtype (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T. and to investigate the presence of selected virulence-associated genes by using the PCR primers and conditions described previously (9).
Statistical analysis of quantitative and qualitative data was performed by using InStat, Version 3.05 (GraphPad Software Inc., San Diego San Diego (săn dēā`gō), city (1990 pop. 1,110,549), seat of San Diego co., S Calif., on San Diego Bay; inc. 1850. San Diego includes the unincorporated communities of La Jolla and Spring Valley. Coronado is across the bay. , CA, USA). A 2-tailed p value of <0.05 indicated statistical significance. For the analysis of clinical features associated with infection, patients whose stools yielded >1 pathogen were excluded.
Frequency of Viral, Parasitic, and Bacterial Pathogens
After exclusion of repeat samples and samples from patients >14 years of age or with cystic fibrosis, chronic inflammatory bowel disease inflammatory bowel disease
n. Abbr. IBD
Any of several incurable and debilitating diseases of the gastrointestinal tract characterized by inflammation and obstruction of parts of the intestine. , leukemia, or a history of recent antimicrobial drug usage, 303 of 972 consecutive fecal samples remained for analysis. Of these, 134 were from the first period of the study, March 1-May 15, when diarrheogenic E. coli were sought together with other enteropathogens, and 169 were from the period May 16-August 31, when E. coli were not sought.
The frequency of bacterial, viral, and parasitic pathogens identified during the 2 phases of the study are shown in Table 2. During the first period, a putative etiologic agent was identified in 88 (66%) of 134 children. Diarrheogenic E. coli were found in 42 (31%) of these children, followed by enteric adenovirus (10%), Salmonella sp. (10%), Campylobacter spp. (9%), Giardia Giardia /Gi·ar·dia/ (je-ahr´de-ah) a genus of flagellate protozoa parasitic in the intestinal tract of humans and other animals, which may cause giardiasis; G. lam´blia (G. intestina´lis) is the species found in humans. sp. (6%), rotavirus (4%), and Cryptosporidium sp. (2%). Of the 42 E. coli isolates, 30 (71%) were EPEC; 6 (14%) were Shiga toxin-producing E. coli (STEC STEC
shiga toxin-producing Escherichia coli. ), of which 3 were EHEC; 4 (10%) were enteroaggregative E. coli (EAEC EAEC
enteroadherent Escherichia coli.
EAEC Enteroadherent Escherichia coli, see there ), 1 (2%) was enterotoxigenic E. coli; and 1 (2%) was enteroinvasive E. coli. Nine children (7%) were infected with > 1 pathogen, including 2 concurrently infected with EPEC and adenovirus or EPEC and rotavirus, and 1 each with EPEC and Giardia sp.; STEC and Campylobacter sp.; STEC and Giardia sp.; EAEC and Campylobacter sp., and EAEC and rotavirus.
All EPEC isolates were atypical EPEC (i.e., PCR negative for bfpA). Determination of the O:H serotype serotype /se·ro·type/ (ser´o-tip) the type of a microorganism determined by its constituent antigens; a taxonomic subdivision based thereon.
v. and intimin subtype of 29 of the 30 EPEC strains (1 was not viable) indicated that they were highly heterogeneous (Table 3). Although 3 strains (R41, R151, and R446) were O-nontypable:H34, intimin-[alpha]2; and 2 (R89 and R104) were O153:H7, intimin [beta], these isolates were neither temporally nor geographically related to each other and showed some differences in their carriage of accessory virulence-related factors (data not shown). Two other isolates (R250 and R436) were O33:H6 but had different intimin types. Ten isolates were O-serogroups that were classified as nontypable because they did not react with any of the available O-typing sera (O1-O181), and 2 isolates could not be serotyped because they were rough. Only 1 isolate (R404) belonged to an E. coli serotype, O128:H2, that is commonly associated with EPEC (4).
During the second period of the study, when E. coli was not sought, putative pathogens were identified in 99 (58.6%) of 169 children; rotavirus was the most frequent (33.7%), followed by Campylobacter (11.8%), adenovirus (7.7%), Salmonella (5.3%), Giardia (1.8%), and Cryptosporidium (0.6%) spp. Four patients were infected with >1 pathogen: 3 concurrently infected with rotavirus and adenovirus and 1 with adenovirus and Salmonella sp. Shigella and Yersinia spp. were not identified during either period of the study. The frequency of rotavirus infection rotavirus infection Virology RI is usually mild, but may be severe in children ≤ 2 yrs due to intense vomiting Morbidity > 870,000 children < age 5 die of rotavirus infection in developing countries, in contrast to 75 to 150 in the US Epidemiology during the second phase of the study was significantly greater than during the first phase (odds ratio [OR] 13.13; 95% confidence interval confidence interval,
n a statistical device used to determine the range within which an acceptable datum would fall. Confidence intervals are usually expressed in percentages, typically 95% or 99%. [CI] 5.08 33.91, p<[10.sup.-6], 2-tailed Fisher exact test), confirming the well-known association of rotavirus with winter diarrhea (12). The frequency of patients in whom no pathogens were identified was not significantly different between the 2 study periods (OR 0.74, 95% CI 0.46-1.18, p>0.2, 2-tailed Fisher exact test), despite the omission of tests for diarrheogenic E. coli during the second period. This finding suggests that E. coli did not account for a large number of cases during the second period of the study and accords with our previous observations that diarrhea due to E. coli is relatively less frequent during winter (9,19).
Comparison of Clinical and Laboratory Findings
The clinical and laboratory features of patients infected with different pathogens were compared (Table 4). Patients infected with >1 pathogen were excluded from this analysis, as were those infected with Giardia or Cryptosporidium spp. or diarrheogenic E. coli other than EPEC because their numbers were too small for the results to be meaningful. For this analysis, only those patients in whom no pathogens were identified from the first study period were considered because of the possibility that some of those studied during the second period were infected with diarrheogenic E. coli.
Patients infected with EPEC were of a similar age (median 16.9 months) to those from whom no pathogens were isolated (median age 11.6 months). Of the various groups of patients defined according to the cause of diarrhea, only those infected with Campylobacter spp. (median age 34.2 months) differed significantly in age from those with EPEC (p = 0.0002, Mann-Whitney U test Mann-Whitney U test,
n.pr See test, Mann-Whitney U. ). Eighteen (72%) of 25 children monoinfected with EPEC were boys compared with 20 (44%) of 46 children in whom no pathogens were identified (OR = 3.34, 95% Cl 1.17-9.55, p = 0.03, 2-tailed Fisher exact test), and with 49 (47%) of 104 children enrolled in phase 1 of the study who were not infected with EPEC (OR = 2.89, 95% CI 1.11 7.5, p = 0.03).
Patients infected with rotavirus or adenovirus were significantly more likely to have a history of vomiting than those with no pathogen identified or those infected with EPEC, Salmonella, or Campylobacter spp. The frequency of vomiting in patients infected with EPEC and in those with no pathogen identified was similar. Abdominal pain was reported significantly more frequently in patients infected with Campylobacter spp. than in those infected with EPEC (p = 0.007, 2-tailed Fisher exact test), adenovirus (p = 0.0005), rotavirus (p = 0.0005), or those in whom no pathogen was detected (p = 0.003).
The duration of diarrhea was significantly longer in patients with EPEC than in those infected with adenovirus (p = 0.002, 2-tailed Student t test), rotavirus (p = 0.0003), Campylobacter (p = 0.0003), Salmonella (p = 0.02), and those without an identifiable pathogen (p = 0.02). Moreover, persistent diarrhea (defined as diarrhea lasting >14 days) was significantly more common in patients infected with atypical EPEC than in those infected with adenovirus, rotavirus, Campylobacter, Salmonella, and those with no pathogen identified (Table 4). Persistent diarrhea also developed in 4 (36%) of 11 patients infected with Giardia sp. The frequency of persistent diarrhea associated with Giardia was significantly greater than that attributable to adenovirus (p = 0.03), rotavirus (p = 0.01), and Camplyobacter, but not Salmonella or atypical EPEC (p>0.1, 2-tailed Fisher exact test).
Fever was significantly more common in patients infected with rotavirus or Salmonella than in those infected with EPEC, adenovirus, or Campylobacter, and those with no pathogen identified. Dehydration of [greater than or equal to] 5% occurred significantly more often in patients infected with rotavirus than in those infected with EPEC, adenovirus, Campylobacter, Salmonella, and those without an identifiable pathogen.
The disease severity score, determined according to the criteria of Ruuska and Vesikari (10), was highest in patients infected with rotavirus followed by Salmonella sp. The mean severity scores in patients infected with EPEC, adenovirus, and Campylobacter sp. and those in whom no pathogen was found were similar. Stools from patients infected with Campylobacter or Salmonella spp. were more likely to contain frank blood, although the differences between patients infected with different etiologic agents were not significant (p>0.05, 2-tailed Fisher exact test). Erythrocytes were more commonly detected on microscopic examination in patients infected with Campylobacter or Salmonella spp. than in those infected with EPEC, adenovirus, rotavirus, or no identifiable pathogen, but the differences were significant with respect to Campylobacter spp. only. Fecal leukocytes were present significantly more often in patients infected with Campylobacter or Salmonella spp. than in those infected with EPEC, adenovirus, rotavirus, or those with no identifiable pathogen.
The principal aims of this study were to compare the frequency of atypical EPEC with frequencies of established enteropathogens in children attending hospital with diarrhea and to determine the clinical and laboratory features associated with each pathogen. During the first part of the study (when pathogenic E. coli was sought), atypical EPEC was the predominant pathogen identified; it was found in 31% of 134 children compared with 10% for adenovirus, 10% for Salmonella sp., 9% for Campylobacter sp., and 4% for rotavirus. In the second period of the study, when EPEC was not sought, rotavirus predominated. In agreement with our findings from a community-based study in Melbourne and reports from investigators in Brazil, Norway, and elsewhere (9,20,21), the atypical EPEC strains obtained in this study were highly heterogeneous in terms of O:H serotype and intimin type, which indicates that the high frequency of atypical EPEC was not due to an outbreak caused by a limited number of strains. Also in agreement with our previous study, we observed that serotypes of EPEC associated with diarrhea differed from those listed by the World Health Organization as being characteristic of EPEC (9).
To determine whether atypical EPEC is a cause of diarrhea, we compared the clinical and laboratory findings of children who were infected with these bacteria with those who were infected with well-established pathogens and those in whom no pathogens were identified. The hypothesis underlying this investigation was that if atypical EPEC is not a pathogen, the symptoms, signs, and laboratory findings in patients infected with these bacteria would be the same as those in patients in whom no pathogens were found. The results showed that diarrhea attributable to atypical EPEC was significantly more common in boys and that it persisted significantly longer than diarrhea in patients without an identifiable pathogen or in those infected with adenovirus, rotavirus, Campylobacter spp., or Salmonella sp. This study also showed that infection with atypical EPEC generally occurred in children <2 years of age, with 72% <24 months of age compared with 55% for the first study group as a whole (OR 3.0, 95% CI 1.17-7.85, p = 0.03, 2-tailed Fisher exact test). Infection with EPEC was associated with vomiting in [approximately equal to] 50% of patients, was generally not accompanied by fever, abdominal pain, or dehydration, and was not characterized by fecal blood or leukocytes, indicating that it was not inflammatory in nature. The reason for the higher frequency of atypical EPEC in boys is not known but confirms our unpublished observations from a community-based study, in which 55 isolates were obtained from 338 male patients, and 34 were obtained from 358 female patients (OR 1.85, 95% CI 1.17-2.92, p = 0.009, 2-tailed Fisher exact test).
The validity of the clinical and laboratory assessments performed in this study was indicated by the confirmation of the well-known associations of specific pathogens with particular parameters: younger age of children infected with EPEC and viruses than those infected with Campylobacter or Salmonella spp.; rotavirus and Salmonella infections with fever; rotavirus with dehydration and an overall greater severity of disease; Campylobacter sp. with fecal blood; and Campylobacter and Salmonella spp. with fecal leukocytes (22).
Persistent diarrhea (lasting more then 14 days) eventually develops in a substantial proportion of children with acute infectious gastroenteritis and may become chronic, leading to malabsorption malabsorption /mal·ab·sorp·tion/ (mal?ab-sorp´shun) impaired intestinal absorption of nutrients.
Defective or inadequate absorption of nutrients from the intestinal tract. , failure to thrive Failure to Thrive Definition
Failure to thrive (FTT) is used to describe a delay in a child's growth or development. It is usually applied to infants and children up to two years of age who do not gain or maintain weight as they should. , and malnutrition (23). A wide range of infectious agents has been implicated im·pli·cate
tr.v. im·pli·cat·ed, im·pli·cat·ing, im·pli·cates
1. To involve or connect intimately or incriminatingly: evidence that implicates others in the plot.
2. in the cause of persistent diarrhea, including viruses, in particular rotavirus; protozoa, such as Giardia and Cryptosporidium spp., and bacteria, including E. coli (23,24). In most cases, however, laboratory investigation of children with persistent diarrhea fails to yield an identifiable cause. The findings of this study suggest that a number of these cases may be caused by infection with atypical EPEC, which is seldom sought in these patients.
Despite the persuasive evidence of a volunteer study and reports of outbreaks of diarrhea attributed to atypical EPEC (25,26), the role of atypical EPEC in disease is controversial. In several reports, however, from countries as diverse as Iran, Norway, Peru, Poland, South Africa South Africa, Afrikaans Suid-Afrika, officially Republic of South Africa, republic (2005 est. pop. 44,344,000), 471,442 sq mi (1,221,037 sq km), S Africa. , the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. , and the United Kingdom (20,27-32), as well as Australia (9,33), atypical EPEC strains have been identified in children with acute diarrheal disease. Atypical EPEC has also previously been reported in association with prolonged diarrhea (34). For example, Hill et al. (35) reported that of 26 children infected with EPEC requiring hospital admission for acute diarrhea, life-threatening, chronic symptoms developed in 6 (23%). Five of these 6 children were infected with EPEC of serogroups O114 or O128, which frequently do not produce Bfp (14,36). Notwithstanding these previous reports, however, the current study is the first to characterize the illness caused by atypical EPEC in a systematic way and to compare the features of atypical EPEC infection with those of other etiologic agents of diarrhea.
The reasons why persistent diarrhea develops more frequently in children infected with atypical EPEC than in those infected with adenovirus, rotavirus, Campylobacter or Salmonella spp. are not known. In a recent study, Mellmann et al. (37) found that only 12 (<9%) of 137 patients who were infected with eae-positive EHEC strains when investigated within 14 days of the onset of diarrhea remained culture-positive when retested 3-16 days later, compared with all 5 patients who were initially infected with eae-positive, stx-negative E. coli (i.e., atypical EPEC) (OR 110.4, 95% CI 5.8-2117.6, p<0.0001, 2-tailed Fisher exact test). These findings indicate that atypical EPEC may have an innate propensity to persist longer in the intestine than varieties of E. coli which cause diarrhea that is more transient in nature. EPEC adheres tightly to epithelial cells and disrupts normal cellular processes (38), and evidence suggests that atypical EPEC may retard apoptosis of intestinal epithelial cells (39), possibly because of the lack of Bfp (40). These features may favor prolonged intestinal colonization by atypical EPEC compared with other intestinal pathogens. Although disease due to atypical EPEC was mild and generally not associated with dehydration, its importance lies in its association with prolonged diarrhea, a major contributor to childhood illness, especially in developing countries. Our findings also suggest that interventions targeted towards atypical EPEC may be beneficial in managing children with prolonged diarrhea.
We are grateful to to K.A. Bettelheim and the staff of the diagnostic microbiology and virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression laboratories at the Royal Children's Hospital for their assistance.
This study was supported by grants to R.R.B. from the Australian National Health and Medical Research Council The National Health and Medical Research Council (NHMRC) is Australia's peak funding body for medical research, with a budget of nearly A$500M a year . The Council was established to develop and maintain health standards and is responsible for implementing the and the Murdoch Children's Research Institute.
Dr Nguyen is head of pediatric pediatric /pe·di·at·ric/ (pe?de-at´rik) pertaining to the health of children.
Of or relating to pediatrics. infectious diseases infectious diseases: see communicable diseases. at An Giang General Hospital in Long Xuyen Long Xuyên City, An Giang, Vietnam. His major research interests are viral infections and the development of methods for the rapid diagnosis of infectious diseases. is a capital city of An Giang Province, Vietnam. It is located at approximately
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Rang N. Nguyen, * (1) Louise S. Taylor, * ([dagger]) Marija Tauschek,* ([dagger]) and Roy M. Robins-Browne * ([dagger])
* University of Melbourne
In 2006, Times Higher Education Supplement ranked the University of Melbourne 22nd in the world. Because of the drop in ranking, University of Melbourne is currently behind four Asian universities - Beijing University, , Parkville, Australia; and ([dagger]) Murdoch Children's Research Institute, Parkville, Victoria, Australia (1) Current affiliation: An Giang General Hospital, Long Xuyen City, An Giang, Vietnam
Address for correspondence: R.M. Robins-Browne, Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria 3010, Australia; fax: 61-3-8344-8276; email: r.browne@ unimelb.edu.au
Table 1. Classification of pathogenic Escherichia coli according to amplicon(s) generated by polymerase chain reaction (PCR) for virulence-associated determinants Gene or virulence-associated determinant * Interpretation ([dagger]) pCVD432 aggA bfpA eae ipaC EAEC ([double dagger]) + + - - - Typical EPEC - - + + - Atypical EPEC - - - + - EIEC - - - - + ETEC ([double dagger]) - - - - - EHEC ([section]) - - - + - STEC not EHEC ([paragraph]) - - - - - Gene or virulence-associated determinant * Interpretation ([dagger]) stlA ItA ehxA stx1 stx2 EAEC ([double dagger]) - - - - - Typical EPEC - - - - - Atypical EPEC - - - - - EIEC - - - - - ETEC ([double dagger]) + + - - - EHEC ([section]) - - + + + STEC not EHEC ([paragraph]) - - - + + Control strain Interpretation ([dagger]) (reference) EAEC ([double dagger]) O42 (13) Typical EPEC E2348/69 (14) Atypical EPEC E128012 (14) EIEC 223/83 (15) ETEC ([double dagger]) H10407 (16) EHEC ([section]) EDL933 (17) STEC not EHEC ([paragraph]) * Factors specified by virulence genes: aggA, aggregative fimbria, AAF/I; bfpA, bundle-forming pilus; eae, intimin; ipaC, invasion plasmid antigen; stlA, heat-stable enterotoxin; ItA, heat-labile enterotoxin; ehxA, EHEC hemolysin; stx, Shiga toxin. ([dagger]) EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli; EIEC, enteroinvasive E. coli; ETEC, enterotoxigenic E. coli; STEC, Shiga toxin-producing E. coli; EHEC, enterohemorrhagic E. coli. ([double dagger]) Either positive by polymerase chain reaction. ([section]) Either eae or ehxA, and either stx1 or stx2 positive. ([section]) Either stx1 or stx2 positive. ([paragraph]) Either stx1 or stx2 positive. Table 2. Frequency of diarrhea-associated pathogens detected during the course of this study 1st period (Mar 1-May 2nd period (May 16-Aug Pathogen 15), n = 134 (%) 31), n = 169 (%) Diarrhepgenic Escherichia coli (all) 42 (31.3) Not done Campylobacter spp. 12 (9.0) 20 (11.8) Salmonella spp. 14 (10.4) 9 (5.3) Adenovirus 14 (10.4) 13 (7.7) Rotavirus 5 (3.7) 57 (33.7) Giardia sp. 8 (6.0) 3 (1.8) Crytosporidium sp. 2 (1.5) 1 (0.6) >1 pathogen 9 (6.7) 4 (2.4) No pathogens identified 46 (34.3) 70 (41.4) Table 3. Characteristics of atypical EPEC identified during this study * Strain no. Serotype Intimin type R41 ONT:H34 [alpha]2 R69 NT [theta] R89 O153:H7 [beta] R104 O153:H7 [beta] R151 ONT:H34 [alpha]2 R154 O71:H6 [alpha]1 R175 O128:H21 [kappa] R176 ONT:H8 [iota] R182 OR:H40 [theta] R215 O117:H2 [epsilon]/[eta] R218 ONT:R [alpha]2 R219 O51:H49 [alpha]1 R227 O170/172:H49 [theta] R228 O88:H- [kappa] R249 O145:H34 [iota] R250 O33:H6 [zeta] R261 O2:H45 [kappa] R278 ONT:H19 [upsilon] R281 O49:H10 [kappa] R380 ONT:H31 [zeta] R392 ONT:H6 [alpha]2 R394 O5/71:H31 [theta] R396 O98:H8 [iota] R404 O128:H2 [beta] R420 ONT:H21 [theta] R436 O33:H6 [beta] R446 ONT:H34 [alpha]2 R447 O28:H45 [zeta] R457 ONT:H- [beta] * EPEC, enteropathogenic Escherichia coli; H-, nonmotile; NT, nontypable (O1-O181; 141-1456); R, rough Table 4. Association between presumed etiologic agent and clinical and laboratory findings in children with diarrhea * Presumed etiologic agent Clinical or laboratory EPEC, parameter n = 25 Age in months, median 16.9 (11.4-28.2) (interquartile range) Sex, no. (% male) 18 (72) ([paragraph]) Vomiting, no. (%) 11 (44) Abdominal pain, no. (%) 5 (20) Days with diarrhea, 12.1 (7.5-16.7) ([paragraph]) mean (95% CI) Diarrhea >14 days, 12 (48) ([paragraph]) no. (%) Temperature [greater than or equal 6 (24) to] 38[degrees]C, no. (%) Dehydration [greater than or equal 1 (4) to] 5%, no. (%) Severity score, mean 9.0 (7.7-10.3) (95% CI) Fecal blood Macroscopic, no. (%) 0 Microscopic only, no. (%) 4 (16) Fecal leukocvtes. no. (%) 5 (20) Presumed etiologic agent Clinical or laboratory Adenovirus, parameter n = 22 Age in months, median 9.5 (4.4-19.5) ([dagger]) (interquartile range) Sex, no. (% male) 14 (64) Vomiting, no. (%) 17 (77) ([dagger]) ([paragraph]) Abdominal pain, no. (%) 2 (9) ([double dagger]) Days with diarrhea, 4.9 (3.6-6.2) ([double dagger]) mean (95% CI) Diarrhea >14 days, 1 (5) ([double dagger]) no. (%) Temperature [greater than or equal 2 (9) to] 38[degrees]C, no. (%) Dehydration [greater than or equal 2 (9) to] 5%, no. (%) Severity score, mean 8.6 (7.5-9.7) (95% CI) Fecal blood Macroscopic, no. (%) 0 Microscopic only, no. (%) 2 (9) Fecal leukocvtes. no. (%) 4 (18) Presumed etiologic agent Clinical or laboratory Rotavirus, parameter n = 55 Age in months, median 15.2 (9.4-28.0) (interquartile range) Sex, no. (% male) 30 (55) Vomiting, no. (%) 49 (89) ([double dagger]) ([section]) Abdominal pain, no. (%) 10 (18) Days with diarrhea, 6.0 (4.9-7.1) ([double dagger]) mean (95% CI) Diarrhea >14 days, 3 (5) ([double dagger]) no. (%) ([paragraph]) Temperature [greater than or equal 30 (55) ([dagger]) ([paragraph]) to] 38[degrees]C, no. (%) Dehydration [greater than or equal 35 (64) ([double dagger]) to] 5%, no. (%) ([section]) Severity score, mean 13.5 (95% CI) (12.6-14.4) ([double dagger]) ([section]) Fecal blood Macroscopic, no. (%) 0 Microscopic only, no. (%) 1 (2) Fecal leukocvtes. no. (%) 10 (18) Presumed etiologic agent Clinical or laboratory Campylobacter, parameter n = 30 Age in months, median 34.2 (interquartile range) (26.9-98.1) ([double dagger]) ([section]) Sex, no. (% male) 17 (57) Vomiting, no. (%) 13 (43) Abdominal pain, no. (%) 17 (57) (#) ** Days with diarrhea, 4.9 (3.9-5.9) ([double dagger]) mean (95% CI) Diarrhea >14 days, 1 (3) ([double dagger]) no. (%) Temperature [greater than or equal 7 (23) to] 38[degrees]C, no. (%) Dehydration [greater than or equal 2 (7) to] 5%, no. (%) Severity score, mean 8.4 (7.5-9.3) (95% CI) Fecal blood Macroscopic, no. (%) 4 (13) Microscopic only, no. (%) 17 (57) ([paragraph]) ([section]) Fecal leukocvtes. no. (%) 23 (77) ([double dagger]) ([section]) Presumed etiologic agent Clinical or laboratory Salmonella, parameter n = 22 Age in months, median 29.8 (9.3-90.9) ([paragraph]) (interquartile range) Sex, no. (% male) 10 (46) Vomiting, no. (%) 7 (32) Abdominal pain, no. (%) 10 (45) Days with diarrhea, 6.5 (4.2-8.6) ([dagger]) mean (95% CI) Diarrhea >14 days, 2 (9) (#) no. (%) Temperature [greater than or equal 17 (77) ([double dagger]) to] 38[degrees]C, no. (%) ([section]) Dehydration [greater than or equal 6 (27) ([dagger]) to] 5%, no. (%) Severity score, mean 11.3 (95% CI) (9.8-12.8) (#) ** Fecal blood Macroscopic, no. (%) 3 (14) Microscopic only, no. (%) 9 (41) Fecal leukocvtes. no. (%) 14 (64) ([paragraph]) (#) Clinical or laboratory NPI, parameter n = 46 Age in months, median 11.6 (3.1- (interquartile range) 52.7) Sex, no. (% male) 20 (44) ([dagger]) Vomiting, no. (%) 23 (50) Abdominal pain, no. (%) 10 (22) ** Days with diarrhea, 6.3 (3.5-9.0) ([dagger]) mean (95% CI) Diarrhea >14 days, 9 (20) ([dagger]) no. (%) Temperature [greater than or equal 13 (28) to] 38[degrees]C, no. (%) Dehydration [greater than or equal 6 (13) to] 5%, no. (%) Severity score, mean 8.3 (7.4-9.2) (95% CI) Fecal blood Macroscopic, no. (%) 1 (2) Microscopic only, no. (%) 9 (20) Fecal leukocvtes. no. (%) 10 (22) * EPEC, Enteropathogenic Escherichia coli; NPI, no pathogen identified; CI, confidence interval. ([dagger]) Significantly different from EPEC (Mann-Whitney U test, Fisher exact test, or Student t-test, 2-tailed), p<0.05, >0.01. ([double dagger]) Significantly different from EPEC (Mann-Whitney U test, Fisher exact test, or Student t test, 2-tailed), p<0.001. ([section]) Significantly different from NPI (Mann-Whitney U test, Fisher exact test, or Student t test, 2-tailed), p<0.001. ([paragraph]) Significantly different from NPI (Mann-Whitney U test, Fisher exact test, or Student t test, 2-tailed), p<0.05, >0.01. (#) Significantly different from EPEC (Mann-Whitney U test, Fisher exact test, or Student Nest, 2-tailed), p<0.01, >0.001. ** Significantly different from NPI (Mann-Whitney U test, Fisher exact test, or Student t test, 2 tailed, p<0.01, >0.001.