Microorganisms Responsible for Neonatal Diarrhea

Nature of the Organism

Although early work on the recognition of E. coli as a potential enteric pathogen focused on biochemical or serologic distinctions, there followed a shift in emphasis to the enterotoxins produced by previously recognized and entirely “new” strains of E. coli . Beginning in the mid-1950s, with work by De and colleagues in Calcutta, E. coli strains from patients with diarrhea were found to cause a fluid secretory response in ligated rabbit ileal loops, analogous to that seen with Vibrio cholerae . Work by Taylor and associates showed that the viable E. coli strains did not always produce this secretory response and that enterotoxin production correlated poorly with classically recognized EPEC serotypes. In São Paulo, Trabulsi made similar observations with E. coli isolated from children with diarrhea, and several veterinary workers showed that ETEC was associated with diarrhea in piglets and calves. A similar pattern was described in 1971, when E. coli strains were isolated from upper small bowel samples of adults with acute undifferentiated diarrhea in Bengal. Such strains of E. coli produced a heat-labile nondialyzable ammonium sulfate–precipitable enterotoxin. Analogous to the usually short-lived diarrheal illnesses of E. coli reported by several workers, a short-lived course of the secretory response to E. coli culture filtrates was described. Similar to responses to cholera toxin, secretory responses to E. coli were associated with activation of intestinal mucosal adenylate cyclase that paralleled the fluid secretory response.

Virulence Factors and Pathogenic Mechanisms

The two types of enterotoxins produced by enterotoxigenic E. coli have been found to be plasmid-encoded but genetically unlinked. The effects of LT follow a lag period required by its intracellular site of action. LT is internalized into target epithelial cells by retrograde vesicular transport. Once inside the cytoplasm, the toxin acts by adenosine diphosphate ribosylation of the G _sα signaling protein. The resulting activation of adenylate cyclase leads to accumulation of cyclic adenosine monophosphate (cAMP), which activates the cystic fibrosis transmembrane conductance regulator(CFTR) chloride channel and inhibits sodium reabsorption by the Na ⁺ /H ⁺ exchanger 3 (NHE3), leading to a net secretion of electrolytes and water. The antigenic similarity of LT and cholera toxin and their apparent binding to the monosialoganglioside GM ₁ have enabled the development of ELISAs for detection of LT and cholera toxin. ST causes an immediate and reversible secretory response. It is a much smaller molecule and is distinct antigenically from LT and cholera toxin. ST increases intracellular intestinal mucosal cyclic guanosine monophosphate (cGMP) concentrations and specifically activates apical plasma membrane–associated intestinal guanylate cyclase. An increase in cGMP leads to activation of the CFTR and inhibition of phosphodiesterase 3, which results in increased concentrations of cAMP. The sum effect for LT is net secretion of electrolytes and water. The receptor for STa responds to an endogenous ligand called guanylin, of which STa is a structural homologue. Both LT and ST may additionally exert some effects on the enteric nervous system that contribute to diarrhea. Because the capacity to produce an enterotoxin may be transmissible between different organisms by a plasmid or even a bacteriophage, interstrain gene transfer is likely to be responsible for occasional toxigenic non– E. coli . Enterotoxigenic Klebsiella and Citrobacter strains have been associated with diarrhea in a few reports, often in patients coinfected with ETEC.

The plasmids encoding LT and ST also encode the colonization factor antigens (CFAs), adhesins required for intestinal colonization, and the regulator that controls CFA expression. At least 25 CFAs have been described for human E. coli isolates, against which local IgA antibody may be produced. CFAs are proteinaceous hairlike fimbriae on the bacterial surface that serve as a bridge between the bacterium and the epithelial membrane. Despite the number of CFAs described, a few appear to be most common, including CFA/1, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS14, CS17, and CS21.

The contribution of additional virulence factors to ETEC pathogenesis continues to be investigated. For example, recently, an adhesin found at the tip of the ETEC flagella, EtpA, was found to be important for initial binding to the epithelial cell surface. EtpA may be useful in vaccine development as an immunoreactive antigen inducing protective immunity.

Epidemiology and Transmission

ETEC are important diarrheal pathogens among infants in developing countries, predominantly affecting children younger than 2 years, This pathogen is estimated to cause 200 million episodes of diarrhea and between 170,000 and 380,000 deaths yearly. Breastfeeding seems to provide some protection to infants. One study from Mexico suggested that breast milk with high LT-ETEC–specific antibodies reduced symptomatic infections by up to 90%. A study in Bangladesh found a relative risk of 0.51 for ETEC-associated diarrhea among infants exclusively breastfed. This effect seems to decrease when additional foods are introduced. ETEC have also been recognized among adults with endemic, cholera-like diarrhea in Calcutta, India, and in Dacca, Bangladesh, and among travelers to areas such as Central and South America, Africa, and South Asia, although rates in Southeast Asia are significantly lower.

The isolation of ETEC is uncommon in sporadic diarrheal illnesses in temperate climates where sanitation facilities are good and where winter viral patterns of diarrhea predominate. ETEC is commonly isolated from infants and children with acute watery summer diarrhea in areas where sanitary facilities are suboptimal, such as Africa, Brazil, Argentina, Bengal, Mexico, and Native American reservations in the southwestern United States. In a multicenter study of acute diarrhea in 3640 infants and children in China, India, Mexico, Myanmar, and Pakistan, 16% of cases had ETEC compared with 5% among 3279 control subjects. A case-control study from northwestern Spain showed a highly significant association of ETEC with neonatal diarrhea (26.5% positivity), often acquired in the hospital. A study conducted at seven sites in Africa and Asia found ETEC to be the most common bacterial pathogen overall in infants younger than 12 months with diarrhea, although the attributable fraction varied by site. The study also noted that the incidence of diarrhea attributed to ETEC decreased with age at all sites. ETEC constitute a major cause of dehydrating diarrhea in infants and young children in these areas. Peaks of illnesses tend to occur in the summer or rainy season, and dehydrating illnesses may be life threatening, especially in infants and young children. Humans are probably the major reservoirs for the human strains of ETEC, and contaminated food and water constitute the principal vectors.

The association of ETEC with outbreaks of diarrhea in newborn nurseries has been documented. Ryder and colleagues isolated ST-producing E. coli from 72% of infants with diarrhea, from the environment, and in one instance from an infant’s formula during a 7-month period in a prolonged outbreak in a special-care nursery in Texas. Another ST-producing E. coli outbreak was reported in 1976 by Gross and associates from a maternity hospital in Scotland.

Clinical Manifestations

The clinical manifestations of ETEC diarrhea tend to be mild and self-limited except in small or undernourished infants, in whom dehydration may be severe and constitute a major threat to life. In many parts of the developing world, acute diarrheal illnesses are the leading recognized causes of death. There is some suggestion that diarrheal illnesses associated with ST-producing ETEC may be more severe than LT-ETEC. Probably the best definition of the clinical manifestations of ETEC infection comes from volunteer studies with adults. In a recent review that identified 27 studies with doses of 10 ⁸ to 10 ¹⁰ ETEC organisms, symptoms included diarrhea, abdominal pain, nausea, headache, and low-grade fever; symptoms were dependent on ETEC strain and dose. Vomiting was less common, occurring after challenge with a few selected strains tested. Incubation periods ranged from 6.8 to 50 hours. Although illness usually resolves spontaneously within 3 to 5 days, it occasionally may persist for 1 week or longer. Diarrhea is noninflammatory, without fecal leukocytes or blood. A study in Spain, including children younger than 3 years with ETEC, of whom 9 of 19 were infants younger than 1 month of age, reported 94.7% of children with diarrhea, 31.6% with fever, 63.7% with vomiting, and 42.1% with dehydration. During outbreaks affecting infants and neonates, diarrhea duration has been similar, with a mean of approximately 4 days.

Pathology

As in cholera, the pathologic changes associated with ETEC infection are minimal. From animal experiments in which intestinal loops were infected with these organisms, and at a time when the secretory and adenylate cyclase responses were present, there was only a mild discharge of mucus from goblet cells and otherwise no significant pathologic change in the intestinal tract. Unless terminal complications associated with severe hypotension occur, ETEC organisms rarely disseminate beyond the intestinal tract. Similar to V. cholerae , ETEC infection is exclusively intraluminal.

Diagnosis

The preliminary diagnosis of ETEC diarrhea can be suspected by the epidemiologic setting and the noninflammatory nature of stool specimens, which reveal few or no leukocytes. ETEC serotypes vary tremendously, and serology is not typically used for diagnosis. The traditional diagnosis of ETEC relies on enterotoxin detection by bioassays, such as tissue culture or ileal loop assays for LT, or the suckling mouse assay for ST; ELISA methods are available for detection of both LT and ST.

More recently, detection of enterotoxin-encoding genes has superseded detection of the toxins themselves. Gene probes and PCR technologies are available. The presence of E. coli genes encoding enterotoxins in patients with diarrhea is generally considered diagnostic, although asymptomatic carriage is known to occur. In epidemiologic studies, it is common to test only three colonies per stool for the presence of ETEC, but it is generally considered that testing additional colonies increases diagnostic sensitivity.

Therapy and Prevention

The cornerstone of treatment for any diarrheal infection is rehydration. This principle especially pertains to ETEC diarrhea, which is an intraluminal infection with high output of fluid and electrolytes. The glucose absorptive mechanism remains intact in E. coli enterotoxin–induced secretion, much as it does in cholera, a concept that has resulted in the major advance of oral glucose–electrolyte therapy. This regimen can usually provide fully adequate rehydration in infants and children capable of tolerating oral fluids, replacing the need for parenteral rehydration in most cases. Use of oral glucose–electrolyte therapy is particularly critical in rural areas and developing nations, where early application before dehydration becomes severe may be lifesaving. The current recommended WHO solution contains 2.6 g of sodium chloride, 1.5 g of potassium chloride, 2.9 g of trisodium citrate dihydrate (or 2.5 g sodium bicarbonate), and 20 g of glucose per 1 L of clean or boiled drinking water. This corresponds to the following concentrations: 75 mmol/L of sodium, 20 mmol/L of potassium, 10 mmol/L of citrate, 65 mmol/L of chloride, and 75 mmol/L of glucose. In a multicenter trial involving 447 children in four countries, a reduced osmolality solution, compared with recommendations before 2002, was found to reduce stool output by 28% and illness duration by 18%. A Cochrane review also found that an oral rehydration solution (ORS) with a total osmolarity of 250 mmol/L or less resulted in decreased stool volume and decreased vomiting. Various recipes for homemade preparations have been described, but unless the cost is prohibitive, the premade standard solution is preferred. Parenteral fluids should be used in cases with severe dehydration and/or if ORS is not tolerated. Zinc supplementation improves outcomes in patients with diarrhea living in resource-limited regions.

The role of antimicrobial agents in the treatment or prevention of ETEC is controversial. This infection usually resolves within 3 to 5 days in the absence of antibacterial therapy. There is concern about the potential for coexistence of enterotoxigenicity and antibiotic resistance on the same plasmid, and cotransfer of multiple antibiotic resistance and enterotoxigenicity has been well documented. Prophylaxis is not recommended except in specific situations for prevention of traveler’s diarrhea. When antibiotic therapy is considered, cefixime, azithromycin, rifaximin, and, in adults, ciprofloxacin are recommended, emphasizing, although, that in infants, antimicrobials are not routinely recommended.

The prevention and control of ETEC infections are similar to those discussed later for EPEC. Breastfeeding should be encouraged. Vaccine development for the different E. coli pathovars has been mostly studied for ETEC. Most promising was a candidate including a mixture of five formalin-inactivated strains expressing different CFAs mixed with recombinant B subunit of cholera toxin (CTb). Unfortunately, in a study among Egyptian children, it was not efficacious in preventing ETEC diarrhea. Investigators are now working on increasing the expression of CFAs in this construct and using a hybrid of LT and cholera toxin with the hope of increasing antigenicity and achieving better protective efficacy. Other approaches have included an LT toxoid dermal patch and the use of attenuated Shigella vectors. A recent Cochrane review concluded that there was insufficient evidence for a benefit from any current vaccine, and thus no current vaccine recommendation can be made. Research is ongoing.

Virulence factors and pathogenic mechanisms

Enteroinvasive E. coli is similar genetically, pathogenetically, epidemiologically, and clinically to shigellosis, although the clinical syndrome associated with EIEC may be milder. EIEC causes diarrhea by means of Shigella -like intestinal epithelial invasion (discussed later). The somatic antigens of these invasive strains have been identified and seem to fall into a few recognized O groups (see Table 11-1 ). Most, if not all, of these bacteria share cell wall antigens with various Shigella serotypes and react with antisera against cross-reacting antigen. Not all strains of E. coli belonging to the serogroups associated with dysentery-like illness are pathogenic because a large (140 MDa) invasive plasmid designated pINV is also required. Additional biologic tests, including the guinea pig conjunctivitis test (Sereny), gene probes for the pINV plasmid, or PCR diagnosis for virulence factors (IpaH, which also detects Shigella spp.) are used to confirm the property of invasiveness.

Epidemiology and Transmission

Little is known about the epidemiology and transmission of this organism in newborns and infants; it is more frequently found in children older than 2 years. Investigations in children with diarrhea often yield a low prevalence of EIEC, although this can vary by geographic area. The pathogen is often associated with outbreaks, and transmission is mostly food- or waterborne. Studies of adult volunteers suggest that attack rates may be lower even after ingestion of inoculums larger than those required to develop Shigella -associated disease. Expression of virulence factors may be decreased in EIEC compared with Shigella .

Clinical Manifestations

Symptoms often include watery diarrhea, although a dysentery-like syndrome with an inflammatory exudate in stool, invasion and disruption of colonic mucosa has been well described.

Descriptions of extensive and severe ileocolitis in infants dying with E. coli diarrhea indicate that neonatal disease also can be caused by invasive strains capable of mimicking the pathologic features of shigellosis. The immunofluorescent demonstration of E. coli together with an acute inflammatory infiltrate in the intestinal tissue of infants tends to support this impression, although it has been suggested that the organisms may have invaded the bowel wall in the postmortem period. Direct evidence for a pathogenic role of invasive strains of E. coli as a cause of neonatal diarrhea is currently lacking. The infrequency with which newborns manifest a dysentery-like syndrome makes it unlikely that this pathogen is responsible for a significant proportion of the diarrheal disease that occurs during the first month of life.

Diagnosis

Enteroinvasive E. coli should be suspected in infants who have an inflammatory diarrhea, as evidenced by fecal neutrophils or bloody dysenteric syndromes from which no other invasive pathogens, such as Campylobacter, Shigella, Salmonella, Vibrio, or Yersinia, can be isolated. In this instance, it may be appropriate to have the fecal E. coli isolated and serotyped or tested for invasiveness in the Sereny test and/or for identification of pINV with genetic probes or by nucleic acid amplification techniques.

Therapy and Prevention

The management and prevention of EIEC diarrhea should be similar to that of acute Shigella or other E. coli enteric infections. Ampicillin has been used in adults, but increasing rates of antibiotic resistance have been documented.

Epidemiology and Transmission

Based on the molecular identification of two virulence factors, EPEC is now classified as either typical, usually associated with classic serotypes, or atypical, which includes a greater diversity of serotypes harboring the characteristic virulence factor. The presence of eaeA , the intimin gene, is a genetic determinant for the EPEC pathotype. Typical EPEC (tEPEC) strains also harbor a functional bfp A gene, which encodes a bundle-forming pilus (BFP), whereas this is absent in atypical EPEC (aEPEC).

Historically, tEPEC have been an important cause of diarrhea in infants in both industrialized and developing countries. However, more recently, outbreaks have become rare. It is unclear why there has been a decrease in the incidence of tEPEC. Some have attributed the change to an increase in breastfeeding, which is thought to be protective. Alternatively, our current diagnostic methods and definition of EPEC rely less heavily on serology but rather on molecular characteristics. Strains diagnosed as EPEC in earlier studies may not have been pathogenic. Noteworthy are the results of the recent comprehensive Global Enteric Multicenter Study (GEMS) performed in seven sites in Africa, India, Pakistan, and Bangladesh, in which infants 0 to 11 months of age had a 2.6-fold increased risk of death 60 days after the diarrheal episode if their presenting diarrhea was associated with typical EPEC infection. The actual cause of death in these children although was unknown.

Socioeconomic conditions play a significant role in determining the incidence of this disease in different populations. It is unusual for newborn infants born in a rural environment to manifest diarrheal disease caused by EPEC; most infections of the GI tract in these infants occur after the first 6 months of life. When living conditions are poor and overcrowding of susceptible infants exists, there is an increase in the incidence of neonatal diarrhea in general and EPEC gastroenteritis in particular.

Newborns can acquire typical EPEC during the first days of life by one of several routes: (1) organisms from the mother ingested at the time of birth; (2) bacteria from other infants or toddlers with diarrheal disease or from asymptomatic adults colonized with the organism, commonly transmitted on the hands of nursery personnel or parents in close and prolonged contact with the infant; (3) airborne or droplet infection; (4) fomites; or (5) organisms present in formulas or solid food supplements. Only the first two routes have been shown conclusively to be of significance in the transmission of disease or the propagation of epidemics. The contours of the epidemiologic curves in nursery and community outbreaks are in keeping with a contact mode of spread. Transmission of organisms from infant to infant occurs by the fecal-oral route in almost all cases, most likely via the hands of individuals attending to their care. Ill infants represent the greatest risk to individuals around them because of the large numbers of organisms found in their stools and vomitus. Cross-infection has also been initiated by infants who were healthy at the time of nursery admission. A respiratory mode of transmission has been suggested but not proven.

Historically, studies into the epidemiology of classic EPEC serotypes have dealt with events that occurred during outbreaks in newborn nurseries. Investigations of this sort frequently regard the epidemic as an isolated phenomenon and ignore the strong interdependence that exists between community-acquired and hospital-acquired illness. The direction of spread is most often from the reservoir of disease within the community to the hospital. When the original source of a nursery outbreak can be established, frequently it turns out to be an infant born of a carrier mother who recently acquired EPEC infection from a toddler living in the home. Cross-infection epidemics also can be initiated by infected newborns that have been admitted directly into a clean nursery unit from the surrounding district or have been transferred from a nearby hospital.

After a nursery epidemic has begun, it generally follows one of two major patterns. Some epidemics are explosive, with rapid involvement of all susceptible infants and a duration that seldom exceeds 2 or 3 months. The case-fatality rate in these epidemics may be very high. Other nursery outbreaks have an insidious onset with a few mild, unrecognized cases; the patients may not even develop illness until after discharge from the hospital. During the next few days to weeks, neonates with an increased number of loose stools are reported by the nurses; shortly thereafter, the appearance of the first severely ill infants makes it apparent that an epidemic has begun.

The nursery can be a source of infection for the community. The release of infants who are in the incubation stages of illness or are convalescent carriers about to relapse may lead to secondary cases of diarrheal disease among young siblings living in widely scattered areas. These children further disseminate infection to neighboring households, involving playmates of their own age, young infants, and mothers As the sickest of these contact cases are admitted to different hospitals, they contaminate new susceptible persons, completing the cycle and compounding the outbreak. This feedback mechanism has proved to be a means of spreading infantile gastroenteritis through entire cities, counties, and provinces. One major epidemic of diarrhea related to EPEC O111:B4 that occurred in the metropolitan Chicago and northwestern Indiana region during the winter of 1961 involved more than 1300 children and 29 community hospitals during a period of 9 months. Almost all of the patients were younger than 2 years, and 10% were younger than 1 month, producing an age-specific attack rate of nearly 4% of neonates in the community. The importance of the hospital as a source of cross-infection in this epidemic was shown through interviews with patients’ families, indicating that a minimum of 40% of infants had direct or indirect contact with a hospital shortly before the onset of illness.

It has been suggested, but not proven, that asymptomatic carriers of EPEC in close contact with a newborn infant, such as nursery personnel or family members, might play an important role in the transmission of the bacterium. Stool culture surveys have shown that at any one time about 1% to 2% of adults and 1% to 5% of young children who are free of illness harbor EPEC strains. Higher percentages have been recorded during community epidemics. Because this intestinal carriage is transitory, the number of individuals who excrete EPEC at one time or another during the year is far higher than the 1% figure recorded for single specimens.

The association of atypical EPEC with acute diarrhea is less well established. Multiple studies have found no association, whereas others have found higher rates of aEPEC identified from symptomatic patients in both industrialized countries and the developing world. GEMS did not identify a statistically significant difference for the isolation of aEPEC from symptomatic children or controls. A study in Baltimore, Maryland and New Haven, Connecticut, was similarly unable to establish a relationship between aEPEC and symptomatic infection. On the other hand, a study in Cincinnati, Ohio, found a higher prevalence of aEPEC (6.5% vs. 3.9%) in children younger than 5 years who were brought to the emergency department. Higher rates of aEPEC compared with tEPEC have additionally been reported from Norway, Denmark, and Australia, among others, and have been associated with outbreaks among school-aged children.

Some of the discrepancy leading to confusion about the significance of aEPEC in diarrheal disease may be due to its significant heterogeneity. By definition, aEPEC must contain eaeA and should be capable of causing typical A/E lesions (see later). However, several studies have described marked heterogeneity in serotypes, with fewer strains belonging to the classic serotypes recognized by the WHO, and heterogeneity in identifiable virulence factors. Of note, there are reports that aEPEC may be associated with prolonged diarrhea (>14 days). Further studies are required for a more complete understanding of the current epidemiology of both typical and atypical EPEC.

Pathogenesis

Bacterial cultures of the meconium and feces of newborns indicate that EPEC can effectively colonize the intestinal tract in the first days of life. Breastfeeding is felt to be protective. Investigators in Misiones, Argentina, noted a lower incidence of EPEC infection, symptomatic or asymptomatic, in breastfed infants younger than 20 months. Similarly, in a recent outbreak in Botswana, where EPEC was commonly isolated from children younger than 5 years, breastfeeding was felt to contribute to improved outcomes. A study in Brazil reported an odds ratio (OR) of 0.1 for breastfeeding in infants with EPEC, suggesting a protective effect. The use of breastfeeding or human milk provided by bottle has shown to be effective in ending nursery epidemics caused by EPEC O111:B4, probably by reducing the incidence of cross-infections among infants.

Although dose-effect studies have not been performed among newborns, severe diarrhea has occurred after ingestion of 10 ⁸ EPEC organisms in very young infants. The high incidence of cross-infection outbreaks in newborn nurseries suggests that a far lower inoculum can often affect spread in this setting. Inocula of 10 ¹⁰ E. coli O142 or O127 organisms caused diarrhea in 8 of 10 adult volunteers.

The mechanism by which EPEC causes diarrhea involves a complex array of plasmid and chromosomally encoded traits. Only uncommonly do EPEC strains invade the bloodstream or disseminate. An A/E lesion is characteristic of EPEC; the lesion is manifested by intimate (about 10 nm) apposition of the EPEC to the plasma membrane of the enterocytes, with dissolution of the normal brush border and rearrangement of the cytoskeleton. In some instances, the bacteria are observed to rise up on pedestal-like structures, which are diagnostic of the infection. Villus blunting, crypt hypertrophy, histiocytic infiltration in the lamina propria, and a reduction in brush border enzyme expression may also be observed.

Two major EPEC virulence factors have been described; strains with both factors are designated as typical EPEC. One such factor is the locus of enterocyte effacement (LEE), a pathogenicity island encoding a type III secretion system. The LEE secretion apparatus injects proteins directly from the cytoplasm of the infecting bacterium into the cytoplasm of the target enterocytes. The injected proteins constitute cytoskeletal toxins, which together elicit the close apposition of the bacterium to the cell and cause the effacement of microvilli. One critical secreted protein, called translocated intimin receptor (Tir), inserts into the plasma membrane of the epithelial cell, where it serves as the receptor for an LEE-encoded EPEC outer membrane protein called intimin, encoded by eaeA . The second major virulence factor of typical EPEC is the BFP, which is encoded on a partially conserved 60-MDa virulence plasmid called EPEC adherence factor (EAF). BFP, a member of the type IV pilus family, mediates aggregation of the bacteria to one another and probably to enterocytes themselves, facilitating mucosal colonization. A BFP mutant was attenuated for virulence in adult volunteers. An array of additional virulence factors have been proposed, including effectors encoded both within and outside of the LEE pathogenicity island. A complete understanding of how the coordination of these virulence mechanisms leads to diarrhea remains somewhat elusive.

More recent epidemiologic data suggest that some virulent EPEC may lack the BFP, although still possessing the eaeA gene and capable of causing A/E lesions; such strains are termed atypical EPEC (aEPEC). These strains exhibit marked variability in serotype and expression of additional virulence factors. Although a complete understanding of virulence mechanisms for aEPEC is still unclear, the presence of some additional virulence factors such as Efa1/LifA, which have adhesive properties and may inhibit lymphocyte proliferation, may be associated with symptomatic infection. Another mechanism for initial attachment to enterocytes is presumed to exist for aEPEC given the absence of BFP.

The role of circulating immunity in the prevention of GI tract disease related to EPEC has not been clearly established. Animal models suggest that a humoral immune response may be protective against subsequent EPEC infection. Such a response to LEE-encoded virulence factors has similarly been noted in human infections but not directly correlated with protection. EPEC is more common in children, although it is unclear whether this is due to an immune response or innate host factors associated with age. In human infants, the frequency of bacteriologic and clinical relapse related to EPEC of the same serotype and the capacity of one strain of EPEC to superinfect a patient already harboring a different strain cast some doubt on the ability of mucosal antibodies to inhibit or alter the course of intestinal infection. Ultimately, the role of an immune response and the potential for an EPEC vaccine continue to be investigated.

Pathology

The principal pathologic lesion in EPEC infection is the A/E lesion, manifest by electron microscopy but not light microscopy. In chronic cases, villus blunting, crypt hypertrophy, histiocytic infiltration of the lamina propria, and reduced brush border enzymes may be seen. Rothbaum and colleagues described similar findings, with dissolution of the glycocalyx and flattened microvilli with the nontoxigenic EPEC strain O119:B14. A wide range of pathologic findings has been reported in infants dying with EPEC gastroenteritis. Most newborns dying with diarrheal disease caused by EPEC show no morphologic changes of the GI tract by gross or microscopic examination of tissues. Bray described such “meager” changes in the intestinal tract that “the impression received was that the term gastroenteritis is incorrect.” At the other extreme, extensive and severe involvement of the intestinal tract, although distinctly unusual among neonates with EPEC diarrhea, has been discussed in several reviews of the pathologic anatomy of this disease. Changes virtually identical to those found in infants dying with necrotizing enterocolitis have been reported. Drucker and coworkers found that among 17 infants with EPEC diarrhea who were dying, “intestinal gangrene, and/or perforation, and/or peritonitis were present in five, and intestinal pneumatosis in five.” The reasons for such wide discrepancies in reported EPEC disease pathology are unclear.

The nonspecific pathologic picture described by some researchers includes capillary congestion and edema of the bowel wall and an increase in the number of eosinophils, plasma cells, macrophages, and mononuclear cells in the mucosa and submucosa. Villous patterns are generally well preserved, although some flattening and broadening of the villi are seen in more severe cases. Almost complete absence of villi and failure of regeneration of small bowel mucosa have been reported in one extreme case. Edema in and around the myenteric plexuses of Auerbach, a common associated finding, may cause the GI tract dilation often seen at autopsy in infants with EPEC infections. In general, the distal small intestine shows the most marked alterations; however, the reported pathologic findings may be found at all levels of the intestinal tract.

Several complications of EPEC infection have been reported. Candidal esophagitis accounted for significant morbidity in two series collected before and during the antibiotic era. Oral thrush has been seen in 50% of EPEC-infected infants treated with oral or systemic antibiotics. A measure of fatty metamorphosis of the liver has been reported by several investigators ; however, these changes are nonspecific and probably result from the poor caloric intake associated with persistent diarrhea or vomiting. Some degree of bronchopneumonia, probably a terminal event in most cases, exists in a large proportion of newborns dying of EPEC infection. In one reported series of fatal infant cases, EPEC was shown by immunofluorescent staining to be present in the bronchi, alveoli, and interalveolar septa.

Mesenteric lymph nodes are often swollen and congested with reactive germinal centers in the lymphoid follicles. Severe lymphoid depletion, unrelated to the duration or severity of the antecedent illness, also has been described. The kidneys frequently show tubular epithelial toxic changes. Various degrees of tubular degeneration and cloudy swelling of convoluted tubules are common findings. Renal vein thrombosis or cortical necrosis may be observed in infants with disseminated intravascular coagulation in the terminal phases of the illness. The heart is grossly normal in most instances but may show minimal vacuolar changes of nonspecific toxic myocarditis on microscopic examination. Candidal abscesses of the heart and kidneys have been described. With the exception of mild congestion of the pia arachnoid vessels and some edema of the meninges, examination of the central nervous system reveals few changes.

Clinical Manifestations

The incubation period after EPEC exposure is quite variable. Its duration has been calculated mostly from evidence in outbreaks in newborn nurseries, where the time of first exposure can be clearly defined in terms of birth or admission dates. In these circumstances, almost all infants show signs of illness 2 to 12 days after exposure, and most cases show signs within the first 7 days. In some naturally acquired and experimental infections with heavy exposure, the incubation period may be only 24 hours; the stated upper limit is 20 days. In adult volunteers fed a high inoculum, the incubation period was less than 3 hours. The first positive stool culture and the earliest recognizable clinical signs of disease occur simultaneously in most infants, although colonization may precede symptoms by 7 to 14 days.

Gastroenteritis associated with typical EPEC infection in the newborn is notable for its marked variation in clinical pattern. Clinical manifestations vary from mild illness manifest only by transient anorexia and failure to gain weight to a sudden explosive fulminating diarrhea causing death within 12 hours of onset. Prematurity, underlying diseases and congenital anomalies often are associated with the more severe forms of illness. The onset of illness is usually insidious, with vague signs such as reluctance to feed, lethargy, spitting up of formula, mild abdominal distention, or weight loss that may occur for 1 or 2 days before the first loose stool is passed. Diarrhea usually begins abruptly. It may be continuous and violent, or in milder infections, it may run an intermittent course with 1 or more days of normal stools, followed by 1 or more days of diarrhea. Prominent and persistent vomiting can be an early finding. Fever is an inconstant feature, and when it occurs, the patient’s temperature rarely is higher than 39° C (>102.2° F). Prolonged hematochezia, distention, edema, and jaundice are ominous signs and suggest an unfavorable prognosis.

The clinical relevance of infection compared with colonization with atypical EPEC is controversial. Clinical symptoms have been described in outbreaks of aEPEC. In a study completed in Melbourne, Australia, the clinical presentation included vomiting in 44% and abdominal pain in 20%, along with diarrhea. A low-grade fever was detected in 24% of patients. No patients had macroscopic blood in their stool, but 16% were Hemoccult positive, whereas 20% were found to have fecal leukocytes.

Prolonged diarrhea has been observed for typical EPEC. Infants with mild illness who receive no treatment can continue to have intermittent loose stools for 1 to 3 weeks. In one outbreak related to EPEC O142:K86, more than one third of untreated or inappropriately treated infants had diarrhea for more than 14 days in the absence of a recognized enteric pathogen on repeated culturing. Recurrence can occur, even after adequate treatment. ^∗

∗ References .

Atypical EPEC has also been associated with prolonged diarrhea lasting more than 14 days. A study in Norway found that 31.6% of patients younger than 2 years with aEPEC had symptoms lasting more than 2 weeks.

Dehydration is the most common and serious complication of gastroenteritis caused by EPEC or a toxin-producing E. coli . Virtually all deaths directly attributable to the intestinal infection are caused by disturbances in fluids and electrolytes. When stools are frequent in number, large in volume, and violent in release, as they often are in severe infections with abrupt onset, a neonate can lose 15% of body weight within a few hours. Rarely, fluid excretion into the lumen of the bowel proceeds so rapidly that reduction of circulating blood volume and shock may intervene before passage of a single loose stool. Before the discovery of the etiologic agent, epidemic diarrhea of the newborn was also known by the term cholera infantum . Even mild disease, however, can lead to fluid and electrolyte imbalance along with nutritional deficiencies. These should be monitored closely, particularly in infants, during the course of illness.

Diagnosis

In 1987, the WHO came to a consensus that O serogroups of E. coli should be classified as EPEC: O26, O55, O86, O111, O114 O119, O125, O126, O127, O128, O142, and O158. Serotyping can be used to identify likely EPEC strains, especially in outbreaks. E. coli, similar to other Enterobacteriaceae members, possesses cell wall somatic antigens (O), envelope or capsular antigens (K), and, if motile, flagellar antigens (H). Many O groups may be divided further into two or more subgroups (a, b, c), and the K antigens are divisible into at least three varieties (B, L, A) based on their physical behavior. Organisms that do not possess flagellar antigens are nonmotile (designated NM). The EPEC B capsular surface antigen prevents agglutination by antibodies directed against the underlying O antigen. Heating at 100° C for 1 hour inactivates the agglutinability and antigenicity of the B antigen. Slide agglutination tests with polyvalent O or OB antiserum may be performed on suspensions of colonies typical of E. coli that have been isolated from infants with diarrhea, especially in nursery outbreaks. Diagnosis by serotyping must be used with caution because it can result in false positives and false negatives. E. coli strains that fall into the above serogroups do not necessarily harbor virulence factors essential for pathogenicity. In addition, aEPEC, which are emerging as a more common etiology of childhood diarrhea than tEPEC, have greater serotype variability and may not fall into the above serogroups. Patients at times have been noted to harbor multiple EPEC serogroups and can be asymptomatic carriers. It is important to consider all possible etiologies of diarrhea because the isolation of one of the above serogroups does not necessarily imply it is the cause of diarrhea.

Classic EPEC has been recovered from the vomitus, stool, or bowel contents of infected newborns. Isolation from bile and the upper respiratory tract has been described in instances in which a specific search has been made. Less commonly, EPEC is isolated from ascitic fluid or purulent exudates ; on occasion, the organism has been recovered from blood cultures, urine, and cerebrospinal fluid.

Stool cultures are generally more reliable than rectal swabs in detecting the presence of enteric pathogens, although a properly obtained swab should be adequate to show EPEC in most cases. Specimens should be obtained as early in the course of the illness as possible because organisms are present in virtually pure culture during the acute phase of the enteritis but rapidly diminish in numbers during convalescence.

After a stool specimen is received, it should be plated as quickly as possible onto noninhibiting media or placed in a preservative medium if it is to be held for longer periods. Deep freezing of specimens preserves viable EPEC when a prolonged delay in isolation is necessary. No biochemical assays readily differentiate between EPEC and nonpathogenic commensal E. coli.

Microscopic examination of stools of infants with acute diarrheal illness caused by these organisms usually, but not always, has revealed an absence of fecal neutrophils, although data on fecal lactoferrin in human volunteers suggest that an inflammatory process may be important in EPEC diarrhea. Serologic methods have not proven to be useful in attempting to establish a retrospective diagnosis of EPEC infection in neonates. Increasing or significantly elevated agglutinin titers rarely could be shown in early investigations ; hemagglutinating antibodies showed a significant response in only 10% to 20% of cases.

Before widespread use of molecular methods, the human epithelial type-2 (HEp-2) cell adherence assay was proposed for EPEC diagnosis. The presence of a focal or localized adherence pattern on the surface of HEp-2 or HeLa cells after 3-hour coincubation is a highly sensitive and specific test for detection of EPEC. The requirement for cell culture and expertise in reading this assay limits its utility to the research setting. An ELISA for BFP has been described but is not readily available. The capacity of localized adherence plus EPEC to polymerize F-actin can be detected in tissue culture cells stained with rhodamine-labeled phalloidin. This fluorescence-actin staining test is cumbersome and impractical for routine clinical use.

The current standard for identification of EPEC is the use of molecular techniques to identify specific virulence factors. By definition, EPEC require the LEE pathogenicity island ( eae gene) and must not produce shiga toxin ( stx gene). Strains that additionally harbor the EPEC EAF plasmid, identified by the presence of the bfpA gene, are considered tEPEC, whereas those missing this gene are considered aEPEC. Specific gene probes and PCR primers for these genes are available. PCR and gene probe analysis can be performed directly on the stools of suspect infants; isolation of the organism should also be attempted. It is important to note that although molecular identification of these genes is the best diagnostic method currently available for EPEC, it is not perfect. E. coli strains that harbor eae may be missing accessory virulence factors needed to cause diarrhea, as evidenced by multiple studies identifying aEPEC in asymptomatic carriers. In addition, the presence of a gene may not ultimately translate to expression of its virulence factor. Experts suggest that whole-genome sequencing may eventually play a role in a more complete understanding of virulence markers associated with symptomatic infection.

Prognosis

The mortality rate recorded previously in epidemics of typical EPEC gastroenteritis is impressive for its variability. During the 1930s and 1940s, when organisms later recognized as classic enteropathogenic serotypes were infecting infants, the case-fatality ratio among neonates was about 50%. During the 1950s and 1960s, about one of every four infected infants still died in many nursery epidemics, but several outbreaks involving the same serotypes under similar epidemiologic circumstances had fatality rates of less than 3%. In the 1970s, reports appeared in the literature of a nursery epidemic with a 40% neonatal mortality rate and of an extensive outbreak in a nursery for premature infants with 4% fatalities ; another report stated that among “243 consecutive infants admitted to the hospital for EPEC diarrheal disease, none died of diarrheal disease per se.” As indicated above, in the GEMS study, the presence of typical EPEC resulted in a 2.6-fold increased risk of death at 60 days not necessarily because of the acute diarrhea episode.

A significant proportion of the infants who died during or shortly after an episode of gastroenteritis already were compromised by preexisting disease or by congenital malformations at the time they acquired gastroenteritis. The overall mortality rate among premature infants with EPEC gastroenteritis has not differed significantly over the years from the mortality recorded for term infants.

Therapy

The management of EPEC gastroenteritis should be directed primarily toward prevention or correction of problems caused by loss of fluids and electrolytes. Most neonates have a relatively mild illness that can be treated with oral rehydration. Infants who appear toxic, infants with voluminous diarrhea and persistent vomiting, and infants with increasing weight loss should be hospitalized for observation and treatment with oral and/or parenteral fluids to carefully maintain fluid and electrolyte balance, and possibly with antimicrobial therapy. The use of atropine-like drugs, paregoric, or loperamide to reduce intestinal motility or cramping should be avoided. Inhibition of peristalsis interferes with an efficient protective mechanism designed to rid the body of intestinal pathogens.

The value of antimicrobial therapy in management of neonatal EPEC gastroenteritis, if any, is uncertain. There are no adequately controlled studies defining the benefits of any antibiotic in eliminating EPEC from the GI tract, reducing the risk of cross-infection in community or nursery outbreaks, or modifying the severity of the illness. Proponents of the use of antimicrobial agents have based their claims for efficacy on anecdotal observations or comparative studies ; some postulate that antimicrobial therapy may shorten the length of illness and aid in the control of nursery outbreaks by decreasing length of shedding. Antibiotic therapy has been used during classic outbreaks and seems to more quickly eradicate EPEC. Oral nonabsorbable antibiotics, such as neomycin, colistin, gentamicin, and polymixin, have been used with some success and apparent decrease in mortality, although not well studied. In the past, oral neomycin has been recommended as initial therapy, when resistance is not a concern, until stools are culture negative for EPEC, often within 2 to 4 days. Parenteral antibiotics can be considered in patients who are systemically ill with concern for sepsis. Relapses can occur, although they do not require therapy, unless they are associated with illness or high epidemiologic risks to other young infants in the household.

When antimicrobial therapy is considered, it is important to consider local patterns of antibiotic susceptibility because EPEC has frequently been found to be resistant to commonly used antibiotics. During an outbreak in Nairobi among preterm neonates, a high resistance level was identified to trimethoprim-sulfamethoxazole (TMP-SMX), chloramphenicol, oxytetracycline, and ampicillin. A recent study in Iran that identified mostly aEPEC strains isolated from children younger than 5 years found all strains to be resistant to ampicillin, chloramphenicol, streptomycin, ciprofloxacin, trimethroprim, and tetracycline, whereas only 7% were resistant to gentamicin. A study in Mexico including 430 children with acute diarrhea found that both tEPEC and aEPEC frequently were multidrug resistant (three or more antimicrobials were tested, including tetracycline, ampicillin, TMP-SMX, and chloramphenicol). All EPEC isolates were sensitive to gentamicin. In Peru, a study of 557 stool samples from children younger than 1 year found EPEC resistance to ampicillin (72%), cotrimoxazole (72%), and tetracycline (50%). Nalidixic acid resistance was 22% among EPEC from symptomatic patients. All strains were susceptible to gentamicin, ciprofloxacin, cefotaxime, and ceftazidime. Empirical choice of antibiotics can be guided by local resistance patterns, and antibiotics should be tailored once the EPEC strain is identified and specific antibiotic susceptibilities are characterized.

Prevention

The transmission for EPEC is fecal-oral. Therefore strict adherence to infection control and appropriate hygiene can help prevent spread. Recommendations for controlling nursery outbreaks come from reported experience, although nursery outbreaks have become rare. Stool cultures can be obtained from infected infants, bearing in mind that most clinical microbiology laboratories do not routinely test for EPEC. If necessary, samples can be sent to a reference laboratory such as the Centers for Disease Control and Prevention (CDC). Infants who are symptomatic or shedding EPEC should be isolated or cohorted to a section of the nursery, and hand hygiene should be scrupulous. Enteral antibiotics can be considered and are thought to have helped curtail spread of the infection in past nursery outbreaks, although this is not well studied. Surveillance of hospital personnel who come into contact with the infants can be undertaken. When the involved infants are discharged, thorough disinfection of the area is warranted. The use of prophylactic antibiotics has been shown to be of no value and can select for increased resistance. An EPEC vaccine is not currently available; research is ongoing.

Epidemiology and Transmission

The mode of transmission of EAEC is not well established. In adult volunteer studies, the infectious dose is high (>10 ⁸ CFU), suggesting that at least in adults, person-to-person transmission is unlikely. Several outbreaks have been linked to consumption of contaminated food. The largest of these outbreaks involved almost 2700 schoolchildren in Japan ; a contaminated school lunch was the source implicated in the outbreak of EAEC. Some studies have shown contamination of condiments or milk, which could represent vehicles of foodborne transmission.

A nursery outbreak of EAEC involved 19 infants in Nis, Serbia in 1995. Because these infants did not ingest milk from a common source, it is presumed that horizontal transmission by environmental contamination or hands of health care personnel was possible. Most of the infants were full term and previously well, and they were housed in two separate nursery rooms.

The earliest epidemiologic studies of EAEC implicated this organism as a cause of endemic diarrhea in developing countries. In this setting, EAEC as defined by the aggregative pattern of adherence to HEp-2 cells can be found in upward of 30% of the population at any one time. Newer molecular diagnostic modalities have revised this figure downward, although the organism remains highly prevalent in many areas. Several studies from the Indian subcontinent implicated EAEC among the most frequent enteric pathogens. Other sites reproducibly reporting high incidence rates include Mexico and Brazil. There is evidence that EAEC may be increasing in incidence. A study from São Paulo, Brazil, identified EAEC as the prevalent E. coli pathotypes in infants, replacing EPEC in this community. Many other sites in developing countries of Africa, Asia, and South America have described high endemic rates. A meta-analysis of 41 studies showed an association between EAEC and acute diarrhea in both the developed and developing world.

Several studies have suggested that EAEC is also a common cause of infant diarrhea in industrialized countries. Using molecular diagnostic methods, a large prospective study in the United Kingdom identified EAEC as the second most common enteric bacterial pathogen after Campylobacter . A similar survey from Switzerland found EAEC to be the most common bacterial enteropathogen. Studies from the United States also have shown a high rate of EAEC diarrhea in infants. Using molecular diagnostic methods, a study in Cincinnati implicated EAEC in 10% and 4.7% of outpatient and inpatient diarrhea cohorts, respectively, of infants younger than 1 year, compared with less than 2% of asymptomatic control infants ( P < .05). Although epidemiologic studies have shown that EAEC can cause diarrhea in all age groups, several studies suggest that the infection is particularly common in infants and children younger than 2 years.

Clinical Manifestations

Descriptions from outbreaks and volunteer studies suggest that EAEC diarrhea is most often watery, with mucus and accompanied by abdominal pain/cramping. Fever, bloody stools, and fecal leukocytes have been reported but are less common.

Early reports of EAEC infection suggested that this pathogen may be associated with persistent diarrhea (>14 days). Later studies suggest, however, that persistent diarrhea may occur in a subset of infected infants and may be linked to malnutrition. In the Serbian outbreak of 19 infected infants, the mean duration of diarrhea was 5.2 days ; diarrhea persisted more than 14 days in only 3 patients. Infants in this outbreak had frequent, green, odorless stools. In 3 cases, the stools had mucus, but none had visible blood. Eleven infants developed temperatures higher than 38° C (>100.4° F); only one had vomiting.

Several clinical studies have suggested that EAEC is associated with subclinical inflammation, including the shedding of fecal cytokines and lactoferrin. Studies in Fortaleza, Brazil suggest that children asymptomatically excreting EAEC may exhibit stunting, compared with uninfected peers. A study from Germany reported an association between EAEC isolation and colic in infants without diarrhea, although this observation has not been reproduced. EAEC should be considered in the differential diagnosis of persistent diarrhea and failure to thrive in infants.

Diagnosis and Therapy

Diagnosis of EAEC requires identification of the organism in the patient’s feces. The HEp-2 adherence assay can be used for this purpose, although the specificity of this assay is decreased compared with molecular diagnosis. Some reports suggest that the adherence phenotype can be observed using formalin-fixed cells, obviating the need to cultivate eukaryotic cells for each assay. PCR for typical EAEC, based on identification of virulence factors, is available in the research setting and is the preferred diagnostic test .

Antibiotic therapy using fluoroquinolones in adult patients has been successful. Studies suggest that azithromycin or rifaximin also may be effective in adults with traveler’s diarrhea. In addition, a small study of azithromycin treatment during the O104:H4 outbreak was associated with decreased length of shedding; fluoroquinolones carry the risk of increased expression of Shiga toxin. Therapy for infected infants has not been well studied and, if deemed necessary, should be guided by the results of susceptibility testing because EAEC organisms are frequently antibiotic resistant.