Acute Infectious Gastroenteritis

Symposium on Infectious Disease

Acute I nfectious Gastroenteritis Robert H. Drachman, M.D. *

Gastroenteritis caused by infectious agents still poses an annoying and sometimes serious problem even in well sanitated populations. Infectious nonbacterial gastroenteritis, after many years of neglect, is now again receiving the investigative attention it deserves. What was said to have been almost a pandemic of this disease in the late 1940's stimulated a good deal of investigative effort at that time here and in Japan. 49 , 72 Lacking a susceptible experimental animal m9del, human volunteer feeding experiments were carried out to study the clinical characteristics of infectious nonbacterial gastroenteritis. Surprising similarities in clinical presentation and even cross protection were demonstrated with fecal filtrates from Japanese and American cases. 44 Recent more intensive studies of infectious nonbacterial gastroenteritis with the "Norwalk agent" obtained from an elementary school outbreak in Norwalk, Ohio, have significantly added to our knowledge of this disease. 9 Histologic study of intestinal mucosa from infected volunteers has provided objective evidence of infection and inflammation,111 Carbohydrate absorption was also found to be impaired. Electronmicroscopy in some studies have revealed virus-like particles in intestinal mucosa biopsies,7 but no replicating agent has yet been directly demonstrated in tissue culture studies. However, fecal preparations from patients with infectious nonbacterial gastroenteritis have been shown by immune electron microscopy to contain virus particles which react with serum antibody obtained during convalescence. 66 Gastroenteritis of bacterial origin, particularly that caused by Salmonella, Shigella, and E. coli, continues to claim attention. One marvels at the rarity of serious Salmonella infections, considering the ubiquity of these organisms. The opportunity for Salmonella contamination of food during commercial preparation is extremely high in light of the many processed foods consumed now. A recent outbreak of Salmonella eastbourne was traced to contaminated chocolate candy.86 Similar instances of contaminated processed foods are numerous. 3 Shigella infections continue to pose a problem in low income populations and after many years' absence from this country Shigella dysenteriae, Shiga's bacillus, has reappeared. It has been associated with

*Associate Professor of Pediatrics, Johns Hopkins University School of Medicine,

Baltimore,

Maryland Pediatric Clinics of North America-Vol. 21, No.3, August 1974

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several reports of severe gastroenteritis. 13 The invasive properties of Shigella and its ability to produce enterotoxin have now been unequivocally demonstrated. 70 As with so many other infectious agents, increased resistance to commonly used antibiotics is ofincreasing concern. In recent years E. coli have less frequently been responsible for the frequent and serious outbreaks of newborn nursery gastroenteritis observed 20 years ago. However, sporadic disease continues affecting not only young children and infants, but adults too. Recent studies demonstrate E. coli capable of invading mucosal cells or elaborating an enterotoxin which may cause a cholera-like disease. 28 Acute infectious gastroenteritis was reviewed in these pages in 1967 by Connor and Barrett-Connor,19 That very excellent review should be referred to as a very thorough overview of the subject up to that time, particularly the initial tabulation of previous efforts at viral isolation in gastroenteritis.

THE PATHOGENESIS OF BACTERIAL DIARRHEA The pathogenesis of acute gastrointestinal disease associated with bacterial pathogens has been examined rather extensively in experimental animal models and to a more limited extent in humans. After the potential bacterial pathogen is ingested, under most circumstances it must then attach to some specific mucosal binding site located in a selected area of the intestinal tract and multiply rapidly in order to attain a significant level of bacterial population in order to incite actual disease. 123 Indigenous bacteria in the gastrointestinal tract are normally quite effective in preventing this initial step from occurring after the potential pathogen is ingested. 10 The normal indigenous Qacteria of the bowel are plentiful and generally tend to be anaerobes or bacteria which are not dependent on oxygen for replication. These bacteria colonize the bowel very early in life and, subsequently, provide a measure of protection to the host by virtue of their ability to interfere with infection by potential bacterial pathogens. 102 Clinically, those factors which are likely to modify the indigenous bacteria of the bowel are likely to predispose to infectious diarrheas. In malnutrition, where bacterial microflora may be altered, this is particularly true, whether the malnutrition results from dietary inadequacies or surgical intervention. 51 Furthermore, where antibiotic therapy has been administered orally, normal protection of the bowel is also reduced. In experimental animals this has been demonstrated repeatedly and antibiotic therapy which reduces the number of certain bacteria, such as anaerobes in the mouse, will result in a marked predisposition to infection by Salmonella which in comparable numbers was incapable of inciting infection in the normal animal. 10 Volatile organic acids produced by indigenous bacteria may also serve to control infection with Salmonella or Shigella organisms. 85 Bactericidal substances produced by some bacteria not only serve to control replication of fungi in the gut, but also may protect against infection with potential pathogens. 109

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Normal intestinal tract flora may also provide protection by virtue of competitive inhibition for combining sites on mucosal surfaces. Of these numerous mechanisms it is not clear which are most significant in protecting the host. However, it is clear that alterations in the normal flora of the intestinal tract as may result from antibiotic use can significantly affect the host's ability to resist infection by potential pathogens.

Mode of Infection by Pathogens Several forms ofinteraction between the pathogen and the intestinal tract may occur in the pathogenesis of diarrheal disease. Three recognized forms of infection are not mutually exclusive and any particular pathogen under certain circumstances may alter its behavior. Savage has summarized these three different forms of interaction which characterize the behavior of the principal bacterial pathogens: 109 1. Attachment without penetration. Bacteria which cause disea~e by the elaboration of an exotoxin may merely attach superficially to the mucosal epithelium. Vibrio cholerae exemplifies this form of interaction and specifically attaches to small intestinal epithelium. Penetration into the epithelium is quite rare and no lesions in the epithelium are detectable by light microscopy. However, replication of the Vibrio cholerae on the intestinal mucosa is accompanied by enterotoxin damage which affects blood vessels and electrolyte cell function in the immediate vicinity.n 3 Certain strains of E. coli may behave similarly and m~y produce disease by virtue of an enterotoxin without penetrating into the mucosa. 28 • 121 Such E. coli diarrheal disease occurs in calves 120 and has been reported in human beings, and the E. coli strain implicated in the disease was shown to produce an enterotoxin. 104 2. Attachment with penetration into epithelial cells. For many years it was believed that Shigella organisms merely lodged superficially on the intestinal epithelium. However, now it is quite clear that after attachment to the intestinal mucosa, Shigella and certain other organisms penetrate into the mucosal cells and multiply within them. 128 They possess a remarkable ability to rapidly penetrate into adjacent cells and substantially increase in number, thereby killing the infected cells and causing the characteristic ulcerations in the intestinal mucosa. 92 The typically bloody stool of bacillary dysentery is produced when bleeding occurs at these ulcerations with resultant red cells and inflammatory cells being swept out in the diarrheal stool. Shigellae initially attach to small bowel mucosa and later in infection attach preferentially to epithelial cells of the colonic mucosa where penetration and ulceration occur.9 In vitro, Shigella can be shown to enter Hela cells in tissue culture and the corneal cells of the guinea pig also. 76 Virulent and avirulent strains of Shigella may be differentiated by their ability to penetrate into epithelial cells. 42 This property is under genetic control and the chromosomal locus for this activity has been mapped. 41 Such genetic information can be transmitted between various strains of E. coli and Shigella flexneri by conjugation. Conceivably, E. coli may be converted into a pathogen which becomes capable of penetrating epithelial cells and producing disease in a fashion similar to that of Shigellae.

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3. Attachment and penetration into submucosal tissues. Salmonella organisms are capable of not only attaching to mucosal cells, but also penetrating through and between epithelial cells to the lamina propria. Polymorphonuclear leukocytes and macrophages are mobilized to the area and ingest the organisms, but may be incapable of killing them. 129 Consequently, some Salmonellae may replicate in macrophages and can be carried elsewhere in the body. The peculiar ability of Salmonellae to survive intracellular residence, even in the presence of effective antibacterial agents, appears to result in microbial persistence. 84 In tissue culture it can be demonstrated that Salmonella remain viable within cells for appreciable periods of time, even in the presence of antibiotics to which the organism is sensitive. The antibiotic enters the cell but merely retards replication of the bacteria, causing it to remain in stationary phase. After withdrawal of the antibiotic the bacteria again are capable of rapid multiplication under usual circumstances.1 18 This phenomenon of microbial persistence probably accounts for the carrier state which often persists for varying periods of time after Salmonella gastroenteritis. Of considerable interest to the immunologic problem presented here is the fact that newborn infants with Salmonella infections are far more likely to remain carriers for long periods of time than are older individuals. 126 Escherichia coli may also penetrate to the lamina propria under certain circumstances, thereby demonstrating the ability of this organism to simulate disease as it might be produced in anyone of these three categories. 124 Production of enterotoxin is also under genetic control and E. coli may become virulent by virtue of this acquired characteris tic. 117 Electron micrographs of infected intestinal mucosa suggest that epithelial cells are stimulated to phagocytize invading bacterial pathogens.1 24 Phagocytic vacuoles lined with cytoplasmic membrane are visible within the cells.1 27 In the case of those pathogens which reach the lamina propria the ingested bacteria leave the epithelial cell after transit through the cell. It is not known what conditions are necessary in order for epithelial cells to be stimulated to ingest bacteria. Needless to say, pathogens remain within the phagocytic vacuoles while in the epithelial cells. Enterotoxic Processes The specific pathogenic mechanisms which result in clinical diarrhea are far clearer now than they were a decade ago. The first mechanism which is of importance has already been alluded to. Pathogens which invade epithelial cells may replicate rapidly and cause destruction of those cells with resultant damage and ulceration of the intestinal tract wall, thereby causing diarrhea. Enterotoxins and their mode of action have been rather intensively studied in the recent past, sparked by an intensified interest in cholera. It now has been very convincingly demonstrated that cholera is principally a local intoxication of the intestinal tract mucosa by means of an enterotoxin elaborated by the Vibrio. 113 The organisms do not penetrate the mucosa and the disease can be controlled with therapy aimed at the

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electrolyte problem. The toxin appears to stimulate copius secretion of intestinal fluid, perhaps by elevating the intracellular levels of cyclic AMP, which enhance succous enteritis secretion. Histologic lesions are not found in the mucosa intoxicated with cholera toxin. The models and concepts developed in the study of cholera toxin have been very useful in the examination of other gastrointestinal diseases which may be enterotoxic enteropathies. 21 Escherichia coli not only has the ability to penetrate mucosal cells, but also have been shown to produce enterotoxins. 28 Much of the work in this area has been carried out with a biological model in which the ligated intestinal loop of a rabbit is injected with the test strain of bacteria. Where enterotoxin is elaborated or other virulence factors are operative, fluid accumulation is readily induced in the ligated section of gut. 131 By this technique and with variations of it, enterotoxin was first identified, associated with E. coli which produced severe diarrhea in young pigs and calves. Subsequently, E. coli isolated from human gastroenteritis have similarly been shown to produce an enterotoxin. The enterotoxin associated with E. coli strains isolated from patients in Calcutta, was found to have toxin which was partially destroyed by heating at 80°C for 30 minutes, or totally destroyed by boiling for two minutes.1° 4 Enterotoxin has similarly been identified in E. coli which were associated with gastroenteritis in young infants in Chicago. 48 It appears that only the small bowel is susceptible to the effect of the E. coli enterotoxin where the pathologic effect is more rapid than with cholera toxin. Therefore, enteropathogenic E. coli may replicate in the large bowel, but will not produce disease unless conditions permit them to penetrate the mucosa or to invade the small bowel and thereby cause illness.4s.133 Genetic control of enterotoxin production may be transferred by means of a plasmid. Consequently, suitable receptor organisms such as other strains of E. coli may be converted to enterotoxin producers by transfer of the plasmid. 122 E. coli and cholera enterotoxin appear to be antigenic ally dissimilar. However, the biochemical defect elicited by these enterotoxins may be similar, although the mucosal binding sites for the two enterotoxins may be different. 21 Recently, stimulation of adenylcyclase by E. coli enterotoxin has been observed, thereby giving further support to the basic similarity between the action of cholera toxin and E. coli enterotoxin. 38 For many years it was known that Shigella dysenteriae produced an exotoxin. However, it was called a neurotoxin because of certain paralytic effects observed in experimental animals after intravenous administration.1 34 However, it is now apparent that the Shigella dysenteriae toxin is an enterotoxin which affects the ligated small intestinal segment of the rabbit by causing fluid accumulation, as with other enterotoxins. Although cholera and Shigella enterotoxins are somewhat similar in that they are heat-labile proteins of comparable molecular size, there are some striking differences. 70 Principal among these is that Shigella toxin alone is capable of causing tissue damage and an inflammatory response. It has already been noted that Shigella are found within epithelial cells in the mucosa of infected hosts. Since shigellae can produce enterotoxin in vitro, it appears that the intracellular site

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is not necessary for the toxin to be elaborated. Craig has commented that the term "invasion" to describe the penetration of shigella into epithelial cells is rather inappropriate, since the organism is non-motile. Be suggests that since the epithelial cell engulfs the Shigella organism the ability of the bacteria to stimulate this type of behavior on the part of normally placid epithelial cells should be called bacterial "seductiveness. "21 Clostridium perJringins, type A, also produces an enterotoxin, although this is principally transmitted by way of contaminated foodstuffs.62 Fluid accumulation in the ligated rabbit gut segment has been associated with diarrhea and vomiting in monkeys and human volunteers. Only those strains positive in the rabbit gut model also cause clinical disease. 125 It is now apparent that E. coli has the potential for producing two different types of diarrheal disease. Cholera-like disease may occur when bacteria do not penetrate epithelial cells but elaborate enterotoxin when superficially attached to intestinal epithelial cells. On the other hand, a bacillary dysentery-like disease may be produced when E. coli invade epithelial cells and cause the type of necrosis and ulceration associated with shigella infection. Indeed, two forms of E. coli gastroenteritis have been described and several E. coli strains have been studied which correspond clinically to the two postulated forms. E. coli strains isolated from American soldiers in Viet N am, who manifested an acute colitis type of disease, had the following characteristics: human volunteers fed these strains manifested fever, severe diarrhea, some with bloody mucoid dysentery. In vitro, guinea pig corneal epithelial cells were invaded, as were Bela cells in tissue culture, and guinea pigs challenged orally with these organisms showed invasion of the lamina propria. E. coli isolates from soldiers with less severe diarrhea caused a choleralike illness in human volunteers with the fever and dysentery characteristics of the other strains. In addition, cell-free culture filtrates from these organisms caused fluid accumulation in rabbit ileal loop preparations and failed to show invasion of the guinea pig corneal epithelial cells, or lamina propria of the guinea pig gut when these animals were challenged per as. In this study the invasive strains of E. coli which resemble virulent shigella strains were of serotypes which were not commonly identified as enteropathogenic E. coli strains in the past.28 The implications of these findings in regard to the virulence of E. coli is of great importance in studying human disease. It is now quite apparent that any specific E. coli serotype associated with diarrheal disease in humans may be characterized by several different virulence factors. Consequently, isolation of any particular serotype does not automatically suggest that this is the etiologic agent. Indeed, so-called enteropathogenic strains of E. coli may be isolated from asymptomatic individuals, even newborn infants who show no evidence of gastrointestinal disease. 2o Virulence can now be associated with genetic information for penetration of epithelial cells or information relative to the elaboration of an enterotoxin. Since these are both biologic tests without simple reproducible chemical methods to parallel them, diagnosis of the etiologic agent in clinical disease will not be made any easier for a while yet.

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INFECTIOUS NONBACTERIAL GASTROENTERITIS Gastroenteritis is one of the more common minor infectious diseases observed among children and adults. Many episodes of local epidemic proportions have been described involving nausea, vomiting, and diarrhea at varying times of the year. In careful bacteriologic studies of many of these outbreaks few could be attributed to bacterial agents. 2,23,47.98 Infectious nonbacterial acute gastroenteritis appears to be a rather mild, self-limited disease which occurs principally during the fall, winter, and spring. Diarrhea, nausea, vomiting, headache, abdominal pain, low grade fever, and malaise in varying combinations characterize this disease, which is usually rather short-lived and may last only 24 to 48 hours.9. 23. 49 Many efforts have been made to identify recognized viruses as etiologic agents in these outbreaks and sporadic cases.1 9 In general, the populations more intensively studied for viral agents have been those with the more severe symptomology, so each study must be examined carefully to make certain of the comparability of study populations. Improved virus identification techniques have made it possible to characterize the indigenous population of known viruses present in the asymptomatic child, as well as the patient with gastroenteritis. Con.trol groups must always be compared with the acutely ill group because of the ubiquity of certain agents. Judging from several appropriately controlled studies of fecal specimens in gastroenteritis it appears that certain viral isolates in a small percentage of cases may be etiologic agents. Of those agents isolated from stool, adenoviruses, coxsackie, polio and ECHO are most often found in gastroenteritis.1 9 ECHO viruses 11, 14 and 18, among others, have been associated with diarrheal disease. An epidemic of diarrhea in premature and older infants was at~ tributed to ECHO virus type 18. 34 Conner et al. have tabulated earlier efforts to characterize viral agents in outbreaks of gastroenteritis. 19 Of the several studies summarized, in 10.5 per cent of controls recognized viral agents were isolated, while in patients acutely ill with gastroenteritis, 14 per cent were found to harbor known viral agents. These results are not very striking since such a large percentage of patients were negative for potential viral pathogens. In marked contrast, studies of children with diarrheal disease in Mexico City revealed unidentifiable viral agents in about 34 per cent of patients while only about 6 per cent of controls were similarly infected. Judging from these studies, presently recognized viral agents appear to be infrequently associated with acute gastroenteritis. The syndrome of acute infectious nonbacterial gastroenteritis is a much milder disease than has been noted in patients studied for known viral agents associated with gastroenteritis. Human volunteer transmission experiments have provided the most useful information about this disease since no suitable experimental animal has been identified. In the late 1940's Gordon studied on outbreaks of nonbacterial gqstroenteritis in New York State. A filterable agent called the Marcy strain of afebrile gastroenteritis was isolated in an institutional outbreak. 49 When volunteers were fed bacteria-free preparations of stool specimens an illness occurred, characterized by watery diarrhea in about two

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thirds of volunteers, accompanied by abdominal cramps, some vomiting and low-grade fever, headache and considerable fatigue. Marcy strain could be passed repeatedly through volunteers without losing virulence, and sixth passage material was still capable of causing symptoms in volunteers fed this material. Marcy strain was capable of inducing immunity in volunteers who, after a first illness, did not respond to subsequent challenges of bacteria-free stool filtrates. The incubation period for Marcy strain ranged from 24 to 120 hours with an average of about 60 hours. The mean duration was about 96 hours. Fever was usually absent or below 101. Constitutional symptoms were mild, as was headache. Nausea was moderate to severe and vomiting occurred in bouts early in the illness. Abdominal pain with cramps was of moderate severity and usually preceded the watery, frequent diarrhea. 49 In the past, it had been observed by Reiman et al. that inhalation of nebulized filtered throat washings from patients with acute gastroenteritis were capable of initiating disease in volunteers.98 Human volunteer experiments with the Marcy strain did not produce disease when human volunteers were challenged by the respiratory route. Storage of the stool specimen filtrates at -70°C. resulted in very little loss of activity and such specimens subsequently thawed and fed to volunteers produced disease. Seventh passage material from volunteers was still capable of inducing disease. At about this same time in late 1947 and 1948 an epidemic of benign diarrheal disease occurred in Japan. Symptoms of gastroenteritis were very similar to those reported for the Marcy strain. However, the incubation period seemed to be somewhat shorter. 72 Human volunteer feeding experiments were done with the material from a Japanese outbreak in Niigata Prefecture. Volunteers were fed bacteria-free stool filtrates or it was administered through a duodenal canula. A short while before diarrhea appeared, volunteers complained of weakness, abdominal pain and nausea. A low-grade fever was noted in some volunteers and symptoms persisted for about 3 to 5 days but recovery was complete in about one week after onset. Volunteers in these same experiments were fed preparations of the Marcy strain and experienced the expected clinical signs and symptoms. Subsequent challenge with the Niigata strain failed to produce disease, suggesting cross-immunity between the Marcy and Niigata strains.44 It is tempting to postulate that the nonbacterial gastroenteritis which occurred in New York State and in Japan were caused by similar agents and that a relatively mild pandemic of infectious nonbacterial gastroenteritis occurred in the late 1940's. It is noted by Fukumi et al. 44 that no similar disease had occurred in Japan during the war or thereafter, until large numbers of American servicemen moved into the country. During the course of the Cleveland family study a febrile type of gastroenteritis was noted among study patients. 63 Conventional efforts at an etiologic diagnosis were unsuccessful. Patients with this disease, identified in the family study as FS strain, experienced an illness of very brief duration, averaging about 24 hours. Significant fever was present; malaise and headache were usually present and frequently rather severe. Nausea and vomiting with abdominal pain were present and often

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described as severe, also. Diarrhea was not at all pronounced and watery stools did not occur, rather they were merely loosely formed. Volunteers remarked that the aftereffects of the FS strain challenge were quite debilitating even after cessation of acute symptoms. Volunteers who became ill after challenge with either the Marcy strain or FS strain were subsequently challenged with the other strain. No protection ,with the heterologous strain was demonstrated, and on second challenge, most volunteers became ill again. 65 Consequently, the rather mild afebrile watery diarrheal disease associated with the Marcy and Niigata strains could be differentiated from the FS strain disease characterized by fever, nausea and vomiting, with no cross-immunity between the two. Efforts to isolate viral agents from the Marcy strain materials were unsuccessful. .50 In October of 1968 in Norwalk, Ohio a winter outbreak of an acute disease occurred, including nausea, vomiting and less frequently, diarrhea and a low-grade fever. About 50 per cent of students in an elementary school became ill. The secondary attack rate was 32 per cent and the disease lasted about 24 hours with an estimated incubation period of 48 hours.2 The epidemiologic studies clearly suggested an infectious etiology with similarities to the previously described outbreaks. Stool specimens were tested for viral and bacterial agents, with no evidence of known pathogens. 71 In addition, enterotoxin could not be demonstrated when stool filtrates were tested in the rabbit ileal loop assay.40 Three serial passages in human volunteers were accomplished with this agent. Symptoms of gastroenteritis were somewhat v'ariable during these experiments. The attack rate in volunteers challenged orally with the Norwalk agent material was about 67 per cent. The incubation period ranged between 16 and 48 hours with a mean of about 37 hours. The disease lasted about 24 to 48 hours with a mean of 33 hours. Combinations of low-grade fever, diarrhea, vomiting, abdominal cramps, malaise and headache occurred in different volunteers and at different passage levels. 9 Norwalk agent did not replicate in tissue culture or in human fetal intestinal organ cultures. 25 Ether treatment of filtrates did not impair infectivity, suggesting a lack of a lipid coat. It was relatively heat stable and resisted acid treatment at pH 2.7. Short-term homologous immunity was also demonstrated by repeat challenges to previously ill volunteers.9 The supernatant fluid from previously incubated human fetal intestinal organ cultures challenged with the Norwalk agent produced illness in a few volunteers. However, this was rather inconstant. Nasopharyngeal washings from acutely ill volunteers did not produce illness in volunteers who received this material orally, thereby suggesting the absence of the agent in the upper respiratory tract. Transient malabsorption of D-xylose and lactose indicating transient enzyme deficiency and steatorrhea were noted in some volunteers infected with the Norwalk agent. Indeed, these observations were made in individuals who developed no clinical signs of gastroenteritis. 9 • 74 In a group of 15 volunteers challenged orally with the Norwalk agent, duodenojejunal mucosal biopsies were obtained before, during and after the induced gastroenteritis. Significant alterations of mucosal

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architecture occurred in all volunteers at 12 to 48 hours after challenge.1 11 Several hours after inoculation shortening of the intestinal villi and hypertrophy of the crypts was noted. Abnormalities of the absorptive cells became prominent and infiltration of the lamina propria with granular leukocytes occurred. Many of these abnormalities persisted for 5 to 6 days, but cleared by several weeks later. Malabsorption of D-xylose and fat was quite consistent with the mucosal pathology.111 In experiments carried out by other investigators duodenal biopsies revealed virus particles in mucosal cells. 7 Kapikian,66 using immune electron microscopy, demonstrated aggregation of viral bodies from fecal materials after incubation in convalescent serum from patients strongly suggesting their etiologic significance. The composite of the above findings suggests very strongly that an infectious agent of small size is capable of damaging the small intestine mucosa and is associated with the typical signs and symptoms of acute infectious nonbacterial gastroenteritis. It is anticipated that new technology in virology isolation techniques will soon provide much more information about this agent.

BACTERIAL AGENTS CAUSING GASTROENTERITIS Bacterial agents mentioned in this section, including Shigella, Salmonella, and Escherichia coli, belong to the family Enterobacteriaceae. Bacteria of this group reside in the intestinal tract of vertebrates and are usually commensals, but on rare occasions, some act as pathogens. Members of the family are closely related and many common biochemical and close serologic relationships exist which result in numerous transitional forms between groups. The clinical importance of this close relationship within Enterobacteriaceae lies in the ability of certain genetic characteristics residing in one group to be transferred to others. For example, the ability to resist the inhibitory or cidal activity of certain antibiotics may be transferred from one bacterial strain to another within the family. This may occur in vivo and probably accounts for changes in antibiotic sensitivity of certain pathogen groups, when selection occurs because of administration of oral antibiotics in particular.

SALMONELLA GASTROENTERITIS

Once bacteria isolated from the stool have been characterized by means of biochemical reactions as Salmonella, the specific serotype can be determined by means of serologic tests. More than a thousand serotypes have now been identified on the basis of antigenic structure. Multiple permutations of many somatic or "0" antigens and two forms of flagella or "H" antigens exist in the numerous serotypes. 33 Specific serotypes are not necessarily associated with virulence, except in the case of the typhoid bacillus. As noted previously, Salmonella organisms produce disease when they possess or acquire the ability to penetrate

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mucosal cells. Consequently, identification of salmonella in the stool of asymptomatic subjects does not necessarily indicate the imminence of gastroenteritis. The patient may be a carrier of long standing, perhaps. This is not to say that suspicion as to its etiologic significance should not be heightened, but an asymptomatic "epidemic" of Salmonella tennessee in a premature nursery139 and other similar observations suggest the importance of intrinsic characteristics of the salmonella organism. Multiple serotypes are of great value to the epidemiologist who can make use of the antigenic markers on the specific serotype for determining sources of infection in epidemic salmonella gastroenteritis. Most human salmonella gastroenteritis occurs by way of contaminated foodstuffs ingested by an individual. 130 Direct, person-to person spread of salmonella organisms does occur, but far less commonly.106 As noted previously, salmonella may penetrate mucosal cells and lodge in the submucosal lamina propria, where a polymorphonuclear leukocyte inflammatory reaction occurs. Clinical gastroenteritis results, but normally the inflammatory cells are capable of ingesting and destroying the organisms so that salmonella gastroenteritis is a self-limited disease. Typhoidal syndrome and salmonella enteric fever result from blood-stream dissemination of these organisms and replication in areas other than the lamina propria.

The Ubiquity of Salmonella Organisms Epidemiologic studies of salmonella gastroenteritis in the past 30 to 40 years have identified several common sources of infection. 3 Often the picture is one of salmonella organisms which reside in domestic animals, causing infection of other animals when brought together before slaughtering or contamination of manufacturing equipment and preparation surfaces which then lead to contamination of finished food products. In one study, for example, 0.5 per cent of calves were found infected with salmonella on the farm. After being kept in holding pens for 2 to 5 days on their way to the abattoir the infection rate reached almost 36 per cent. 5 This experience has been observed repeatedly in the literature with other types of domestic animals. 3 Poultry constitute the largest reservoir of salmonella. 61 The vast majority of salmonella cultures isolated from animals during recent years have come from domestic fowl.9 6 Poultry flocks may suffer extensive outbreaks with high mortality rates ranging to more than 80 per cent. Very often poultry outbreaks will precede human outbreaks as occurred in an epidemic of Salmonella reading.27 Salmonella typhimurium is the serotype most commonly isolated from poultry, and also is most frequently isolated in human disease. Transmission of salmonella in poultry may be transovarian or may be by way of the intestinal tract, and chicks are readily infected. Baby chicks sold at Easter time as pets have also transmitted the disease to humans. 4 In several salmonella epidemics traced to poultry, processing plants have been carefully investigated. Frequent contamination with salmonella was detected at many points in the slaughtering and processing procedures. Many difficult to reach surfaces and machine parts yielded positive cultures for salmonella. I!. 82 Only a small number of salmonella

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positive birds might infect such a plant. Bacterial multiplication then occurs on machine surfaces and in the plant environs thereby serving as an amplifier to cause contamination of other fowl going through processing. Considering that many thousands of fowl may pass through a large processing plant, the magnitude of this amplification process becomes apparent. Egg products, both dried and fresh, have been implicated in many outbreaks of salmonella infection in man. Since dried egg preparations go into many cake and bread mixes, the potential for contamination is significant. 132 Highly processed meat products, such as sausage may be contaminated, a Florida investigation reporting 8 to 58 per cent of samples contaminated. The occurrence of salmonella in meat and poultry products is obviously a large one. 43 The problem of contaminated domestic animals is in part due to a more basic problem; mainly, the fact that animal feeds have been found to harbor salmonella also, thereby setting off a predictable chain of events. 3, 13 Quantitative counts of salmonella in many foodstuffs prepared for human consumption indicates that the number of organisms is often quite small. Nevertheless, under conditions of inadequate refrigeration or handling the bacteria may multiply and reach numbers significant to cause human disease when ingested. Feeding experiments with human volunteers have indicated a wide range of dosage requirement for inciting infection. However, as few as 12,000 salmonellae of certain serotypes were capable of causing infection.15 Patients experiencing salmonella gastroenteritis may provide a source of organisms which can infect other individuals by direct contact or contamination of food and pose serious problems in the newborn nursery particularly.37 Of household pets sporadically implicated as salmonella carriers small pet turtles have been a recurrent problem. 67 Young children are most likely to carry salmonella from infected turtle droppings to their mouths. A ban on the sale of pet turtles is currently under study by the FDA.

Clinical Disease After ingestion of an infecting dose of salmonella the average incubation period before clinical signs and symptoms is about 24 to 48 hours. Fever and diarrhea are the most common symptoms in salmonella gastroenteritis, with vomiting occurring early.35.103 Transient bacteremia may occur in some cases of self-limited salmonella gastroenteritis, but the organisms normally are readily cleared without localization outside of the intestinal tract. Great variability in the severity of symptomatology has been noted in children. Stools are often loose or watery, on occasion with mucous and blood, but rarely contain gross pus. The temperature range is usually between 37 and 38°C and median leukocyte counts range between 12,000 and 15,000. 16 Salmonella gastroenteritis is by far the most common type of salmonella infection occurring in more than 75 per cent of infected patients. The illness is usually self-limited and abates in 2 to 5 days from onset. Pneumonitis and isolation of salmonella from the respiratory tract has been observed. Whether this represents primary respiratory route

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infection or superinfection of the lung parenchyma secondary to bacteremia, is not clear. However, this complication is characterized by acute respiratory distress and radiographic evidence of pulmonary infiltration. 8 Salmonella meningitis is a rare complication, but may occur in the neonatal period. It may occur subsequent to gastroenteritis or can follow bacteremia particularly in the neonatal age group. The association of salmonella osteomyelitis and hemoglobinopathies, particularly sickle cell disease, is well known. 64 Explanation of the increased susceptibility of such patients to salmonella infections is not entirely clear. However, in experimental animals with immunologically induced hemolytic anemias, salmonella infections were far more virulent than in controls. Salmonella appeared to be ingested normally by macrophages, but persisted for much longer periods of time in the macrophages of hemolysing animals. 68 Areas of aseptic necrosis in the bones of sickle cell disease patients might also provide a convenient site where blood borne salmonella might lodge and multiply, thereby causing disease. Another group of patients with unusual susceptibility to salmonella infections are those with recent intestinal tract surgery. In a recent interstate hospital epidemic of Salmonella derby, these patients were at unusual risk.t o8 Enteric fever may occur without prior significant gastroenteritis and represents extension and multiplication of organisms in RES and other extraintestinal tissues rather than in submucosal areas as in primary infection. In enteric fever, organisms lodge in reticuloendothelial tissue and periodically may send out showers of salmonella with persistence at these sites for several days or longer. 93 Fever and signs and symptoms of sepsis are clinically evident although at times the presentation may be deceptively mild. Despite the large number of salmonella serotypes, a small number account for the majority of infections. These types invariably include S. typhimurium, S. newport, S. enteritidis, S. infantus, S. heidelberg, S. St. Paul and S. Thompson. Salmonella typhimurium alone usually accounts for more than one quarter of all reported infections. 3

Treatment Salmonella gastroenteritis is usually a self-limited disease and specific therapy has little influence on the course of the illness. 6 In fact, by the time culture reports are received the patient may be asymptomatic. Indeed, patients are more likely to become carriers when uncomplicated salmonella gastroenteritis is treated with antibiotics. In addition, the appearance of antibiotic resistant organisms is more likely. This is not surprising in view of the previous observations on microbial persistence, where it was demonstrated that salmonella may persist within cells despite adequate levels of antibiotics to which the organisms normally were sensitive.t 18 However, antibiotic therapy is clearly indicated in enteric fever, salmonella bacteremia, meningitis, osteomyelitis, and other localized infections. In addition, neonates and the very young deserve therapy, in order to prevent extraintestinal seeding of salmonella. However, it should be noted that neonates are more likely to become chronic salmonella carriers and perhaps this state is facilitated

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by antibiotic therapy which paradoxically may be necessary for acute treatment of the infant. 126 In studies of rural near-Eastern children with salmonella enteric fever, it was demonstrated that they responded more readily to chloramphenicol, suggesting this drug should be considered for severe extraintestinal disease.lO o Some have found ampicillin to be effective and have suggested this antibiotic for treatment of non typhoidal salmonella infections.l, 36 Where essential that chloramphenicol be used in premature and full term newborn infants, up to 2 weeks of age the dose suggested is 25 mg. per kg. per 24 hours. In infants over 2 weeks of age 50 mg. per kg. per 24 hours is suggested. However, in light of the serious potential toxicity of this antibiotic other antibiotics should be used first wherever possible. Frequent chloramphenicol blood levels should be obtained to make certain that they do not rise above 10 to 20 mcg. per ml. In older children 50 to 100 mg. per kg. per 24 hours of chloramphenicol is suggested. In uncomplicated cases about 7 days of therapy is usually adequate. However, for localized disease, several weeks of therapy may be necessary after the acute signs of inflammation have subsided. Where there are pressing reasons for treating salmonella gastroenteritis or in severe extraintestinal salmonella infections, and when the organism is sensitive to ampicillin, this far less toxic antibiotic may be used. However, an increasing number of salmonella strains are reported to be resistant. 112 Assuming that ampicillin is reserved for severe infections 200 mg. per kg. per 24 hours, in 4 to 6 divided doses, may be used by any route. In older children with serious infection up to about 14 gm. per 24 hours may be used. Other antibiotics which may be of value in specific salmonella infections where antibiotic sensitivities are known include kanamycin, gentamicin, and colistimethate. 53 Aserkoff and Bennett6 studied the effect of both chloramphenicol and ampicillin on post-convalescent excretion of salmonellae. All patients had experienced acute salmonella gastroenteritis following ingestion of contaminated turkey sandwiches. At 31 days after exposure 11.5 per cent of those who had received no antibiotic therapy were still positive. Of those who had been treated with antibiotic 27 per cent were still positive. In addition, resistant strains were isolated from about 10 per cent of patients treated with antibiotics, while none was found in untreated patients. There was no difference in complications between the two groups. Many multiply resistant E. coli containing transferable R factors have been identified in normal stool cultures. 78 By means of conjugation, antibiotic resistance can be transferred to salmonella organisms, but principally in the presence of antibiotics. 54 This probably explains the absence of resistant salmonella in untreated patients in the Aserkoff, Bennett study.6

Management of the N ontyphoid Salmonella Carrier Following an outbreak of salmonella gastroenteritis some minority of those infected may continue to excrete salmonella for weeks or many months. Antibiotic treatment of such chronic excretors has generally been unsuccessful or the experiment inconclusive. 53, 94,105 However,

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many local or State public health regulations require three negative cultures before a salmonella excretor is allowed to return to work. Adherence to such rules may cause considerable hardship for both employee and employer or for a school child. Since antibiotics are of little value, spontaneous cessation of positive cultures must be awaited and this might take many months. The awkwardness of this situation is obvious and for noncritical jobs, not the nurse in a premature nursery for example, some have unofficially modified the rules. Taylor and McCoy130 suggest careful instructions to the salmonella excretor regarding hand washing and personal hygiene before returning him to work or school fairly promptly. This seems to be an eminently reasonable plan for most occupations but as yet does not have official sanction in this country.

BACTERIAL GASTROENTERITIS: CULTURES FOR ENTERIC PATHOGENS

The likelihood of a positive culture in a patient with bacterial gastroenteritis depends on many factors having to do with the peculiarities of the intestinal flora, nature of the specimens, the handling of these and the bacteriologic isolation techniques used. Where multiple techniques have been used to identify bacterial pathogens, deficii:mcies in single specimen cultures become apparent. Several rectal swab specimens were necessary to identify the total population of salmonella carriers in one study. The likelihood of detecting such carriers with a single rectal swab specimen was only about 50 per cent.80 In general, only salmonella survive well in transported stool specimens and where E. coli or shigella are suspected, specimens should be plated as rapidly as possible. Shigella and E. coli are adversely affected by many elements in stool, including other bacteria, bacteriophage and limitation of nutrients. Buffered glycerol saline solution has been frequently recommended for transport of stool specimens containing shigella. 33 However, the yields from specimens transported in this fashion are less than when freshly obtained specimens are processed immediately. 107 The reliability of three different types of specimens obtained for shigella culture were studied during the Korean War in a prisoner of war camp.60 Patients with dysentery were cultured by means of bacteriologic examination of rectal swabs, freshly passed stool and specimens obtained from areas of bowel involvement observed on proctoscopy. All three specimens were obtained at about the same time and cultured promptly. Specimens positive for shigella included 608 sigmoidoscopic specimens, 509 rectal swab specimens, and 496 freshly passed stools. Of those specimens positive by one procedure only which included 363 specimens, the positives included 95 stools, 98 rectal swabs, and 170 sigmoidoscopic specimens. Two procedures were positive for 307 or 34 per cent and all three were positive in only 223 or 25 per cent. These observations emphasize the questionable reliability of single specimens obtained from patients with gastroenteritis. The need formul-

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tiple specimens probably should be emphasized where it is desirable to identify the maximum number of patients positive with bacterial pathogens. In addition, specimens should be processed promptly and where clinical findings suggest otherwise, negative bacteriologic results should be viewed with skepticism.

SHIGELLOSIS

Four principal subgroups make up the genus and include Shigella dysenteriae, formerly Shiga's bacillus, S. fiexneri, S. boidii, and S. sonnei. At present about 80 per cent of all shigella isolates from clinical disease are S. sonnei. 115 Formerly flexneri 2A was by far the most common type. Man and primates are the principal reservoirs of shigella and the route of infection is usually person to person or less commonly contaminated food or water. However, several extensive water-borne outbreaks of shigellosis have been described and shigella may be one of the principal etiologic agents in so-called "sewage" poisoning, which occurs when water supplies are contaminated with effluent containing human waste. 116 Small numbers of shigella are capable of causing infection in human volunteers,114 In institutions for the mentally retarded shigellosis is a common problem which is extremely difficult to control because of limited ability to impose reasonable standards of sanitary conditions. 29 Shigellosis tends to be endemic in such institutions. The largest population group afflicted with endemic shigellosis are lowincome families living under conditions of extremely poor environmental sanitation. 110 Many observations have clearly established that the accessibility of water and sanitary facilities are inversely proportional to high shigella morbidity. In studies of shigella morbidity in preschool children in Kentucky, the prevalence of shigella was highest where water and toilet facilities were located outside the dwelling. The prevalence was lowest in those homes where water and flush toilets were located inside. Flies also are a significant mode of transport for shigella, particularly where they have access to open privies or surface collections of human excretion. Where fly control has been carried out significant reductions in shigellosis in children at risk have been observed. 79 Waterborne outbreaks of shigellosis have generally occurred where water supplies have become contaminated through faulty plumbing or where unchlorinated surface supplies have been contaminated by known or unknown excretors of shigella,116 However, the vast majority of shigellosis seen in low-income urban areas, particularly in the southern parts of the country, is disseminated by direct contact between children. ss Several outbreaks of shigellosis in day care nurseries have emphasized the ease with which shigella can be transmitted in such settings. Foodstuffs contaminated by infected food handlers have been identified as vehicles far less often.12 Shigella dysenteriae gastroenteritis caused by Shiga's bacillus was rarely reported in this country until about 1968 when travelers to Mexico

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and Central American acquired shigella infections with these organisms. Since 1969 there has been a continued increase in the number of Shiga's bacillus infections in the United States, but the number is still a very small percentage of the total reported cases. 13

Clinical Illness In human volunteers challenged with virulent shigellae, large numbers of viable shigellae are detectable in jejuneal and ileal contents 8 to 40 hours after ingestion. Diarrhea and fever appear and dysenteric stools with blood and mucous occur somewhat later when the shigella penetrate mucosal cells of the large bowel. 31 Necrosis of epithelial cells and ulceration of the intestinal mucosa is characteristic of the pathogenesis of shigellosis. 92 As few as 200 virulent shigellae or less are capable of causing illness in volunteers. The frequency of contact spread is probably dependent on this striking infectivity of a few organisms. 31 • 77 Children under the age of about 5 to 8 are usually susceptible to shigellosis, and this is observed frequently in common source outbreaks where all age groups in a population may be exposed. Attack rates and illness are more common and more severe in children. 77 Increased susceptibility in young children is probably due to a lack of coproantibody or mucosal antibody which is most effectively elicited by direct contact with pathogenic bacteria or related antigens in other organisms. Secretory IGA synthesized in the intestinal mucosa probably protects the individual if the dose of virulent bacteria is not too large. As in viral respiratory disease serum antibody is of little value in predicting susceptibles and nonsusceptibles, mucosal antibody being far more predictable in this respect. Live shigella adIninistered by mouth induces immunity, but circulating antibody often cannot be detected. However, subsequent challenge usually shows protection suggesting the importance of mucosal coproantibody in the intestine. 32 Shigella gastroenteritis usually begins rather abruptly, with diarrhea and fever occurring about the same time or with some slight delay in the appearance of the fever. VoIniting may occur but is less frequent. Gross blood in the stool is not as common as suggested in classical descriptions and may only appear in less than half of patients.103 Fecal smears may reveal polymorphonuclear leukocytes and red blood cells, although this may occur wherever mucosal cell invasion and necrosis is apparent, as with certain of E. coli infections which involve the colon. This description characterizes the disease associated with Shigella flexneri and S. sonnei, currently the most prevalent types.u 5 Shigella dysenteriae, Shiga's bacillus, was rarely seen in this country before 1968. However, as of November, 1972, 81 Shiga infections were reported. 13 About half the reported infections occurred in children and almost all were associated with infection outside of the country, either in Central America or Mexico. The reported illnesses were far more severe than commonly seen shigellosis. Grossly bloody diarrhea with tenesmus was noted in 88 per cent. VoIniting was common and 54 per cent of patients were hospitalized with one death in the reported group. An exotoxin produced by Shiga's bacillus has been described as

j

1

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a neurotoxin in the past. However, more recent investigations find this to be an enterotoxin with striking effects on the intestinal mucosa, in addition to an ability to adversely affect capillary endothelium. The neurotoxic effects after intravenous administration are now thought to result from this latter effect.77 Increasing numbers of Shiga's bacillus infections should be looked for in the near future. Febrile convulsions may occur early in shigella infections, sometimes preceding the appearance of diarrhea. Rarely, they occur without fever. The incidence of seizures varies widely, but may occur in 5 to 10 per cent of patients. Seizures are readily controlled with medication or are self limited. 26 , 75, 103 The principal complication of shigellosis is dehydration, particularly in the young child. Where diarrhea is profuse or continues for some time without adequate replacement, dehydration may be severe, with marked hyponatremia. A definitive diagnosis of shigellosis can be made by isolation of the organism from the stool. Because of the fragile nature of the organism in transport, rectal swabs or stool cultures should be plated immediately. A history of diarrhea in other family members is fairly commonly elicited with milder signs and symptoms in older children and adults. Peripheral white cell counts have been reported to range from 2200 to 33,000. However, Poh has noted that in shigellosis the percentage of immature versus segmented neutrophils in the differential count was 85 per cent compared to about 20 per cent or less in E. coli or other types of diarrheal disease. 95 Shigella are rarely isolated from blood cultures and Connor noted only three positive among 141 such cultures from patients with shigellosis. '9 Goscienski and Haltalin have recently reported the association of rose spots with Shigella ftexneri 4A. 52 Nelson and Haltalin have assembled the signs and symptoms of diarrheal diseases to facilitate differential diagnosis of these conditions. 91 They suggest the watery, relatively ordorless stools with blood-tinged mucus are pathognomonic of shigellosis. Using these criteria for etiologic diagnosis physicians were correct in 67 per cent of 144 patients. However, 29 per cent of those thought to have non bacterial diarrhea had positive bacterial cultures for pathogens. Treatment

Replacement of fluid and electrolyte losses in the patient with shigellosis occupies first priority in therapy. Antibiotic therapy, in contrast to its use in salmonella gastroenteritis, has been demonstrated to be of considerable value in that the persistence of diarrhea and duration of stool positive for shigella are shortened. In Korean prisoner of war camp studies carried out in the early 1950's, 1408 individuals with positive cultures for shigella were studied. 46 Sulfa resistance was widespread among the shigellae isolated. Chloramphenicol and tetracycline therapy were found to produce negative stool cultures more rapidly, about 10 per cent at 2 days, versus 73 per cent positive cultures in untreated controls. Sigmoidoscopy at 4 days revealed active colitis in about half of controls, while only 4 per cent of appropriately treated groups were still positive at that time. Others have similarly found that appro-

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priate antibiotic therapy is of considerable benefit to the child with shigellosis. 55. 58 It is also clear that only absorbable antibiotics are effective since those which primarily pass unabsorbed through the gut have little effect on the course of disease. 56 By the late 1940's sulfa drugs which originally had been effective against shigella became almost wholly useless in therapy because of extensive antibiotic resistance. Tetracyclines were subsequently used until increasing resistance and other objections to its use, as well as the availability of new antibiotics modified its use. Ampicillin came into wide use in the 1960's and Haltalin et al. demonstrated its beneficial effect on the bacteriologic clearing of stools, with cultures turning negative in about 48 hours, and its beneficial clinical effect as well. 55 At that time ampicillin resistance was noted in only about 3 per cent of shigella isolates, a percentage also noted elsewhere. However, by 1969, Japanese, investigators reported that more than 50 per cent of shigella isolates from Japan were resistant to ampicillin. Similar observations were made in this country, although the incidence of ampicillin resistance was quite variable from one area to the next. 59 Where shigella are sensitive to ampicillin it should be used for therapy. However, if because of resistance, the toxic antibiotic chloramphenicol must be used in treating shigellosis, some have raised questions as to whether it should be reserved for more severe illnesses since the disease is usually self-limited and the carrier state rare. The rapid emergence in recent years of multiply resistant strains of shigella poses a serious problem in therapy. Antibiotic resistance is associated with a bit of extrachromosomal genetic material called an episome which carries the necessary resistance information.1 35 The so-called "R" factors are readily transmissible between various members' of Enterobacteriacae. Episomes associated with antibiotic resistance may be readily transmitted from shigella to recipient E. coli or vice versa by means of bacterial conjugation. Consequently, the use of antibiotics represents a selective pressure on sensitive shigella strains, which may be converted to resistant forms by acquisition of the appropriate episome from intestinal bacteria. 136 Symptomatic therapy of shigellosis or other diarrheal diseases often has included medications to inhibit intestinal motility in order to control diarrhea. DuPont and Hornick 30 have demonstrated in human volunteers with shigellosis that Lomotil (diphenoxylate with atropine) prolongs fever and tends to nullify antibiotic effectiveness. Intestinal motility is an important host defense against invasive gut pathogens which do not penetrate mucosal cells when rapidly swept along the intestine. Tampering with this defense appears to be to the disadvantage of the patient.

ESCHERICHIA COLI GASTROENTERITIS

In a previous section recent findings describing various forms of E. coli gastroenteritis were presented. E. coli may cause gastroenteritis by elaboration of an exotoxin or by invasion of epithelial cells producing

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a dysentery-like disease. So-called enteropathogenic serotypes of E. coli have been identified primarily in epidemic situations involving infants with diarrhea. 33 However, these same strains have been found in asymptomatic individuals on a rather sporadic basis varying from 1 to 10 per cent. ZOo 69 It is apparent now that serotyping whereby somatic, flagella and envelope antigens are characterized only tells a part of the story in regard to etiology. There may be a dissociation between socalled enteropathogenic E. coli serotypes and those organisms that are identified as etiologic agents in some cases of diarrhea. Gorbach and Khurana studied E. coli isolates from infants admitted to the Cook County Hospital with acute diarrhea. 48 Standard serotyping of these isolates revealed 31 per cent among the conventional enteropathogenic E. coli serotypes. However, they also performed biologic tests of enterotoxin production by inoculating the E. coli strains isolated from the infants into ligated ileal loops of infant rabbits. This biologic model is quite sensitive to enterotoxin, causing large amounts of fluid to be secreted into the bowel when present. When isolates were studied in this fashion 24 of the 29 infants were found to be carrying E. coli which elaborated an exotoxin causing injury to the ileal loop wall. The investigators note that many colonies should be examined in carrying out such studies. Fortunately, however, the virulent E. coli are usually present in large numbers when they are responsible for the disease. These findings provide very little laboratory assistance for the clinician, since the biologic assay for enterotoxin is not suitable for a diagnostic laboratory procedure. Toxin-producing strains of E. coli will not produce disease unless they replicate in the small intestine where the mucosa appears to be sensitive to the toxin. 124. 133 They may reside in the colon without producing any clinical disease. The "0" groups which have been associated with enterotoxin production include 0:06, 0:15, 0:25, 0:55, 0:78, 0:111, 0:119, 0:125, 0:128, and 0:148. The last named serotype was only recently assigned a number and was isolated from British soldiers struck with traveler's diarrhea shortly after they arrived in Aden. Evidently, serotypes responsible for newborn nursery epidemics of gastroenteritis are primarily of the toxigenic type. 48 Other E. coli strains capable of producing disease in humans include the serotypes 0:28, 0:112, 0:115, 0:124, 0:136, 0:143, 0:144, 0:147, 0:152. The invasive strains which penetrate epithelial cells are associated with a dysentery-like illness associated with fever and diarrhea with bloody mucous. Consequently, two rather distinct types of disease may be caused by E. coli strains, depending on their pathogenic potential.

Epidemiology Enteropathogenic strains of E. coli principally came to attention because of rather severe outbreaks of gastroenteritis in newborn nurseries. 73 Indeed, it was thought that most E. coli gastroenteritis occurred in children under 2 years of age. Spread in these epidemics was quite rapid and the mode of nursery transmission was by way of contaminated hands, food or equipment. Airborne spread of E. coli was demon-

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strated by Rogers.1 01 Organisms may be carried by mothers who infect their infants at delivery, and thereby, provide access to other newborn infants.2o In older children and adults direct contact and contamination of food are probable sources of transmission. A recent widespread outbreak of sporadic cases of invasive E. coli, 0:124, was traced to imported cheese, infected with this serotype. 87

Clinical Illness Clinical manifestations of E. coli gastroenteritis depend upon whether or not the disease was caused by invasive or toxigenic organisms. Descriptions of E. coli disease in the group less than 2 years of age suggest the toxigenic type of disease where onset is somewhat gradual with loose mucoid foul-smelling stools with a green color.91 Newborn infants may develop very severe manifestations of the disease. Older individuals are also affected by the toxigenic organisms, but the clinical manifestations vary markedly in degree of severity. Adults are generally only mildly ill, with watery diarrhea, and are afebrile. Human volunteers challenged with E. coli eventually have many of the same serotype in the stool and also show an antibody response. In invasive E. coli gastroenteritis leukocytes may be found in fecal smears since penetrated intestinal mucosal cells necrose and leave ulcerations in the gut wall. Obviously, this picture is comparable to that seen with shigellosis. 28 The diagnosis of E. coli gastroenteritis will remain extremely difficult until diagnostic procedures paralleling results with the ileal loop rabbit model can be developed. However, where large numbers of E. coli are isolated from the stool culture of a child with gastroenteritis they should be serotyped and virulence tests carried further if possible. Some enteropathogenic E. coli may be inhibited on conventional enteric media and other less inhibitory media may be necessary for isolation. In addition to the serotyping of E. coli isolates by agglutination tests, fluorescent antibody techniques are widely used. 14, 18 Fluorescine dye is conjugated with specific antibody and the yellow-green fluorescence of bacteria with similar antigens can be detected in direct smears of positive stool. This technique is quite convenient and has been used for processing large numbers of specimens. However, the reservations which introduced this section must be kept in mind and probably only a portion of potentially virulent E. coli are detected by serotyping.

Treatment As mentioned previously, attention to fluid and electrolyte losses, particularly in the very young infant are essential as a first consideration of therapy. Antibiotics have a useful place in the young child and neonate with E. coli gastroenteritis, particularly where mucosal invasion or sepsis is suspected. Both clinical and bacteriologic signs improved more rapidly with antibiotic therapy. 57 Indeed, the neonate and the premature infant are both at considerable risk from E. coli infections with a significant mortality rate in some outbreaks. 73 Neomycin, orally administered, was long the first drug for treatment of E. coli diarrhea in the neonatal period. However, resistance to this antibiotic among E.

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coli has been increasing in recent years. In the 1960's, kanamycin was more extensively used, but 22 to 30 per cent of E. coli have recently been found to be resistant. 81 Resistance to ampicillin, another agent used for suspected E. coli gastroenteritis, has similarly increased. Streptomycin also has lost its effectiveness. However, colistin resistance remains low and this antibiotic has been used successfully in E. coli gastroenteritis. 89 Gentamicin also retains considerable effectiveness against E. coli, with 90 to 100 per cent of E. coli strains showing susceptibility to gentamicin. Consequently, this probably should be the first drug of choice in treating severely ill neonates before specific sensitivity information is available. Riley used gentamicin orally with excellent results. 99 Parenteral gentamicin may also be advisable in the seriously ill neonate where a question of sepsis exists. Nelson studied the duration of neomycin therapy necessary to adequately control E. coli gastroenteritis. 90 Two groups of infants were treated with neomycin orally, one group for 10 days and the other for 3 days. Bacteriologic relapse without return of symptoms occurred in 14 out of 57 in the long-term group and 7 of 56 babies in the short-term group. Symptoms were found to be prolonged in the 10-day therapy group. This observation is comparable to that regarding therapy with other antibiotics. 17 Current information suggests that neonatal E. coli gastroenteritis should probably be treated orally with one of the antibiotics mentioned ideally based on sensitivity data. Where extension to tissues or sepsis is suspected parenteral antibiotic should be given.

REFERENCES w. D., Holmes, L. F., et aI.: A Salmonella newport outbreak in a premature nursery with a one year follow-up. Effect of ampicillin following bacteriologiC failure of response to kanamycin. Pediatrics, 37 :616, 1966. Adler, J. L., and Zickl, R.: Winter vomiting disease. J. Infect. Dis., 119:668-673, 1969. An Evaluation of the Salmonella Problem. Committee on Salmonella, National Research Council. Washington, D. C., National Academy of Sciences, 1969, pp. 207. Anderson, A. S., Bauer, H., and Nelson, C. B.: Salmonellosis due to Salmonella typhimurium with Easter chicks as likely source. J.A.M.A., 158:1153-1155, 1955. Anderson, E. S., Galbraith, N. S., and Taylor, C. E. C.: An outbreak of human infection due to Salmonella typhimurium, phage type 20A associated with infection in calves. Lancet, 1 :854-8'58, 1961. Aserkoff, B., and Bennett, J. V.: Effect of antibiotic therapy in acute salmonellosis on the fecal excretion of salmonellae, New Eng. J. Med., 281 :636-640, 1969. Bishop, R. F., Davidson, G. P., Holmes, I. H., et al.: Virus particles in epithelial cells of duodenal mucosa from children with acute non bacterial gastroenteritis. Lancet, 2:1281-1283,1973. Black, p, H., Kenny, L. J., and Swartz, M. N.: Salmonellosis-A review of some unusual aspects. New Eng. J. Med., 262 :811-817,864-870, 921-927, 1960. Blacklow, N. R., Dolin, R., Fedoon, D. S., et al.: Acute infectious nonbacterial gastroenteritis: Etiology and Pathogenesis. Ann. Int. Med., 76:993-1008, 1972. Bohnhoff, M., and Miller, C. P.: Enhanced susceptibility to salmonella infection in Streptomycin-treated mice. J. Infect. Dis., 111 :117-127, 1962. Brobst, D" Greenberg, J" and Gezon, H. M.: Salmonellosis in poultry and poultry processing plants in Western Pennsylvania. J.A.V.M.A., 133 :435-437, 1958. Bryan, F. L.: Infections due to miscellaneous microorganisms. In Riemann, H., ed.: Food-Borne Infections and Intoxications. New York, Academic Press, 1969, p. 260. Cases of Shigas' Bacillus Infection, Shigella Surveillance, Center for Disease Control, No. 30, Nov., 1972, pp, 4-8. Cherry, W. B., Thomason, B. M., Pomales-Lebron, A., et al.: Rapid presumptive identi-

1. Abrams, I. F., Cochran,

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