Virology

Rotaviruses are nonenveloped RNA viruses that belong to the family Reoviridae. They are approximately 100 nm in diameter and are composed of three separate shells (capsids) that surround and encase the viral genome consisting of 11 double strands of RNA. This structure gives the virus its characteristic appearance of a wide-rimmed wheel with spokes radiating from the hub, from which its name was derived ( rota is Latin for “wheel”).

Rotaviruses are classified into various serotypes based on the antigenic differences of two outer layer structural proteins coded for by the RNA viral genome, VP4 and VP7. These structural proteins are the target for natural antibodies and have been the targeted approach to vaccine development. VP4 is designated as the P antigenic protein because it is cleaved by the protease trypsin at the intestinal level. VP7 is designated as the G antigenic protein because it is a glycosylated structure. There are at least 42 different G/P strains with different serotype combinations. However, five serotypes—G1P8, G2P4, GP8, G4P8, and G9P8—are the predominant circulation rotavirus G/P serotypes and are responsible for approximately 95% of infections worldwide. Among these serotypes there are substantial geographic differences. For example, in a global study, G1P8 was responsible for more than 70% of infections in North America, Australia, and Europe, but less than 30% in South America, Asia, and Africa.

Epidemiology

Although rotavirus infection occurs in developing countries as well as industrialized countries, the seasonality is varied. In temperate climates, incidence peaks more commonly during the winter months. Tropical climates demonstrate a less specific seasonality. Transmission is primarily from person to person, through contact with feces or contaminated fomites, and is highly contagious, since only very few infectious virions are needed to cause disease in susceptible hosts.

Although the virus may affect all age groups, it most commonly produces disease in children between 6 and 24 months of age. Before vaccination, most children were infected at least once by the age of 3 to 5 years and developed rotavirus antibodies by the age of 2 years, which helps to explain the observed decreased incidence of rotavirus infection later in childhood. Children residing in low income countries contract infection at a younger age (median age 6 to 9 months) compared to those children living in higher income countries (median age 2 to 5 years).

Factors associated with increased risk for hospitalization for rotavirus gastroenteritis among U.S. children include lack of breast-feeding, low birth weight, day-care attendance, the presence of another child younger than 24 months in the household, and having Medicaid or no medical insurance.

Clinical Manifestations

The clinical presentation of rotavirus infections varies widely from mild loose stools to severe diarrhea accompanied by vomiting, which can lead in some instances to dehydration and shock, electrolyte disturbances, and even death. For most infections, an abrupt onset of watery and explosive diarrhea follows an incubation period that ranges from 1 to 3 days. The duration of illness is most commonly 3 to 7 days but can be as long as 2 to 3 weeks. Fever and vomiting usually resolve after 24 to 48 hours. In the neonatal population, abdominal distension with mucoid, bloody stools may occur, even though rotavirus is not though to infect the colon. An association between rotavirus infection and necrotizing enterocolitis has also been observed.

A typical, less severe form of infection with mild diarrhea and lack of systemic symptoms is common among children who are immunocompromised. However, children and adults with congenital immunodeficiency or who have received bone marrow or solid organ transplantation often have a more severe and sometimes fatal rotavirus-induced gastroenteritis. In addition, rotavirus replication can occur in the liver and kidney, at least in immunocompromised hosts. The severity of rotavirus disease among children infected with human immunodeficiency virus (HIV) is thought to be similar to that among children without HIV infection.

Shedding of virus into the intestinal lumen begins about 3 days after infection and may persist for as long as 3 weeks. Prolonged shedding (>3 weeks) is common in immunocompromised individuals. A comparison of the characteristics of rotavirus infections with those of other enteric viruses is presented in Table 38-1 .

Pathophysiology

Rotavirus invades primarily mature villus intestinal epithelial cells of the small intestine. Other portions of the gastrointestinal tract such as the stomach and rectum are likely spared, as studies in patients with known rotavirus disease have yielded normal gastric and rectal biopsies. After invasion and replication, there is epithelial cell death and sloughing. Histologically, this is manifested as blunting of the intestinal villi and crypt hypertrophy in response to the loss of villus cells. The lytic infection of highly differentiated absorptive enterocytes and the sparing of undifferentiated crypt cells results in both a loss of absorptive capacity with “unopposed” crypt cell secretion (causing secretory diarrhea) and loss of brush border hydrolase activity (causing osmotic diarrhea). Hence, the picture of rotavirus infection is that of a mixed secretory/osmotic diarrhea.

Another possible mechanism for rotavirus diarrhea also has been demonstrated. The rotavirus nonstructural glycoprotein NSP4 has been shown to mediate age-dependent intestinal secretion in mice. The relevance of this novel viral enterotoxin to human rotavirus infection is uncertain. Other models, including vasoactive inflammatory agents, have also been proposed.

Diagnosis

Rotavirus was initially linked to acute gastroenteritis through electron-microscopic evidence of viral particles in biopsy specimens of affected patients. The need for specialized personnel and equipment renders this method clinically impractical. Consequently, a variety of immunoassays have been developed for detecting group A rotavirus antigen in stool to include latex agglutination and enzyme immunoassay techniques. Enzyme immunoassay techniques to detect fecal rotavirus antigen are most commonly used with a sensitivity of 95% and a specificity of 99% in some studies. Other diagnostic methods that have been studied have comparable sensitivity and specificity rates such as the immunochromatographic method for rapid detection of rotavirus in stool. Multiplex real time polymerase chain reaction (PCR) panels or Luminex-based assays with universal sample preparation may allow streamlined, rapid diagnosis of rotavirus and other common viral, bacterial, and parasitic gastrointestinal pathogens in the future.

Treatment

Currently, supportive care with oral or intravenous rehydration is the mainstay of therapy. Although novel antisecretory therapies have been reported, no antiviral agents effective against rotavirus have yet been developed. However, probiotic therapy has been shown to be effective in preventing and treating rotavirus infection. Treatment with Lactobacillus rhamnosus GG has been shown to shorten the course of rotavirus diarrhea by at least 1 day. Additional studies have demonstrated that use of probiotics as a single species ( Lactobacillus acidophilus, Saccharomyces boulardii ) or polyspecies ( Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and S. boulardii) decreased duration of diarrhea by 30% as well as duration of fever and vomiting in children ranging in age from 1 to 23 months of age. Antiemetics may reduce the need for intravenous rehydration because of vomiting and the number of hospital admissions. However, the main antiemetic, ondansetron, has a warning for potentially severe side effects, and data on cost effectiveness for the routine use of ondansetron treatment for acute gastroenteritis is lacking.

Prevention

In infants, natural rotavirus infection confers protection against subsequent infection. This protection increases with each new infection and reduces the severity of diarrhea. Immunity can also be conferred following vaccination with oral live vaccines. The first licensed rotavirus vaccine in the United States was the RotaShield Wyeth Lederle Vaccine in 1998. Although vaccine efficacy against severe rotavirus induced gastroenteritis approached 90%, the vaccine was withdrawn from the market secondary to an increased rate of intussusception. Two different rotavirus vaccine products were subsequently developed and are currently licensed and widely used in infants in the United States since 2006.

RotaTeq is an oral, live pentavalent [G1, G2, G3, G4, and P(8)] human-bovine reassortant vaccine. It is administered orally as a three-dose series beginning as early as 6 weeks of age and dosed in 4-week intervals. The maximum age for the last dose in the series is 8 months. In several large randomized placebo controlled trials, RotaTeq was found to be 98% effective against severe rotavirus gastroenteritis during the first full rotavirus season following vaccination. Efficacy against rotavirus in subsequent seasons was 74%.

Rotarix is a live, oral monovalent (G1P1A) vaccine that has been licensed in the United States since 2008. The vaccine is to be administered orally in a two-dose series at ages 2 and 4 months with the same minimum and maximum age ranges and intervals as RotaTeq. Studies have revealed the efficacy to be 84% against severe rotavirus gastroenteritis, with an 85% reduction in hospitalizations.

Early success from the vaccines has been documented. The National Respiratory and Enteric Virus Surveillance System (NREVSS) and the New Vaccine Surveillance Network (NVSN) indicated that the onset and peak of the 2008 rotavirus season were delayed by 15 and 8 weeks, respectively, as compared with the six previous consecutive seasons. Further data indicate that the number of tests positive for rotavirus during the 2008 season decreased by more than two-thirds as compared with the seven preceding rotavirus seasons. The overall impact of these vaccines has been evaluated both in the United States and globally. Visits to the emergency department as well as hospitalizations have been reduced by almost 90% in some studies. Although the protection from rotavirus infection in developing countries is not as good, due to the higher burden of disease, the absolute benefit is higher in these settings.

Caliciviruses

“Winter vomiting disease” was thought to be caused by nonbacterial gastroenteritis for decades before an etiologic agent was identified from an outbreak, in 1968, in Norwalk, Ohio. In this outbreak, only some of the patients had diarrhea. The predominant clinical manifestation was vomiting and nausea. Virus particles were visualized by immunoelectron microscopy on fecal material derived from the Norwalk outbreak. This represented the first definitive association between a specific virus (Norwalk virus) and acute gastroenteritis. Subsequently a number of similar etiologic agents were identified. Before the cloning of the prototype Norwalk virus genome, these viruses, which were a group of morphologically diverse, positive-stranded RNA viruses that caused acute gastro­enteritis, were identified as Norwalk-like agents. These organisms were also named for the communities in which they were first isolated (e.g., Montgomery County, Hawaii, Snow Mountain, Taunton, Otofuke, and Sapporo viruses). Based on reverse transcription–polymerase chain reaction (RT-PCR), the sequence structure of these viruses has enabled their classification as human caliciviruses (HuCV). Human caliciviruses are now recognized as a leading cause of diarrhea worldwide among persons of all ages.

With the use of molecular tools, HuCV have now been preliminarily classified into four genotypes, represented by Norwalk virus, Snow Mountain agent, Sapporo virus, and hepatitis E virus. The nomenclature of two genotypes has changed, renaming Norwalk virus as norovirus and Sapporo virus as sapovirus. This HuCV classification system may allow the further development of assays based on recombinant HuCV antigens or PCR products rather than the current cumbersome classification schemes that rely on human reagents (convalescent outbreak sera) of varying sensitivity and specificity. Molecular tools have already allowed the identification of HuCV as agents of both pediatric and adult viral gastroenteritis in foodborne outbreaks as well as outbreaks in nursing homes, hospitals, and a university setting. Despite the potential for future understanding of the contribution of individual HuCV to outbreaks of nonbacterial gastroenteritis, Norwalk virus still remains the prototypic agent of HuCV.

Norovirus

Enteric Adenovirus

The enteric adenoviruses are among the more recently recognized viral pathogens that cause acute gastroenteritis. Adenoviruses are a large group of viruses long recognized for their role in the pathogenesis of respiratory infections and keratoconjunctivitis. Most of the 47 serotypes are known to be shed in the feces of infected patients. In patients with predominantly gastrointestinal symptoms, the organisms are detectable by electron microscopy of stool samples; however, they fail to grow in standard tissue culture conditions. Their unique cell culture requirements allow for the differentiation of nonenteric adenoviruses from the enteric serotypes (Ad40 and Ad41), which are recognized to be among the common causes of viral childhood gastroenteritis.

Infection with enteric adenoviruses apparently occurs throughout the year, with only slight seasonal variation. Children are predominantly affected, with most patients being younger than 2 years of age. Enteric adenovirus is spread by the fecal-oral route. Transmission of the disease to family contacts is unusual.

Diarrhea is the most commonly reported symptom of enteric adenoviral infection. In contrast with diarrhea from other viral enteritides, diarrhea from enteric adenovirus typically persists for a prolonged period, sometimes as long as 14 days. Vomiting frequently occurs, but is usually mild and of a much shorter duration than is the diarrhea. Dehydration has been seen in approximately half of affected patients, and hospitalization is sometimes necessary.

The diagnosis of enteric adenovirus was previously made by electron microscopy, immunoelectron microscopy of stool samples, or from intestinal biopsy specimens. Enzyme-linked immunosorbent assay (ELISA) and PCR techniques are now used successfully in the diagnosis of enteric adenovirus, and multiplex PCR-based assays that are in development for a broad range of stool pathogens commonly include adenovirus. Treatment is mainly supportive, and oral rehydration solutions are useful in cases of dehydration.

Astrovirus

Astrovirus, similar to HuCV, is a single-stranded RNA virus grouped with the small, round, structured viruses. However, unique features of the astrovirus RNA genome are sufficient to garner its own family classification, Astroviridae. Astrovirus is worldwide in distribution and tends to infect mainly children in the 1- to 3-year age group. In controlled studies in Thailand, astrovirus infection was the second most common cause of enteritis, after rotavirus infection, in symptomatic children. Astrovirus infection occurred in 9% of children with diarrhea, compared with 2% of controls. In children younger than 5 years of age with acute gastroenteritis, astrovirus was identified in 4.9% of stool specimens compared to 3% of controls. Vomiting, diarrhea, abdominal pain, and fever all are commonly seen with infection, and symptoms typically last from 1 to 4 days. Spread of the virus may occur via the fecal-oral route from person-to-person contact or through contaminated food or water.

Other Viruses

A variety of other viruses are being studied to determine what role, if any, they may play in the pathogenesis of human enteric infections. With the exception of those viruses discussed previously in detail, insufficient data are available to ascertain clinical and epidemiologic differences, if any, among the various small round viruses. In a surveillance study of children with acute gastroenteritis in the United States, sapovirus, parechovirus, bocavirus, and Aichivirus were detected in the stool specimens of 5.4%, 4.8%, 1.4%, and 0.2% of patients and 4.2%, 4.4%, 2.4%, and 0% of healthy controls, respectively.

Pestivirus, a single-stranded RNA virus of the togavirus family, has been found in the feces of 24% of children living on an American Indian reservation who had diarrhea attributable to no other infectious agent. These children experienced only mild diarrhea but had more severe respiratory complaints.

Coronavirus is known to cause an upper respiratory illness in humans and has been shown to cause diarrhea in some animals. The role of this agent in human diarrheal disease is unclear, and at least one study found coronavirus more commonly in children without diarrhea than in those who were ill.

Toroviruses are pleomorphic viruses recognized to cause enteric illness in a variety of animals. Members of this group, originally described in Berne, Switzerland, and Breda, Iowa, and named for those cities, have been seen in the feces of humans with diarrheal disease. Because of the pleomorphic structure of toroviruses, electron microscopy was inadequate to prove an etiopathogenic role of these viruses in diarrheal disease. Additional findings of torovirus-like particles by immunoassay, using validated anti-Breda virus antiserum, lends additional weight to the hypothesis that these are agents of human gastroenteritis. Their causative role in human disease, however, remains unproved. Similarly, picobirnavirus is known to cause disease in animals and has been isolated from stools of humans with diarrheal illness.

Cytomegalovirus has been associated with enteritis and colitis. Except for Ménétrier’s disease, caused by gastric cytomegalovirus infection, enteritis and colitis seem to occur almost exclusively in immunocompromised patients. In this population, cytomegalovirus causes viremia and is carried by the bloodstream to a variety of sites, including organs of the gastrointestinal tract. Diagnosis may be made by virus detection in feces, by demonstration of typical cytomegalic inclusion cells, or by in situ hybridization.

Microbiology

Salmonella is a motile, gram-negative bacillus of the family Enterobacteriaceae. It can be identified on selective media because it does not ferment lactose. Three distinct species of Salmonella are recognized: Salmonella enteritidis, Salmonella choleraesuis, and Salmonella typhi. S. enteritidis is further subdivided into approximately 1700 serotypes. Each serotype is referred to by its genus and serotype names (e.g., Salmonella typhimurium ) rather than the formally correct S. enteritidis , serotype typhimurium. S. choleraesuis and S. typhi are known to have only one serotype each. The most common serotypes in infants are Typhimurium, Newport, Javiana, Enteritidis, and Heidelberg.

Epidemiology

Salmonella is estimated to cause 1 to 2 million gastrointestinal infections each year in the United States. In 2012, the CDC estimated the incidence of foodborne Salmonella infections to be 16 per 100,000 persons in the United States. This overall incident rate was found to be unchanged when compared to previously measured rates from 2006 to 2008. At Cincinnati Children’s Hospital Medical Center, salmonellae are the second most commonly isolated bacterial enteropathogens ( Figure 38-1 ). The highest attack rate for salmonellosis is in infancy, with a lower incidence of symptomatic infection in patients older than 6 years of age. Nontyphoidal Salmonella is usually spread via contaminated water supplies or foods, with meat, fresh produce, fowl, eggs, and raw milk frequently implicated.

Bacterial enteropathogens isolated at Cincinnati Children’s Hospital Medical Center (CCHMC) in the year 2013. A total of 116 specimens (2.9%) were positive of the 3997 stool cultures submitted. In addition to the stool cultures, 451 specimens (14.2%) tested positive for C. difficile by toxin assay of the 3226 tests ordered in 2013.

(Data from Infection Control Office, CCHMC.)

A wide variety of foods have caused outbreaks of salmonella (i.e., one large outbreak involved contaminated alfalfa sprouts shipped worldwide ). Most of the egg-associated outbreaks have involved products such as mayonnaise, ice cream, and cold desserts, in which salmonella can multiply profusely and which are eaten without cooking after the addition of, or contamination by, raw egg. Although “shell” eggs are frequently contaminated, the number of bacteria in infected eggs is often near or below the human infective dose. In contrast, with a generation time of 80 minutes at 20°C, one bacterium can become a billion in 40 hours, and with a generation time of 40 minutes at 25°C, it can do so in 20 hours.

Although any of these food sources may become contaminated through contact with an infected food handler, the farm animals themselves are often infected. Pets, notably cats, turtles, lizards, snakes, and chicks, may also harbor Salmonella . Turtles in particular continue to be a source of salmonella infections in the United States despite a ban on the sale of turtles with shell lengths <4 inches long issued by the U.S. Food and Drug Administration (FDA) in 1975. Person-to-person spread of infection also occurs and is especially common in cases involving infants. A population-based case-control study was done in infants younger than 1 year of age and identified the following risk factors: (1) travel outside the United States, (2) attending day care with a child with diarrhea, (3) riding in a shopping cart next to meat or poultry; and (4) exposure to reptiles. Breast-feeding was found to be protective.

Pathogenesis

Patients in whom host defenses are diminished are more likely to develop clinical manifestations of the disease. This has been demonstrated in patients who have reduced levels of gastric acid. Patients with lymphoproliferative diseases and hemolytic diseases, especially sickle cell anemia, are more likely to experience severe disease and develop complications from Salmonella infection. The mechanisms for this increased susceptibility may involve altered macrophage function, defective complement activation, or damage to the bones from thromboses.

Having overcome host defenses, Salmonella produces disease through a process that begins with colonization of the ileum and the colon. The organisms next invade enterocytes and colonocytes and proliferate within epithelial cells and in the lamina propria ( Figure 38-2 ). From the lamina propria, Salmonella may then move to the mesenteric lymph nodes and eventually to the systemic circulation, causing bacteremia. Because these organisms invade enterocytes and colonocytes, both enteritis, with watery diarrhea, and colitis, with bloody diarrhea, may result. This multistage infection of the host is directed by Salmonella -mediated delivery of an array of specialized effector proteins into the eukaryotic host cells via two distinct secretion systems. Additional secretion systems appear to be functional and contribute to virulence but are not currently well characterized.

Interaction of Salmonella with the intestinal epithelium enterocyte and M cell. Following this interaction, Salmonella organisms traverse the epithelium and invade the host, activating a number of processes including macrophage apoptosis and inflammatory cascades.

Clinical Manifestations

After an incubation period of 12 to 72 hours, Salmonella usually produces a mild, self-limited illness characterized by fever and watery diarrhea. Blood, mucus, or both, are commonly present in the stool. Bacteremia occurs in approximately 6% of Salmonella infections in children but much less frequently in adults. Patients may develop nonintestinal sequelae after Salmonella infection, including pneumonia, meningitis, and osteomyelitis. Fecal excretion of Salmonella organisms can occur for approximately 3 to 7 weeks following resolution of symptoms and is prolonged in children younger than five years of age and in the immunocompromised.

Diagnosis and Treatment

Diagnosis of Salmonella infection can be made through stool or blood culture. Owing to the increased risk of developing the carrier state, antimicrobial treatment of uncomplicated cases of Salmonella gastroenteritis is not recommended. In a Cochrane review, treatment with antibiotics does not shorten duration of illness and can actually prolong fecal excretion of Salmonella organisms. Treatment is recommended in patients at high risk for the development of disseminated disease, including those who are immunocompromised, those with hematologic disease, patients with artificial implants, those with severe colitis, and pregnant women. Treatment is also recommended for patients at any age who appear toxic. There is no evidence of benefit for antibiotics in treating nontyphoid Salmonella diarrhea in otherwise healthy people not at the extremes of age.

Treatment of all children younger than 1 year of age with salmonellosis remains controversial because of the risk of bacteremia and secondary infections. Antimicrobial therapy is recommended for infants with Salmonella bacteremia. Parenteral antibiotics are recommended for any infant (younger than 3 months of age) with a stool culture that is positive for Salmonella.

Most Salmonella are sensitive to a wide variety of antibiotics, including ampicillin (100 mg/kg/day [maximum 1 g] divided every 6 hours, orally or intramuscularly, for 5 days), chloramphenicol (120 mg/kg/day [maximum 1 g], given every 6 hours, orally, for 10 days), trimethoprim-sulfamethoxazole (trimethoprim, 5 mg/kg [maximum 160 mg], plus sulfamethoxazole, 25 mg/kg [maximum 800 mg] per dose, given every 12 hours, orally, for 14 days), and the third-generation cephalosporins. Resistance to ampicillin is increasing. Ceftriaxone, cefotaxime, or a fluoroquinolone (not approved for use in children younger than 18 years of age) are often effective when resistance to other agents is demonstrated.

A follow-up stool culture usually is not warranted unless the patient is employed in the preparation of food. If evidence of a “cure” is necessary, two to three consecutive negative stool cultures, obtained 1 to 3 days apart, are sufficient.

Microbiology

Shigella is a gram-negative, nonmotile, non–lactose-fermenting aerobic bacillus, closely related to members of the genus Escherichia . The organisms are classified into four species or groups each with corresponding serotype(s): Shigella dysenteriae (group A with 15 serotypes), Shigella flexneri (group B with 14 serotypes), Shigella boydii (group C with 20 serotypes) , and Shigella sonnei (group D with only 1 serotype). S. sonnei continues to be the most commonly recovered Shigella species in developed countries, accounting for 77% of all infections. In developing countries S. flexneri (60%) and S. sonnei (15%) account for the majority of infections.

Epidemiology

Shigella is worldwide in its distribution, and the incidence and severity of shigellosis span an equally broad range. Globally, Shigella is thought to be responsible for up to 15% of all reported cases of diarrhea, and is the most common organism found at Cincinnati Children’s Hospital Medical Center on routine stool cultures (see Figure 38-1 ). In 2008, FoodNet calculated the incidence of Shigella infection in the United States to be 6.59 per 100,000. Although Shigella occurs much less frequently in the developed world, in some studies it is the second most common pathogen identified in cases of bacterial diarrhea in children aged 6 months to 10 years. It may also be the most common bacterial cause of outbreaks of diarrhea in day-care settings. Outbreaks of shigellosis have also been described in residential institutions and on cruise ships. This disease is endemic on American Indian reservations in the Southwest.

Shigella is predominantly spread via the fecal-oral route, with person-to-person contact the most likely method. Secondary spread to household contacts may occur. The infection may be spread through contamination of food and water, as often occurs in areas of poor sanitation and inadequate personal hygiene.

Risk exposures for cases include international travel in the week before symptom onset, attending or working in day care, contact with a child or household member with diarrheal illness, using untreated drinking water or recreational water, and sexual contact with someone with diarrhea. Shigellosis should still be considered in patients with watery diarrhea progressing to mucopus even without a contact history.

Clinical Manifestations

Patients infected with Shigella can experience a variety of symptoms that differ according to age of the patient and serogroup of the organism. For example, S. sonnei typically causes mild disease with watery diarrhea of several days’ duration, whereas other serogroups including S. dysenteriae and S. flexneri produce bloody diarrhea associated with high fevers. This pattern of bloody, mucus-containing stools is referred to as dysentery . One review of more than 300 children demonstrates the differences observed between infants and children in the various clinical manifestations. In this review, infants were more commonly found to have watery diarrhea and a shorter duration of illness compared to children. However, infants were also more likely to have hyponatremia, acidosis, and a higher mortality rate. Bacteremia is an uncommon feature of this illness, but several other complications have been reported, including seizures (in children), arthritis, purulent keratitis, and hemolytic-uremic syndrome (HUS). Nonsuppurative arthritis and seizures are among the most commonly occurring extraintestinal complications of shigellosis. Patients who carry the human leukocyte antigen HLA-B27 are predisposed to the development of this complication as well as to the development of Reiter’s syndrome. The association of seizures with shigellosis was earlier attributed to the neurotoxic effect of the Shigella toxin (Shiga toxin). It now seems likely, however, that the seizures may simply represent a subgroup of common febrile seizures and have no direct relation to the effects of Shiga toxin.

Pathogenesis

The Shigella organism is potent, with an inoculum containing as few as 10 organisms able to cause disease in a healthy adult. Patients infected with Shigella may excrete 10 ⁵ to 10 ⁸ organisms per gram of feces. This high rate of excretion and the relatively low number of organisms required to produce disease make possible the widespread distribution of disease.

For Shigella to exert its pathologic effect on a host, the bacteria must first come into contact with the surface of an intestinal epithelial cell and induce cytoskeletal rearrangements resulting in phagocytosis. Studies with S. flexneri have provided further insight into the pathogenesis of infection. Microfold cells (M cells) are specialized epithelial cells that continuously sample particles from the lumen, delivering them to the underlying mucosal lymphoid tissue. Shigella uses and triggers M cells for uptake and transcytosis across the epithelial layer. The newly arrived bacteria are then engulfed by resident macrophages. The bacteria then secrete enzymes that degrade the phagosome membrane, releasing the bacteria into the host cytoplasm. Intracytoplasmic bacteria move rapidly, in association with a comet tail made up of host-cell actin filaments. When moving bacteria reach the cell margin, they push out long protrusions with the bacteria at the tips that are then taken up by neighboring cells, allowing the infection to spread from cell to cell ( Figure 38-3 ).

Shigella invades and crosses the intestinal epithelium via the M cell where inflammatory pathways are stimulated. Macrophages and neutrophils are intimately involved, leading to an increase in permeability along the basolateral surface of enterocytes, which facilitates cell-to-cell spread of Shigella organisms and continued inflammation.

(From Hodges K, Gill R. Infectious diarrhea: cellular and molecular mechanisms. Gut Microbes 2010; 1 (1):4–21, with permission.)

Among all Shigella strains, three enterotoxins have been identified. All four species produce ShET2, which is plasmid encoded. S. flexneri produces a chromosomally encoded ShET1 enterotoxin, whereas Shiga toxin (Stx) is produced by S. dysenteriae . Structurally, Stx is a hexamer composed of five receptor-binding B subunits that encircle an α-helix of an active A subunit. The B subunits bind to cell-specific receptors and are taken up by endocytosis. Within the cells, the B subunits are cleaved away, and the remaining A subunit is shortened by proteolysis. The A subunit has N-glycosidase activity and is responsible for cell death through inhibition of protein synthesis by acting at the level of the 28S ribosomal RNA. An enterotoxic effect of Shiga toxin in the ileum may account for the watery diarrhea seen early in the course of illness.

Diagnosis and Treatment

In patients with signs and symptoms of colitis, the diagnosis of shigellosis should be considered. Stool culture provides the only definitive means to differentiate this organism from other invasive pathogens. Shigella may be cultured from stool specimens or rectal swabs, especially if mucus is present, but there may be a delay of several days from the onset of symptoms to the recovery of organisms. Sigmoidoscopy or colonoscopy typically reveals a friable mucosa, possibly with discrete ulcers. Rectal biopsy may be useful to differentiate shigellosis from ulcerative colitis.

In addition to rehydration, antimicrobial therapy has been recommended by the World Health Organization (WHO) for the treatment of any diarrhea that is bloody including Shigella. Treatment has been found to (1) shorten the course of the disease, (2) decrease the period of excretion of the organisms, and (3) decrease the secondary attack rate, because humans provide the only reservoir for the organism. However, handwashing, rather than use of antimicrobials, is the most effective method to prevent person-to-person spread. Those clinicians who advise against the routine treatment of shigellosis with antibiotics argue that (1) the disease is most often self-limited and (2) the use of antibiotics may facilitate the development of resistant strains and may increase the likelihood of developing HUS.

A wide range of antibiotics have been used to treat Shigella, necessitated by the development of resistant strains. Resistance to conventional treatment is emerging, and multidrug-resistant strains have been documented in in Latin America, central Africa, and Southeast Asia. Alternative agents for resistance organisms include ceftriaxone and ciprofloxacin according to the Antimicrobial Resistance Monitoring System for Enteric Bacteria from the CDC. Treatment for children can include azithromycin 10 mg/kg/day in a single daily dose for 3 days.

Ciprofloxacin is not approved for treatment of Shigella infection in children, and the American Academy of Pediatrics notes that the systemic use of ciprofloxacin in children should be restricted to infections caused by multidrug-resistant pathogens or when no safe or effective alternatives are available. Children can be treated with 15 mg/kg/dose orally twice daily for 3 days. Adults can be treated with 500 mg twice daily orally for 3 days. Second line agents include pivmecillinam (children, 20 mg/kg/dose orally 4 times daily for 5 days; adults, 100 mg/dose orally 4 times daily for 5 days) and ceftriaxone (children only, 50 to 100 mg/kg once daily intramuscularly for 2 to 5 days). WHO no longer recommends the use of ampicillin or nalidixic acid for the treatment of Shigella infection .

Development of a vaccine for shigellosis continues to be a challenge. These efforts include vaccines using a modified E. coli strain; one using a mutant strain of S. flexneri , which lacks the ability to proliferate intracellularly; and one based on a strain with mutations in its virulence genes. Vaccine development continues to be limited by the lack of a suitable animal model. Identification of the correlates of protection is arguably the most crucial catalyst needed to accelerate the development of effective Shigella vaccines.

Epidemiology

Campylobacter is worldwide in distribution and is responsible for approximately 8.4% of the total diarrhea burden globally each year as documented by WHO. In developing countries, Campylobacter is the most common cause of bacterial gastroenteritis. Infection rates are highest in two different age groups: children younger than 5 years old and young adult males age 20 to 29 years. Campylobacter is the most common cause of bacterial enteric infections in the United States, causing an estimated 2 million infections annually.

Campylobacter may be spread by direct contact or through contaminated sources of food and water. Milk, meat (chicken in particular), and eggs, especially if undercooked, have been implicated in outbreaks. These sources may be contaminated from human fecal shedding, or the organisms may be harbored in the asymptomatic farm animals. Campylobacter is commonly spread among populations of children in day-care centers. A population-based case-control study showed that risk factors for campylobacteriosis were drinking well water, eating fruits and vegetables prepared in the home, having a pet in the home with diarrhea, visiting or living on a farm, riding in a shopping cart next to meat or poultry, and traveling outside of the United States. Infants with campylobacteriosis were less likely to be breast-fed or to be in a household where hamburger was prepared.

Pathogenesis

The mechanisms through which Campylobacter produces disease involve the following events: (1) penetration though the intestinal mucus barrier via flagella-based motility and the corkscrew morphology of the capsule; (2) intestinal epithelial cell invasion and release of cytolethal distending toxin; (3) and interaction with pro-inflammatory interleukin 8 (IL-8) and subsequent activation of inflammatory pathways involving macrophages, dendritic cells, and neutrophils. Diarrhea and other symptoms occur as a result.

Clinical Manifestations

Disease from Campylobacter ranges from mild diarrhea to frank dysentery. Typically, patients experience fever and malaise followed by diarrhea, nausea, and abdominal pain that may mimic appendicitis or inflammatory bowel disease. Symptoms usually last less than one week. Bacteremia may rarely occur, with some species implicated more often than are others. Campylobacter is also known to cause meningitis, abscesses, septic abortions, pancreatitis, and pneumonia. Guillain-Barré syndrome (GBS) and Reiter’s syndrome are documented to occur as sequelae of Campylobacter infection. The majority of cases (up to 70%) of GBS are preceded by infection. Gastrointestinal illnesses comprise 26% of these infections, with C. jejuni being the most commonly implicated agent. Evidence also supports C. jejuni as the most common antecedent of Miller-Fisher syndrome, a neuropathy associated with ataxia, areflexia, and ophthalmoplegia. Although evidence for molecular mimicry is still preliminary, it is likely that peripheral nerves share epitopes with C. jejuni. Therefore, the immune response initially mounted to attack C. jejuni is misdirected to peripheral nerves. Symptoms consistent with GBS usually manifest 10 days to 3 weeks after the onset of diarrhea. After the resolution of symptoms, patients may continue to shed organisms for as long as 7 weeks.

Diagnosis and Treatment

Culture of the organisms, the gold standard for diagnosis, is accomplished in most laboratories if selective media are used and cultures are incubated at 42°C. Because disease caused by Campylobacter is usually mild and self-limited, supportive treatment alone should suffice. The need for antibiotic therapy has been questioned based in part on several studies demonstrating a decrease in the duration of excretion of Campylobacter after antibiotic treatment but no decrease in the duration of symptoms. In cases of severe disease, erythromycin (10 mg/kg [maximum 500 mg] per dose, given every 6 hours for 5 to 7 days) has been traditionally recommended. However, a randomized parallel-group assessor-blind trial in children compared standard erythromycin dosing to single-dose azithromycin regimens (20 mg/kg and 30 mg/kg). Azithro­mycin dosed at 30 mg/kg once orally was superior to a 5-day course of erythromycin in the eradication and clinical improvement with infection. For cases of Campylobacter septicemia, gentamicin (1.5 to 2.5 mg/kg per dose, intramuscularly or intravenously, given every 8 hours) is recommended, with chloramphenicol and erythromycin acceptable as alternatives. Ciprofloxacin is an effective alternative agent but is not approved for use in children younger than 18 years of age, and there has been increasing resistance to fluoroquinolones in developing countries.

Microbiology

The genus Yersinia includes the species Yersinia pestis , which causes plague; Yersinia pseudotuberculosis , known to cause pseudoappendicitis, mesenteric adenitis, and gastroenteritis; and Yersinia enterocolitica , recognized with increasing frequency as a cause of bacterial diarrhea. Yersinia is a non–spore-forming gram-negative rod-shaped or coccoid bacillus that can be grown in both aerobic and anaerobic cultures. The bacteria are flagellated and all are motile with the exception of Y. pestis at 22 to 30°C but nonmotile at 37°C.

Epidemiology

Yersinia was initially thought to occur with greater frequency in countries with cooler climates but is now recognized to be worldwide in distribution. It is estimated that the incidence of Y. enterocolitica in the United States is 100,000 cases per year. It is rarely isolated at Cincinnati Children’s Hospital Medical Center, where it is included among the organisms sought in a routine stool culture (see Figure 38-1 ). The major reservoir for Y. enterocolitica are pigs, as has been implicated in various outbreaks including one in the Fulton County, Georgia, outbreak in 1990, in which chitterlings were found to be the vehicle of infection. Outbreaks due to Yersinia have also been associated with spread through contaminated water and foods, including bean sprouts, tofu, and chocolate milk. Infection rates are higher in the winter months, and infection is transferred by direct contact with infected animals or contaminated food products and/or water. Young black and Asian children (<5 years old) have the highest rates of infection. A case-control study from Sweden reported that risk factors for acquiring Y. enterocolitica in children younger than 6 years of age were foods prepared from unprocessed raw pork products and treated sausages. Other factors were the use of pacifiers and contact with domestic animals.

Pathogenesis

Y. enterocolitica constitutes a heterogeneous group of six biotypes (1A, 1B, 2, 3, 4, and 5) with several serotypes, all with identified virulence factors. Known pathogenic strains include 4/O:3 and 2/O:9A, whereas the most well-characterized nonpathogenic strain is 1A. After ingestion of contaminated food or water, Y. enterocolitica first colonizes the intestinal tract, more commonly the terminal ileum and proximal colon. This is followed by attachment and penetration of the mucosal layer and eventual incorporation into the M cells of Peyer’s patches. The bacteria are then transported across the intestinal epithelial cell layer and expelled from the basolateral side, where they are phagocytized by resident macrophages. Replication then ensues within the macrophages and spreads to lymph nodes, liver, and spleen as macro­phages migrate and enter the bloodstream. Symptoms of abdominal pain and diarrhea soon follow. Virulence of this organism is attributed to the presence of a 70-kb pYV (plasmid for Yersinia virulence) plasmid and some chromosomally encoded virulence factors. The plasmid-encoded factors include Yersinia adhesion A (YadA) and Ysc-Yop type III secretion system (TTSS). The chromosomally borne virulence genes include ail (attachment and invasion locus) and inv (invasion). Y. enterocolitica has also been found to produce a heat-stable enterotoxin known as yst , which is thought to contribute to diarrheal disease. However, not all strains carry the yst gene, leading to controversy as to the degree of the toxin’s direct involvement in diarrheal illness.

Different serotypes exhibit different degrees of virulence. For example, in the United States, serogroup O:8 has been associated with a more aggressive presentation, whereas serogroup O:3 is generally considered more mild.

Clinical Manifestations

The most frequent clinical syndrome caused by Y. enterocolitica is gastroenteritis, which typically affects young children. After an incubation period of 1 to 11 days, patients develop diarrhea, fever, and abdominal pain. A marked increase in the leukocyte count is common. The symptoms usually resolve in 5 to 14 days, but they have been known to persist for several months. Symptoms also vary depending on the biotype of the Y. enterocolitica strain involved. For example, in a report of more than 400 patients (adults and children), those infected with pathogenic strains were more often found to have fever, diarrhea, and abdominal cramping. Vomiting was rare in these individuals. In those infected with nonpathogenic strains, fever was less common whereas vomiting was more prevalent, in addition to diarrhea and cramping. Several complications, including appendicitis, have been documented after Y. enterocolitica infection. However, in older children and young adults, Yersinia is more likely to produce the pseudo-appendicular syndrome in which the signs and symptoms mimic appendicitis. In this same age group, there has also been an association of Y. enterocolitica with nonspecific abdominal pain. Radiographic changes in the terminal ileum more often associated with Crohn’s disease, namely, mucosal thickening and aphthous ulcers, have been seen with yersiniosis in young adults.

Yersinia bacteremia has also been documented, and despite therapy with appropriate antibiotics, has a case-fatality rate of 34% to 50%. The finding of Yersinia in blood from asymptomatic donors, however, makes the possibility of transient bacteremia seem likely to occur as well.

Extraintestinal manifestations of Yersinia infection are rare and include erythema nodosum and reactive arthropathy. These are more commonly seen in adults and tend to involve the weight-bearing joints of the lower extremities and have been noted to occur most often in Yersinia patients who carry the histocompatibility antigen HLA-B27.

Diagnosis and Treatment

Yersinia may be cultured with the use of selective media, preferably with “cold enrichment.” Despite the best of methods, culture of Yersinia may require as long as 4 weeks. In addition to diagnosis by culture, Yersinia may also be detected serologically with the use of agglutinin titers. These measurements appear to be useful only in conjunction with cultures, because agglutinin titers may be affected by a number of factors, including the patient’s age, the underlying disease, and previous use of antibiotics and immunosuppressive agents. These titers may also be more useful in Europe and Japan, where infection is caused by a restricted number of serotypes.

Antibiotics have not proved effective in alleviating symptoms of Yersinia or in shortening the duration of illness in both children and adults. In cases of severe disease and in patients with underlying illness, treatment is recommended. Trimethoprim-sulfamethoxazole, aminoglycosides, chloramphenicol, and third-generation cephalosporins are generally recommended. Tetracycline and quinolones are alternatives for adult patients. Gentamicin or chloramphenicol is recommended for treatment of septicemia. Because septicemia may be associated with an iron-overload state, cessation of iron therapy is also recommended during infection.

Microbiology

Vibrio cholerae is a gram-negative, motile, curved bacillus that is free-living in bodies of salt water. V. cholerae is classified on the basis of lipopolysaccharide antigens. Until recently, all epidemic strains of V. cholerae were of the O1 serotype. Group O1 is further subdivided into two biotypes: classic and El Tor. Other serotypes were thought to cause sporadic cases of diarrhea but not epidemic disease. This dictum was discarded after the development of an ongoing epidemic in Asia and South America caused by a new serotype, O139, synonym Bengal. Although the pathogenesis and clinical features of O139 cholera are identical to those of O1 cholera, persons having immunity to serotype O1 are not immune to the Bengal serotype. This lack of immunity is primarily a result of the unique O139 cell surface antigen.

Epidemiology

V. cholerae is estimated to be responsible for at least 120,000 deaths annually worldwide, with the majority occurring in children 1 to 5 years of age. Cholera has been categorized as one of the “emerging and reemerging infections” owing to the number of recurring outbreaks in developing countries. V. cholera is spread via contamination of food and water supplies. There is no evidence of an animal reservoir, but humans may serve as transient carriers. Owing to the nature of its spread, persons living in areas with adequate sanitation are at minimal, if any, risk for encountering cholera. Cholera does occur in the United States, but usually as a result of imported food brought back by returning international travelers. Travelers from the United States to endemic areas are at low risk (incidence of 1 per 30,000 travelers). Cholera has also been isolated from oysters in the Gulf Coast. However, owing to the frequency of international travel, it is important for the clinician who encounters a patient with severe cholera symptoms (dehydration and rice-water stools) to suspect this infection even in nonendemic areas.

Pathogenesis

V. cholerae enters its potential host through the oral route, usually in contaminated food or water. Volunteer studies have shown that a relatively large number of organisms (approximately 10 ¹¹ ) must be ingested to produce symptoms. Similar to other ingested organisms, V. cholerae must survive the acidic gastric environment. The importance of gastric acidity as a host-protective factor is borne out by the increased occurrence of cholera in patients with absent or reduced gastric acidity.

The organisms travel to the small intestine, where they adhere to the epithelium. This process may be aided by production of mucinase. The intestinal epithelium remains intact with normal morphology. Vibrio species produce a toxin that is composed of a central subunit (A) surrounded by five B subunits; the latter bind to a ganglioside, GM ₁ , which serves as the toxin receptor. This binding facilitates the transfer of the A subunit across the cell membrane, where it is cleaved into two components, denoted A ₁ and A ₂ . The disulfide linkage between A ₁ and A ₂ is reduced to liberate an active A ₁ peptide, which acts as a catalyst to facilitate the transfer of adenosine diphosphate-ribose from nicotinamide adenine dinucleotide to a guanyl nucleotide-binding regulatory protein (G _s ). G _s then stimulates adenylate cyclase, located on the basolateral membrane, thereby increasing cyclic adeno­sine monophosphate. This result in turn leads to chloride secretion and a net flux of fluid into the intestinal lumen (see Figure 38-4 ).

Although this mechanism of toxin action adequately explains the clinical symptoms of cholera, similar symptoms have been noted in research subjects infected with strains that do not produce the classic cholera toxin. This has led to the recognition that V. cholerae harbors additional virulence factors in the bacterial genome that may contribute to diarrheal disease and must be considered in the design of a nonreactigenic vaccine. These include zonula occludens toxin and the accessory cholera toxin.

Clinical Manifestations

After an incubation period, commonly 1 to 3 days, the symptoms of cholera usually begin abruptly with profuse, watery diarrhea and sometimes with vomiting. The stool soon becomes clear, with bits of mucus giving it the so-called rice-water appearance. Patients do not experience tenesmus but rather a sense of relief with defecation. Typically, there is no fever. The rate of fluid loss with cholera can be remarkable in severe disease, with purging rates in excess of 1 L per hour reported in adult patients. Despite the dramatic presentation and health risk of “ cholera gravis ,” most patients with cholera infection are asymptomatic or experience mild symptoms. In addition to people with reduced gastric acidity, people with blood group O are at increased risk for more severe disease. Other host factors that predispose to increased purging are less clear, but there is great variability in clinical symptoms after infection.

Diagnosis and Treatment

V. cholerae is identified by colonial morphology and pigmentation on selective agar (e.g., thiosulfate citrate bile salt-sucrose agar). Further identification depends on biochemical markers (e.g., positive oxidase reaction) and motility of the organism. Specific serotyping is used to confirm the identification.

The mainstay of cholera treatment is rehydration. In cases in which the disease is less severe and is recognized early, oral rehydration solutions are appropriate and effective. When purging is excessive (more than 10 mL/kg per hour), intravenous rehydration is required.

Antibiotics have been shown to cause a decrease in duration of the diarrhea, total amount of fluid lost, and length of time organisms are excreted. Single-dose ciprofloxacin has also been shown to be effective in the treatment of V. cholerae O1 or O139, although this drug is not approved for use in children. A single 1 g dose of azithromycin treatment is effective in adults, and a randomized controlled trial showed that a single dose of azithromycin 20 mg/kg was superior to ciprofloxacin for treating cholera in children. Ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and doxycycline may also be used.

Despite much progress, an ideal cholera vaccine is not yet available. An ideal vaccine would provide a high level of long-term protection, even to those at high risk for severe illness (e.g., people with blood group type O), and this protection would commence shortly after administration of a single oral dose. New oral vaccines have been developed for cholera, including both killed vaccines and live attenuated strains. Center for Vaccine Development (CVD) 103-HgR, a vaccine strain with a 94% deletion of the ctxA , proved efficacious against experimental challenge with V. cholerae El Tor Inaba 3 months after inoculation, suggesting it may be useful for travelers to endemic areas. Unfortunately, CVD 103-HgR was not effective in a field trial. Peru-15, a nonmotile strain that colonizes better than CVD 103-HgR, has been shown to be highly effective in volunteer studies. A reformulated bivalent ( V. cholerae O1 and O139) killed whole-cell oral vaccine was also found to be safe and immunogenic in a cholera-endemic area in India. Other live attenuated O1 oral cholera vaccines are in earlier stages of development, including VA1.3 vaccine from India, IEM 108 from China, and an intranasal vaccine.

Other Vibrios

In addition to V. cholerae , more than 100 Vibrio species are ubiquitous in the environment. Among these, there have been 12 species isolated from humans including Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio mimicus, Vibrio hollisae, Vibrio furnissii, and Vibrio vulnificus. V. parahaemolyticus is found in marine environments and is a common seafood contaminant that can cause gastroenteritis, contaminated wound infections, and even septicemia. V. vulnificus is also found in estuarine and marine environments and is the leading cause of shellfish-associated deaths in the United States. Other manifestations of this species include diarrhea and wound infections, in addition to septicemia.