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Shigellosis
SECTION II: PATHOGENS
PART A: Bacterial and Mycobacterial Infections
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CHAPTER 18 Shigellosis Gerald T. Keusch • Mohammed A. Salam • Dennis J. Kopecko
INTRODUCTION Shigellosis is an infection of the large bowel caused by one of the many serotypes of the genus Shigella. While it is a disease typically associated with poverty, poor hygiene, and crowded living conditions in developing countries, as well as an important contributor to childhood malnutrition where such conditions prevail, it also remains a frequent cause of diarrhea in developed countries.1 It has been difficult to reliably determine the burden of shigellosis because etiological data are infrequently collected from representative populations around the world. In addition, the organism is fragile and may not survive long enough to be identified in clinical samples when they are obtained.2 Studies in rural Peru have demonstrated that active surveillance increases yield by 20-fold compared to passive methods.3 Previous retrospective analysis of published data estimated the annual global burden at >200 million infections and 1.1 million deaths, 99% in the developing world.4 In such settings, Shigella species cause as many as 5–15% of all diarrhea cases and most episodes of bloody diarrhea or dysentery in infants and children seeking care in clinics or hospitals. In Bangladesh, the isolation rate in rural Matlab is approximately twice that of urban Dhaka. While these mortality figures are likely an over estimate, Shigella are responsible for a significant portion of the residual 1–1.5 million non-rotavirus diarrhea deaths annually, even in settings where dehydration is effectively treated early with oral rehydration therapy (ORT). In contrast, in developed countries shigellosis typically presents as mild to moderate watery diarrhea, indistinguishable from other etiologies. These patients are not investigated microbiologically, and thus go unreported. The number of laboratory confirmed Shigella isolates reported to the US Centers for Disease Control and Prevention (CDC), around 10 000–15 000, fluctuates year by year. The isolation rate appears to be slowly trending downward from an average of 6.4/100 000 per year between 1968 and 1988 to 5.6/100 000 per year between 1989 and 2002.5 It reached an all-time low of 3.5/100 000 in 2006, the latest year reported;6 this is several hundred-fold lower than the incidence in developing countries. Seventy-five to 80% of infections in infants and young children are due primarily to S. sonnei, and are commonly acquired at day-care centers. The true incidence of shigellosis in the United States is estimated by CDC as around 444 000 cases/year.7 For reasons not well understood, there is a recent trend to less severe disease in some developing regions of the world.8 This may be related to reduced transmission of and mortality due to the most virulent serotype, S. dysenteriae type 1, which occurs almost exclusively in developing countries. Systematic surveillance in both rural and urban Bangladesh documents its virtual disappearance since outbreaks in 1984 and 1994. Previous large-scale epidemics due to S. dysenteriae type 1, especially during humanitarian emergencies, have been associated with high attack rates (6–39%) and case-fatality rates (1.5–9%).9 Whether the current trends will be sustained is not yet clear. Improved hygiene, sanitation, nutrition, and access to health services in many developing countries are
encouraging developments; however, Shigella epidemiology has always been unpredictable and full of surprises.
THE AGENT Shigella was identified by Kiyoshi Shiga just over a century ago during a severe outbreak of bloody diarrhea in Japan,10 and ultimately named in his honor. They are Gram-negative highly host-adapted bacilli within the family Enterobacteriaceae, infecting only humans and some nonhuman primates.11 Shigella cannot be differentiated from Escherichia coli by DNA relatedness, and were they discovered today would be classified within the genus Escherichia.12 It remains a separate genus because of the distinctive clinical illnesses Shigella cause. Unlike most E. coli and Salmonella, Shigella do not possess flagella and are nonmotile. There are biochemical differences between Shigella and other Enterobacteriaceae, but aside from the inability to ferment lactose, these are of little importance for diagnosis or, apparently, for virulence. Shiga’s original isolate is now known as S. dysenteriae type 1. At least 14 additional S. dysenteriae serotypes have been described, some associated with clinical disease and outbreaks.13,14 There are three additional species, flexneri (14 serotypes/subtypes), boydii (20 serotypes), and sonnei (1 serotype, multiple phage/colicin types).1 Although S. dysenteriae can be distinguished from other Shigella by its inability to ferment mannitol, in practice all species are identified initially by their inability to utilize lactose or produce hydrogen sulfide, and then are distinguished serologically.
EPIDEMIOLOGY Shigella are present wherever humans are. Direct person-to-person spread is the major route of transmission, facilitated by the very low inoculum required for infection. In experimental human studies just 10–100 S. dysenteriae type 1 cause infection and clinical disease in 10–20% of nonimmune subjects.15 This is one reason why Shigella is so readily transmitted within clinical microbiology laboratories.16 Large numbers of organisms are excreted in stool; post-infection carriage in stool is selflimited, although occasional chronic carriers are reported. Further transmission is fecal-oral, typically due to contamination of the hands with infected feces. Failure to wash hands after defecation often leads to transfer of organisms directly or via an intermediate object (fomite) to the mouth of a susceptible individual. Shigella may also contaminate food or water directly or indirectly from feces, resulting in common-source outbreaks. Flies attracted to human feces can also transfer Shigella to food or water, and, especially where open defecation is practiced, the use of traps to reduce the fly population can diminish the incidence of shigellosis.17 Shigella can also be sexually transmitted via anal-oral contact. In the United States, men (76%) and S. flexneri (52.6%) predominate, although the proportion of S. sonnei (46.1%) has been increasing.18 Because significant environmental or animal reservoirs do not exist, shigellosis
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Section II
Shigellosis
PART A: Bacterial and Mycobacterial Infections
Susceptibles Children and adults Ingestion of Shigella via water, food, fomites
Bloody diarrhea or bacillary dysentery
Dire
ct pe
Contamination of environment
rson
-to-p
erso
n sp
read Fecal shedding of Shigella
Figure 18.1 Life cycle/pathogenesis of Shigella species in urban and rural populations. Shigella are highly adapted to humans, and do not have a reservoir outside of the human intestine. The cycle is therefore from the stool of an infected individual to the mouth of a susceptible individual, generally referred to as fecal-oral transmission. Because the inoculum necessary to transmit infection is so small, the common pathway is direct person-to-person contact; however, stool can directly contaminate food or water or be transferred by flies, or organisms can be transmitted through anal-oral sex or through inanimate objects handled by individuals whose hands have been in contact with infected material.
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can theoretically be controlled by effective environmental sanitation and personal hygiene. However, chlorinated drinking water and environmental sanitation have not yet eliminated shigellosis from the United States. The life cycle for Shigella infection is illustrated in Figure 18.1. Incidence is highest in children 1–5 years of age, presumably because good personal hygiene is much more difficult to achieve in the young and they have not yet acquired specific immunity. In many disease-endemic countries shigellosis peaks during the hot dry season, when water for handwashing and household hygiene is in short supply. Outbreaks also occur in the rainy season, presumably because feces are washed from the environment into drinking water sources. In the United States, S. sonnei infection peaks in late summer and early fall. Shigellosis is very common in day-care centers, where susceptible infants and toddlers cluster. Poor sanitation on native American reservations has, for many years, resulted in especially high rates of shigellosis and infant mortality; federal programs to construct sanitary facilities and improve access to clean water have reduced this disparity.5 S. dysenteriae type 1 was the dominant species when originally discovered in 1896, but was replaced by S. flexneri after World War I and virtually disappeared. Following World War II, S. sonnei gradually became the principal isolate in industrialized countries while S. flexneri, particularly serotypes 2a, 2b and 3a, has persisted in developing countries (Fig. 18.2).19 There are a few exceptions, such as Thailand, where S. sonnei has also replaced S. flexneri as the most common isolate.20 It has been
suggested that contamination of water in some developing countries with Pleisiomonas shigelloides, which expresses the same O antigen as S. sonnei, induces a cross-reactive O-antigen-specific immunity to S. sonnei.21 The fourth species, S. boydii, is present primarily in the Indian subcontinent and has always been uncommon elsewhere. Specific Shigella clones can rapidly move around the world, evidenced by food-associated outbreaks of an identical S. sonnei in Denmark and Australia, associated with baby corn imported from Thailand.22 S. dysenteriae type 1, the only species known to cause epidemics, reemerged during 1969–1972 in Mexico and Central America, affecting children and adults alike, with high mortality rates.1 Similar outbreaks in South Asia and Africa followed; in the past decade, however, incidence has sharply diminished. Shigella has evolved relatively recently from ancestral E. coli serotypes,12 and its major virulence properties, encoded on a plasmid, have not yet stabilized genetically by integration into the chromosome. The continuing detection of new Shigella serotypes in South Asia suggests that the genus is still evolving, which may be partly responsible for the changing epidemiology of shigellosis.
PATHOGENESIS AND IMMUNITY Shigella cause a superficial invasive colitis involving the epithelium and lamina propria, in contrast to noninvasive enterotoxigenic pathogens such
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Shigellosis
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Year
Figure 18.2 Isolation of Shigella species in a systematic 2% microbiological sampling of fecal specimens at ICDDR,B Hospital in Dhaka (A) and from all patients presenting to the rural ICDDR,B Matlab Hospital (B). S. flexneri accounts for approximately 75% of all isolates in both settings. In contrast (not shown), the vast majority of isolates in developed countries are S. sonnei. (Source: ICDDR, B.)
as Vibrio cholerae or systemic invaders such as Salmonella enterica serovar Typhi.25 When excreted in stool Shigella are noninvasive but resistant to low pH, due to activation of a glutamate-dependent acid-resistance system transcriptionally upregulated during the stationary growth phase.23 This property allows the organisms to tolerate exposure to a pH below 2.5 for several hours and facilitates their survival through the stomach. Shigella genes for cell invasion are activated in the small bowel, in advance of reaching the preferred target, the terminal ileum and colon. The combination of acid resistance and efficient host cell invasion contributes to the low inoculum needed for infection and disease. Invasion of the intestinal epithelium proceeds through a complex and intricate mechanism, mediated by both microbial and host proteins.24,25 Intimate biochemical “cross-talk” signals between Shigella and various host cell types activate specific bacterial and host genes necessary for disease pathogenesis, particularly a bacterial type 3 secretion system (TTSS) and invasion mediators, which trigger essential host cell responses, cytoskeletal rearrangements, and release of inflammatory cytokines.26,27 Our understanding of microbial invasion and the ensuing hostmediated pathogenesis of disease has undergone dramatic advancement in recent decades. The pathologic consequences of Shigella are now thought to be due largely to a microbially induced exaggerated host inflammatory response resulting in intestinal mucosal damage. The mucus-covered gut epithelium is a major defense barrier against most enteric microbes, and is endowed with intracellular and extracellular receptors to recognize microbial invasion and activate the innate immune system. Indeed, nonmotile Shigella cannot penetrate mucus-covered intestinal cells. Instead, using its unique molecular mechanisms, Shigella initially traverse the mucosa through non-mucin-covered specialized antigen-processing microfold (M) cells, without disrupting them.28 They encounter the underlying lymphoid follicles within the mucosa, are ingested by resident macrophages and lyse the phagosomal vacuole and enter the cytoplasm where they multiply. The consequence is apoptotic death of the infected macrophage29 and release of intracellular stores of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18.25,30 Invasive pathogens like Shigella also bind host Toll-like receptors (TLRs) on the basolateral epithelial cell membrane, upregulating proinflammatory cytokine genes and increasing secretion of IL-8 and tumor necrosis factor-α (TNF-α). Invasion of colonic cells in vitro is associated with upregulation of IL-1, IL-4, IL-6, IL-8, TNF-α, and gamma interferon (IFN-γ ).31 This has been confirmed in vivo using rectal biopsy samples from acutely ill or convalescing shigellosis patients.32 Severe illness is
associated with markedly increased IL-1β, IL-6, TNF-α, and IFN-γ expression. IL-1β and IL-8 both induce migration and activation of polymorphonuclear neutrophils (PMNs), leading to recruitment of PMNs and their transmigration between epithelial cells into the gut lumen.33 Shigella lipopolysaccharide (LPS) is another critical signal for PMN adherence and migration. LPS translocated from the luminal to the basal side of intestinal epithelial cells during Shigella invasion interacts with circulating host LPS-binding protein and is recognized by the PMN ligand, CD14. This leads to upregulation and activation of the PMN adherence ligand CD11b/CD18, required for transepithelial PMN migration.34 Migration of PMNs between intestinal epithelial cells disrupts the integrity of interepithelial cell tight junctions and allows Shigella in the lumen to gain entry to the submucosa and the basolateral epithelial surface where they trigger TLRs and amplify the production of proinflammatory cytokines. Invasion of the colonic mucosa is divisible into three stages: primary translocation of Shigella across M cells and infection of follicular macrophages, secondary release from dead macrophages of bacteria that now invade the basolateral surface of adjacent epithelial cells, and tertiary amplification of epithelial cell invasion following disruption of the tight junction (Fig. 18.3). All three are essential to disease pathogenesis. In a rabbit model, blocking IL-1 by pretreatment with IL-1 receptor antagonist, or inhibiting neutrophil chemotaxis and migration by antibody to PMN CD18, prevents progression of infection and clinical disease.35 Attraction and activation of PMNs triggers degranulation and damage of surrounding tissue,36 even though organisms are efficiently killed by a mechanism that does not require oxygen.37 Shigella pathogenesis is a double-edged sword, engaging innate immune responses that are both defensive (PMN-mediated killing) and offensive (destabilization of epithelial cohesion and mucosal damage) in nature. The imbalance of the response to Shigella, to some degree, mimics the excessive immunologic response of inflammatory bowel diseases. Invasion of intestinal M cells and epithelial cells depends on the TTSS, a microbial needle-like secretion apparatus that injects effector proteins, invasion plasmid antigens (Ipas), through the host cell membrane into the cytoplasm24 (Fig. 18.3) . IpaA modulates the activities of Cdc42, Rac, and Rho, small GTPases that control actin polymerization and cytoskeletal alterations.39 This leads to the internalization of Shigella within a hostmembrane bound endosome (Fig. 18.4). Subsequent lysis of the endosomal membrane is due to IpaB and IpaC, releasing organisms into the cytoplasm. Mutations that inhibit endosomal lysis also block bacterial
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M-cell
Mucin glycoprotein Absorptive epithelial cell
Stage 1: TTSS-mediated entry into M-cell
N
N
N
Lymphocytes
IL-8 (enlists PMNs)
Macrophage apoptosis
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PART A: Bacterial and Mycobacterial Infections 140
= Shigella
Apical/luminal surface Microvilli
Actin tail
Translocated LPS
PATHOGENS
PMN
IL-1β
Cytokine release
N
Colonic epithelium
TLRs
Basolateral surface
Stage 2: TTSS-mediated entry of released Shigella
IL-8
IL-18
Figure 18.3 Stages of Shigella pathogenesis. The colonic mucosal epithelial cells are coated with mucin glycoprotein at the apical surface. Specialized microfold (M) cells, not covered by mucin, overlay lymphoid follicles filled with macrophages, dendritic cells, and lymphocytes, to sample entering antigens/pathogens and present them to the immune system for a response. Shigella entry into the mucosa involves three discrete stages. In stage 1, ingested Shigella utilize their type 3 secretion system (TTSS) needle to trigger invasion across the apical surface of M cells. This entry process involves a reorganization of actin filaments which results in the entry of Shigella into the cytoplasm. The bacteria pass through the M cells where they encounter and are engulfed by macrophages. The consequence is macrophage apoptosis, resulting in the release of IL-1b, IL-18, and viable organisms. In stage 2, the released Shigella interact with Toll-like receptors (TLRs), upregulating the production of IL-8, which is further enhanced by Shigella lipopolysaccharide (LPS) translocated during infection. Shigella, released from dead macrophages, invade the basolateral surface of absorptive epithelial cells utilizing their TTSS, quickly lyse the endosomal vacuole and reach the cytoplasm where they can multiply, triggering more inflammation. Intracellular Shigella also nucleate an actin tail at one pole of the rod-shaped bacterium. This constantly growing tail of contractile proteins serves as a “motor” pushing the bacteria to the plasma membrane and into the adjacent cell, now surrounded by a double membrane. When these bacteria escape into the cytoplasm the process repeats as infection proceeds laterally without exiting the epithelial cells. The third stage of invasion involves IL-8 mediated enlistment of polymorphonuclear neutrophils (PMNs) and their paracellular migration between adjacent epithelial cells, breaching the normally tight intercellular junctions. This allows bacteria in the gut lumen to pass between host cells to the basolateral space where they interact with TLRs and further upregulate inflammation. Shigella are effectively killed by PMNs; however, this causes more tissue damage due to the ensuing degranulation. Disease pathogenesis is due to the death and sloughing of small patches of colonic epithelial cells creating focal microulcerations, with leakage of underlying blood/ lymphatic vessels in the lamina propria and the loss of serum protein. N, nucleus.
Figure 18.4 Invasion of cultured HeLa cell monolayers by Shigella flexneri. The bacterial cell at the top of the figure has initiated the reorganization of epithelial cell cytoskeleton, and the actin-based movement of the host cell membrane to form a phagocytic vacuole is evident. The two pseudopods of host cell origin will fuse at the top, engulfing the bacterium within a membrane-bounded endosome, as shown at the lower portion of the cell (white arrowhead).
multiplication and are avirulent. Multiplying organisms express IcsA, an adenosine triphosphatase, energizing the polymerization of actin filaments at one end of the bacterium, forming an actin tail and propelling the organisms forward.40 IcsA-negative strains cannot generate an actin tail or move intracellularly, and are greatly reduced in virulence.41 The actin polymerization effect is often referred to as the “actin motor.” It can drive Shigella to the plasma membrane, creating membrane-bound protrusions at the host cell intermediate junction (Fig. 18.5), leading to invasion of the adjacent host cells within a double-membrane endosome.42 When this vesicle is lysed, organisms released into the cytoplasm continue the process. Invasion requires host cell E-cadherin, a calcium-dependent cell adhesion molecule localized to intermediate junctions below the tight junction.43 The clinical correlate of cell-to-cell invasion, bacterial multiplication, and necrosis and sloughing of infected epithelial cells, is focal microulcerations, occurring primarily in the terminal ileum and colon, with the greatest intensity in the distal colon and rectum. Shigellosis persists longer in children than in adults, perhaps reflecting immunologic immaturity in young children. Clearance of infection likely involves a combination of normal intestinal cell turnover, microbial death due to innate defenses (e.g., PMNs), down-modulation of inflammation, and the development of specific local immunity. Strong epidemiologic evidence indicates that immunity to Shigella is serotype-specific and based on LPS O antigens, although its precise nature, whether mucosal IgA, serum IgG, or some cellular immune mechanism, is not known. Shiga toxin (Stx)-producing S. dysenteriae type 1 is epidemiologi cally linked to the development of hemolytic-uremic syndrome (HUS). The importance of Stx is supported by the ability of serotypes of E. coli
Chapter 18 Shigellosis
Figure 18.5 Intracellular movement of Shigella flexneri within HeLa cells stained with fluorescein-labeled phalloidin, which specifically binds to polymerized actin. The actin tails, labeled by the dye, appear as bright streaks within the cytoplasm, and as they are created the organism is propelled forward by the force of actin contraction, the “actin motor.” Organisms that reach the plasma membrane protrude beyond the plane of the cell (arrow), a display termed fireworks, and can be seen approaching the plasma membrane of adjacent cells.
Figure 18.7 Bangladeshi child with rectal prolapse, a consequence of intense proctitis due to invading Shigella and the resulting inflammatory response.
Figure 18.6 Typical dysenteric stool is a small-volume mix of blood and pus. Such stools may be passed 30 or more times per day, often with increased pain (tenesmus).
(such as O157:H7) that produce structurally and biochemically similar toxins (Stx1 and Stx2) to cause HUS.44 Shiga toxins are known to bind glycolipid receptors on endothelial cells, essential in the pathogenesis of HUS.45
Figure 18.8 Bangladeshi child with toxic megacolon caused by Shigella dysenteriae type 1 infection. The dilated loops of bowel are clearly discernible beneath the abdominal wall.
THE DISEASE Clinical Manifestations and Complications Shigellosis typically begins with malaise and fever, 24–72 hours after ingestion of the organism. This is followed by diarrhea, usually watery at the outset, but containing many leukocytes when examined microscopically. Diarrhea may become bloody or progress to dysentery, a syndrome characterized by the triad of small volume, grossly bloody, mucopurulent stools (Fig. 18.6), abdominal cramping, and tenesmus, a painful straining with the urge to pass stool. Shigellosis is often referred to as bacillary dysentery to distinguish it from amebic dysentery due to the protozoan Entamoeba histolytica. These manifestations result from inflammation, intense proctocolitis, and ulcerations of the colonic mucosa. Dysentery is not an invariable consequence of Shigella, and is in large part determined by the virulence of the infecting strain, the infectious dose, and the susceptibility of the individual. Most patients with S. sonnei infections never develop grossly bloody diarrhea or dysentery, whereas most patients with S. dysenteriae type 1 infection do. Host factors, some undoubtedly genetic, others related to nutritional status or other co-morbidities such as HIV/AIDS, also determine severity in individuals.46,47 Shigella dysentery is associated with profound anorexia.48 Because the stomach and small bowel are not directly involved in shigellosis, neither
vomiting nor severe dehydration is prominent, although mild-moderate dehydration occurs due to stool water losses, increased insensible water loss from fever, and reduced food and fluid intake. The intensity of the proctitis can be so severe that rectal prolapse occurs, especially in young children with S. dysenteriae type 1 or S. flexneri infection49 (Fig. 18.7). Functional intestinal obstruction and toxic megacolon (Fig. 18.8) may develop with severe inflammation, most commonly with S. dysenteriae type 1 infections.50 Although infection with Shigella usually does not progress beyond the lamina propria, colonic or distal ileal perforation can occur, most typically in neonates or malnourished children.51 Bacteremia due to the infecting Shigella itself or another luminal organism, especially in malnourished or immunocompromised patients, is a complication in 5–10% of patients in Bangladesh.52 Bacteremic infection in the United States is extremely uncommon, and >99% of isolates reported to CDC are from feces.5 Shigellosis is associated with a number of systemic complications. Patients may present with a generalized motor seizure, especially young children with high fever and S. flexneri or S. dysenteriae type 1 infection.53 More than one seizure is rare; however, patients can become obtunded or even comatose, usually in association with metabolic aberrations such as severe hypoglycemia or hyponatremia.54 Hypoglycemia, due to lack of food intake and an inadequate gluconeogenic response, may be profound
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Section II PATHOGENS
PART A: Bacterial and Mycobacterial Infections
(plasma glucose <1 mmol/L). Hyponatremia is associated with dysentery, and is due to sodium loss in the stool plus secretion of antidiuretic hormone in amounts inappropriate for the serum sodium concentration, possibly triggered by hypoalbuminemia and decreased intravascular oncotic pressure in patients with severe dysentery. In Bangladesh serum sodium concentrations less than 125 mmol/L are present in approximately 50% of all patients with severe S. dysenteriae type 1 and 25% of those with S. flexneri infection. The most dramatic systemic complication of shigellosis is HUS, characterized by microangiopathic hemolytic anemia, thrombocytopenia, and oliguric renal failure. HUS occurs exclusively in S. dysenteriae type 1 infection55 and is usually first noted 1–5 days after dysentery develops, often as intestinal manifestations are subsiding. Renal failure and hemolytic anemia can be severe; thrombocytopenia is usually less marked, with platelet counts in the range of 25 000–100 000/mm3 and no bleeding manifestations other than in the gut. HUS may be incomplete, with any manifestation occurring in isolation.56 Shigellosis has a profound effect on nutrition, and wherever shigellosis is hyperendemic it is a major contributor to the high prevalence of malnutrition in children. First, energy requirements are increased due to high fevers.57 Second, inflammatory cytokines released in the host response58 cause muscle protein catabolism, altered priorities for acute phase protein synthesis, and anorexia with decreased food intake. Third, these patients lose considerable amounts of protein due to transudation of serum across the damaged gut mucosa.59 These changes persist long into convalescence. In developing countries, severe Shigella infections due to S. dysenteriae type 1 and S. flexneri almost double the subsequent rate of persistent (greater than 14 days) diarrhea and acute malnutrition, and increase mortality by 10-fold in such patients.60 Shigella (and other invasive enteric bacteria such as nontyphoidal Salmonella, Campylobacter, and Yersinia) can initiate reactive (sterile) arthritis, sometimes accompanied by tendinitis, conjunctivitis, uveitis, urethritis, or erythema nodusum.61 This clinical constellation, previously termed Reiter’s syndrome, is an autoimmune process occurring 2–3 weeks post infection. It is highly associated with the HLA-B27 haplotype, and in these individuals arthritis can be acute or chronic, and can result in severe joint damage.
DIAGNOSIS
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Although all O-antigens of Shigella can now be detected by DNA microarray methods,62 in practice specific diagnosis still depends on stool culture. This is best done directly from fresh stool samples, which increases yield compared to rectal swabs.1 If culture cannot be done rapidly (best at the bedside) stool should be inoculated to transport media such as buffered glycerol-saline or Cary–Blair medium. Use of a moderately selective medium for Gram-negative organisms, such as MacConkey or deoxycholate citrate agar (DCA), and a more highly selective medium such as xylose-lysine-deoxycholate (XLD), Hektoen enteric (HE), or SalmonellaShigella (SS) agar, optimizes yields. Shigella, as well as Salmonella, does not change the color of the pH indicator in these media because they cannot ferment lactose. Lactose-negative colonies can be identified and subcultured to triple sugar iron (TSI) agar or Kligler iron agar (KIA). On these media Shigella produce an alkaline slant, confirming lactose negativity, and an acid butt due to anaerobic fermentation of glucose, are nonmotile and do not produce hydrogen sulfide or gas. Putative isolates fulfilling these criteria can be serologically confirmed, speciated and serotyped with specific antisera.1 It has been suggested that S. dysenteriae type 1 could be rapidly identified by testing for catalase activity, since it is catalase negative, compared to other Shigella and most Enterobacteriaceae which are catalase positive.63 In many areas of the tropics, microbiology is not readily available and diagnosis is based on clinical and simple laboratory features. While some practitioners in endemic areas still believe that dysentery is commonly amebic in etiology, there is ample evidence that patients with dysentery are far more likely to be infected with Shigella than with E. histolytica.64
Figure 18.9 Fecal smear of shigellosis.
Clinical and laboratory features in amebic and bacillary dysentery differ sharply. Shigellosis is suggested by a short pre-hospitalization illness, high fever, and the presence of abundant fecal leukocytes, greater than 50 neutrophils per high-power field (Fig. 18.9).65 Amebiasis is more often chronic, and leukocytes are few or absent in the stool because the parasite lyses PMNs. Mere presence of amebic cysts in stool is not diagnostic, as the vast majority are nonpathogenic strains of E. dispar. Virulent E. histolytica are suggested by identification of motile trophozoites containing ingested erythrocytes in fresh stool preparations.66 More specific diagnosis is possible with ELISA or PCR, and sharply reduces the false positives with microscopy alone. In the absence of confirmed amebiasis, patients with frank dysentery should be presumed to have shigellosis and treated empirically for this infection. In developing countries the diagnosis of clinical dysentery can often be established by community health workers who either observe the characteristic stool or obtain a history of bloody stools from the mother, who are reliable historians in reporting bloody diarrhea.67 Rapid methods have been developed to diagnose shigellosis, including fluorescent antibody staining for S. dysenteriae type 1, which has high sensitivity (92%) and specificity (93%),68 immunomagnetic isolation of bacteria followed by PCR69 or monoclonal antibody70 confirmation, and isotope- or enzyme-labeled DNA probes for virulence markers specific for Shigella, although some of the latter are also present in the rare Shigella-like enteroinvasive E. coli.71,72 A rapid simple dipstick method has been devised for S. flexneri 2a, the most common serotype in developing countries.73 Although these may become commercially available, cost will remain a major deterrent to their use in resource-limited settings. The only reliable rapid commercially available method in routine use is an enzyme immunoassay for Shiga toxin in stool. A positive test is indicative of S. dysenteriae type 1 or an Stx-producing E. coli serotype, such as O157. This test is used primarily in developed countries for rapid diagnosis of Stx-producing E. coli.74,75 Serologic testing for O-antigen antibody is useful for epidemiologic studies of shigellosis but not for diagnosis of acute disease, especially in endemic areas where the majority of the population may be seropositive from prior infections.76
TREATMENT AND PROGNOSIS Except for mild-moderate watery diarrhea due to S. sonnei in wellnourished individuals, for which antibiotics are not warranted, antimicrobial therapy is the cornerstone of treatment for shigellosis. In the absence of effective antimicrobial therapy, mortality from bloody diarrhea or dysentery due to Shigella is appreciable; S. dysenteriae type 1 and S. flexneri in developing countries have been associated with mortality rates in excess of 10%, particularly in the young and the elderly.77 In this setting, even S. sonnei can be lethal, especially among individuals sick enough to seek hospital care. Effective antimicrobial therapy given within 72 hours of symptoms not only brings prompt clinical resolution but also reduces the likelihood of HUS.78 All persons who have dysentery and presumed Shigella infection should therefore be treated with an appropriate anti microbial agent when first seen; the results of culture and susceptibility testing, if available, can then be used subsequently to modify treatment when necessary.
% resistant
70 60
Ampicillin
50
TMP-SMX
40
Nalidixic Acid
30
Mecillinam
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Ciprofloxacin
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90
90 80 70 % resistant
To be useful in resource-poor settings, an antimicrobial agent should be safe for use in children under 5 years, proven clinically effective in controlled trials, administered orally, and affordable.79 Because of the ever rising prevalence of multidrug-resistant strains, selection of an appropriate agent has become increasingly difficult.80 Older oral drugs, such as chloramphenicol and tetracycline, and trimethoprimsulfamethoxazole and ampicillin, until recently the drugs of choice for shigellosis, are no longer first-line choices because of drug resistance (Fig. 18.10).81 Some agents effective in vitro, such as oral extended-spectrum cephalosporins (e.g., cefixime), are ineffective in vivo.82 Older nonabsorbable agents, such as furazolidone, are ineffective, despite advertising claims to the contrary. A newer nonabsorbable agent, rifamixin, has been shown to be effective when given prophylactically to prevent illness in human challenge experiments with S. flexneri. If safe and useful for established infection and substantiated in field trials, rifamixin may become another option for multidrug-resistant infections.83 Unfortunately, current options for treating Shigella infections in developing countries are limited and recommendations are subject to change as drug resistance develops (Table 18.1). Nalidixic acid, pre viously useful against multiresistant strains, is no longer reliable because of increasing resistance.84 Newer fluoroquinolones, such as ciprofloxacin, the macrolide azithromycin, and the β-lactam pivamdinocillin, remain effective,85,86 although emerging resistance to ciprofloxacin and azithromycin is of concern.87,88 Short course (3-day) ciprofloxacin is proven effective, and reduces the cost of treatment.89 The parenteral agent ceftriaxone is also effective, even when given for just 2 days.90 The major constraints currently are cost and the need for parenteral administration, which precludes its use at the community level. It is a good initial option for individuals with severe illness treated in health care facilities wherever multidrug resistance is common. Unfortunately, ceftriaxone resistance is also beginning to emerge.91 Depending on the drug, a course of treatment in Bangladesh can cost the equivalent of 1 day to 3 weeks’ earnings for the poorest, those with an income less than US$1 per day. If empiric antimicrobial treatment for severe shigellosis does not result in clinical improvement within 48 hours, infection with a drug-resistant strain or another organism should be considered, and therapy modified. It is essential to know local drug resistance patterns to devise an empirical strategy for first- and second-line drugs. This necessitates a surveillance system and adequate laboratory capacity, neither of which may be available in developing countries. Because patients with shigellosis are rarely severely dehydrated, intravenous rehydration is not necessary. There are no contraindications to
60
Ampicillin
50
TMP-SMX
40
Nalidixic Acid
30
Mecillinam
20
Ciprofloxacin
10 0
2000 2001 2002 2003 2004 2005 2006 2007 2008
Year
B
Figure 18.10 Antimicrobial sensitivity of strains isolated in Dhaka (urban) and Matlab (rural) Bangladesh. These isolates were obtained through a 2% systematic surveillance of patients seen at the ICDDR,B Hospital in Dhaka, Bangladesh (A) and from the culture of all patients seen at the Matlab Hospital (B). Over the past decade, more than 70% of strains have become resistant to nalidixic acid, more than 60% to trimethoprim-sulfamethoxazole, and 30–60% of strains to ampicillin. Of concern is the recent emergence of resistance to mecillinam and ciprofloxacin. (Source: ICDDR, B.)
Table 18.1 Current Antimicrobial Agents for Treatment of Shigellosis in Tropical Countries Agent
Adult Dose
Pediatric Dosea
Frequency
Duration
Total Treatment Cost in Dhakab (Pediatric/Adult)
Pivamdinocillinc Ciprofloxacin Azithromycin
400 mg 500 mg 500 mg on day 1, 250 mg/day thereafter 2 g 160 mg
20 mg/kg 10 mg/kg 10 mg/kg on day 1, 5 mg/kg thereafter
4 times daily Twice daily Once daily
5 days 3 daysd 5 days
US$3.50/7.00 US$1.30/1.30 US$2.50/1.75e
100 mg/kg 4 mg/kg
Once daily Twice daily
5 days 5 days
800 mg 500 mg
20 mg/kg 25 mg/kg
US$11–14/18–22 Most S. dysenteriae type 1 and S. flexneri strains are resistant. May still be useful for S. sonnei if drug resistance is limited
4 times daily
5 days
Most S. dysenteriae type 1 and S. flexneri strains are resistant. May still be useful for S. sonnei if drug resistance is limited
Ceftriaxone Trimethoprim and Sulfamethoxazole Ampicillin
a
Maximum pediatric dose is the adult dose. Calculated on the basis of packaged drugs in Dhaka, and for a 10 kg child. c Not available in many countries, including the United States. d Prolonged use in children is contraindicated because of risk of arthritis. This is not an issue for treatment of acute shigellosis. e Because of the packaging of pediatric formulation of azithromycin, it costs more to purchase a pediatric course of treatment than a full course for an adult. b
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PART A: Bacterial and Mycobacterial Infections
ORT, and it is useful to manage mild-moderate dehydration that may occur. Severe hyponatremia, a serious complication, can be treated with either intravenous normal saline or a bolus of 3% NaCl; close clinical monitoring is required. Increasing serum sodium concentration by 6–8 mmol/L in 24 hours is considered safe.92 Patients with shigellosis should be fed to the extent they are willing to eat (breast milk or solid foods, depending on age) to prevent hypoglycemia and malnutrition. Anorexia responds to effective antimicrobial therapy.93 Restoration of nutritional status is most rapidly achieved if sufficient calories and highquality protein are provided during convalescence; however, it takes considerably longer to replenish nutrient stores than it does to create the deficit during infection. Parents of a child recovering from shigellosis must be encouraged to feed their child more than normal during convalescence until satisfactory weight gain has occurred. This may be difficult in settings of poverty and food insecurity. Treatment of complications is largely supportive. Patients with seizures do not need anticonvulsant therapy, since more than one seizure is uncommon. HUS may require transfusion and peritoneal dialysis; however, mortality from S. dysenteriae-associated HUS remains higher than observed in HUS due to other etiologies,94,95 perhaps because the disease is more severe, the host is more malnourished, or because facilities for managing this complication are limited where S. dysenteriae type 1 outbreaks occur. Toxic megacolon due to transmural colitis and micro vascular thrombosis is an ominous complication, with mortality rates as high as 50%. Colectomy can be lifesaving but often not feasible in developing countries, because complex postoperative support is unavailable and subsequent long-term care for a child with a colectomy is difficult to ensure. Conservative management with antibiotics, fluids, nasogastric suction, and limited surgical intervention if intestinal perforation occurs – with or without megacolon – may be the better strategy.96,97 Rectal prolapse resolves on its own as the inflammation wanes; in the interim, the prolapsed tissues should be kept moist and protected against injury. Suspected bacteremia should be treated with parenteral, broad-spectrum antibiotics.
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PREVENTION AND CONTROL Because of the low inoculum and person-to-person transmission, the primary method of preventing shigellosis remains good personal hygiene,98,99 although access to potable water and sanitary disposal of feces is often lacking where the disease is most prevalent. Caretakers of children should be encouraged to wash their hands after contact with stool or soiled clothes, and always before preparing food. Handwashing with soap is preferred, but if unavailable then traditional practices using sand or ash, or even with water alone, are better than not washing at all. In addition to resistance to change of established behaviors, the lack of water in or near the household is a major handicap. Where open defecation is practiced, simple fly control can reduce the prevalence of Shigella infection.100 While immunization could have significant impact, it has proved difficult to develop safe and effective Shigella vaccines, in part because the mechanisms of protective immunity remain unclear and because there are many antigenically distinct serotypes involved.101 Pioneering early vaccine studies demonstrated that protective immunity against shigellosis is serotype-specific.102 Experimental human shigellosis models support this concept, as prior infection is highly protective against reinfection with the same species and serotype.103,104 Because traditional parenteral killed vaccines have been ineffective,105 attention has shifted to alternative approaches, including oral immunization with live attenuated Shigella106 or Salmonella Typhi expressing Shigella O-antigens,107 isolated Shigella cell surface components,108 or parenteral O-polysaccharide-protein conjugates.109 A limited multivalent vaccine of the most common serotypes would be very useful. To date, no acceptably safe and sufficiently effective vaccine has been developed; too often the most immunogenic vaccines have been the most reactogenic, while other vaccine approaches with acceptable reactogenicity have not been very immunogenic. The lack of a good animal model of shigellosis has also limited Shigella vaccine development. Finally, despite the lack of success thus far, the search for common protective component(s) for a serotype-independent vaccine should not be abandoned.
PART A: Bacterial and Mycobacterial Infections
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CHAPTER 18 Shigellosis Gerald T. Keusch • Mohammed A. Salam • Dennis J. Kopecko
INTRODUCTION Shigellosis is an infection of the large bowel caused by one of the many serotypes of the genus Shigella. While it is a disease typically associated with poverty, poor hygiene, and crowded living conditions in developing countries, as well as an important contributor to childhood malnutrition where such conditions prevail, it also remains a frequent cause of diarrhea in developed countries.1 It has been difficult to reliably determine the burden of shigellosis because etiological data are infrequently collected from representative populations around the world. In addition, the organism is fragile and may not survive long enough to be identified in clinical samples when they are obtained.2 Studies in rural Peru have demonstrated that active surveillance increases yield by 20-fold compared to passive methods.3 Previous retrospective analysis of published data estimated the annual global burden at >200 million infections and 1.1 million deaths, 99% in the developing world.4 In such settings, Shigella species cause as many as 5–15% of all diarrhea cases and most episodes of bloody diarrhea or dysentery in infants and children seeking care in clinics or hospitals. In Bangladesh, the isolation rate in rural Matlab is approximately twice that of urban Dhaka. While these mortality figures are likely an over estimate, Shigella are responsible for a significant portion of the residual 1–1.5 million non-rotavirus diarrhea deaths annually, even in settings where dehydration is effectively treated early with oral rehydration therapy (ORT). In contrast, in developed countries shigellosis typically presents as mild to moderate watery diarrhea, indistinguishable from other etiologies. These patients are not investigated microbiologically, and thus go unreported. The number of laboratory confirmed Shigella isolates reported to the US Centers for Disease Control and Prevention (CDC), around 10 000–15 000, fluctuates year by year. The isolation rate appears to be slowly trending downward from an average of 6.4/100 000 per year between 1968 and 1988 to 5.6/100 000 per year between 1989 and 2002.5 It reached an all-time low of 3.5/100 000 in 2006, the latest year reported;6 this is several hundred-fold lower than the incidence in developing countries. Seventy-five to 80% of infections in infants and young children are due primarily to S. sonnei, and are commonly acquired at day-care centers. The true incidence of shigellosis in the United States is estimated by CDC as around 444 000 cases/year.7 For reasons not well understood, there is a recent trend to less severe disease in some developing regions of the world.8 This may be related to reduced transmission of and mortality due to the most virulent serotype, S. dysenteriae type 1, which occurs almost exclusively in developing countries. Systematic surveillance in both rural and urban Bangladesh documents its virtual disappearance since outbreaks in 1984 and 1994. Previous large-scale epidemics due to S. dysenteriae type 1, especially during humanitarian emergencies, have been associated with high attack rates (6–39%) and case-fatality rates (1.5–9%).9 Whether the current trends will be sustained is not yet clear. Improved hygiene, sanitation, nutrition, and access to health services in many developing countries are
encouraging developments; however, Shigella epidemiology has always been unpredictable and full of surprises.
THE AGENT Shigella was identified by Kiyoshi Shiga just over a century ago during a severe outbreak of bloody diarrhea in Japan,10 and ultimately named in his honor. They are Gram-negative highly host-adapted bacilli within the family Enterobacteriaceae, infecting only humans and some nonhuman primates.11 Shigella cannot be differentiated from Escherichia coli by DNA relatedness, and were they discovered today would be classified within the genus Escherichia.12 It remains a separate genus because of the distinctive clinical illnesses Shigella cause. Unlike most E. coli and Salmonella, Shigella do not possess flagella and are nonmotile. There are biochemical differences between Shigella and other Enterobacteriaceae, but aside from the inability to ferment lactose, these are of little importance for diagnosis or, apparently, for virulence. Shiga’s original isolate is now known as S. dysenteriae type 1. At least 14 additional S. dysenteriae serotypes have been described, some associated with clinical disease and outbreaks.13,14 There are three additional species, flexneri (14 serotypes/subtypes), boydii (20 serotypes), and sonnei (1 serotype, multiple phage/colicin types).1 Although S. dysenteriae can be distinguished from other Shigella by its inability to ferment mannitol, in practice all species are identified initially by their inability to utilize lactose or produce hydrogen sulfide, and then are distinguished serologically.
EPIDEMIOLOGY Shigella are present wherever humans are. Direct person-to-person spread is the major route of transmission, facilitated by the very low inoculum required for infection. In experimental human studies just 10–100 S. dysenteriae type 1 cause infection and clinical disease in 10–20% of nonimmune subjects.15 This is one reason why Shigella is so readily transmitted within clinical microbiology laboratories.16 Large numbers of organisms are excreted in stool; post-infection carriage in stool is selflimited, although occasional chronic carriers are reported. Further transmission is fecal-oral, typically due to contamination of the hands with infected feces. Failure to wash hands after defecation often leads to transfer of organisms directly or via an intermediate object (fomite) to the mouth of a susceptible individual. Shigella may also contaminate food or water directly or indirectly from feces, resulting in common-source outbreaks. Flies attracted to human feces can also transfer Shigella to food or water, and, especially where open defecation is practiced, the use of traps to reduce the fly population can diminish the incidence of shigellosis.17 Shigella can also be sexually transmitted via anal-oral contact. In the United States, men (76%) and S. flexneri (52.6%) predominate, although the proportion of S. sonnei (46.1%) has been increasing.18 Because significant environmental or animal reservoirs do not exist, shigellosis
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PATHOGENS
Section II
Shigellosis
PART A: Bacterial and Mycobacterial Infections
Susceptibles Children and adults Ingestion of Shigella via water, food, fomites
Bloody diarrhea or bacillary dysentery
Dire
ct pe
Contamination of environment
rson
-to-p
erso
n sp
read Fecal shedding of Shigella
Figure 18.1 Life cycle/pathogenesis of Shigella species in urban and rural populations. Shigella are highly adapted to humans, and do not have a reservoir outside of the human intestine. The cycle is therefore from the stool of an infected individual to the mouth of a susceptible individual, generally referred to as fecal-oral transmission. Because the inoculum necessary to transmit infection is so small, the common pathway is direct person-to-person contact; however, stool can directly contaminate food or water or be transferred by flies, or organisms can be transmitted through anal-oral sex or through inanimate objects handled by individuals whose hands have been in contact with infected material.
138
can theoretically be controlled by effective environmental sanitation and personal hygiene. However, chlorinated drinking water and environmental sanitation have not yet eliminated shigellosis from the United States. The life cycle for Shigella infection is illustrated in Figure 18.1. Incidence is highest in children 1–5 years of age, presumably because good personal hygiene is much more difficult to achieve in the young and they have not yet acquired specific immunity. In many disease-endemic countries shigellosis peaks during the hot dry season, when water for handwashing and household hygiene is in short supply. Outbreaks also occur in the rainy season, presumably because feces are washed from the environment into drinking water sources. In the United States, S. sonnei infection peaks in late summer and early fall. Shigellosis is very common in day-care centers, where susceptible infants and toddlers cluster. Poor sanitation on native American reservations has, for many years, resulted in especially high rates of shigellosis and infant mortality; federal programs to construct sanitary facilities and improve access to clean water have reduced this disparity.5 S. dysenteriae type 1 was the dominant species when originally discovered in 1896, but was replaced by S. flexneri after World War I and virtually disappeared. Following World War II, S. sonnei gradually became the principal isolate in industrialized countries while S. flexneri, particularly serotypes 2a, 2b and 3a, has persisted in developing countries (Fig. 18.2).19 There are a few exceptions, such as Thailand, where S. sonnei has also replaced S. flexneri as the most common isolate.20 It has been
suggested that contamination of water in some developing countries with Pleisiomonas shigelloides, which expresses the same O antigen as S. sonnei, induces a cross-reactive O-antigen-specific immunity to S. sonnei.21 The fourth species, S. boydii, is present primarily in the Indian subcontinent and has always been uncommon elsewhere. Specific Shigella clones can rapidly move around the world, evidenced by food-associated outbreaks of an identical S. sonnei in Denmark and Australia, associated with baby corn imported from Thailand.22 S. dysenteriae type 1, the only species known to cause epidemics, reemerged during 1969–1972 in Mexico and Central America, affecting children and adults alike, with high mortality rates.1 Similar outbreaks in South Asia and Africa followed; in the past decade, however, incidence has sharply diminished. Shigella has evolved relatively recently from ancestral E. coli serotypes,12 and its major virulence properties, encoded on a plasmid, have not yet stabilized genetically by integration into the chromosome. The continuing detection of new Shigella serotypes in South Asia suggests that the genus is still evolving, which may be partly responsible for the changing epidemiology of shigellosis.
PATHOGENESIS AND IMMUNITY Shigella cause a superficial invasive colitis involving the epithelium and lamina propria, in contrast to noninvasive enterotoxigenic pathogens such
150
S. dysenteriae 2–12 S. flexneri
100
S. sonnei
50 0
S. boydii 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
All Shigella
Year
A 250
S. dysenteriae 1
No. Isolates
200
S. dysenteriae 2–12
150
S. flexneri S. sonnei
100
S. boydii
50 0 B
Chapter 18
S. dysenteriae 1
Shigellosis
No. Isolates
200
All Shigella 2000 2001 2002 2003 2004 2005 2006 2007 2008
Year
Figure 18.2 Isolation of Shigella species in a systematic 2% microbiological sampling of fecal specimens at ICDDR,B Hospital in Dhaka (A) and from all patients presenting to the rural ICDDR,B Matlab Hospital (B). S. flexneri accounts for approximately 75% of all isolates in both settings. In contrast (not shown), the vast majority of isolates in developed countries are S. sonnei. (Source: ICDDR, B.)
as Vibrio cholerae or systemic invaders such as Salmonella enterica serovar Typhi.25 When excreted in stool Shigella are noninvasive but resistant to low pH, due to activation of a glutamate-dependent acid-resistance system transcriptionally upregulated during the stationary growth phase.23 This property allows the organisms to tolerate exposure to a pH below 2.5 for several hours and facilitates their survival through the stomach. Shigella genes for cell invasion are activated in the small bowel, in advance of reaching the preferred target, the terminal ileum and colon. The combination of acid resistance and efficient host cell invasion contributes to the low inoculum needed for infection and disease. Invasion of the intestinal epithelium proceeds through a complex and intricate mechanism, mediated by both microbial and host proteins.24,25 Intimate biochemical “cross-talk” signals between Shigella and various host cell types activate specific bacterial and host genes necessary for disease pathogenesis, particularly a bacterial type 3 secretion system (TTSS) and invasion mediators, which trigger essential host cell responses, cytoskeletal rearrangements, and release of inflammatory cytokines.26,27 Our understanding of microbial invasion and the ensuing hostmediated pathogenesis of disease has undergone dramatic advancement in recent decades. The pathologic consequences of Shigella are now thought to be due largely to a microbially induced exaggerated host inflammatory response resulting in intestinal mucosal damage. The mucus-covered gut epithelium is a major defense barrier against most enteric microbes, and is endowed with intracellular and extracellular receptors to recognize microbial invasion and activate the innate immune system. Indeed, nonmotile Shigella cannot penetrate mucus-covered intestinal cells. Instead, using its unique molecular mechanisms, Shigella initially traverse the mucosa through non-mucin-covered specialized antigen-processing microfold (M) cells, without disrupting them.28 They encounter the underlying lymphoid follicles within the mucosa, are ingested by resident macrophages and lyse the phagosomal vacuole and enter the cytoplasm where they multiply. The consequence is apoptotic death of the infected macrophage29 and release of intracellular stores of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18.25,30 Invasive pathogens like Shigella also bind host Toll-like receptors (TLRs) on the basolateral epithelial cell membrane, upregulating proinflammatory cytokine genes and increasing secretion of IL-8 and tumor necrosis factor-α (TNF-α). Invasion of colonic cells in vitro is associated with upregulation of IL-1, IL-4, IL-6, IL-8, TNF-α, and gamma interferon (IFN-γ ).31 This has been confirmed in vivo using rectal biopsy samples from acutely ill or convalescing shigellosis patients.32 Severe illness is
associated with markedly increased IL-1β, IL-6, TNF-α, and IFN-γ expression. IL-1β and IL-8 both induce migration and activation of polymorphonuclear neutrophils (PMNs), leading to recruitment of PMNs and their transmigration between epithelial cells into the gut lumen.33 Shigella lipopolysaccharide (LPS) is another critical signal for PMN adherence and migration. LPS translocated from the luminal to the basal side of intestinal epithelial cells during Shigella invasion interacts with circulating host LPS-binding protein and is recognized by the PMN ligand, CD14. This leads to upregulation and activation of the PMN adherence ligand CD11b/CD18, required for transepithelial PMN migration.34 Migration of PMNs between intestinal epithelial cells disrupts the integrity of interepithelial cell tight junctions and allows Shigella in the lumen to gain entry to the submucosa and the basolateral epithelial surface where they trigger TLRs and amplify the production of proinflammatory cytokines. Invasion of the colonic mucosa is divisible into three stages: primary translocation of Shigella across M cells and infection of follicular macrophages, secondary release from dead macrophages of bacteria that now invade the basolateral surface of adjacent epithelial cells, and tertiary amplification of epithelial cell invasion following disruption of the tight junction (Fig. 18.3). All three are essential to disease pathogenesis. In a rabbit model, blocking IL-1 by pretreatment with IL-1 receptor antagonist, or inhibiting neutrophil chemotaxis and migration by antibody to PMN CD18, prevents progression of infection and clinical disease.35 Attraction and activation of PMNs triggers degranulation and damage of surrounding tissue,36 even though organisms are efficiently killed by a mechanism that does not require oxygen.37 Shigella pathogenesis is a double-edged sword, engaging innate immune responses that are both defensive (PMN-mediated killing) and offensive (destabilization of epithelial cohesion and mucosal damage) in nature. The imbalance of the response to Shigella, to some degree, mimics the excessive immunologic response of inflammatory bowel diseases. Invasion of intestinal M cells and epithelial cells depends on the TTSS, a microbial needle-like secretion apparatus that injects effector proteins, invasion plasmid antigens (Ipas), through the host cell membrane into the cytoplasm24 (Fig. 18.3) . IpaA modulates the activities of Cdc42, Rac, and Rho, small GTPases that control actin polymerization and cytoskeletal alterations.39 This leads to the internalization of Shigella within a hostmembrane bound endosome (Fig. 18.4). Subsequent lysis of the endosomal membrane is due to IpaB and IpaC, releasing organisms into the cytoplasm. Mutations that inhibit endosomal lysis also block bacterial
139
Section II
M-cell
Mucin glycoprotein Absorptive epithelial cell
Stage 1: TTSS-mediated entry into M-cell
N
N
N
Lymphocytes
IL-8 (enlists PMNs)
Macrophage apoptosis
Stage 3: (PMN transmigration)
PART A: Bacterial and Mycobacterial Infections 140
= Shigella
Apical/luminal surface Microvilli
Actin tail
Translocated LPS
PATHOGENS
PMN
IL-1β
Cytokine release
N
Colonic epithelium
TLRs
Basolateral surface
Stage 2: TTSS-mediated entry of released Shigella
IL-8
IL-18
Figure 18.3 Stages of Shigella pathogenesis. The colonic mucosal epithelial cells are coated with mucin glycoprotein at the apical surface. Specialized microfold (M) cells, not covered by mucin, overlay lymphoid follicles filled with macrophages, dendritic cells, and lymphocytes, to sample entering antigens/pathogens and present them to the immune system for a response. Shigella entry into the mucosa involves three discrete stages. In stage 1, ingested Shigella utilize their type 3 secretion system (TTSS) needle to trigger invasion across the apical surface of M cells. This entry process involves a reorganization of actin filaments which results in the entry of Shigella into the cytoplasm. The bacteria pass through the M cells where they encounter and are engulfed by macrophages. The consequence is macrophage apoptosis, resulting in the release of IL-1b, IL-18, and viable organisms. In stage 2, the released Shigella interact with Toll-like receptors (TLRs), upregulating the production of IL-8, which is further enhanced by Shigella lipopolysaccharide (LPS) translocated during infection. Shigella, released from dead macrophages, invade the basolateral surface of absorptive epithelial cells utilizing their TTSS, quickly lyse the endosomal vacuole and reach the cytoplasm where they can multiply, triggering more inflammation. Intracellular Shigella also nucleate an actin tail at one pole of the rod-shaped bacterium. This constantly growing tail of contractile proteins serves as a “motor” pushing the bacteria to the plasma membrane and into the adjacent cell, now surrounded by a double membrane. When these bacteria escape into the cytoplasm the process repeats as infection proceeds laterally without exiting the epithelial cells. The third stage of invasion involves IL-8 mediated enlistment of polymorphonuclear neutrophils (PMNs) and their paracellular migration between adjacent epithelial cells, breaching the normally tight intercellular junctions. This allows bacteria in the gut lumen to pass between host cells to the basolateral space where they interact with TLRs and further upregulate inflammation. Shigella are effectively killed by PMNs; however, this causes more tissue damage due to the ensuing degranulation. Disease pathogenesis is due to the death and sloughing of small patches of colonic epithelial cells creating focal microulcerations, with leakage of underlying blood/ lymphatic vessels in the lamina propria and the loss of serum protein. N, nucleus.
Figure 18.4 Invasion of cultured HeLa cell monolayers by Shigella flexneri. The bacterial cell at the top of the figure has initiated the reorganization of epithelial cell cytoskeleton, and the actin-based movement of the host cell membrane to form a phagocytic vacuole is evident. The two pseudopods of host cell origin will fuse at the top, engulfing the bacterium within a membrane-bounded endosome, as shown at the lower portion of the cell (white arrowhead).
multiplication and are avirulent. Multiplying organisms express IcsA, an adenosine triphosphatase, energizing the polymerization of actin filaments at one end of the bacterium, forming an actin tail and propelling the organisms forward.40 IcsA-negative strains cannot generate an actin tail or move intracellularly, and are greatly reduced in virulence.41 The actin polymerization effect is often referred to as the “actin motor.” It can drive Shigella to the plasma membrane, creating membrane-bound protrusions at the host cell intermediate junction (Fig. 18.5), leading to invasion of the adjacent host cells within a double-membrane endosome.42 When this vesicle is lysed, organisms released into the cytoplasm continue the process. Invasion requires host cell E-cadherin, a calcium-dependent cell adhesion molecule localized to intermediate junctions below the tight junction.43 The clinical correlate of cell-to-cell invasion, bacterial multiplication, and necrosis and sloughing of infected epithelial cells, is focal microulcerations, occurring primarily in the terminal ileum and colon, with the greatest intensity in the distal colon and rectum. Shigellosis persists longer in children than in adults, perhaps reflecting immunologic immaturity in young children. Clearance of infection likely involves a combination of normal intestinal cell turnover, microbial death due to innate defenses (e.g., PMNs), down-modulation of inflammation, and the development of specific local immunity. Strong epidemiologic evidence indicates that immunity to Shigella is serotype-specific and based on LPS O antigens, although its precise nature, whether mucosal IgA, serum IgG, or some cellular immune mechanism, is not known. Shiga toxin (Stx)-producing S. dysenteriae type 1 is epidemiologi cally linked to the development of hemolytic-uremic syndrome (HUS). The importance of Stx is supported by the ability of serotypes of E. coli
Chapter 18 Shigellosis
Figure 18.5 Intracellular movement of Shigella flexneri within HeLa cells stained with fluorescein-labeled phalloidin, which specifically binds to polymerized actin. The actin tails, labeled by the dye, appear as bright streaks within the cytoplasm, and as they are created the organism is propelled forward by the force of actin contraction, the “actin motor.” Organisms that reach the plasma membrane protrude beyond the plane of the cell (arrow), a display termed fireworks, and can be seen approaching the plasma membrane of adjacent cells.
Figure 18.7 Bangladeshi child with rectal prolapse, a consequence of intense proctitis due to invading Shigella and the resulting inflammatory response.
Figure 18.6 Typical dysenteric stool is a small-volume mix of blood and pus. Such stools may be passed 30 or more times per day, often with increased pain (tenesmus).
(such as O157:H7) that produce structurally and biochemically similar toxins (Stx1 and Stx2) to cause HUS.44 Shiga toxins are known to bind glycolipid receptors on endothelial cells, essential in the pathogenesis of HUS.45
Figure 18.8 Bangladeshi child with toxic megacolon caused by Shigella dysenteriae type 1 infection. The dilated loops of bowel are clearly discernible beneath the abdominal wall.
THE DISEASE Clinical Manifestations and Complications Shigellosis typically begins with malaise and fever, 24–72 hours after ingestion of the organism. This is followed by diarrhea, usually watery at the outset, but containing many leukocytes when examined microscopically. Diarrhea may become bloody or progress to dysentery, a syndrome characterized by the triad of small volume, grossly bloody, mucopurulent stools (Fig. 18.6), abdominal cramping, and tenesmus, a painful straining with the urge to pass stool. Shigellosis is often referred to as bacillary dysentery to distinguish it from amebic dysentery due to the protozoan Entamoeba histolytica. These manifestations result from inflammation, intense proctocolitis, and ulcerations of the colonic mucosa. Dysentery is not an invariable consequence of Shigella, and is in large part determined by the virulence of the infecting strain, the infectious dose, and the susceptibility of the individual. Most patients with S. sonnei infections never develop grossly bloody diarrhea or dysentery, whereas most patients with S. dysenteriae type 1 infection do. Host factors, some undoubtedly genetic, others related to nutritional status or other co-morbidities such as HIV/AIDS, also determine severity in individuals.46,47 Shigella dysentery is associated with profound anorexia.48 Because the stomach and small bowel are not directly involved in shigellosis, neither
vomiting nor severe dehydration is prominent, although mild-moderate dehydration occurs due to stool water losses, increased insensible water loss from fever, and reduced food and fluid intake. The intensity of the proctitis can be so severe that rectal prolapse occurs, especially in young children with S. dysenteriae type 1 or S. flexneri infection49 (Fig. 18.7). Functional intestinal obstruction and toxic megacolon (Fig. 18.8) may develop with severe inflammation, most commonly with S. dysenteriae type 1 infections.50 Although infection with Shigella usually does not progress beyond the lamina propria, colonic or distal ileal perforation can occur, most typically in neonates or malnourished children.51 Bacteremia due to the infecting Shigella itself or another luminal organism, especially in malnourished or immunocompromised patients, is a complication in 5–10% of patients in Bangladesh.52 Bacteremic infection in the United States is extremely uncommon, and >99% of isolates reported to CDC are from feces.5 Shigellosis is associated with a number of systemic complications. Patients may present with a generalized motor seizure, especially young children with high fever and S. flexneri or S. dysenteriae type 1 infection.53 More than one seizure is rare; however, patients can become obtunded or even comatose, usually in association with metabolic aberrations such as severe hypoglycemia or hyponatremia.54 Hypoglycemia, due to lack of food intake and an inadequate gluconeogenic response, may be profound
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Section II PATHOGENS
PART A: Bacterial and Mycobacterial Infections
(plasma glucose <1 mmol/L). Hyponatremia is associated with dysentery, and is due to sodium loss in the stool plus secretion of antidiuretic hormone in amounts inappropriate for the serum sodium concentration, possibly triggered by hypoalbuminemia and decreased intravascular oncotic pressure in patients with severe dysentery. In Bangladesh serum sodium concentrations less than 125 mmol/L are present in approximately 50% of all patients with severe S. dysenteriae type 1 and 25% of those with S. flexneri infection. The most dramatic systemic complication of shigellosis is HUS, characterized by microangiopathic hemolytic anemia, thrombocytopenia, and oliguric renal failure. HUS occurs exclusively in S. dysenteriae type 1 infection55 and is usually first noted 1–5 days after dysentery develops, often as intestinal manifestations are subsiding. Renal failure and hemolytic anemia can be severe; thrombocytopenia is usually less marked, with platelet counts in the range of 25 000–100 000/mm3 and no bleeding manifestations other than in the gut. HUS may be incomplete, with any manifestation occurring in isolation.56 Shigellosis has a profound effect on nutrition, and wherever shigellosis is hyperendemic it is a major contributor to the high prevalence of malnutrition in children. First, energy requirements are increased due to high fevers.57 Second, inflammatory cytokines released in the host response58 cause muscle protein catabolism, altered priorities for acute phase protein synthesis, and anorexia with decreased food intake. Third, these patients lose considerable amounts of protein due to transudation of serum across the damaged gut mucosa.59 These changes persist long into convalescence. In developing countries, severe Shigella infections due to S. dysenteriae type 1 and S. flexneri almost double the subsequent rate of persistent (greater than 14 days) diarrhea and acute malnutrition, and increase mortality by 10-fold in such patients.60 Shigella (and other invasive enteric bacteria such as nontyphoidal Salmonella, Campylobacter, and Yersinia) can initiate reactive (sterile) arthritis, sometimes accompanied by tendinitis, conjunctivitis, uveitis, urethritis, or erythema nodusum.61 This clinical constellation, previously termed Reiter’s syndrome, is an autoimmune process occurring 2–3 weeks post infection. It is highly associated with the HLA-B27 haplotype, and in these individuals arthritis can be acute or chronic, and can result in severe joint damage.
DIAGNOSIS
142
Although all O-antigens of Shigella can now be detected by DNA microarray methods,62 in practice specific diagnosis still depends on stool culture. This is best done directly from fresh stool samples, which increases yield compared to rectal swabs.1 If culture cannot be done rapidly (best at the bedside) stool should be inoculated to transport media such as buffered glycerol-saline or Cary–Blair medium. Use of a moderately selective medium for Gram-negative organisms, such as MacConkey or deoxycholate citrate agar (DCA), and a more highly selective medium such as xylose-lysine-deoxycholate (XLD), Hektoen enteric (HE), or SalmonellaShigella (SS) agar, optimizes yields. Shigella, as well as Salmonella, does not change the color of the pH indicator in these media because they cannot ferment lactose. Lactose-negative colonies can be identified and subcultured to triple sugar iron (TSI) agar or Kligler iron agar (KIA). On these media Shigella produce an alkaline slant, confirming lactose negativity, and an acid butt due to anaerobic fermentation of glucose, are nonmotile and do not produce hydrogen sulfide or gas. Putative isolates fulfilling these criteria can be serologically confirmed, speciated and serotyped with specific antisera.1 It has been suggested that S. dysenteriae type 1 could be rapidly identified by testing for catalase activity, since it is catalase negative, compared to other Shigella and most Enterobacteriaceae which are catalase positive.63 In many areas of the tropics, microbiology is not readily available and diagnosis is based on clinical and simple laboratory features. While some practitioners in endemic areas still believe that dysentery is commonly amebic in etiology, there is ample evidence that patients with dysentery are far more likely to be infected with Shigella than with E. histolytica.64
Figure 18.9 Fecal smear of shigellosis.
Clinical and laboratory features in amebic and bacillary dysentery differ sharply. Shigellosis is suggested by a short pre-hospitalization illness, high fever, and the presence of abundant fecal leukocytes, greater than 50 neutrophils per high-power field (Fig. 18.9).65 Amebiasis is more often chronic, and leukocytes are few or absent in the stool because the parasite lyses PMNs. Mere presence of amebic cysts in stool is not diagnostic, as the vast majority are nonpathogenic strains of E. dispar. Virulent E. histolytica are suggested by identification of motile trophozoites containing ingested erythrocytes in fresh stool preparations.66 More specific diagnosis is possible with ELISA or PCR, and sharply reduces the false positives with microscopy alone. In the absence of confirmed amebiasis, patients with frank dysentery should be presumed to have shigellosis and treated empirically for this infection. In developing countries the diagnosis of clinical dysentery can often be established by community health workers who either observe the characteristic stool or obtain a history of bloody stools from the mother, who are reliable historians in reporting bloody diarrhea.67 Rapid methods have been developed to diagnose shigellosis, including fluorescent antibody staining for S. dysenteriae type 1, which has high sensitivity (92%) and specificity (93%),68 immunomagnetic isolation of bacteria followed by PCR69 or monoclonal antibody70 confirmation, and isotope- or enzyme-labeled DNA probes for virulence markers specific for Shigella, although some of the latter are also present in the rare Shigella-like enteroinvasive E. coli.71,72 A rapid simple dipstick method has been devised for S. flexneri 2a, the most common serotype in developing countries.73 Although these may become commercially available, cost will remain a major deterrent to their use in resource-limited settings. The only reliable rapid commercially available method in routine use is an enzyme immunoassay for Shiga toxin in stool. A positive test is indicative of S. dysenteriae type 1 or an Stx-producing E. coli serotype, such as O157. This test is used primarily in developed countries for rapid diagnosis of Stx-producing E. coli.74,75 Serologic testing for O-antigen antibody is useful for epidemiologic studies of shigellosis but not for diagnosis of acute disease, especially in endemic areas where the majority of the population may be seropositive from prior infections.76
TREATMENT AND PROGNOSIS Except for mild-moderate watery diarrhea due to S. sonnei in wellnourished individuals, for which antibiotics are not warranted, antimicrobial therapy is the cornerstone of treatment for shigellosis. In the absence of effective antimicrobial therapy, mortality from bloody diarrhea or dysentery due to Shigella is appreciable; S. dysenteriae type 1 and S. flexneri in developing countries have been associated with mortality rates in excess of 10%, particularly in the young and the elderly.77 In this setting, even S. sonnei can be lethal, especially among individuals sick enough to seek hospital care. Effective antimicrobial therapy given within 72 hours of symptoms not only brings prompt clinical resolution but also reduces the likelihood of HUS.78 All persons who have dysentery and presumed Shigella infection should therefore be treated with an appropriate anti microbial agent when first seen; the results of culture and susceptibility testing, if available, can then be used subsequently to modify treatment when necessary.
% resistant
70 60
Ampicillin
50
TMP-SMX
40
Nalidixic Acid
30
Mecillinam
20
Ciprofloxacin
10 0
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
Year
A
Chapter 18
80
Shigellosis
90
90 80 70 % resistant
To be useful in resource-poor settings, an antimicrobial agent should be safe for use in children under 5 years, proven clinically effective in controlled trials, administered orally, and affordable.79 Because of the ever rising prevalence of multidrug-resistant strains, selection of an appropriate agent has become increasingly difficult.80 Older oral drugs, such as chloramphenicol and tetracycline, and trimethoprimsulfamethoxazole and ampicillin, until recently the drugs of choice for shigellosis, are no longer first-line choices because of drug resistance (Fig. 18.10).81 Some agents effective in vitro, such as oral extended-spectrum cephalosporins (e.g., cefixime), are ineffective in vivo.82 Older nonabsorbable agents, such as furazolidone, are ineffective, despite advertising claims to the contrary. A newer nonabsorbable agent, rifamixin, has been shown to be effective when given prophylactically to prevent illness in human challenge experiments with S. flexneri. If safe and useful for established infection and substantiated in field trials, rifamixin may become another option for multidrug-resistant infections.83 Unfortunately, current options for treating Shigella infections in developing countries are limited and recommendations are subject to change as drug resistance develops (Table 18.1). Nalidixic acid, pre viously useful against multiresistant strains, is no longer reliable because of increasing resistance.84 Newer fluoroquinolones, such as ciprofloxacin, the macrolide azithromycin, and the β-lactam pivamdinocillin, remain effective,85,86 although emerging resistance to ciprofloxacin and azithromycin is of concern.87,88 Short course (3-day) ciprofloxacin is proven effective, and reduces the cost of treatment.89 The parenteral agent ceftriaxone is also effective, even when given for just 2 days.90 The major constraints currently are cost and the need for parenteral administration, which precludes its use at the community level. It is a good initial option for individuals with severe illness treated in health care facilities wherever multidrug resistance is common. Unfortunately, ceftriaxone resistance is also beginning to emerge.91 Depending on the drug, a course of treatment in Bangladesh can cost the equivalent of 1 day to 3 weeks’ earnings for the poorest, those with an income less than US$1 per day. If empiric antimicrobial treatment for severe shigellosis does not result in clinical improvement within 48 hours, infection with a drug-resistant strain or another organism should be considered, and therapy modified. It is essential to know local drug resistance patterns to devise an empirical strategy for first- and second-line drugs. This necessitates a surveillance system and adequate laboratory capacity, neither of which may be available in developing countries. Because patients with shigellosis are rarely severely dehydrated, intravenous rehydration is not necessary. There are no contraindications to
60
Ampicillin
50
TMP-SMX
40
Nalidixic Acid
30
Mecillinam
20
Ciprofloxacin
10 0
2000 2001 2002 2003 2004 2005 2006 2007 2008
Year
B
Figure 18.10 Antimicrobial sensitivity of strains isolated in Dhaka (urban) and Matlab (rural) Bangladesh. These isolates were obtained through a 2% systematic surveillance of patients seen at the ICDDR,B Hospital in Dhaka, Bangladesh (A) and from the culture of all patients seen at the Matlab Hospital (B). Over the past decade, more than 70% of strains have become resistant to nalidixic acid, more than 60% to trimethoprim-sulfamethoxazole, and 30–60% of strains to ampicillin. Of concern is the recent emergence of resistance to mecillinam and ciprofloxacin. (Source: ICDDR, B.)
Table 18.1 Current Antimicrobial Agents for Treatment of Shigellosis in Tropical Countries Agent
Adult Dose
Pediatric Dosea
Frequency
Duration
Total Treatment Cost in Dhakab (Pediatric/Adult)
Pivamdinocillinc Ciprofloxacin Azithromycin
400 mg 500 mg 500 mg on day 1, 250 mg/day thereafter 2 g 160 mg
20 mg/kg 10 mg/kg 10 mg/kg on day 1, 5 mg/kg thereafter
4 times daily Twice daily Once daily
5 days 3 daysd 5 days
US$3.50/7.00 US$1.30/1.30 US$2.50/1.75e
100 mg/kg 4 mg/kg
Once daily Twice daily
5 days 5 days
800 mg 500 mg
20 mg/kg 25 mg/kg
US$11–14/18–22 Most S. dysenteriae type 1 and S. flexneri strains are resistant. May still be useful for S. sonnei if drug resistance is limited
4 times daily
5 days
Most S. dysenteriae type 1 and S. flexneri strains are resistant. May still be useful for S. sonnei if drug resistance is limited
Ceftriaxone Trimethoprim and Sulfamethoxazole Ampicillin
a
Maximum pediatric dose is the adult dose. Calculated on the basis of packaged drugs in Dhaka, and for a 10 kg child. c Not available in many countries, including the United States. d Prolonged use in children is contraindicated because of risk of arthritis. This is not an issue for treatment of acute shigellosis. e Because of the packaging of pediatric formulation of azithromycin, it costs more to purchase a pediatric course of treatment than a full course for an adult. b
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Section II PATHOGENS
PART A: Bacterial and Mycobacterial Infections
ORT, and it is useful to manage mild-moderate dehydration that may occur. Severe hyponatremia, a serious complication, can be treated with either intravenous normal saline or a bolus of 3% NaCl; close clinical monitoring is required. Increasing serum sodium concentration by 6–8 mmol/L in 24 hours is considered safe.92 Patients with shigellosis should be fed to the extent they are willing to eat (breast milk or solid foods, depending on age) to prevent hypoglycemia and malnutrition. Anorexia responds to effective antimicrobial therapy.93 Restoration of nutritional status is most rapidly achieved if sufficient calories and highquality protein are provided during convalescence; however, it takes considerably longer to replenish nutrient stores than it does to create the deficit during infection. Parents of a child recovering from shigellosis must be encouraged to feed their child more than normal during convalescence until satisfactory weight gain has occurred. This may be difficult in settings of poverty and food insecurity. Treatment of complications is largely supportive. Patients with seizures do not need anticonvulsant therapy, since more than one seizure is uncommon. HUS may require transfusion and peritoneal dialysis; however, mortality from S. dysenteriae-associated HUS remains higher than observed in HUS due to other etiologies,94,95 perhaps because the disease is more severe, the host is more malnourished, or because facilities for managing this complication are limited where S. dysenteriae type 1 outbreaks occur. Toxic megacolon due to transmural colitis and micro vascular thrombosis is an ominous complication, with mortality rates as high as 50%. Colectomy can be lifesaving but often not feasible in developing countries, because complex postoperative support is unavailable and subsequent long-term care for a child with a colectomy is difficult to ensure. Conservative management with antibiotics, fluids, nasogastric suction, and limited surgical intervention if intestinal perforation occurs – with or without megacolon – may be the better strategy.96,97 Rectal prolapse resolves on its own as the inflammation wanes; in the interim, the prolapsed tissues should be kept moist and protected against injury. Suspected bacteremia should be treated with parenteral, broad-spectrum antibiotics.
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144
PREVENTION AND CONTROL Because of the low inoculum and person-to-person transmission, the primary method of preventing shigellosis remains good personal hygiene,98,99 although access to potable water and sanitary disposal of feces is often lacking where the disease is most prevalent. Caretakers of children should be encouraged to wash their hands after contact with stool or soiled clothes, and always before preparing food. Handwashing with soap is preferred, but if unavailable then traditional practices using sand or ash, or even with water alone, are better than not washing at all. In addition to resistance to change of established behaviors, the lack of water in or near the household is a major handicap. Where open defecation is practiced, simple fly control can reduce the prevalence of Shigella infection.100 While immunization could have significant impact, it has proved difficult to develop safe and effective Shigella vaccines, in part because the mechanisms of protective immunity remain unclear and because there are many antigenically distinct serotypes involved.101 Pioneering early vaccine studies demonstrated that protective immunity against shigellosis is serotype-specific.102 Experimental human shigellosis models support this concept, as prior infection is highly protective against reinfection with the same species and serotype.103,104 Because traditional parenteral killed vaccines have been ineffective,105 attention has shifted to alternative approaches, including oral immunization with live attenuated Shigella106 or Salmonella Typhi expressing Shigella O-antigens,107 isolated Shigella cell surface components,108 or parenteral O-polysaccharide-protein conjugates.109 A limited multivalent vaccine of the most common serotypes would be very useful. To date, no acceptably safe and sufficiently effective vaccine has been developed; too often the most immunogenic vaccines have been the most reactogenic, while other vaccine approaches with acceptable reactogenicity have not been very immunogenic. The lack of a good animal model of shigellosis has also limited Shigella vaccine development. Finally, despite the lack of success thus far, the search for common protective component(s) for a serotype-independent vaccine should not be abandoned.