Pathophysiology of viral diarrhea

In the classic and simple view, the pathogenesis of diarrhea may be divided into osmotic and secretory (Figure 9.1). Viral diarrhea was originally believed to be caused by cell invasion and epithelial destruction by enteropathogenic agents, therefore being the result of endoluminal fluid accumulation osmotically driven by non-absorbed nutrients. It is now known that several mechanisms are responsible for diarrhea, depending on the specific agents and the host features. In addition, selected viruses possess multiple virulence pathways that act synergistically to induce diarrhea.

The mechanisms of diarrhea induced by group A rotaviruses have been extensively investigated and provide a paradigm of the pathophysiology of viral diarrhea. Rotavirus has tissue- and cell-specific tropisms, infecting the mature enterocyte of the small intestine. The first step is virus binding to specific receptors located on the cell surface, the GM1 ganglioside. However, different rotavirus strains bind in either a sialic acid-dependent or an -independent fashion.9 Most rotaviruses, including all human strains, infect polarized enterocytes through both the apical and the basolateral side, in a sialic acid-independent manner, suggesting the presence of different receptors.10 The early stages of rotavirus binding involve the viral protein (VP)4 spike attachment and cleavage. This outer capsid protein contains ligand sequences for a201 and a401 integrins, and for complement receptor 4, located on the entero-cyte surface. After binding, the rotavirus enters into the cell by a multistep process that requires both VP7 and VP4 proteins. The route of internal-ization remains controversial. Two mechanisms have been proposed: direct penetration through the cell membrane, which could be mediated by the VP5 cleavage product of VP4, or a receptor-primed calcium-dependent endocytosis.

Infection of the villous enterocyte leads to cell lysis, compromising nutrient absorption and driving water into the intestinal lumen through an osmotic mechanism (Figure 9.1). However, the destruction of villus-tip cells induces a compensatory proliferation of crypt cells. These immature enterocytes physiologically maintain a secretive tone, thus contributing to diarrhea with ion secretion, as the result of the imbalance between absorptive villous and secretory crypt cells. Thus, the cytopathic action by rotavirus results in both osmotic and secretory diarrhea.

Table 9.2 Epidemiological features and associated impact of viral diarrhea in specific settings

Setting

Developing countries

Features high frequency

Impact high mortality

Viruses rotavirus

Industrialized countries

high frequency

high costs

all

Day-care centers

high frequency

high costs for the society

rotavirus + others

Seasonal pattern

major outbreaks

winter/early spring

rotavirus

Food-related transmission

food poisoning

massive attack rates

calicivirus, rotavirus, Aichi virus

Age

maximal incidence in children <5 years

increased severity in younger infants

rotavirus

Children at risk

severity increased in immunocompromised malnourished, other chronic diseases

poor outcome

rotavirus, CMV, EBV, adenovirus, astrovirus, picornavirus

Protection

neonates, breast-fed infants, previous infections

protected from severe clinical course more than infection

rotavirus

Persistent diarrhea

occasional (prolonged shedding described)

more frequent in malnourished/ immunocompromised children

rotavirus, CMV

Intractable diarrhea

rare (but described) in industrialized countries

high case/fatality ratio in children at risk but also with no risk

rotavirus

Nosocomial

second most frequent cause of nosocomial infections

increase in hospital stay and associated costs

rotavirus (group B), calicivirus, astrovirus

CMV, cytomegalovirus;

EBV, Epstein-Barr virus

Histological changes induced by rotavirus infection occur within 24h of infection in animal models.11,12 The proximal small intestinal wall appears thinner with villous atrophy, blunting and conversion to a cuboidal epithelium, without extensive pathological changes. This observation led to the hypothesis of a neurovascular mechanism with a role of a local villous ischemia induced by a vasoactive agent from rotavirus-

infected neuronal cells.13 The enteric nervous system may also play a direct role in inducing fluid secretion, similar to that induced by cholera toxin and other intestinal secretagogs.14

The molecular mechanisms of fluid secretion have also been investigated. Rotavirus induces an increase in intracellular calcium levels,15 which is responsible for the disassembly of microvillar F-

Figure 9.1 Diagram of osmotic and secretory mechanisms of viral diarrhea. The arrows indicate the water's movements and their volumes. In osmotic diarrhea, water is driven into the intestinal lumen by the osmotic force of non-absorbed nutrients. In secretory diarrhea, ions are actively pumped within the lumen and are passively followed by water. From Guandalini S. Acute diarrhea. In Walzer WA et al. Pediatric Gastrointestinal Disease, 3rd edn. Hamilton, Ontario: BC Decker Inc. 2000, with permission.

actin, the perturbation of cellular protein trafficking, the damage of tight-junctions, with disruption of cell-cell interaction and cytolysis.16,17 This is reflected by the loss of epithelial integrity, as shown by the progressive decrease in tissue resistance measured in Caco-2 cell monolayers mounted in Ussing chambers18 (Figure 9.2).

In children with rotavirus infection, the onset of diarrhea is abrupt and occurs in the absence of histological changes, even if oral feeding is withdrawn, suggesting that a secretory pathway is responsible for diarrhea, at least in the initial phases of infection. A major advancement in the understanding of rotavirus pathophysiology came from the identification of the non-structural protein NSP4 as a viral enterotoxin, defined by its ability to cause fluid secretion, but not epithelial changes.19

NSP4 is a multifunctional virulence factor, as it possesses the following features (Figure 9.3):

  • 1) It is released from infected cells;20
  • 2) It enters the cells through a specific receptor;21-24
  • 3) It causes calcium-dependent chloride secretion, with an age-dependent pattern;19
Caco Rotavirus

Figure 9.2(a) Cytopathic effects induced by rotavirus (5 PFU/cell) in a model based on the morphology of Caco-2 cell monolayers. a, Uninfected Caco-2 cell monolayers; b, Caco-2 cells at 48 h post-infection: cellular vacuolization, opening of intracellular junction and spotting cell detachments are observed; c, Caco-2 cells at 96 h post-infection: extensive cellular detachment is observed with only a few picnotic cells yet present. From reference 18.

Osmotic Diarrhea

Figure 9.1 Diagram of osmotic and secretory mechanisms of viral diarrhea. The arrows indicate the water's movements and their volumes. In osmotic diarrhea, water is driven into the intestinal lumen by the osmotic force of non-absorbed nutrients. In secretory diarrhea, ions are actively pumped within the lumen and are passively followed by water. From Guandalini S. Acute diarrhea. In Walzer WA et al. Pediatric Gastrointestinal Disease, 3rd edn. Hamilton, Ontario: BC Decker Inc. 2000, with permission.

  • Virus-free control
  • Virus-free control
Secretory Diarrhea

Figure 9.2(b) Cytopathic effects of rotavirus infection in a Caco-2 cell model, as measured by transepithelial electrical resistance (TEER). The decrease of TEER reflects progressive cell damage, which is shown in Figure 9.3. Increasing loads of virus induce an earlier and steeper fall of TEER, with a clear relationship with virus multiplicity. The TEER value decreases below a detectable level within approximately 36 h postinfection with 25 PFU/cell, 60-72 h post-infection with 5 PFU/cell, and 96 h post-infection with 1 PFU/cell. From reference 18.

Figure 9.2(b) Cytopathic effects of rotavirus infection in a Caco-2 cell model, as measured by transepithelial electrical resistance (TEER). The decrease of TEER reflects progressive cell damage, which is shown in Figure 9.3. Increasing loads of virus induce an earlier and steeper fall of TEER, with a clear relationship with virus multiplicity. The TEER value decreases below a detectable level within approximately 36 h postinfection with 25 PFU/cell, 60-72 h post-infection with 5 PFU/cell, and 96 h post-infection with 1 PFU/cell. From reference 18.

Pathophysiology Diarrhea

Figure 9.3 Combined effects by NSP4 in the pathophysiology of rotavirus diarrhea. Rotavirus infects epithelial cells of the small intestine, replicates, and induces cell lysis. NSP4 is released by infected cells and functions as a Ca2+-dependent enterotoxin triggering Cl- secretion. It decreases fluid and electrolyte transport by inhibiting Na-glucose symport SGLT1 and, possibly Na-K ATPase. It also impairs disaccharidase expression. Furthermore, rotavirus and/or NSP4 may diffuse underneath the intestinal epithelium activating secretory reflexes in the enteric nervous system. Late during the infection, an inflammatory response in the lamina propria may be detected, and the production of inflammatory substances and cytokines may further contribute to the increase of intestinal permeability and diarrhea.

Figure 9.3 Combined effects by NSP4 in the pathophysiology of rotavirus diarrhea. Rotavirus infects epithelial cells of the small intestine, replicates, and induces cell lysis. NSP4 is released by infected cells and functions as a Ca2+-dependent enterotoxin triggering Cl- secretion. It decreases fluid and electrolyte transport by inhibiting Na-glucose symport SGLT1 and, possibly Na-K ATPase. It also impairs disaccharidase expression. Furthermore, rotavirus and/or NSP4 may diffuse underneath the intestinal epithelium activating secretory reflexes in the enteric nervous system. Late during the infection, an inflammatory response in the lamina propria may be detected, and the production of inflammatory substances and cytokines may further contribute to the increase of intestinal permeability and diarrhea.

  • 4) It alters plasma membrane permeability and is cytotoxic;25-27
  • 5) It is sensitive to specific antibody, which prevents or reduces diarrhea;28,29

NSP4 is the only rotavirus gene product capable of eliciting intracellular calcium mobilization.30 NSP4 was demonstrated to stimulate a calcium-dependent chloride secretion, in mouse small intestinal mucosa sheets mounted in Ussing chambers, suggesting that this enterotoxin triggers diarrhea in the early phase of infection in animal models.14,31 NSP4 further contributes to diarrheal pathogenesis by directly altering enterocyte actin distribution and paracellular permeability.32 Finally, NSP4 plays a role in the inhibition of the Na+-dependent glucose transporter SGLT-1.33 Glucose absorption is impaired in rotavirus diarrhea as well as disaccharidase activities, whereas the Na/amino acid co-transporters are not involved.

Rotavirus diarrhea may also have an inflammatory component. The induction of cytokines is important in developing an inflammatory and immune response, especially in intestinal infection caused by bacteria. In rotavirus infection, limited inflammation is detected by histological studies, suggest ing that cytokines are effective in inducing a host immune response to rotavirus diarrhea. However, it has been shown that the rotavirus-infected ente-rocyte activates NF-kB and the production of chemokines interleukin (IL)-8, Rantes and GRO-a, and of cytokines interferon (IFN)a and granulo-cyte/macrophage-colony-stimulating factor (GM-CSF).34'35

In conclusion, the primary target of rotavirus is the enterocyte, which is induced to secrete fluids and is subsequently destroyed. On the other hand, the enterocyte acts as a sensor to the mucosa with the production of viral and endogenous factors and the activation of other cell types including nerves. Thus, rotavirus-induced diarrhea is a multistep and multifactorial event, in which fluid secretion and cell damage are observed in sequence, as shown in an intestinal cell line-based experimental model (Figure 9.4). A summary of the multiple mechanisms involved in the rotavirus-intestine interaction is given in Table 9.3.

Clinical signs and symptoms

The predominant symptoms of vomiting and diarrhea are common to enteric infections, regardless of the causative role of more than 20 different

Viral Diarrhoea Mechanism
Hours after infection

Figure 9.4 Biphasic effect of rotavirus in Caco-2 cells. Rotavirus induces a biphasic response, in an in vitro model of infection in Caco-2 enterocytes mounted in Ussing chambers. An early secretion is evident in the first few hours of infection, with a peak at 2 h post-infection, as shown by the increase in short circuit current (Isc, - -). Subsequently, rotavirus exerts a cytotoxic effect with a loss of tissue integrity, as demonstrated by the fall of transepithelial resistance (Ohm/cm2, - -) which is evident at 24h post-infection. The results suggest that rotavirus diarrhea is initially the result of an early secretory mechanism and of a subsequent osmotic pathway, due to cell damage and loss of functional absorptive surface, leading to nutrient malabsorption. From G. De Marco et al, unpublished data.

Table 9.3 Pathogenesis of rotavirus diarrhea

Key process

Pathway

Consequences

Villous cell destruction

cytoskeleton disruption, cell lysis

nutrient malabsorption, osmotic diarrhea

Crypt cell proliferation

compensatory secretory cell proliferation

secretory diarrhea

NSP4 enterotoxin

increase in intracellular calcium, chloride secretion

secretory diarrhea

NSP4-induced

inhibition of SGLT-1

osmotic diarrhea

glucose malabsorption

Neuromediated

neurotransmitter microcirculation

secretory diarrhea

vascular ischemia

impairment

Inflammation

NF-kB, IL-8, Rantes

osmotic/secretory diarrhea

microbial agents, including bacteria, parasites and viruses.

Usually, viral diarrhea lasts for 3-5 days. In selected cases, viral diarrhea may be persistent and even become life-threatening. In a series of children with intractable diarrhea syndrome, rotavirus was detected in five out of 38 children who had no evident risk factors.36

The history may provide valuable clinical information. Diarrheal onset may be abrupt or progressive. The child's age, admission to a daycare center, the time of year, exposure to diarrheal contacts, previous antibiotic courses, and ingestion of contaminated food such as eggs or water should be evaluated to define the origin of diarrhea. Risk factors for severe diarrhea include malnutrition, immune derangement and AIDS, and history of repeated episodes of diarrhea. Recent weaning from breast milk or introduction of feedings other than milk may be associated with a more severe course of the disease (Table 9.2).

Dehydration is the key symptom to define the severity of the disease and the need for fluid replacement. The degree of dehydration should be evaluated at first observation and followed up to evaluate the ongoing losses and the efficacy of fluid intake to replace them. The gold standard for determining dehydration is acute weight loss. Because a patient's pre-illness weight is rarely known, an estimate of fluid deficiency is made on clinical grounds.

Vomiting may be associated with diarrhea and induce additional fluid losses. In addition, vomiting may prevent oral rehydration, thus requiring parenteral fluid replacement.

Body temperature may be elevated, as a consequence of dehydration or reflecting an inflammatory response. Abdominal pain is not frequent and suggests colonic involvement, but may also indicate a surgical problem.

Stool characteristics should be carefully considered: large volumes of watery stools indicate small bowel involvement and are frequently associated with dehydration, whereas frequent outputs of a small amount of mucus or bloody stools are associated with colitis. In severe cases the entire intestine is involved.

Colonic involvement is more often associated with bacterial rather than viral etiology. Some features may help in distinguishing viral from bacterial diarrhea (Table 9.4).37 Viral diarrhea is more frequently associated with vomiting and dehydration. It affects younger children compared to

Table 9.4 Main clinical features associated with the most frequent enteric pathogens (modified from reference 37)

Salmonella species

Campylobacter species

Rotavirus

NLV

Giardia lamblia

Fever

++++*

++++*

++++*

+

+

Blood in stool

++

++++*

+

++

+++

Abdominal cramps

++++*

++

+

+

Vomiting

+

+

++++ *

++++*

+

>6 stools per day

+

++++*

++

+

+

Duration of

7

7

<4

<4

symptoms (days)

NLV, Norwalk-like virus *p <0.05

Table 9.5 Individual symptoms and signs of acute gastroenteritis according to etiological agents in subjects younger than 2 years; mixed infections excluded (modified from reference 38)

Rotavirus

Adenovirus

Astrovirus

NLV

SLV

(n=189)

(n=35)

(n=34)

(n=115)

(n=44)

Duration of diarrhea (days)

4

5

1

2

3

Maximum number of diarrhea

6

5

4

4

4

episodes in 24 h

Duration of vomiting (days)

2

1

1

1

1

Maximum number of vomiting

3

1

1

4

1

episodes in 24 h

Temperature (°C)

38.8

38.4

37.9

37.9

37.8

Severity score

10

7

5

8

6

Data are medians of findings

NLV, Norwalk-like virus; SLV, Sapporo-like virus

bacterial diarrhea. Selected features are more frequently associated with specific enteric viruses (Table 9.5).38 However, signs and symptoms in the individual child do not reliably allow identification of specific etiological agents of gastroenteritis.

Diagnosis

Although acute diarrhea is generally the manifestation of an enteric infection, it may be associated with other illnesses, such as food poisoning or intolerance, extraintestinal infections or surgical disease.39 Acute diarrhea may be a side-effect of various drugs including antibiotics. History and clinical evaluation may aid in the differential diagnosis. However, close follow-up is required.

Generally, microbiological investigations are not necessary in children with acute gastroenteritis. Several studies have reported a low yield and cost/efficacy ratio per identified agent even in hospitalized children. Microbiological examination should be considered: in cases of persistent diarrhea; when a specific antimicrobial treatment is considered, such as for children belonging to a group at risk; when an intestinal infection must be excluded in order to support a different etiology; and to investigate an outbreak.

The search for enteric viruses is made by several techniques, including culture in sensitive cells, electron microscopy, immune-based assays and molecular probes (Table 9.6). Virus culture is the gold standard, but it is cumbersome and the results are available only with delay, limiting its clinical applications. Immune-based methods are widely used, but a progressive increase in the use of polymerase chain reaction (PCR) techniques is leading to a shift of diagnostic techniques. The choice of a specific technique is based on the clin ical need, the availability of technical skills and the efficacy ratio. In most clinical institutions, immune-based assays are available to detect rotavirus and less frequently enteric adenovirus. The search for other viruses is generally available in reference centers or research institutions.

However, trying to identify the etiology may not be clinically useful, as treatment of diarrhea is relatively independent of the responsible agent. Rather, it is the evaluation of the child's clinical condition that provides information for case management.

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Responses

  • MATHIAS HAHN
    Is viral diarrhea secretory or osmotic?
    6 years ago
  • AMI
    Is rotavirus diarrhea secretory?
    6 years ago
  • gilly
    Is rotavirus a secretory or osmotic diarrhea?
    5 years ago
  • Kathrin
    How does cholera cause disease?
    4 years ago

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