Persistent Diarrhea: Still a Serious Public Health Problem in Developing Countries (Part 2)

Pathophysiology

There are multiple and complex pathophysiological mechanisms that may be involved in and determine the perpetuation of an acute diarrheic episode. Small bowel injury has been incriminated as the central mechanism in PD [27, 28]. The status of the mucosal permeability barrier and the ability of the host in the clearance of the enteropathogenic agent have a direct influence on the persistence of these lesions [25].

It is well known that some enteropathogenic agents cause diarrhea by damaging the small bowel mucosa and/or through the secretion of enterotoxins that act on the enterocytes triggering the secretion of water and electrolytes, and even by the production of some cytotoxins that induce cell damage [29, 30].

EAEC and EPEC, among the several other agents, are usually the most frequent enteropathogenic agents isolated in the stools of infants with PD. The mechanisms of EAEC and EPEC infections are also the ones known and studied in most detail in the medical literature [29–31].

EAEC infection represents an important cause of PD in developing countries [30]. The investigation of the role of the EAEC strains in the persistence of diarrhea has shown the ability of this microorganism to adhere to both the small bowel and the colonic mucosa.

Hicks et al. [31] examined the interaction between EAEC and the human intestine using the in vitro organ culture model from biopsies obtained from infants with diarrhea. The EAEC strains were able to adhere to the jejunal, ileal, and colonic mucosa; most of the bacteria were associated with the mucus layer above the intestinal mucosa, and few of them were found in close association with the mucosal surface. Andrade et al. [32] studied the interaction of EAEC strains that were isolated from the stools of infants with PD, with the human fragments of ileal and colonic mucosa in vitro, utilizing the transmission electron microscopy, and showed the occurrence of bacterial aggregates colonizing and provoking cytotoxic effects in the ileal and colonic mucosa.

Aggregative adherent fimbriae that are encoded on a 60 mda virulence plasmid called paa create a mucus biofilm that seems responsible for the small bowel damage leading to malabsorption of nutrients and persistence of diarrhea [33].

In contrast to the limited importance of EPEC in developed countries, EPEC is a major cause of infantile diarrhea in developing countries [12] and, in many cases, is responsible for PD [34]. Proximal small intestinal mucosal biopsy specimens show, but not always, intimately adherent bacteria and the classic attaching and effacing (A/E) histopathology [35]. Cantey and Blake [36] were the first authors to describe this new pattern of adherence of an EPEC strain in rabbits, which was characterized by adherence of bacteria to the apical portion of the enterocyte, with cuplike pedestal formation and subsequent effacement of the brush border. This effect was designated by Moon et al. as attaching and effacing (A/E) [37]. Rothbaum et al. [38] reported on the adherence of EPEC O119:B14 associated with effacement of microvilli, leading to the formation of a cuplike pedestal on the enterocytes of infants with PD. The determinants of the A/E lesion have been localized to a large island of pathogenicity on EPEC chromosomes, termed the locus of enterocyte effacement, or LEE [39]. Fagundes-Neto et al. [40] reported the presence of bacteria within the enterocytes from an infant with acute diarrhea that evolved into PD caused by EPEC O111:H2. Fagundes-Neto et al. [28] studied patients with PD in whom EPEC strains were isolated in the jejunal fluid secretion and in the stools, utilizing the scanning electron microscopy. At low magnification (150×), most of the villi showed mild to moderate stunting, but on several occasions, there was subtotal villous atrophy. At higher magnification (7,500×), photomicrographs showed derangement of the enterocytes; on several occasions, cell borders were not clearly defined, and very often microvilli decreased in number and height; in some areas there was complete disappearance of the microvilli. Moreover, several baciliform microorganisms tightly adhering to the enterocytes were seen, and lymphocytes and fat droplets overlay the surface of the enterocytes as well. In half of the patients, a mucous-fibrinoid pseudomembrane partially coating the enterocytes was observed. This mucus coating may hamper absorption of nutrients of a diet due to mechanical block, thus leading to osmotic diarrhea and nutritional aggravation. This hypothesis may be supported by the finding off at droplets accumulated on the apical surfaces of the enterocytes. These ultrastructural changes may arise from a combined interaction of the enteropathogenic agent that causes the A/E lesion associated with disarrangement of the digestive–absorptive enzyme system, leading to malabsorption of nutrients [29]. These pathophysiological events determine the onset of food intolerance that is responsible for the perpetuation of diarrhea and nutritional aggravation.

Bacterial overgrowth in the small bowel lumen of the colonic microflora may occur in up to 68 % of patients with PD, and it is another factor that will perpetuate the damage of the intestinal mucosa [28]. It is particularly associated with anaerobic bacteria, suchas Veillonella and Bacteroides species, and predisposes to intestinal mucosal injury [41]. Pathologic changes occur due to the ability of the anaerobic bacteria to induce deconjugation and 7α-dehydroxylation of the primary bile acids cholic and chenodeoxycholic acid, converting them into their respective secondary bile acids (deoxycholic and lithocholic acid), which are highly damaging to the jejunal mucosa, provoking a decrease in the absorption surface and functional lesions with a deficiency of enterokinase [42] and of the ATPase (Na+K+) enzyme [43]. When present in the intestinal lumen, these secondary, unconjugated bile acids induce water and sodium secretion and glucose malabsorption and can also lead to a breakdown of the intestinal permeability barrier, facilitating the penetration of potentially allergenic macromolecules (food proteins, intact or partially hydrolyzed), leading, as a consequence, to an allergy to the proteins of the diet (cow milk, soybean protein) [44].

The presence of secondary and unconjugated bile salts in the small bowel prevents the formation of the mixed micelles, which play an essential role in ensuring solubilization of dietary fats. The consequent decrease of bile salts pool will lead, first, to malabsorption of fats in a diet, having as a result steatorrhea, which will deprive the patient of an important caloric offer [45]. Second, the excretion of bile salts will induce the appearance of cholereic diarrhea, due to the direct toxic action of the bile salts on the colonic mucosa [46].

Finally, the synergistic effects of these pathophysiologic events become responsible for the perpetuation of the diarrheal process and for the aggravation of the nutritional status, with a high risk of death.

Diagnosis

In order to establish an accurate diagnosis of the respective etiology and potential complications, detailed information must be obtained on the following topics: a comprehensive clinical history extending as far back as the onset of the diarrheal illness; prior dietary history; breastfeeding history; socioeconomic status and living conditions; and prior medical history, including prior infectious diseases and family history. History and physical examination can provide an outline of the profile of a patient’s nutritional status and other consequences of the diarrheal illness. The laboratory investigation should include a stool culture and a search for ova and parasites in fresh stool specimens, a detection of fecal pH and reducing substances search in the stools, a search for leukocytes and occult blood in the stools, and a determination of fecal α1 anti-trypsin and steatocrit.

Considering the high prevalence of carbohydrate intolerance in the diet reported in PD patients as a perpetuating factor of diarrhea, the approach should include overload tests with the various carbohydrates commonly used in the diet, such as lactose, glucose, and fructose. The lactulose load test should also be carried out in order to detect a possible bacterial overgrowth in the small intestine. All these tests should preferably be carried out by the technique of the H2 breath test, because this is a noninvasive method with high sensitivity and specificity [47].

If possible, the determination of fecal electrolytes should also be done, which will distinguish osmotic from secretory diarrhea [48]. In many cases, a small bowel biopsy should be performed to evaluate the mucosal architecture and the inflammatory infiltrate in the lamina propria, to investigate specific causes, and to demonstrate the extent of intestinal damage [49]. The knowledge of the intensity and extension of morphological damage enables the appropriate dietary management approach. When concurrent rectal bleeding occurs, it may be necessary to perform a rectal biopsy to evaluate the degree and type of inflammation [50].