COURSE OBJECTIVES:
Upon completion of this course the participant will be able to:

• Discuss the protective role of normal microbial flora
• Explain the role of Clostridium difficile in hospital-acquired diarrhea
• List principal characteristics of C. difficile, including the virulence factors
• Describe the reservoirs of infection
• List factors that contribute to susceptibility to infection with C. difficile
• Describe the course of disease, including complications and relapses
• Outline laboratory diagnostic procedures
• Summarize the therapy protocol for C. difficile-associated disease

INTRODUCTION

Protective role of normal microbial flora
The size of the microbial population of the intestinal tract far exceeds that of all other microbial communities associated with the body. The adult human intestinal tract contains close to 100 trillion microorganisms. This represents 10 times as many microbial cells as there are human cells in the adult body. More than 800 species of bacteria are found in the intestinal tract. Most of these bacteria have not been cultured in the laboratory and represent previously unrecognized microbial species. Information about these organisms comes from culture-independent molecular studies, such as the sequencing of bacterial 16S ribosomal NA genes.

The microbial population of the gut may contain more than a hundred times the number of genes present in the human genome, thus providing additional functions, features, and traits not present in the human host. In spite of the tremendous density of the intestinal flora only nine of the fifty-five known bacterial divisions have been identified to date. The predominant bacterial genera in the intestinal tract are Bacteroides, Clostridium and Eubacterium. One member of Archaea, Methanobrevibacter smithi, is also prevalent. These bacteria and related members of the Cytophaga-Flavobacterium division comprise close to 60% of intestinal bacteria. There is, however, considerable variability in the distribution of intestinal microorganisms: microbial populations of mucosal surfaces in the gut are not identical to those found in the feces. The composition of the intestinal microbial flora also varies with the individual’s age, diet, and health status.

Gut flora communicates with surrounding tissues to stimulate the development of an environment beneficial to microbial co-existence with the host. An illustration of gut flora function is provided by studies with germ-free animals. Germ-free rats lacking gastrointestinal tract bacteria require 30% more calories to maintain body mass than normal rats, thus providing evidence that resident intestinal bacteria contribute to host nutrition, most probably by generating and liberating simplified carbohydrates, amino acids, and vitamins. An example is Bacteroides thetaiotaomicron, one of the most abundant microorganisms in the human gastrointestinal tract. A large portion of the genome of this bacterium is devoted to regulation of its environment: the bacterial genome encodes pathways for breaking down complex carbohydrates, such as xylan and pullullan found in the unabsorbed remains of the host diet. Additional enzymes for metabolizing host-derived carbohydrates, including mucin, hyaluronic acid, and heparin, are also encoded. The breakdown of complex carbohydrates into simple sugars benefits the host as well as the microbial population.

Additional studies with germ-free animals (mice) have demonstrated underdeveloped lymphoid tissue in the mucosa of the intestinal tract, an absence of germinal centers, and a deficiency in IgA plasma cells and T lymphocytes. Apparently, an active intestinal immune system depends on the presence of resident microorganisms.
A number of other functions are performed by the normal flora in the intestinal tract. These functions include regulating host fat storage, stimulating vascularization, and facilitating the development of intestinal villi. Of major importance is the contribution of the gut microbial flora to host defense by limiting colonization of the gastrointestinal tract by pathogens. Antibiotic therapy, particularly the use of broad-spectrum antibiotics, disrupts the resident bacterial flora microorganisms and allows colonization by pathogenic microbial species.

Antibiotic Therapy and Opportunistic Infections
Extensive use in hospitals of antibiotics and of indwelling devices has resulted in an increasing rate of hospital-acquired infections. It is estimated that, at least, 5 to 10% of hospital patients acquire an infection not present on admission to the hospital. The microorganisms involved vary with current antibiotic use and with clinical practices since a number of factors combine to alter the patient’s normal colonizing microbial flora. These factors include, in addition to antimicrobial therapy, acquisition of potential pathogens from environmental sources, from hospital staff, and through patient-to-patient contamination.

A major health problem is presented by antibiotic-associated diarrhea. The frequency of this complication varies with antimicrobial agents, ranging from 2% to 25%, depending on the specific antibiotics used in therapy. Diarrhea may occur in 5 to 10% of patients who are treated with ampicillin, 10 to 25% of those treated with amoxicillin-clavulanate, 15-20% of patients given cefixime, and 2 to 5% of those who receive cephalosporins, fluoroquinolones, azithromycin, clarithromycin, erythromycin and tetracycline. Symptoms may range from frequent loose and watery stools without other complications (simple diarrhea) to antibiotic-associated colitis with abdominal cramping, fever, leukocytosis, hypoalbuminemia, colon pathology, and other major complications. A relatively small proportion of the cases of antibiotic-associated diarrhea without complications are due to infection with C. difficile. Other enteric pathogens may be involved or the diarrhea may be caused by direct effects of antimicrobial agents on the intestinal tract mucosa as well as by the metabolic consequences of reduced concentrations of fecal flora. However, the majority of cases of colitis associated with antibiotic therapy are caused by C. difficile. Antibiotic therapy with clindamycin, expanded-spectrum penicillins, cephalosporins, and more recently fluoroquinolones is especially likely to initiate C. difficile-associated disease (CDAD) (2).

C. difficile: HISTORICAL BACKGROUND AND PRINCIPAL CHARACTERISTICS OF THE BACTERIUM

Historical Background
In 1935 Hall and O’Toole recognized a previously undescribed anaerobe in the stools of newborn infants. The bacterium was difficult to culture in the laboratory and, therefore, was named Bacillus difficilis. Almost 40 years later the use of antibiotics was linked to diarrheal disease and to a rare but serious complication, pseudomembranous colitis. The bacterium, now called Clostridium difficile, was identified as the etiological agent of these conditions. All cases of pseudomembranous colitis and 15 to 25% of cases of antibiotic-associated diarrhea appear to be caused by C. difficile infection.

Principal Bacterial Characteristics
This anaerobic Gram positive rod is pleomorphic, produces subterminal spores, and is motile by peritrichous flagella. Three toxins (Toxin A, Toxin B, and binary toxin) are produced by this bacterium although toxin production varies with specific strains. In 1999 a genome sequencing project of C. difficile strain 630 was begun at Sanger Centre in the United Kingdom. The results generated by this project are available on http://www.sanger.ac.uk/Projects/C. difficile/

Bacterial Virulence Factors
C. difficile virulence factors fall into two categories: (1) surface proteins that facilitate adherence and penetration of host cells and (2) bacterial toxins which damage host cells and initiate clinical symptoms of disease.

(1) Surface Proteins
Flagellar proteins:
FLIC – major structural component of the flagellar filament
FLID – flagellar cap protein with surface- adherent properties and capacity for attachment to mucus
Surface layer (S layer) proteins that form the crystalline surface layer (two peptides):
These proteins facilitate attachment to host cells and colonization and are antiphagocytic.
Cell wall proteins:
Cwp 66: functions as an adhesin
Cwp 84: has proteolytic activity

(2) Toxins:
Pathogenicity of C. difficile is related to production of exotoxins. Toxins A and B belong to the group of so-called “large clostridial exotoxins.” Both toxins disrupt the cytoskeleton of intestinal epithelial cells by acting on regulatory proteins involved in actin polymerization. Although the two toxins are related they have a number of distinguishing characteristics. Toxin A is thought to be responsible for most of the gastrointestinal symptoms associated with the disease. It has been termed an enterotoxin because it causes extensive tissue damage to the gut mucosa. Toxin B is highly cytotoxic to a range of mammalian tissue culture cells. It has a high level of cytotoxic activity but appears to require Toxin A to initiate tissue damage, providing Toxin B with access to intestinal cells.

Toxins A and B are encoded by two genes, tcd A and tcd B, located within the region called the “pathogenicity locus”. Based on variations in these genes C. difficile strains can be divided into 24 groups called toxinotypes. Two regulatory genes are also located in the pathogenicity locus. These genes regulate expression of the toxin genes. A partial deletion of one of the regulatory genes may therefore lead to increased production of Toxin A and Toxin B.

Binary Toxin:
This toxin is distinct from Toxins A and B. It belongs to the iota group of clostridial toxins and can be neutralized by antiserum to iota toxin. Binary toxin is composed of two independent, unlinked protein chains: CDTa (enzymatic component) and CDTb (binding component). The binding component recognizes a cell-surface receptor, resulting in internalization of the enzymatic component which catalyzes ribosylation of actin. This leads to disorganization of the cytoskeleton of the affected cell.

Binary toxin is encoded by genes cdt A and cdt B located on a chromosome outside the pathogenicity locus. Binary toxin genes are present in 1.6% to 20.8% of C. difficile isolates. Approximately 1.9% of Toxin A and B negative strains produce binary toxin. Strains that produce binary toxin appear to be responsible for severe gastrointestinal disease that may require hospitalization. These strains are more commonly community-acquired than those producing only Toxin A and Toxin B.
Toxin production patterns of C. difficile:

The majority of strains produce only A and B toxins but variant strains have been identified with a range of toxin- producing capability. Five different toxin production patterns have been identified (3), (4).

NATURAL HABITAT AND RESERVOIR OF INFECTION
C. difficile has been isolated from various environmental sources such as soil, hay, and sand. It can also be isolated from the gastrointestinal tracts of 50% of infants during the first year of life where it is present without any clinical signs of disease. The carriage rate decreases in older children and young adults, reaching a rate of 2-3% in the healthy adult population. The bacterium is present in the spore phase in the intestinal tract of carriers. There is no toxin production or clinical symptoms.
C. difficile has also been isolated from many animal species including household pets, cows, horses, donkeys, dogs, ostriches, rabbits, cats, rodents, and pigs. The prevalence of C. difficile in the feces of dogs and cats ranges from 6-40%. Horses have carriage rates of 2-29%. It has been reported that approximately 25% of isolates from humans are indistinguishable from isolates from one or more animal species (5). This suggests a possibility of interspecies transmission of C. difficile.
In hospitals C. difficile is frequently found on various surfaces in the patients’ rooms, in the toilet areas and on the floors. Colonized health care workers have also been implicated as a source of infection. The rate of isolation of C. difficile from hospital patients has been reported to be 26% in the general medical wards and 30% from geriatric patients.

EPIDEMIOLOGY OF C. difficile INFECTION
C. difficile is responsible for both sporadic cases of antibiotic-associated diarrhea and epidemic outbreaks. Most cases are hospital-acquired (nosocomial) infections, but community acquired C. difficile-associated disease (CDAD) is being increasingly recognized. Recent occurrence of outbreaks at multiple sites suggests the emergence of new strains of increased virulence. It is known that certain strains of C. difficile are more frequently involved in outbreaks, particularly multi-state outbreaks in health care facilities. These outbreak-associated strains are apparently more virulent and are frequently resistant to antimicrobial agents, such as clindamycin or the fluoroquinolones (levofloxacin, moxifloxacin, and gatifloxacin). The increasing use of these antibiotics in health care facilities may provide a selective advantage for these epidemic strains and promote their widespread emergence.

A recent epidemiologic study reviewed 1,721 cases of CDAD over the past 13 years. This study showed that the rate of occurrence of CDAD increased by a factor of four during the study period and that the cases were increasingly more severe. Major risk factors for CDAD in this group of patients were age over 65 years and treatment with fluoroquinolones.

RISK FACTORS
The patient’s age and antibiotic therapy are known as the two principal factors for acquiring C. difficile-associated disease.
Treatment with broad-spectrum antibiotics disrupts the normal resident microflora. After treatment there is a long period of susceptibility to colonization with other microbial species. It may take as long as three months for the normal microbial population to fully recover after antibiotic exposure. Some antibiotics are involved more frequently than others in antibiotic-associated diarrhea.

Clindamycin was the most commonly implicated agent in the 1970s followed by cephalosporins in the 1980s. More recently fluoroquinolones have assumed an important role as inducers of C. difficile-associated disease (CDAD). In general, almost any antibiotic that interferes with the normal microbial flora may precipitate CDAD.

Role of age as a risk factor for CDAD:
A number of epidemiological studies have reported an increase in the incidence of CDAD with age. The number of cases of CDAD per 1000 hospital admissions increases from 20 for the 51 to 60 age group to 74.4 for patients over 90. The mortality rate also increases with age, reaching 14% for patients over 90 years of age.

Additional risk factors:
The presence or absence of these factors contributes to the outcome of CDAD and explains the wide range of observed clinical symptoms.

1. Immune status of the patient: an immune response to bacterial adhesins (surface antigens) may be important in host defense. In addition, the course of the disease is affected by the patient’s ability to generate an antibody response to C. difficile toxins.
2. Antineoplastic therapy has an adverse effect on the patient’s immune system and is considered a risk factor.
3. The nature of underlying disease: vascular and heart disease as well as lung and kidney disorders are strongly associated with CDAD.
4. The duration of hospitalization: in the hospital environment symptomatic patients and asymptomatic carriers shed C. difficile cells and spores. New sporadic cases as well as outbreaks occur through patient-to-patient or patient-to-staff contact. This may take place through hand transmission or indirectly by exposure to contaminated objects in the hospital environment. The spores of C. difficile can remain in the hospital environment for many months, providing a reservoir for new infections. Hospitalized patients have rates of colonization of 20% to 30% compared to a rate of 3% in outpatients.
5. Enteral feeding: the use of nasal feeding tubes may affect the resident microbial flora or disrupt some of the natural barriers to invasion by opportunistic pathogens.
6. Treatment with proton pump inhibitors to reduce gastric acidity: treatment within preceding eight weeks changes the composition of the gut microflora.
7. Gender: female gender has been identified as one of the risk factors for CDAD in hospitalized patients.
8. Gastrointestinal surgery: surgery leads to disruption of normal protective tissue barriers and increases the susceptibility of the patient to infection with opportunistic pathogens.
9. Virulence of the infecting strain: the course of the disease is affected by the ability of the bacterial strain to produce and to release any of the C. difficile toxins (toxin A, toxin B, or binary toxin)
.
Guidelines have been developed for controlling C. difficile infection in hospitals and in long-term facilities. These guidelines are
1. Personnel should use soap and water to wash hands; frequent washing is recommended.
2. Disposable gloves should be used by all medical personnel.
3. Environmental surfaces should be cleaned with sporicidal agents.
4. Symptomatic patients should be placed in private rooms.
5. The use of rectal thermometers should be discouraged.
6. In case of outbreaks, restriction of the use of antibiotics may be necessary.

PATHOGENESIS AND CLINICAL COURSE OF DISEASE
Pathogenesis
Disruption of the intestinal barrier by trauma or the suppression of protective microflora by antibiotic treatment allows spores of C. difficile to germinate and vegetative cells to multiply in the intestinal tract. Bacterial flagella assist in invasion of host cells through chemotaxis and adhesion to host surfaces. Some of the bacterial proteins found in the S layer and in the cell wall also act as adhesins. Invasion of cells lining the intestinal tract is accompanied by synthesis of bacterial toxins, host cell damage, and signs of disease.

Clinical course of disease:
Symptoms may range from loose and watery stools (“nuisance diarrhea”) with no other complications, to clinical manifestation of antibiotic-associated colitis: abdominal cramping, fever, leukocytosis, fecal leukocytes, hypoalbuminemia, profuse diarrhea with mucus and characteristic odor and a yellow-greenish cast. An extreme form of CDAD, pseudomembranous colitis, is characterized by the presence of a pseudomembrane in the colon consisting of mucosal plaques and presenting a typical endoscopic appearance. Other complications may include toxic megacolon (an acute toxic colitis with dilatation of the colon) and sepsis or septic shock.
Sepsis and septic shock:
Sepsis is a serious cause of morbidity and mortality. It is estimated that in the United States close to 700,000 persons annually are affected by sepsis, with over 200,000 deaths. According to recent reports the incidence of sepsis is rising at rates of 1.5% to 8.0% per year.
A clear definition of sepsis has been difficult to develop. Historically, sepsis had been defined as the host’s systemic response to infection. The spectrum of responsible microorganisms has shifted from predominantly Gram negative bacteria in the late 1970s to mostly Gram positive bacteria at present. Fungi also have become increasingly common causes of sepsis.

Originally sepsis was believed to be associated with the presence of bacteria in the blood (bacteremia) and the terms “sepsis” and “septicemia” were frequently interchanged in the clinical setting. However, clinical signs of sepsis were observed in patients without demonstrable bacteremia. In 1992 the term “systemic inflammatory response syndrome” was introduced to describe the sepsis syndrome. The terms “severe sepsis” and “septic shock” are used to differentiate between different stages of disease.

A patient suffering from septic shock may exhibit either fever or hypothermia, an altered mental state, very rapid heart beat, leukocytosis or leukopenia, organ dysfunction and organ failure, hypotension that does not respond to treatment, as well as a number of other abnormal findings. Mortality rate is very high, particularly in the elderly.

IMMUNE RESPONSE AND VACCINE STUDIES
Clinical studies have shown a protective effect of the host’s immune response to C. difficile toxins and to bacterial surface proteins. A reduced risk of CDAD is associated with a high level of IgM antibody to the predominant surface antigens, the S layer proteins. Convalescent patients may have a strong IgG antibody response to the S proteins. These findings suggest that a vaccine may offer protection against CDAD and vaccine development studies are in progress.
An antitoxin vaccine is also one of the experimental projects under consideration. However, this type of vaccine would not prevent colonization with C. difficile.
In other laboratories, bacterial surface antigens are being studied in animal models as possible vaccine candidates.

LABORATORY DIAGNOSIS
The laboratory diagnosis of CDAD is based on two types of procedures: fecal culture and toxin detection. A number of different methods are available for detection of toxins.

Culture methods:
Fecal specimens for culture should be liquid or, at least, unformed and cultured within two hours of collection for optimal recovery of bacteria. If the fecal specimen cannot be cultured promptly it can be refrigerated for up to two days. Spores will survive but there will be a large decrease in the number of viable vegetative cells of C. difficile.

A selective culture medium such as cycloserine cefoxitin fructose agar (CCFA) is suitable for isolation of C. difficile. The culture medium should be fresh or prepared and stored under anaerobic conditions. The antibiotics cycloserine and cefoxitin inhibit the growth of most anaerobic bacteria other than C. difficile. Fructose is metabolized by C. difficile more actively than is glucose. Neutral red acts as an indicator to detect proteolysis of the medium. Colonies of C. difficile break down proteins in the medium resulting in production of alkaline by-products that turn neutral red a yellow color. Culture plates are incubated anaerobically for 24 to 48 hours at 37C. After incubation distinctive colonies of C. difficile on CCFA are approximately 4 mm in diameter, yellow, ground-glass in appearance, and circular with a slightly filamentous edge. The medium surrounding the colonies is often changed to yellow. The colonies have a characteristic “horse stable” odor.
C. difficile can be further identified by Gram stain and by the chartreuse fluorescence of colonies under ultraviolet light. Isolated colonies can be inoculated into thioglycollate broth for subsequent end-product analysis by gas liquid chromatography (GLC).

Tests for toxin production:
1. Fecal-cytotoxin immunoassay
A sterile fecal filtrate is prepared and applied to monolayers of cultured mammalian cells. A control set of cultured cells pretreated with polyclonal antiserum to toxin A and/or toxin B is included. Cultures are incubated and observed for cytopathic effect after 24 and 48 hours.

2. Toxigenic culture
Toxigenicity tests can be done on isolated colonies of C. difficile. Several characteristic colonies are subcultured to chopped-meat-carbohydrate or brain infusion broth and incubated anaerobically at 35C to 37C for 24 hours. The isolates are tested for in vitro toxin production by performing a cytotoxin assay on filtered 24-hour broth cultures.

A number of commercial immunoassay kits are also available for toxin detection. In the procedure used with the Oxoid C. difficile Immunoassay kit bacterial colonies are picked from a selective medium and suspended in the sample diluent. After centrifugation a sample of the supernatant is inoculated on commercial immunoassay plates. Results can be recorded as soon as 30 minutes after inoculation of samples. A variety of other immunoassay kits are available for detection of C. difficile toxins. Some of these kits have higher sensitivity than others
.
Detection of proteins associated with C. difficile: tests for glutamate dehydrogenase
Latex agglutination test and enzyme immunoassays (EIA) are available for detection of this bacterial enzyme. These tests do not distinguish between toxigenic and non-toxigenic strains. The enzyme immunoassay tests are more sensitive than the latex agglutination test.

There is a great deal of variation in diagnostic tests used by different laboratories. A large number of laboratories limit the diagnosis to a single toxin detection test on fecal specimens using cultured cells or immunoassays. A recent study in Belgium of 10,552 patients with antibiotic-associated diarrhea showed that culture is the most sensitive method of detecting C. difficile. However, not all of the cultured isolates are toxigenic and, therefore, are not responsible for disease symptoms. Compared with culture, the direct cytotoxin assay has low sensitivity. The results of the Belgian study suggest that both culture and direct toxin assay should be performed on every diarrheal specimen. In case of a positive culture but a negative stool toxin result, C. difficile colonies should be tested for toxin production in vitro. A toxigenic culture would be required.

Methods used in epidemiological studies:
Various molecular methods have been used for identification of infecting strains. These methods include:
1. Sequence typing of gene (slpA) encoding the surface layer protein (SlpA): the surface layer proteins of C. difficile vary in their composition. This variability can be detected by sequencing the variable region of the slpA gene using the polymerase chain reaction (PCR).
2. “S-typing”: this is a phenotypic typing method for S proteins. This method utilizes the high degree of variation in the molecular mass of S-layer proteins. These proteins are extracted using 5M guanidine hydrochloride and visualized on SDS-PAGE (polyacrilamide gel electrophoresis). Molecular mass of the S-layer proteins is calculated using special software. Seven different S-types have been identified in a recent epidemiologic study of 865 patients in hospital geriatric units.
3. PCR analysis: this method has been used for identifying ribotypes of C. difficile isolates from both domestic animals and humans. Twenty-three ribotypes were identified among 133 bacterial isolates. Overall, 25% of isolates from humans were the same as the isolates from one or more animal species. Genes encoding toxins A and B were detected in all human, equine, and bovine isolates and in 69% of canine isolates. PCR ribotyping is also frequently used in studies of epidemic strains of C. difficile and is considered a standard for typing methods.
4. Additional methods: these include restriction-endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE), toxinotyping, and Western blot analysis. PFGE has proved to be discriminatory and reproducible for typing C. difficile and has traditionally been considered the gold standard for this procedure. Western blot analysis has been used for identification of binary toxin in a small epidemiologic study.

TREATMENT OF C. difficile DISEASE
To be effective, patient treatment must accomplish several things: it must eliminate or reduce the presence of C. difficile in the intestine, restore the normal bacterial flora and improve the patient’s resistance to colonization by pathogens.

Traditional treatments have relied on the use of two antibiotics that are effective against C. difficile: vancomycin and metronidazole. These antibiotics are bactericidal against this pathogen when the bacterium is in the vegetative phase of growth but not in the spore phase. Although effective as therapeutic agents, vancomycin and metronidazole do not restore the normal resistance to colonization by opportunistic pathogens or prevent re-infection with bacterial spores from the environment.

A standard treatment protocol includes the following:
1. Discontinuance of antibiotic therapy that provoked CDAD and, if necessary, substitution of an antibiotic with a more narrow range of antimicrobial activity. This step is often sufficient to resolve symptoms of mild diarrhea. However, most patients need treatment with one of the two standard antibiotics, vancomycin or metronidazole.
2. Supportive therapy with hydration and electrolyte replacement, particularly for young children or when diarrhea is severe. Anti-peristaltic drugs are not recommended as they may slow the clearance of bacteria and toxins from the intestine and precipitate toxic megacolon.
3. Initiation of treatment with vancomycin or metronidazole, generally for 14 days. This results in improvement in diarrheal symptoms within several days. Both antibiotics are effective in treatment of CDAD but vancomycin is more costly.

Standard antibiotic treatment: vancomycin compared with metronidazole.
Metronidazole:
Oral metronidazole is used to treat initial cases of CDAD. Generally a dose of 250 mg to 500 mg four times a day for 7 to 14 days is administered. Several treatment guidelines suggest this protocol as first choice treatment for initial cases of CDAD. Response to treatment is generally excellent with improvement in 95% of patients. The response is lower in extremely ill patients. Use of metronidazole in recurrent CDAD may result in the development of antibiotic resistance. Metronidazole resistance had been reported in 6.3% of C. difficile strains isolated from patients undergoing extended treatment for recurrent CDAD. In addition, some patients are not able to tolerate metronidazole due to side effects such as nausea, metallic taste, and rare neurotoxic complications.

Vancomycin:
The efficacy of oral vancomycin therapy for initial CDAD had been demonstrated in several trials. The drug is usually given at doses of 125 mg or 500 mg four times a day for 5 to 14 days. An initial cure rate of over 90% had been reported for patients receiving the 500 mg dose. Vancomycin has no systemic adsorption and few side effects except for rash. Some of the negative aspects of vancomycin therapy are its very high cost and the selection of vancomycin-resistant bacteria.
Comparative studies of metronidazole and vancomycin report similar cure rates for initial cases of CDAD (98% and 99% respectively). The lower cost of metronidazole makes it the treatment of choice for initial cases of CDAD. Vancomycin should be reserved for more severe cases or for patients who cannot tolerate metronidazole.

Recurrent CDAD:
It is estimated that from 12 to 24% of patients develop recurrent CDAD after the initial episode. The median time for recurrence of infection is 7 days after completion of treatment, with 97% of relapses occurring within 4 weeks post-treatment. Recurrent CDAD may be caused by the original strain of bacteria, or by re-infection with a new strain. Most studies show that half are re-infections and half are relapses. The frequency of further relapses increases with each episode. Repeated antibiotic therapy may eventually cure the patient of recurrent CDAD but this may require months and even years of antibiotic treatment.

Treatment of recurrent CDAD:
Studies of vancomycin or metronidazole therapy in patients with recurrent CDAD showed similar recurrence rates for patients treated with either antibiotic. However, vancomycin cleared both vegetative cells and toxins in 89% of patients. Metronidazole eliminated vegetative cells but only 59% of patients were toxin-negative at the end of treatment. Upon completion of treatment spores were found in 50% of patients in either antibiotic group. Spores can germinate once therapy has ended, resulting in rapid re-emergence of vegetative bacterial cells. If the normal intestinal flora has not re-established itself, C. difficile can colonize the intestinal wall, produce toxins, and initiate symptoms of CDAD.

The recurrence rate of CDAD can be decreased through the use of different dosing methods: vancomycin tapering and vancomycin pulsed dosing. An example of this dosing procedure is the administration of a standard 10-day course of vancomycin followed by a 4-week to 8-week tapered or pulsed dosing protocol. A tapered protocol employs decreasing doses of vancomycin for a period of several weeks, while a pulsed method uses antibiotic treatment every third day. The reason why this method is more effective than the standard course of treatment is that vegetative cells of C. difficile but not the spores are affected by the antibiotic. The tapered and pulsed antibiotic protocols gradually clear infection by eradicating bacteria as spores germinate. At the same time the normal protective microbial flora is in the process of being restored. A disadvantage of this treatment method is that it may encourage resistant strains of C. difficile to develop.

Treatment of CDAD with antibiotics other than vancomycin and metronidazole:
A number of other antibiotics have been tested for treating CDAD. These include bacitracin, fusidic acid, rifampicin, and teichoplanin. These antibiotics have been compared to vancomycin and to metronidazole in clinical trials. Initial cure rates were similar for all drugs, ranging from 93% to 96%. Patients treated with vancomycin or metronidazole had a recurrence rate of 16%. The recurrence rate for the teichoplanin group was 7%. Patients treated with fusidic acid had a recurrence rate of 28%. No adverse reactions were noted.

Bacitracin
Bacitracin is a relatively expensive, non-adsorbable antibiotic which has an unpleasant taste. When its effectiveness was compared to that of vancomycin, the cure rate for patients treated with bacitracin was 80% and 76% in two trials while that for vancomycin-treated patients ranged from 86% to 93%.
Fusidic acid
Treatment of patients with fusidic acid resulted in cure rates similar to those in patients treated with vancomycin or metronidazole. The rate of recurrent CDAD in the fusidic acid group was 28%.
Teicoplanin
Clinical trials of teicoplanin showed high clinical response with cure rates of 70% to 96%, depending on the dose. A low recurrence rate was reported (7.7%), although this was not statistically significant when compared to vancomycin. Teicoplanin appears to be more effective against recurrent CDAD than fusidic acid.
Rifampicin
Rifampicin had been given to a limited number of patients and there are no reports of clinical trials. One case report of a patient with CDAD and a lymphoma reported success with rifampicin treatment after previous treatment with vancomycin and metronidazole failed.
Studies of different antibiotics for the treatment of CDAD showed no overall superiority of any antibiotic over vancomycin and metronidazole. Since all antibiotics disrupt normal microbial flora, alternate treatment strategies are of considerable interest. These treatments include the use of probiotics, bacteriotherapy, use of adsorbents, and immunotherapy.

Restoration of protective microflora in the intestinal tract: use of probiotics
Up to 30 days may be required to re-establish normal flora after antibiotic treatment. The indigenous flora may be improved by the use of probiotics. These are live pure or mixed cultures of bacteria that tend to facilitate the establishment of normal microflora. Probiotics also stimulate the immune response and promote production of enzymes that degrade toxic metabolites. Various bacteria have been tested for use as probiotics. For instance, cultures of Saccharomyces boulardii and different species of lactobacilli (Lactobacillus rhamnosus and Lactoabacillus plantarum) have shown a beneficial effect when used concurrently with antibiotic treatment. The success of probiotic treatment appears to depend on a number of factors, such as the patient’s age, immune status, number of recurrences of CDAD, current antimicrobial therapy for non-CDAD conditions, and gastric acid-suppressive therapy. Adverse effects due to probiotics are mild or moderate and infections caused by probiotics have not been reported.

Adsorbents:
Another treatment strategy is to bind the toxins of C. difficile in the colonic lumen before they damage intestinal cells. Several types of adsorbents have been tested, such as oligosaccharides, various polymers, and ion-exchange resins.

Ion-exchange resins:
Clinical trials of ion-exchange resins for binding toxins have shown variable results. Two resins, cholestyramine and colestipol, were investigated. The use of these resins in animal studies (hamsters) showed favorable results. However, a clinical trial of colestipol in 38 patients with post-operative diarrhea showed no difference in fecal excretion of C. difficile toxin. Cholestyramine binds vancomycin and teichoplanin, thus its use may lead to suboptimal levels of the antibiotic.
Polymers:
Several polymers were investigated for their ability to bind clostridial toxins. These polymers include Synsorb 90, an oligosaccharide bound to an inert polymer matrix, and the polymer Tolevamer. A clinical trial of Tolevamer showed that CDAD was resolved in 83% of patients by day ten of treatment with 6 grams per day of this polymer.
Immune therapy:
A pilot study investigated the use of immune whey in prevention of CDAD relapses. Cows were immunized with inactivated C. difficile cells and toxins. Immune whey protein from the milk of these cows contained a high concentration of specific antibody (IgA class), and was effective in neutralizing the cytotoxic effect of C. difficile toxins in cell assays in vitro. Immune whey concentrate also protected hamsters from otherwise lethal C. difficile infection. When given to patients with CDAD, the immune whey protein was well tolerated and did not cause any adverse effects. Patients received immune whey protein for two weeks after completion of a standard course of antibiotic therapy. In all but one patient, bacterial toxins had disappeared from the feces upon completion of treatment. During a follow-up period of 35 days to one year, none of the patients developed recurrent CDAD.

Fecal enemas and bowel irrigation:
Fecal enemas prepared from healthy stools have been used in an effort to replace the normal microorganisms disrupted by recurrent CDAD and antibiotic treatments. Most of the patients reported no further CDAD recurrence.

CONCLUSION
C. difficile has distinctive features: it causes gastrointestinal disease in association with previous antibiotic treatment of the patient, it produces toxins in the host only in the colon, and it is a major pathogen in the hospital setting. A review of CDAD for the past dozen years shows an increase in the number of cases as well as in their severity.

In addition, epidemics of CDAD are occurring that involve multiple institutions. Of particular concern is the very high incidence and mortality rate associated with increasing age of the patients. It seems that control of CDAD would depend on more effective efforts at prevention, recognition of cases, and improved management of disease. This would involve a change in treatment protocol and a greatly improved method of infection control in hospitals and nursing facilities.

SUMMARY
Current extensive use of antibiotics is both beneficial and harmful. Antibiotic therapy eliminates the pathogen as well as some of the protective normal microbial flora, allowing the multiplication of opportunistic pathogens. C. difficile is a major cause of antibiotic-associated diarrhea. The disease may be self-limited or severe, with major complications and high mortality. Patients over the age of 60 are particularly vulnerable and the severity of disease increases with age. Hospitalized patients are particularly at risk.

C. difficile-associated disease (CDAD) is diagnosed by demonstration of the presence of bacterial toxins in the diarrheal stools, by culture of fecal specimens and bacterial isolation and identification, and by performing toxigenic cultures for in vitro toxin production. Molecular diagnostic techniques are used in investigation of epidemics.

Treatment of CDAD relies on the use of oral metronidazole or vancomycin for a period of two weeks or longer. This treatment eliminates current infection but relapses of disease are common.

Both the incidence and the severity of CDAD have been showing an increase for the past several years.

REFERENCES
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2. Bartlett JG. Antibiotic-associated diarrhea. N.Engl.J.Med. 2002;346:334-339.
3. Wren BW. <em>Clostridium difficile</em>. In: Sussman M, ed. Molecular Medical Microbiology. Academic Press; 2002:1153-1159.
4. Rupnik M, Dupuy B, Fairweather,NF, et al. Revised Nomenclature of C. difficile toxins and associated genes. J.Med.Microb. 2005;54:113-117.
5. Arroyo LG, Kruth S, Willey BM, Staempfli HR, Low DE, Weese JS. PCR ribotyping of C. difficile isolates originating from human and animal sources. J.Med.Microb. 2005;54:163-166.

1. Pseudomembranous colitis is caused by
   a. C. difficile
   b. C. botulinum
   c. C. tetani
   d. C. perfringens
   
2. Healthy human gastrointestinal tract
   a. contains about 100 billion microorganisms
   b. may harbor C. difficile without symptoms of disease
   c. has Escherichia coli as the predominant organism
   d. breaks down xylan for Bacteroides to use
   
3. Functions of the normal intestinal flora include all but which of the following?
   a. contribute to the host nutrition by generating some vitamins
   b. limit colonization of the G.I. tract by pathogens
   c. aid the development and maintenance of intestinal immunity
   d. cause the host to consume 30% more calories
   
4. Toxins A, B and binary toxin
   a. have identical mode of activity
   b. are produced only by Gram-negative rods
   c. are produced by C. difficile
   d. are all members of the iota group of clostridial toxins
   
5. Toxin A and Toxin B
   a. act by causing ribosylation of actin
   b. work independently of each other
   c. are the same in all C. difficile strains
   d. expression is regulated by genes in the pathogenicity locus
   
6. C. difficile toxins are:
   a. responsible for major symptoms of CDAD
   b. primarily adhesins to host cells
   c. frequently found in healthy individuals
   d. all called enterotoxins
   
7. A person infected with C. difficile
   a. may not show any symptoms of disease
   b. always develops diarrhea
   c. always develops pseudomembranous colitis
   d. cannot transmit infection to another person
   
8. Colonization with C. difficile has been shown to occur
   a. in dogs but not in cats
   b. in the spore phase in carriers
   c. with strains specific to each animal species
   d. in less than 2% of human infants
   
9. A major risk factor for CDAD is
   a. current viral infection
   b. anti-inflammatory drug therapy
   c. antibiotic therapy
   d. excessive exercise
   
10. Symptoms of CDAD may include
    a. a severe cough
    b. muscle pain
    c. nasal discharge
    d. watery diarrhea with mucus and abdominal cramps
    
11. Symptoms of sepsis may include
    a. hypotension that does not respond to treatment
    b. very slow heart beat
    c. sneezing
    d. eye irritation
    
12. Immune response to C. difficile
    a. does not play any role in the course of the disease
    b. may be protective
    c. shows high levels of IgM anti-S proteins in convalescent patients
    d. is directed only against the bacterial toxins
    
13. Isolation of C. difficile from stools is facilitated by
    a. the use of MacConkey agar as culture medium
    b. incubation of cultures aerobically at 20C
    c. use of fresh specimens and cycloserine-cefoxitin fructose medium
    d. use of Shigella-Salmonella agar
    
14. Which of the following groups of characteristics is typical of C. difficile?
    a. anaerobic, strongly fructose positive, horse stable odor
    b. colonies fluoresce chartreuse, aerobic growth, gram positive rod
    c. inhibited by cycloserine, anaerobic growth, white colonies
    d. horse stable odor, strongly glucose positive, gram positive rod
    
15. Fecal cytotoxin immunoassay is used for
    a. isolation of bacteria from feces
    b. determining whether an isolate is toxinogenic
    c. determining the patient’s antibody titer
    d. identification of biochemical reactions
    
16. Toxigenic culture is
    a. not important in laboratory diagnosis of CDAD
    b. a one–step procedure that produces results in 30 minutes
    c. a test for in vitro toxin production
    d. a standard diagnostic procedure in all laboratories
    
17. Latex agglutination test for glutamate dehydrogenase
    a. is the most sensitive test for diagnosis of CDAD
    b. distinguishes between toxigenic and non-toxigenic strains
    c. is a DNA sequencing test for selected genes
    d. is less sensitive than EIA for detection of the enzyme glutamate dehydrogenase
    
18. Standard treatment for CDAD is
    a. metronidazole or vancomycin
    b. rifampicin
    c. clindamycin
    d. fluoroquinolones
    
19. Vancomycin pulsed dosing refers to
    a. administering vancomycin when the patient’s pulse is below 70
    b. administering vancomycin every third day
    c. administering vancomycin in very low doses for 4 days
    d. administration of one very high dose of vancomycin
    
20. Probiotics are
    a. live cultures of non-pathogenic bacteria
    b. antibiotics, such as penicillin
    c. medications commonly given for respiratory disease
    d. cultures of pathogenic bacteria