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There are two medically important Bacillus species: Bacillus anthracis and Bacillus cereus. Important features of pathogenesis by these two Bacillus species are described in Table 17–2.
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1. Bacillus anthracis
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Bacillus anthracis causes anthrax (Figure 17–1), which is common in animals but rare in humans. Human disease occurs in three main forms: cutaneous, pulmonary (inhalation), and gastrointestinal. In 2001, an outbreak of both inhalation and cutaneous anthrax occurred in the United States. The outbreak was caused by sending spores of the organism through the mail. There were 18 cases, causing five deaths in this outbreak.
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Bacillus anthracis is a large gram-positive rod with square ends, frequently found in chains (Figure 17–2). Its antiphagocytic capsule is composed of D-glutamate. (This is unique—capsules of other bacteria are polysaccharides.) It is nonmotile, whereas other members of the genus are motile. Anthrax toxin is encoded on one plasmid, and the polyglutamate capsule is encoded on a different plasmid.
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Spores of the organism persist in soil for years. Humans are most often infected cutaneously at the time of trauma to the skin, which allows the spores on animal products, such as hides, bristles, and wool, to enter. Spores can also be inhaled into the respiratory tract. Pulmonary (inhalation) anthrax occurs when spores are inhaled into the lungs. Gastrointestinal anthrax occurs when contaminated meat is ingested.
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Inhalation anthrax is not communicable from person to person, despite the severity of the infection. After being inhaled into the lung, the organism moves rapidly to the mediastinal lymph nodes, where it causes hemorrhagic mediastinitis. Because it leaves the lung so rapidly, it is not transmitted by the respiratory route to others.
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Pathogenesis is based primarily on the production of two exotoxins, collectively known as anthrax toxin. The two exotoxins, edema factor and lethal factor, each consists of two proteins in an A–B subunit configuration. The B, or binding, subunit in each of the two exotoxins is protective antigen. The A, or active, subunit has enzymatic activity.
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Edema factor, an exotoxin, is an adenylate cyclase that causes an increase in the intracellular concentration of cyclic adenosine monophosphate (AMP). This causes an outpouring of fluid from the cell into the extracellular space, which manifests as edema. (Note the similarity of action to that of cholera toxin.)
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Lethal factor is a protease that cleaves the phosphokinase that activates the mitogen-activated protein kinase (MAPK) signal transduction pathway. This pathway controls the growth of human cells, and cleavage of the phosphokinase inhibits cell growth, leading to apoptosis, cell death, and the necrotic skin lesion (black eschar). Protective antigen forms pores in the human cell membrane that allows edema factor and lethal factor to enter the cell. The name protective antigen refers to the fact that antibody against this protein protects against disease.
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The typical lesion of cutaneous anthrax is a painless ulcer with a black eschar (crust, scab) (see Figure 17–1). Local edema is striking. The lesion is called a malignant pustule. Untreated cases progress to bacteremia and death.
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Pulmonary (inhalation) anthrax, also known as “wool-sorter’s disease,” begins with nonspecific respiratory tract symptoms resembling influenza, especially a dry cough and substernal pressure. This rapidly progresses to hemorrhagic mediastinitis, bloody pleural effusions, septic shock, and death. Although the lungs are infected, the classic features and X-ray picture of pneumonia are not present. Mediastinal widening seen on chest X-ray is an important diagnostic criterion. Hemorrhagic mediastinitis and hemorrhagic meningitis are severe life-threatening complications. The symptoms of gastrointestinal anthrax include vomiting, abdominal pain, and bloody diarrhea.
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Smears show large, gram-positive rods in chains (see Figure 17–2). Spores are usually not seen in smears of exudate because spores form when nutrients are insufficient, and nutrients are plentiful in infected tissue. Nonhemolytic colonies form on blood agar aerobically. Colonies on blood agar typically have a characteristic flared “comet’s tail” appearance.
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In case of a bioterror attack, rapid diagnosis can be performed in special laboratories using polymerase chain reaction (PCR)-based assays. Another rapid diagnostic procedure is the direct fluorescent antibody test that detects antigens of the organism in the lesion. Serologic tests, such as an enzyme-linked immunosorbent assay (ELISA) test for antibodies, require acute and convalescent serum samples and can only be used to make a diagnosis retrospectively.
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Ciprofloxacin is the drug of choice. Doxycycline is an alternative drug. No resistant strains have been isolated clinically.
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Ciprofloxacin or doxycycline was used as prophylaxis in those exposed during the outbreak in the United States in 2001. People at high risk can be immunized with cell-free vaccine (BioThrax) containing purified protective antigen as immunogen. The vaccine is weakly immunogenic, and six doses of vaccine over an 18-month period are given. Annual boosters are also given to maintain protection. An immune globulin preparation containing a monoclonal antibody against protective antigen (raxibacumab, Anthrasil) is available for prevention in people at risk of inhalational anthrax. Incinerating animals that die of anthrax, rather than burying them, will prevent the soil from becoming contaminated with spores.
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Bacillus cereus causes gastroenteritis (food poisoning).
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Spores on grains such as rice survive steaming and rapid frying. The spores germinate when rice is kept warm for many hours (e.g., reheated fried rice). The portal of entry is the gastrointestinal tract.
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Bacillus cereus produces two enterotoxins. The mode of action of one of the enterotoxins is the same as that of cholera toxin (i.e., it adds adenosine diphosphate [ADP] ribose, a process called ADP-ribosylation, to a G protein, which stimulates adenylate cyclase and leads to an increased concentration of cyclic AMP within the enterocyte). The mode of action of the other enterotoxin resembles that of staphylococcal enterotoxin (i.e., it is a superantigen).
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There are two syndromes. (1) One syndrome has a short incubation period (4 hours) and consists primarily of nausea and vomiting, similar to staphylococcal food poisoning. (2) The other has a long incubation period (18 hours) and features watery, nonbloody diarrhea, resembling clostridial gastroenteritis.
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This is not usually done.
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Only symptomatic treatment is given.
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There is no specific means of prevention. Rice should not be kept warm for long periods.
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There are four medically important species: Clostridium tetani, Clostridium botulinum, Clostridium perfringens (which causes either gas gangrene or food poisoning), and Clostridium difficile. All clostridia are anaerobic, spore-forming, gram-positive rods (Figure 17–3). Important features of pathogenesis and prevention are described in Table 17–3.
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1. Clostridium tetani
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Clostridium tetani causes tetanus (Figure 17–4).
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Spores are widespread in soil. The portal of entry is usually a wound site (e.g., where a nail penetrates the foot), but the spores can also be introduced during “skin-popping,” a technique used by drug addicts to inject drugs into the skin. Germination of spores is favored by necrotic tissue and poor blood supply in the wound. Neonatal tetanus, in which the organism enters through a contaminated umbilicus or circumcision wound, is a major problem in some developing countries.
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Tetanus toxin (tetanospasmin) is an exotoxin produced by vegetative cells at the wound site. This polypeptide toxin is carried intra-axonally (retrograde) to the central nervous system, where it binds to ganglioside receptors and blocks release of inhibitory mediators (e.g., glycine and γ-aminobutyric acid [GABA]) at spinal synapses. Tetanus toxin is encoded by a plasmid, unlike botulinum toxin which is encoded by a lysogenic bacteriophage.
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Tetanus toxin and botulinum toxin (see later) are among the most toxic substances known. They are both proteases that cleave the proteins involved in mediator release from the neurons.
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Tetanus toxin has one antigenic type, unlike botulinum toxin, which has eight. There is therefore only one antigenic type of tetanus toxoid in the vaccine against tetanus.
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Tetanus is characterized by strong muscle spasms (spastic paralysis, tetany). Specific clinical features include lockjaw (trismus) due to rigid contraction of the jaw muscles, which prevents the mouth from opening; a characteristic grimace known as risus sardonicus; and exaggerated reflexes. Opisthotonos, a pronounced arching of the back due to spasm of the strong extensor muscles of the back, is often seen (see Figure 17–4). Respiratory failure ensues. A high-mortality rate is associated with this disease. Note that in tetanus, spastic paralysis (strong muscle contractions) occurs, whereas in botulism, flaccid paralysis (weak or absent muscle contractions) occurs.
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There is no microbiologic or serologic diagnosis. Organisms are rarely isolated from the wound site. Clostridium tetani produces a terminal spore (i.e., a spore at the end of the rod). This gives the organism the characteristic appearance of a “tennis racket.”
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Tetanus immune globulin (tetanus antitoxin) is used to neutralize the toxin. The role of antibiotics is uncertain. If antibiotics are used, either metronidazole or penicillin G can be given. An adequate airway must be maintained and respiratory support given. Benzodiazepines (e.g., diazepam [Valium]) should be given to prevent spasms.
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Tetanus is prevented by immunization with tetanus toxoid (formaldehyde-treated toxin) in childhood and every 10 years thereafter. Tetanus toxoid is usually given to children in combination with diphtheria toxoid and the acellular pertussis vaccine (DTaP).
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When trauma occurs, the wound should be cleaned and debrided, and tetanus toxoid booster should be given. If the wound is grossly contaminated, tetanus immune globulin, as well as the toxoid booster, should be given and penicillin administered. Half of the immune globulins should be infiltrated into the wound and the other half given intramuscularly at a site separate from the tetanus toxoid.
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Tetanus immune globulin (tetanus antitoxin) is made in humans to avoid serum sickness reactions that occur when antitoxin made in horses is used. The administration of both immune globulins and tetanus toxoid (at different sites in the body) is an example of passive–active immunity.
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2. Clostridium botulinum
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Clostridium botulinum causes botulism.
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Spores, widespread in soil, contaminate vegetables, and meats. When these foods are canned or vacuum-packed without adequate sterilization, spores survive and germinate in the anaerobic environment. Toxin is produced within the canned food and ingested preformed. The highest-risk foods are (1) alkaline vegetables such as green beans, peppers, and mushrooms and (2) smoked fish. The toxin is relatively heat-labile; it is inactivated by boiling for several minutes. Thus, disease can be prevented by sufficient cooking.
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Botulinum toxin is absorbed from the gut and carried via the blood to peripheral nerve synapses, where it blocks release of acetylcholine. It is a protease that cleaves the proteins involved in acetylcholine release. The toxin is a polypeptide encoded by a lysogenic phage. Along with tetanus toxin, it is among the most toxic substances known. There are eight immunologic types of toxin; types A, B, and E are the most common in human illness. Botox is a commercial preparation of exotoxin A used to remove wrinkles on the face. Minute amounts of the toxin are effective in the treatment of certain spasmodic muscle disorders such as torticollis, “writer’s cramp,” and blepharospasm.
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Descending weakness and paralysis of cranial nerves, including diplopia, dysphagia, ptosis, and respiratory muscle failure, are seen. No fever is present. In contrast, Guillain-Barré syndrome is an ascending paralysis (see Chapter 66).
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Two special clinical forms occur: (1) wound botulism, in which spores contaminate a wound, germinate, and produce toxin at the site and (2) infant botulism, in which the organisms grow in the gut and produce the toxin there. Ingestion of honey containing the organism is implicated in transmission of infant botulism. Affected infants develop weakness or paralysis and may need respiratory support but usually recover spontaneously. In the United States, infant botulism accounts for about half of the cases of botulism, and wound botulism is associated with drug abuse, especially skin-popping with black tar heroin.
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The organism is usually not cultured. Botulinum toxin is demonstrable in uneaten food and the patient’s serum by mouse protection tests. Mice are inoculated with a sample of the clinical specimen and will die unless protected by antitoxin. Enzyme-linked immunoassay (EIA) tests are also used to detect the toxin, and PCR tests are used to detect the DNA encoding the toxin.
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The heptavalent antitoxin containing all seven types (A to G) is preferred to the trivalent antitoxin containing types A, B, and E. Respiratory support is provided as well. The antitoxin is made in horses, and serum sickness may occur. A bivalent antitoxin (types A and B) purified from the plasma of humans immunized with botulinum toxoid is available for the treatment of infant botulism.
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Proper sterilization of all canned and vacuum-packed foods is essential. Food must be adequately cooked to inactivate the toxin. Swollen cans must be discarded (clostridial proteolytic enzymes form gas, which swells cans).
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3. Clostridium perfringens
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Clostridium perfringens causes two distinct diseases, gas gangrene and food poisoning, depending on the route of entry into the body.
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Disease: Gas Gangrene
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Gas gangrene (myonecrosis, necrotizing fasciitis) is one of the two diseases caused by C. perfringens (Figure 17–5). Necrotizing fasciitis is often called the “flesh-eating” disease. In addition to C. perfringens, Streptococcus pyogenes, and methicillin-resistant Staphylococcus aureus (MRSA) are important causes. The clinical aspects of necrotizing fasciitis are described in Chapter 77.
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Gas gangrene is also caused by other histotoxic clostridia such as Clostridium histolyticum, Clostridium septicum, Clostridium novyi, and Clostridium sordellii. (C. sordellii also causes toxic shock syndrome in postpartum and postabortion women.)
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Spores are located in the soil; vegetative cells are members of the normal flora of the colon and vagina. Gas gangrene is associated with war wounds, automobile and motorcycle accidents, and septic abortions (endometritis).
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Organisms grow in traumatized tissue (especially muscle) and produce a variety of toxins. The most important is alpha toxin (lecithinase), which damages cell membranes, including those of erythrocytes, resulting in hemolysis. Degradative enzymes produce gas in tissues.
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Pain, edema, cellulitis, and gangrene (necrosis) occur in the wound area (see Figure 17–5). If crepitus is palpated in the affected tissue, it indicates gas in the tissue. This gas is typically hydrogen produced by the anaerobic bacteria. Hemolysis and jaundice are common, as are blood-tinged exudates. A foul-smelling, bloody vaginal discharge can occur in endometritis. Shock and death can ensue. Mortality rates are high.
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Smears of tissue and exudate samples show large gram-positive rods. Spores are not usually seen because they are formed primarily under nutritionally deficient conditions. The organisms are cultured anaerobically and then identified by sugar fermentation reactions and organic acid production. C. perfringens colonies exhibit a double zone of hemolysis on blood agar. The colonies also produce a precipitate in egg yolk agar caused by the action of its lecithinase. Serologic tests are not useful.
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Penicillin G plus clindamycin is the treatment of choice. Wounds should be debrided. Incision and drainage of focal lesions should be done.
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Wounds should be cleansed and debrided. Penicillin may be given for prophylaxis. There is no vaccine.
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Disease: Food Poisoning
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Food poisoning is the second disease caused by C. perfringens.
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Spores are located in soil and can contaminate food. The heat-resistant spores survive cooking and germinate. The organisms grow to large numbers in reheated foods, especially meat dishes.
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C. perfringens is a member of the normal flora in the colon but not in the small bowel, where the enterotoxin acts to cause diarrhea. The mode of action of the enterotoxin is the same as that of the enterotoxin of S. aureus (i.e., it acts as a superantigen).
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The disease has an 8- to 16-hour incubation period and is characterized by watery diarrhea with cramps and little vomiting. It resolves in 24 hours.
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This is not usually done. There is no assay for the toxin. Large numbers of the organisms can be isolated from uneaten food.
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Symptomatic treatment is given; no antimicrobial drugs are administered.
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There are no specific preventive measures. Food should be adequately cooked to kill the organism.
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4. Clostridium difficile (Clostridioides difficile)
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C. difficile causes antibiotic-associated pseudomembranous colitis (Figure 17–6). C. difficile is the most common nosocomial (hospital-acquired) cause of diarrhea. It is the leading infectious cause of gastrointestinal-associated deaths in the United States. The name C. difficile has recently been changed to Clostridioides difficile but for this edition the previous name will be used.
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The organism colonizes the large intestine of approximately 3% of the general population and up to 30% of hospitalized patients. Note that most people are not colonized, which explains why most people who take antibiotics do not get pseudomembranous colitis. C. difficile is transmitted by the fecal–oral route. Either the spores or the bacterial organism itself can be transmitted.
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The majority of cases occur in hospitalized patients, but about one-third of cases are community-acquired. The hands of hospital personnel are important intermediaries.
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Antibiotics suppress drug-sensitive members of the normal flora of the colon, allowing C. difficile to multiply and produce large amounts of exotoxins A and B. Both exotoxin A and exotoxin B are glucosyltransferases (i.e., enzymes that glucosylate [add glucose to] a G protein called Rho GTPase). The main effect of these exotoxins is to cause depolymerization of actin, resulting in a loss of cytoskeletal integrity, apoptosis, and death of the enterocytes. Exotoxin B is thought to play the leading role in producing the signs and symptoms of human disease.
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Clindamycin was the first antibiotic to be shown to predispose to pseudomembranous colitis, but many antibiotics are known to predispose to this disease. At present, third-generation cephalosporins are the most common because they are so frequently used. Ampicillin and fluoroquinolones are also commonly implicated. In addition to antibiotics, cancer chemotherapy and proton pump inhibitors predispose to pseudomembranous colitis. C. difficile rarely invades the intestinal mucosa.
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C. difficile causes diarrhea associated with pseudomembranes (yellow-white plaques) on the colonic mucosa (see Figure 17–6). (The term pseudomembrane is defined in Chapter 7). The diarrhea is usually not bloody, and neutrophils are found in the stool in about half of the cases. Fever and abdominal pain often occur. The organism rarely enters the bloodstream and rarely causes metastatic infection.
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The pseudomembranes are visualized by sigmoidoscopy. Toxic megacolon can occur, and surgical resection of the colon may be necessary. Pseudomembranous colitis can be distinguished from the transient diarrhea that occurs as a side effect of many oral antibiotics by testing for the presence of the toxin in the stool. Even with adequate treatment, the organism may not be eradicated from the colon, and recurrences occur at a rate of approximately 15%−20%.
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In 2005, a new, more virulent strain of C. difficile emerged. This hypervirulent strain causes more severe disease, is more likely to cause recurrences, and responds less well to metronidazole than the previous strain. The strain is also characterized by resistance to quinolones. It is thought that the widespread use of quinolones for diarrheal disease may have selected for this new strain.
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The presence of exotoxins in the filtrate of a patient’s stool specimen is the basis of the laboratory diagnosis. It is insufficient to culture the stool for the presence of C. difficile because people can be colonized by the organism and not have disease.
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There are two types of tests used to make the laboratory diagnosis. One detects the exotoxin itself, and the other detects the genes that encode the exotoxin. To detect the exotoxin itself, an ELISA test employing antibody to the exotoxin is used. To detect the genes that encode the exotoxin, a PCR assay to determine the presence of the toxin gene DNA is used. The DNA-based test has greater sensitivity and specificity than the ELISA test. However, these nucleic acid amplification tests (NAATs) should be interpreted with caution because a person may only be colonized by C. difficile and be recorded as positive when, in fact, C. difficile is not the cause of the patient’s disease.
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The causative antibiotic should be withdrawn. Oral vancomycin is the drug of choice for the initial episode and for recurrences. Fidaxomicin can also be used. If vancomycin is not available, metronidazole can be used for the initial episode of non-severe cases. Vancomycin can be given orally or per rectum or, in very severe cases, both ways. In life-threatening cases, surgical removal of the colon may be required.
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In many patients, treatment does not eradicate the carrier state, and recurrent episodes of colitis can occur. Fidaxomicin is used both in the treatment of pseudomembranous colitis and in preventing relapses of this disease. It is effective in life-threatening cases. Bezlotoxumab, a monoclonal antibody against exotoxin B of C. difficile, is effective in preventing relapses.
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Fecal transplantation is another possible therapeutic approach. It involves administering bowel flora from a normal individual either by enema or by nasoduodenal tube to the patient with pseudomembranous colitis. This approach is based on the concept of bacterial interference (i.e., to replace the C. difficile with normal bowel flora). Very high cure rates are claimed for this technique, but aesthetic considerations have limited its acceptance.
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There are no preventive vaccines or drugs. Because antibiotics are an important predisposing factor for pseudomembranous colitis, they should be prescribed only when necessary. Fidaxomicin is useful in preventing relapses of this disease. In the hospital, strict infection control procedures, including rigorous handwashing, are important. Probiotics, such as the yeast Saccharomyces, may be useful to prevent pseudomembranous colitis.