Anaerobic Bacterial Infections

Epidemiologic Features

Clostridium difficile is a spore-forming gram-positive anaerobic bacillus that can be found in the stools of 5% of healthy adults and in 30% to 70% of infants. The organism was first isolated in 1935 from stools of healthy neonates and named Bacillus difficilis because it was difficult to grow in culture; it was not linked to antibiotic-associated diarrhea until the 1970s. The C. difficile spores can survive for prolonged periods of time in the environment. Spores are ingested but are resistant to the acid in the stomach and convert into the vegetative form in the intestine. The majority of hospital patients in whom C. difficile can be found are asymptomatic but they serve as reservoirs contaminating the environment. It is only when there is an imbalance of the intestinal microbiota, primarily due to antibiotics such as clindamycin, cephalosporins and fluoroquinolones, that the vegetative form causes disease by producing two exotoxins: TcdA and TcdB. TcdA causes recruitment of inflammatory cells and release of interleukins by intestinal epithelial and inflammatory cells, whereas TcdB has cytotoxic effects and is essential for virulence. A third toxin has been described, CDT ( C. difficile transferase) or binary toxin, which is believed to depolymerize actin in the cytoskeleton and increase adherence of the bacteria to epithelial cells. In addition to the duration, type, and number of antibiotics used, other risk factors for having C. difficile disease include being hospitalized in an acute care or long-term care facility, advanced age, use of proton pump inhibitors or H2 antagonists, immunosuppressive treatments, and gastrointestinal diseases such as inflammatory bowel disease. However, the number of patients with community-acquired C. difficile infection has increased, and they tend to be younger, healthier females who have not taken antibiotics.

During the first decade of the 21st century, there was a dramatic increase in the incidence and severity of C. difficile infections in North America and Europe. In 2008, in the Southeastern United States, C. difficile infections surpassed health care–associated infections caused by methicillin-resistant Staphylococcus aureus. Similarly, in England and Wales, C. difficile infections surpass methicillin-resistant S. aureus health care–related infections. The increase has been attributed to a hypervirulent strain that produces higher amounts of TcdA and TcdB. Normally, the two toxins are down-regulated by the TcdC protein; however, in the hypervirulent strain the TcdC protein is truncated, which leads to unregulated toxin production. The hypervirulent strain is known as group BI by restriction endonuclease analysis, NAP1 by pulse-field gel electrophoresis, and 027 by ribotype. In addition, the 027 strains are resistant to fluoroquinolones and some have the CDT toxin, increasing the virulence. Although many outbreaks have been attributed to the 027 strain, other strains have also been implicated.

Clinical Features

A frequently used definition of C. difficile infection includes the presence of more than three unformed stools in 24 hours, with a positive test (toxins or toxin genes) or the colonoscopic/histopathologic presence of pseudomembranes. However, C. difficile causes a broad spectrum of gastrointestinal diseases from mild diarrhea to toxic megacolon with perforation. Symptoms usually start shortly after the antibiotic treatment, but they may occur several weeks after antibiotics were given. Some patients present with mild to moderate watery diarrhea that may be accompanied with lower abdominal cramps and resolves after the antibiotic is discontinued. The most frequent presentation consists of fever, malaise, nausea, anorexia, abdominal pain, and watery diarrhea, which may have some blood. In the affected patients there is endoscopic evidence of erythematous colitis without the presence of pseudomembranes.

Pseudomembranous colitis is a well-known presentation of C. difficile infection. It consists of fever, abdominal pain and tenderness, severe diarrhea, and laboratory findings of leukocytosis and hypoalbuminemia. Raised yellow plaques (pseudomembranes) are seen scattered in the mucosa of the rectosigmoid area. Although less frequent, involvement can also be found in the right colon, even in the small intestine. However, it needs to be remembered that not all pseudomembranous colitis infections are due to C. difficile.

The most severe presentation of C. difficile disease is toxic megacolon, which can be complicated with perforation. Affected patients present with fever, chills, hypotension, tachypnea, and diffuse abdominal pain, and they may or may not have diarrhea. They show marked leukocytosis. In these patients, imaging shows ileus. Concomitant with the surge of the hypervirulent strains, there has been an increase in the mortality associated with C. difficile infections reported in different surveillance systems in North America and Europe. Although C. difficile appears to be an important contributor to death, in the setting of elderly, hospitalized patients with multiple comorbidities, it may not be the only contributing factor.

Note that most patients with antibiotic-associated diarrhea do not have C. difficile infection (see the section regarding differential diagnosis). Important clues to diagnosis of C. difficile infection include fever and leukocytosis. White blood cell counts (WBC) of more than 20,000 cells/μL are associated with increased mortality. Other predictors of severe disease include albumin less than 2.5 g/dL, creatinine above 2 mg/dL, and the presence of ileus or bowel obstruction. For treatment purposes the Infectious Diseases Society of America and the Society for Hospital Epidemiology of America define severe disease when WBC are above 15,000 cells/μL or there is a creatinine above 1.5 times the baseline, and severe complicated disease if there is presence of shock, hypotension, or ileus.

The epidemiology of C. difficile infection in children has not been well studied. This bacterium is frequently found as a colonizer in children younger than 1 year old and it is not recommended to test this age group. However, older children with comorbidities such as cancer, transplants, gastrostomies, or jejunostomies are at increased risk of the disease.

Pathologic Features

Few studies correlate the evidence of the presence of C. difficile or its toxins in stools and endoscopic and histopathologic findings. In adults with C. difficile –associated diarrhea, pseudomembranes were detected in 51% to 89% of patients by endoscopy and in 63% by histology. In patients with the presence of C. difficile toxin in stool, approximately one third had pseudomembranous colitis histologically, whereas 42% have suspicious histology. Correlation of positive C. difficile polymerase chain reaction (PCR) assays, the presence of toxin in stool, and histopathologic changes in biopsies have also shown a range of morphologies including no significant changes to ulcers covered by pseudomembranes ( Figure 17-1 ). The histopathology of children with positive C. difficile toxin in stools has ranged from minimal pathology in the majority of cases to moderate colitis, granulomatous inflammation, intestinal necrosis, and pseudomembranous colitis. Immunohistochemistry and PCR testing of the pediatric tissues only showed evidence of clostridia in the patient with pseudomembranes.

Pseudomembranous colitis is characterized by discrete yellow plaques in the mucosa of the colon and rectum. Histologically the colonic crypts are dilated due to abundant inflammatory cells and debris, and as they discharge this exudate into the colonic lumen they appear as erupting volcanoes. In severe cases of C. difficile –associated diarrhea, the presence of pseudomembranes may not occur if the patient is neutropenic or immunosuppressed and thus unable to produce the inflammatory response that creates the characteristic pseudomembranes. The lack of pseudomembrane formation with evidence of C. difficile toxins has been described in patients who have received hematopoietic stem cell transplantation and those with ulcerative colitis.

Ancillary Studies

Diagnosis of toxin-producing C. difficile strains has evolved from cytotoxic culture assays to enzyme immunoassays (EIA) to molecular testing. If immunoassays are to be used because of cost constraints, a tiered approach with two tests is suggested. Use an EIA for glutamate dehydrogenase (GDH) to screen, as this is an enzyme uniquely produced by C. difficile . All positive stools then should be tested with a second EIA that detects toxins A/B. Alternatively, the more expensive PCR tests that detect the toxin genes can be used. PCR tests have sensitivities above those found for EIA assays and have much shorter turnaround times compared to cultures. The American Society of Microbiology has established some guidelines for the use of PCR tests: they should only be performed in loose or liquid stools of symptomatic patients and should not be repeated in a week. The PCR assays are very sensitive and detect colonization in asymptomatic patients as well as the presence of the toxin in cured patients. Facilities should expect an increase in the frequency of C. difficile infections when transitioning from use of EIA to PCR.

Differential Diagnosis

Nosocomial diarrhea is defined as having three or more unformed stools during one day arising 3 or more days after having been hospitalized. Although C. difficile is a frequent concern in patients with nosocomially acquired diarrhea, it accounts for less than 20% of nosocomial diarrheas. Most instances of diarrhea in hospitalized patients are due to medications, enteral feeding, and underlying illnesses. The use of antibiotics may alter the gut flora and remove bacteria that produce butyrate, which is an important epithelial cell nutrient, or remove bacteria that break down complex carbohydrates or bile salts, leading to diarrhea that is not associated with C. difficile infections. Most nosocomial diarrheas are clinically mild to moderate. However, severe diarrhea can be caused by medications in patients receiving chemotherapy for neoplasias and those who have been transplanted, or those caused by noroviruses or toxin-producing Clostridium perfringens and Klebsiella oxytoca .

The great majority of cases with pseudomembranous colitis are associated with C. difficile infection; however, other infectious agents have been found to show similar macroscopic and microscopic features including viruses (cytomegalovirus), parasites (Entamoeba histolytica), and other bacteria (enterotoxin-producing C. perfringens , S. aureus, Shigella dysenteriae, Escherichia coli O157:H7, and K. oxytoca ). It is interesting to note that before C. difficile was associated with diarrhea due to antibiotics, S. aureus was thought to be the etiologic agent of antibiotic-associated diarrhea and presence of pseudomembranes in the colon and rectum. Some authors have commented that when non– C. difficile pseudomembranes occur, these are in reality ulcerated lesions covered by inflammatory membranes. In addition, in the case of cytomegalovirus and amoeba, the presence of viral inclusions or the protozoan should alert the pathologist to the appropriate diagnosis.

Prognosis and Treatment

Treatment for C. difficile diarrhea begins with discontinuation of the causative antibiotic. For patients with mild to moderate disease, metronidazole is suggested, whereas for those with severe disease, vancomycin is the drug of choice. Fidaxomicin, a macrocyclic antibiotic with minimal systemic absorption that has anti– C. difficile coverage but little effect in the normal flora, has been used as an alternative to vancomycin. In some instances, a colectomy may be necessary; however, mortality is very high in patients requiring this treatment. Asymptomatic carriage of C. difficile decreases the risk of having infection, suggesting there is natural immunity that may be conferred by nontoxigenic strains, and monoclonal antibodies against toxins A and B have been used for treatment. Other treatment modalities have included the use of probiotics and transplant of microbiota.

Patients using a room that had been previously occupied by another patient with C. difficile are at a high risk of acquiring the disease. Multiple measures have to be implemented to prevent transmission of the disease in hospitals. These include isolation of symptomatic patients, use of barrier precautions and disposable patient equipment, targeted cleaning and disinfection of rooms and all the surfaces in them, and antimicrobial stewardship.

Patients with mild and moderate disease have a good prognosis with cure rates above 90%. Patients with severe disease have lower cure rates (around 70%) with more recurrences. Also associated with the recurrence of disease is the continued use of antibiotics either during treatment for C. difficile infections or immediately after finishing treatment.

Epidemiologic Features

Clostridium perfringens is a spore-forming, gram-positive anaerobic bacillus that is notorious for its fast growth (it has a generation time of less than 10 minutes). It is widely distributed in nature with its spores highly prevalent in soil and the intestinal tract of humans and animals. A study of food items associated with foodborne disease outbreaks in France showed that C. perfringens was the most frequently cultured bacteria, accounting for 15.7% of isolates. C. perfringens was found primarily in meats, and dishes or meats with sauce. Of gram-positive anaerobes cultured in hospitals, C. perfringens is the Clostridium most frequently encountered, accounting for 20% to 30% of the isolates. There are five strain types, which are divided according to the toxin produced with more than 15 toxins known. The alpha (α) and theta (θ) exotoxins have been implicated as the major toxins causing damage in patients with C. perfringens gas gangrene infections. The α toxin acts as a phospholipase C, a sphingomyelinase and a potent platelet agonist causing intravascular thrombosis. The θ toxin is a cholesterol-dependent cytolysin, which results in cell lysis and modulates inflammatory response. The β toxin has been associated with necrotizing enterocolitis and causes endothelial cell damage by creating pores in the plasma membranes. Similarly, the enterotoxin produced by several C. perfringens strains causes cell damage by attaching to claudin-3 and -4 receptors and forming a pore that allows the influx of calcium.

Historically, gas gangrene has been an important killer during wars, as treatment of wounded soldiers has only recently included surgical debridement and use of antitoxin and antibiotics. Gas gangrene has been less common in soldiers who were wounded in wars that occurred in deserts compared to those occurring in fertile lands. Gas gangrene also occurs after natural disasters such as earthquakes and in persons who inject drugs using dirty needles. Once a wound occurs, various types of bacteria and foreign material are introduced, which in addition to causing tissue necrosis, in particular muscle necrosis, create a hypoxic environment that allows C. perfringens to grow. As the clostridia proliferate, they produce α toxin, which further decreases the local pH and the redox potential and induces thrombus formation. Vessel thrombosis does not allow neutrophils to reach the site of infection, giving rise to the usual histopathologic picture: necrosis and the presence of gram-positive bacilli but no inflammation. Lastly, the α and θ toxins are responsible for systemic toxicity and shock.

The most common niche of C. perfringens in humans is in the gastrointestinal tract, as it is found in approximately two thirds of asymptomatic healthy subjects. Enterotoxin-producing C. perfringens are found in healthy animals such as cattle, poultry, sheep, and swine, as well as in humans. Clostridium perfringens enterotoxin is responsible for food poisoning in many industrialized countries. Enterotoxin-producing strains have also been implicated in antibiotic-associated diarrhea and sporadic diarrhea; however, in the affected patients the amount of C. perfringens found is low compared to the amount needed to cause food poisoning. It is believed that the plasmid encoding the enterotoxin gene (cpe) is transferred to cpe -negative normal gut bacterial populations, thus causing the diarrheic episode.

Clostridium perfringens type C produces β toxin and causes necrotizing enteritis, an entity classically described in malnourished patients that ingest a poorly cooked contaminated meal. Occasionally, C. perfringens type A can cause necrotizing enterocolitis. The foods associated with this entity include pork, sweet potatoes, and peanuts. Other risk factors for development of necrotizing enterocolitis include diabetes mellitus, low-protein diets, a sudden intake of proteinaceous meals (usually involving pork), and high amounts of trypsin inhibitors.

Clinical Features

Clostridium perfringens causes a varied range of diseases in humans and animals. In humans, these include gas gangrene (myonecrosis), alimentary intoxication, gastroenteritis, necrotizing enteritis, acute cholecystitis, uterine gangrene, and septic shock. Gas gangrene is one of the most fulminant gram-positive infections in humans, and the predisposing conditions include crush injuries, open fractures, lacerations, knife or gunshot wounds, and injections that are contaminated with soil or in which the gastrointestinal contents contaminate the surrounding tissues. The wound site is usually abruptly painful, foul smelling, and has a serosanguineous discharge and gas bubbles. As the infection progresses, a blister forms, and the tissue liquefies and sloughs off. The necrotic tissue advances rapidly (inches per hour), and sepsis may ensue if there is no appropriate treatment. Radiologic studies show gas-filled spaces in the affected tissue such as muscle and different facial planes.

There is a clinical spectrum of gastrointestinal diseases associated with C. perfringens. These range from self-limited, enterotoxin-mediated food poisoning to fulminant, life-threatening necrotizing enterocolitis. Patients with food poisoning present with abdominal cramps, nausea, vomiting, and diarrhea that occur 8 to 12 hours after ingestion of food that is contaminated, chilled, and reheated. Patients usually recuperate in 24 hours.

Necrotizing enterocolitis, also known as pigbel, Darmbrand, or fire belly, is an often fatal syndrome characterized by ischemic necrosis of the intestines, primarily the jejunum but in some cases the colon. A chronic pigbel syndrome has been described in patients with malnutrition or obstructive strictures. The symptoms occur between 24 hours to a week after having ingested the contaminated or poorly cooked food. Patients present with abdominal pain and distention that may be accompanied by nausea, vomiting, and diarrhea. The vomitus and diarrhea may show blood. Patients are usually afebrile and may show a normal white blood cell count. On abdominal exam there is an acute abdomen with radiographic evidence of ileus. The patients may progress to perforation in the area of the necrotic intestine or shock, which is attributed to sepsis or electrolyte imbalance.

A study of 18 patients with positive blood cultures for C. perfringens showed that disease in the hepatobiliary system, such as gallbladder stones or hepatic carcinoma, was the most frequent primary focus. Others have found that cirrhosis is a predisposing condition for bacteremia. Similarly, a study of 38 patients with clostridial bacteremias showed that of 30 patients with C. perfringens 12 (40%) had acute cholecystitis or acute cholangitis, whereas cirrhosis was present in 3 patients (10%), and gangrene preceded the bacteremia in 3 patients (10%). Altered mental status is a frequent symptom in patients with systemic inflammatory response syndrome in addition to digestive symptoms. Hemolysis and low fibrinogen can accompany shock in patients with C. perfringens septicemia. Meningitis and encephalitis can occur after the patient has had bacteremia.

Pathologic Features

Presumptive diagnosis of gas gangrene can be performed by finding large, boxcar-shaped, gram-positive bacilli with minimal inflammatory infiltrate in tissue or fluid samples of the affected site. When examining tissues microscopically, the necrosis and cell damage extends beyond what is expected on gross examination. The devitalized structures show coagulative necrosis (cell architecture intact with pale eosinophilic cytoplasm and absent or pyknotic nuclei or ghost cells) with some degree of hemorrhage but little inflammation for the degree of tissue injury observed. Bubbles of air observed macro- and microscopically in solid tissues indicate the production of gas. Gram stains of the tissue with necrosis show large, boxcar-shaped gram-positive bacilli with inconspicuous spores as the organisms are in the vegetative stage. In autopsy, material spores are noticeable. Animal models in which C. perfringens is injected intramuscularly show similar changes regarding the coagulative necrosis that is explained by the presence of thrombi in vessels upstream from the injection site.

Gross examination of C. perfringens necrotizing enterocolitis specimens demonstrates patchy, hemorrhagic, ulcerated, and ischemic areas of the intestine ( Figure 17-2 ). Thrombosis of the portal vein and mesenteric vessels has been observed. Microscopically there is severe hemorrhage with necrosis that may range from only the mucosa to the entire thickness of the intestinal wall (see Figure 17-2 ). As would be expected, perforation occurs when the entire wall is necrotic. The presence of inflammation varies. Mesenteric lymph nodes may show focal hemorrhage. Immunohistochemically, clostridia are noted in the mucosal surface and lamina propria of the affected portion of the intestine, whereas the β toxin can be identified in endothelial cells. Necrotizing enterocolitis can occur in a variety of animals including poultry, foals, dogs, and piglets with similar pathologic characteristics.

In the occasional patients with endophthalmitis caused by C. perfringens after trauma or a bacteremic episode, necrosis, neutrophilic inflammatory infiltrate, hemorrhage, and the presence of abundant gram-positive bacilli in different planes can be seen in enucleated eyes. The necrosis can be so intense that structures such as the retina can be difficult to recognize.

Ancillary Studies

Diagnosis requires the integration of the clinical features including radiologic evidence of the presence of gas, evidence of necrosis in the surgical specimen, and microbiologic findings. Anaerobic cultures are the reference standard. On blood agar plates, C. perfringens colonies show a characteristic double zone of hemolysis: a narrow zone of complete hemolysis surrounded by a wider zone of incomplete hemolysis. Other biochemical characteristics, such as the production of lecithinase and the inability to produce catalase and indole, are also used for identifying C. perfringens . Once isolated, few laboratories can differentiate between the different C. perfringens subtypes or can identify the toxins.

When found in blood cultures, C. perfringens is usually isolated from the anaerobic bottle but occasionally it can be isolated from the aerobic bottle. It should be noted that not all Clostridium spp. isolated from blood are clinically relevant. A study of 68 clostridial isolates from blood determined that only 38 were clinically relevant bacteremias, and of these, 30 were C. perfringens. Some authors have used primers for 16S rRNA and toxin encoding genes in PCR reactions to detect C. perfringens in formalin-fixed, paraffin-embedded tissues.

Differential Diagnosis

Gas gangrene must be differentiated from cellulitis and other soft tissue infections caused by other bacteria. Some organisms such as streptococci can cause extensive soft tissue infections with myonecrosis; however, they rarely produce gas. Septic shock due to other bacterial infections is also in the differential diagnosis. Similarly meningitis or encephalitis has to be distinguished from those caused by other organisms particularly when a primary focus is not evident, which occurs frequently in cases with meningitis.

Gastrointestinal diseases caused by C. perfringens have to be differentiated from other infectious and noninfectious causes of gastrointestinal disease. The clinical history, including recall of food intake and epidemiologic features, needs to be taken into consideration.

Prognosis and Treatment

The single best treatment and prevention of gas gangrene is debridement or amputation of the tissue with necrosis, leaving a wide surgical margin of unaffected tissues. Reduced mortality in war theaters has been achieved by early evacuation of injured soldiers so that surgical intervention can take place. Antibiotic treatment is not enough to halt the infection. The antibiotics of choice are those that inhibit toxin production such as clindamycin and tetracycline. Most clostridia are susceptible to chloramphenicol, piperacillin, metronidazole, imipenem, and combinations of β-lactams with β-lactam inhibitors. Antitoxin has been used, but it is not commercially available. Hyperbaric oxygen therapy has also been used, but its benefit has not been completely established.

Treatment of necrotizing enterocolitis is primarily supportive, correcting the electrolyte imbalance and using antibiotics (chloramphenicol and metronidazole). Surgery is controversial in cases where the entire bowel wall is not involved. In Papua New Guinea where pigbel was the most common cause of mortality in children in the 1960s, a vaccination trial with β toxin showed protection against the disease. By 1987, a high proportion of children had been vaccinated, and, although a booster was necessary in some children, a striking decrease in the frequency of pigbel was noted.

The mortality of patients that have bacteremia ranges from 26% to 50%.

Epidemiologic Features

Clostridium botulinum is a spore-forming gram-positive anaerobic bacillus; the heat-resistant spores produced can survive in minimally or incorrectly processed foods. Although C. botulinum produces the most deadly neurotoxin known to humans, the toxin is also used as a powerful treatment for involuntary muscular disorders such as tics, dystonias, and spasticity of the urinary bladder and esophagus as well as an antiaging agent. The neurotoxin blocks the release of acetylcholine at the neuromuscular junction and causes flaccid paralysis. Botulism typically occurs after ingestion of neurotoxin-contaminated food; however, inhalation or introduction of the bacteria into a laceration can also result in disease in humans or animals.

Each C. botulinum serotype secretes a different botulinum toxin, and each of these toxins cleave the SNARE proteins in the synaptic junction at different locations. The neurotoxins consist of a heavy chain and a light chain linked by disulfide bonds. These two chains are synthesized as one chain that is posttranslationally modified (i.e., nicked). The heavy chain component is involved in binding, internalizing, and translocating the toxin into the nerve cytoplasm and synaptic vesicles, whereas the light chain component is an endopeptidase that cleaves different proteins involved in neurotransmission. These chains are coupled to nontoxic components so as to protect the neurotoxin from the environment.

Clinical Features

Four types of botulism have been described: (1) wound botulism occurs when spores are introduced into deep subcutaneous tissues (lacerations, injections); (2) infant botulism takes place when children younger than 6 months old ingest the spores, and these colonize the bowel lumen and produce the neurotoxin that is then absorbed; (3) foodborne botulism occurs when C. botulinum colonizes the gastrointestinal tract and the secreted neurotoxin is absorbed; and (4) inhalational botulism takes place when the neurotoxin is inhaled. Symptom onset and severity depend on the amount of toxin in the blood and the type of toxin produced. Symptoms include blurring of vision, dropping eyelids, slurring of speech, difficulty swallowing, dry mouth, and muscle weakness. In infants there is lethargy, poor feeding, constipation, poor muscle tone, and a weak cry. If untreated, the condition progresses to paralysis of extremities and respiratory muscles. Of note, patients do not have altered mental status or fever. These symptoms may start between 6 hours to 10 days after exposure and do not subside until new motor axons reenervate the affected muscles. As the toxin is a bioterrorism category A agent, clinical suspicion of the disease needs to be reported immediately to the state health department.

Pathologic Features

The pathologic changes observed in cases of botulism are nonspecific: central nervous system hyperemia and microthrombosis of small vessels. However, if there is suspicion of a potential bioterrorist act, an autopsy is needed to obtain samples for definitive testing of neurotoxin and as part of the medicolegal death investigation. From the epidemiologic perspective, sampling of tissues may also be necessary to examine if the toxin’s route of entry to the body can be determined.

Ancillary Studies

Clinical suspicion is paramount so that definitive diagnosis by identification of the toxin can be performed. Specimens in which this can be done include serum, vomit, gastric aspirate, and stool. The reference standard test for diagnosis of the presence of neurotoxin is the mouse bioassay in which dilutions of the specimen are injected intraperitoneally into the mouse to see if paralysis occurs. If paralysis takes place in 48 hours, a botulinum antitoxin is administered together with the lowest dilution of toxin that produced symptoms in the mouse to define specificity. This assay takes 4 days or more to render results and requires having a mouse colony and personnel proficient in this bioassay. Several variations of the bioassay and new techniques are being developed, including immunoassays for the toxin (ELISA, lateral flow rapid detection tests, chemiluminescence detection, immune-PCR, biosensors, and others). Detection of the endopeptidase activity by mass spectrometry has also been developed at the Centers for Disease Control and Prevention (CDC).

As it is the toxin that produces the symptoms and only a small amount of this is needed, detection of the organism using culture techniques is usually not useful.

Differential Diagnosis

A variety of neuromuscular disorders can be confused with botulism including Guillain-Barré syndrome, myasthenia gravis, and strokes.