Toxin Information and Courses from MediaLab, Inc.

Beavers C, Kern W, Blick K. Isolated acute thrombocytopenia in a 21-year-old caucasian male. Lab Med. June 2009;40(6):337-339.Bromberg MB. Immune thrombocytopenic purpura, the changing therapeutic landscape. N Engl J Med. 2006; 355:1643-1645. Glassy EF. ed. Color Atlas of Hematology. Northfield, IL: College of American Pathologists; 1998.Kwon JY, Shin JC, Lee JW. Predictor of idiopathic thrombocytopenic purpura in pregnant women presenting with thrombocytopenia. Int J Gynacol Obstet. 2007;85-88. Taghizadeh, M. An update on immune-mediated thrombocytopenia. Lab Med. 2008;39(1):51-54.Tarr PI, Gordon CA, Chandler WE. Shiga like toxin producing Escherichia coli and hemolytic uremic syndrome. Lancet. 2005;365:1073-86.Woelke C , Eichler P. Washington G, etal. Post transfusion purpura in a patient with HPA-1a and GP1a/11a antibodies. Transfus Med 2006;16:69-72. Wyrick-Glatzel J.Thrombotic thrombocytopenic purpura and ADAMTS-13: New insights into pathogenesis, diagnosis and therapy. Lab Med. 2004;35(12):733-737.

Atherosclerosis is one of the leading causes of heart disease and its presence is an important risk factor for events leading to acute myocardial infarction (AMI). In the past, atherosclerosis was described as a cholesterol and lipid storage event. Now we know it is a chronic inflammatory disorder of the arterial vessels with lipid components. Atherosclerosis begins with damage to the cells that line the blood vessels. Some possible causes of this cell injury are bacterial infection, hyperlipidemia, hypertension, glycosylated products of diabetes, cytokines from adipose tissue, or exposure to toxins such as pollution and second-hand smoke. Monocytes and lymphocytes adhere to the injured site; macrophages enter and ingest proteins and, along with modified lipoproteins, create foam cells. An inflammatory milieu results as cytokines and other inflammatory molecules become involved; foam cells and white blood cells begin secreting cytokines and metalloproteinases. Myeloperoxidase is also released by degranulated white blood cells and macrophages. As inflammation and accumulation of these products continues, fatty dots and streaks are formed on the vessel lining and the formation of plaque begins. As the atherosclerotic process continues, involved cells proliferate forming a complex extracellular matrix and a fibrous cap. If development continues, possibly over decades, the plaque formations are distributed throughout various vessels, become calcified or collagenized and make the vessel walls rigid. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

Tuomanen EI.: Pathogenesis of pneumococcal inflammation: otitis media Vaccine. 19 Suppl 1:S38-40, 2000 Pneumococci cause damage to the ear in otitis media with an association with bacterial meningitis. The pathogenesis of injury involves host response to cell wall constituents and the pore-forming toxin, pneumolysin. Release of cell wall constituents, particularly during antibiotic-induced bacterial lysis, leads to an influx of leukocytes and subsequent tissue injury. The signal transduction cascade for this response is becoming defined and includes CD14, Toll-like receptor 2, NFkB, and cytokine production. The second source of injury is the cytotoxicity of the pore forming toxin, pneumolysin. Decreasing the sequelae of otitis can be achieved by an increased understanding of the site-specific mechanisms of pneumococcal-induced inflammation.

A 63-year-old man was seen in the emergency room with the complaints of sudden onset of fever, chills, and abdominal pain, accompanied by mild diarrhea. The blood pressure was 140/84, the pulse rate 82/minute, and the body temperature 39.8C. A blood sample was drawn for a complete blood count, and a blood culture. A second blood culture was drawn from the opposite arm, with 10 mL of blood being placed into each an aerobic and an anaerobic bottle, following customary practice. The complete blood count revealed a hemoglobin of 15.8 mg/dL, a hematocrit of 45%, and a white blood count of 4.2/L. The neutrophils were 39%, lymphocytes 45%, monocytes 10%, eosinophils 4% and basophils 2%. The platelet count was 255/L. The patient was admitted to the hospital for further work-up and empiric antibiotic therapy. Within 24 hours after admission, the body temperature had decreased to 38.2C, although the mild diarrhea persisted. A stool toxin test for Clostridium difficile was negative and neither enteric pathogens nor Campylobacter species were recovered in stool culture after 24 hours incubation. Fecal neutrophils were not seen on direct examination. The anaerobic blood culture became positive 36 hours after inoculation.

Biological agents are organisms or toxins that can kill or incapacitate people, live stock, and crops. The three basic groups of biological agents that would likely be used as weapons are bacteria, viruses, and toxins. Biological agents can be dispersed as aerosols or airborne particles.

Category B agents include:Q Fever (Coxiella burnetii)Brucellosis (Brucella sp.)Glanders (Burkholderia mallei)Venezuelan encephalomyelitisEastern and western equine encephalomyelitisRicin toxin from castor beans (Ricinuscommunis)Epsilon toxin of Clostridium perfringensStaphylococcus enterotoxin B

There are four primary agents that could possible be used in a chemical attack: Lung-damaging or choking agents Blood agents Blister agents Nerve agentsOthers that might be used include: incapacitating agents, riot-control agents, heavy metals, volatile toxins, pesticides, dioxins, explosive nitro compounds and oxidizers, flammable industrial gases and liquids, plus corrosive industrial acids and bases.

Clostridium difficile is the cause of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (PMC). PMC is an inflammatory disease of the colon caused by toxins of C. difficile.C. difficile produces two potent toxins: Toxin A (TcdA), an enterotoxinToxin B (TcdB), a cytotoxin It is the production of these toxins in the gastrointestinal tract that ultimately leads to disease. There is a relationship between toxin levels, the development of pseudomembranous colitis (PMC), and the duration of diarrhea. For many years, toxin A was regarded as more important than toxin B in the disease process. Later on, disease producing strains producing only toxin B were identified. These strains produced serious disease, and toxin B was found to be responsible for more serious damage to intestinal cells.

Toxin assaysThe most common laboratory tests for the detection of C. difficile are enzyme immunoassays (EIA) for the detection of C. difficile toxin A and toxin B. The immunoassays are simple to perform, provide rapid results, and are easily incorporated into the workflow of most laboratories. Sensitivities of these tests do NOT compare favorably to culture, cell cytotoxicity neutralization assay (CCNA), or molecular methods. There are many test kits commercially available for detection of C. difficile toxins. Results are available in 15 minutes to 2 hours, depending on the assay. Initially, toxin A was thought to be the toxin responsible for the majority of the effects of C. difficile disease, so most early kits only detected toxin A. With the realization that there are strains that produce aberrant or no toxin A (A-) that are known to produce infection, and more recently toxin B negative (B-) strains, it is now recommended to use kits detecting BOTH toxins.Glutamate Dehydrogenase (GDH) assaysPublished studies have indicated that toxin immunoassays, by themselves, may not provide adequate sensitivity of detection. GDH assays initially attracted attention as a possible means to provide a rapid but more sensitive means for screening for C. difficile.GDH is an enzyme produced by C. difficile. EIAs negative for the GDH antigen have been associated with high negative predictive values. However, positive results are not necessarily associated with a toxin producing strain. A second assay on GDH positive samples is required to confirm the presence of a toxigenic strain. Initially, CCNA assays were recommended as the confirmatory method of choice; molecular methods (PCR for the toxin gene) were subsequently explored for this purpose.

Staphylococci are non-motile, non-spore-forming, gram-positive organisms occurring singly, in pairs, tetrads or in clusters resembling grapes. More than 20 species have been identified; three species are significant in their interactions with humans - S. aureus, S. epidermidis and S. saprophyticus.The staphylococci are members of the normal flora of the skin and mucous membranes of humans and warm-blooded animals. Colonization of the nares (nostrils) and skin can provide large reservoirs of organisms for transmission. Approximately 25-30% of the general population are colonized by Staphylococcus aureus, mainly in the nasal passages, but the organism can be found in most anatomical sites including the skin, oral cavity and GI tract.Infections are frequently acquired when the colonizing strain gains access to a normally sterile site as a result of trauma or abrasion to skin or mucosal surface. S. aureus infections range from superficial, localized skin infections, such as folliculitis, to deeper, more serious skin lesions and the more serious toxin mediated conditions – scalded skin syndrome and toxic shock syndrome.

In general, the infection that develops is dependent on the virulence of the particular strain, the inoculum size, and immune status of the host. Staphylococcal infections are typically suppurative, producing abscesses filled with pus and damaged leukocytes surrounded by necrotic tissue. Skin infections range from superficial - boils, carbuncles and furuncles, to bullous impetigo; largely opportunistic infections that develop as a result of previous injury e.g., cuts, burns, surgical wounds - and scalded skin syndrome (extensive exfoliative dermatitis; also known as Ritter's Disease). Other major infections include pneumonia, osteomyelitis (localized infection of bone), and septic arthritis. S. aureus also causes food poisoning as a result of ingestion of food contaminated with an enterotoxin producing strain (enterotoxins A&D) and the potentially fatal toxic shock syndrome, a multisystem disease most often associated with the use of highly absorbent tampons. Toxic shock syndrome is attributed to another toxin (enterotoxin F – TSST1) released by certain strains of S. aureus.Human staphylococcal infections usually remain localized by the normal host defenses. Foreign objects (fomites) such as sutures or intravenous (IV) lines - are readily colonized by S. aureus from skin and can allow the organism to spread systemically via the blood stream – bacteremia/septicemia - leading to more serious infections. Staphylococcal pneumonia is becoming a frequent complication of influenza. Whatever the mode of entry, the invasive nature of S. aureus always poses the threat of more serious deeper tissue invasion and/or bacteremia and hematogenous spread.

Several laboratory methods are currently available to aid in the detection of C. difficile including culture for toxigenic C. difficile (considered the "gold standard" for viable C. difficile detection), detection of Toxin A, B, or both, and molecular detection methods. These methods differ in their sensitivity and specificity and should always be used in conjunction with clinical considerations. To make the diagnosis, it is usually only necessary to submit 1-2 diarrheic (non-formed) stools per episode. Once positive for C. difficile by any laboratory method, there is no need for follow-up assays to make sure the organism or toxins are absent from the initial episode. If assays are performed for subsequent episodes, culture or tissue culture assay for Toxin B are probably most appropriate to avoid the possibility of detecting the initial antigen, toxin, or gene.

Stool culture is very effective in detecting C. difficile. Unfortunately, non-toxigenic strains will also grow, requiring strains to be tested for toxin production. The greatest disadvantage to culture is the length of time that is needed before results are available, which may be up to four days. However, antibiotic sensitivity testing following culture is useful for strain-typing that would provide necessary epidemiological information during nosocomial outbreaks.Colonies of C. difficile will appear white, flat, and spreading on blood agar (see top image on the right). Cycloserin- cefoxitin-fructose agar(CCFA) is a selective media that is used for isolation of C. difficile. There is however, no distinction between pathogenic and commensal strains, which all produce yellow colonies with a characteristic "ground glass" appearance. as shown in the bottom image on the right. The characteristic odor of "horse manure" aids in identification of C. difficile. Stool samples are directly inoculated onto CCFA and incubated in an anaerobic atmosphere at 37°C for 48 hours. Large, thin, gram-positive bacilli with spores will be observed on a Gram stain of a typical colony, as shown below.

The Cell Cytotoxicity Neutralization Assay (CCNA) was developed to detect the presence of C. difficile toxin in fecal samples.In this assay, a filtrate of stool sample is prepared and inoculated onto sensitive tissue culture cells. Typically human fibroblast cells are utilized; if toxin is present in the filtrate, it causes the fibroblasts to round up in a characteristic cytopathic effect.To verify that the cytopathic effect is in fact caused by C. difficile toxin (and not by some other toxic component or viral agent) the filtrate is also inoculated in parallel onto a second set of tissue culture cells, to which C. difficile specific anti-toxin has been added.Absence of the cytopathic effect in the second set of cell cultures provides evidence that the cellular changes in the first set were caused by C. difficile toxin.Although CCNA is considered a gold standard for the detection of C. difficile toxin, it is labor intensive, requires the use of cell cultures, and requires at least 48 hours incubation.

Clostridium are gram-positive or gram-variable, spore-forming, catalase-negative anaerobic bacilli. More than 100 species are currently recognized, though relatively few are encountered in properly collected clinical specimens from humans. There are three types of infection associated with Clostridium species: Non-invasive: Toxin-mediated Invasive: Progressive infection with tissue destruction Purulent disease: Closed space (e.g., in the peritoneal cavity) mixed infection with multiple organisms.Clostridium are well known as the agents of these classic toxin-mediated diseases : DISEASE TOXIN INVOLVED CAUSATIVE ORGANISM Tetanus or "lock jaw" Tetanospasmin Clostridium tetani Myonecrosis/Gas gangrene Exotoxins Clostridium perfringens Botulism (severe food poisoning) Botulin Clostridium botulinum

Clostridial toxins are among the largest bacterial toxins reported to date and C. difficile produces two potent toxins: Toxin A ((TcdA), an enterotoxin and Toxin B (TcdB), a cytotoxin. It is the production of these toxins in the gastrointestinal tract that ultimately leads to disease. There is a relationship between toxin levels, the development of pseudomembranous colitis (PMC), and the duration of diarrhea. Levels of Immunoglobulin G against TcdA correlate directly with protection from disease following colonization, suggesting that a robust immune response is sufficient for protection from C. difficle-associated diarrhea (CDAD). The role of TcdB is not as well understood. Naturally occurring Toxin A negative/Toxin B positive (TcdA-TcdB+) strains have been identified from clinical isolates, which are capable of causing disease, even extensive PMC, suggesting a role for TcdB in CDAD. Toxin A had always been regarded as more important than Toxin B in infection. However, recent work utilizing mutant C. difficile, strains which did not, or could not produce Toxin A, and which were capable of producing very serious disease has led researchers to completely rethink the roles of Toxin A and Toxin B in CDAD. Toxin B was found to be responsible for the more serious damage to intestinal cells. In addition to the primary virulence factors (Toxin A and Toxin B ), Clostridium difficile also produces a third toxin, binary toxin (CDT). The prevalence of CDT in clinical isolates varies widely and its clinical relevance and role in pathogenicity are still not well defined.

In the early 2000's researchers in Quebec, Canada noticed an increase in the number of colectomies being performed as a result of an increase in the frequency and severity of CDAD. At around the same time, doctors at the Centers for Disease Control and Prevention (CDC) were receiving reports of increased frequency and severity of disease in the United States. There were also reports of more disease and more severe forms of C. difficile infection in other areas of the world, suggesting that the experience was very widespread and possibly global. In 2004, analysis of this hypervirulent strain showed a very characteristic strain that had previously been rare but was responsible for the majority of the more serious outbreaks. This strain – BI/NAP1 /027 – has several designations depending on which biological property was examined :- BI: Restriction Endonuclease Analysis (USA)- NAP1: North American PFGE Type 1 based on polyacrylamide gel electrophoresis (USA) - 027: Ribotype 027 by polymerase chain reaction (Europe)There are 5 unique features associated with this strain – It produces the classic toxins A & B, but faster and at much higher levels than other strains. It is Toxinotype III in contrast to the more typical clinical isolates, which tend to be Toxinotype 0. tcdC is deleted from the PaLoc, possibily explaining the observed increase in toxin production. It produces the binary toxin CDT, but its role is still unclear. It exhibits high level in vitro resistance to fluoroquinolones.

Each facility that stores and transports hazardous materials must have a written, detailed security plan. The Select Agents and Toxins Security Information Document that was prepared by the Centers for Disease Control and Prevention (CDC) and the U.S. Department of Agriculture, Animal and Plant Health Inspection Service (APHIS) is an excellent resource to use for developing a security plan that would apply to category A infectious substances. This document can be found at http://www.selectagents.gov/resources%5CSecurity%20Information%20Document.pdfThe current version, dated March 8, 2007, is available in this course as a resource. However, because the document does undergo revisions, it is recommended that the URL given above be checked periodically for document updates.

Drugs and toxins including therapeutic drugs and drugs of abuse may be present in the plasma. Other substances too numerous to mention are also present in plasma.

Toxin Comment Most Likely Means of Dissemination Primary Route of Entry General Signs and Symptoms Laboratory Testing Botulism toxin: Gram stained image of C. botulinum courtesy of CDC Produced by Clostridium botulinum Could be purified and used in a bioterrorist event to contaminate food or aerosolized to cause disease Aerosol Food contamination Inhalation Ingestion Difficulty speaking or swallowing Blurred or double vision Drooping eyelids (ptosis) Dilated pupils Dry mouth, decreased gag reflex Weakening of the reflexes (hyporeflexia) Abnormal sensations such as numbness, tingling, and progressive arm or leg weakness Flaccid paralysis Culture, anaerobic Digoxigen-labeled IgG ELISA to detect A, B, E, and F toxins Mouse Bioassay for all toxin types and to confirm DIG ELISA Ricin toxin: Extracted from Castor beans Inhibits protein synthesis Causes death approximately 72 hours after initial exposure As an aerosol Inhalation Fever Cough Chest tightness Dyspnea Cyanosis Gastroenteritis Necrosis Antibody detection in clinical specimens Clinical testing not performed unless known exposure has occurred

Brucellosis: Organisms survive well in aerosols, resist drying, and are highly infectious As few as 10-50 cells can be an infectious dose Mellioidosis/Glanders: Only a few aerosolized organisms are needed to trigger diseases that could potentially carry a high mortality rate Poisoning (Ricin Toxin): Source material, castor beans, is widely available Aerosolized or introduced contamination of a food supply could lead to mass casualties