SPRING 2010 dialog

Scientific Support Clostridium Species Reported in Stool Analysis at DDI by Chuck Masur, MD and David Quig, PhD

any DDI clients have responsiveness. Rao AV et al with a diagnosis of regressive- asked for more (2009) reported that, in patients onset autism, and who had a information on the with chronic fatigue syndrome, history of antimicrobial therapy, M various clostridium there was a significant decrease with vancomycin 500 mg/d species that may occupy large in anxiety symptoms (Beck for 8 weeks. Communication David Quig, PhD portions of the ecosystem that Anxiety Inventories) in patients and behavior improved but, Vice President is the human gastrointestinal taking probiotics compared to unfortunately, did not endure— tract. In response, DDI will controls (p=0.01). two weeks after the trial ended, assist the clinician with a Members of the clostridium most had regressed and by 8 stepped approach to addressing species may be shown to play months all but one had returned this issue. Non-pathogenic yet-to-be-elucidated roles in to baseline levels. clostridium species now are health and disease, for example, Clostridium is an anerobic reported in the “beneficial/ as mediators in the gut-brain genus of bacteria and, as such, expected” section of our connection, in the evolution of does not grow in the more standard microbiology panel. psychiatric illness (e.g. anxiety aerobic environment found We will continue to offer our disorders), in the prevention in the distal colon. However, Clostridium difficile Toxins A of immune dysfunction and in all clostridia do produce and B panel as an accurate acquired neurodevelopmental endospores, many of which can Chuck Masur, MD assessment for the presence of delay (e.g. autism and the survive for long periods under C. difficiledisease. In addition, autism spectrum disorders, or aerobic conditions. The new DDI is pleased to announce the ASD). The basic fact remains, DDI Comprehensive Clostridium roll-out of the Comprehensive however, that most clostridia Culture uses special media and Clostridium Culture panel that are non-pathogenic and are a anaerobic culture conditions to includes identification of more “commensal” component of the isolate and identify over twenty than twenty expected and gastrointestinal microbiota. clostridium species. Clostridia dysbiotic clostridium species. Some authors (e.g. Bolte also produce specific toxins, These cultures are performed E, 1998) believe that certain two of which (C. difficile toxins under anerobic conditions using species of clostridium produce A and B) are specifically special media optimally suited one or more toxins that may analyzed at DDI. for the growth of clostridia. be factors in the development There are approximately Barb Berta, MS, RD Non-pathogenic (commensal) of some cases of autism. 100 different clostridium bacteria in the human gut Interestingly, clostridia code for species, most of which are play significant roles in the their toxins on genes located — continued on next page Doctor’s Data, Inc. maintenance of health as in plasmids; these short bits of 3755 Illinois Avenue well as in protecting the body DNA can be easily transferred CONTENTS St. Charles, IL 60174-2420 against disease. For example, from one organism to another Clostridium Species Reported 1 800.323.2784 (US & Canada) Huang et al (2005) showed and can thereby confer on a new 630.377.8139 (Elsewhere) that commensal microbiota organism the ability to make Behind the Scenes at DDI 3 44.(0)8712.180.052 (UK) influence steps in CD4 T cell toxins where no such ability What’s New at DDI 3 630.587.7860 (fax) differentiation moderating basal existed previously. Sandler et [email protected] TCR signaling and immune al (2000) treated 11 children Frequently Asked Questions 4 www.doctorsdata.com SPRING 2010 DOCTOR’S DATA INC.

Clostridium Species Reported — continued from page 1

benign inhabitants of the C. difficile can be a C. bolteae, C. histolyticum, normal human gut, although a commensal organism in up C. limosum, C. bifermentans, few are well-known pathogens. to 5% of the population; long- C. novyi A, C. sordellii, C. Clostridia are best known for term institutional care is an subterminale and others their pathogenicity in disease independent risk factor for that are included in the DDI entities such as food-borne colonization. Pathogenic C. Comprehensive Clostridium illnesses, wound infections, difficile strains produce several Culture. For further pseudomembranous colitis and toxins, the best known being discussion regarding the gas gangrene. The four main toxins A and B, both of which potential role of clostridium species that are pathogenic in cause diarrhea and inflammation species in autism and the humans are: and both of which can be tested autism spectrum disorders, • C. botulinum—produces a for at DDI by using the “C. the work of Finegold SM toxin found in food and in difficile toxins A and B” test as et al (e.g. 2003. 2004, wounds; causes botulism; a stand-alone or as an add-on 2008) provides excellent Non-pathogenic • C. difficile—found normally to other stool tests. background in that regard. in small quantities in the gut C. perfringens (once called Clostridia generally are clostridium but can overgrow, especially C. welchii) is ubiquitous in nature resistant to antibiotics, and, species now are with prolonged use of and is the third most common in most cases, antibiotic some antibiotics; the most cause of food-borne illness in the treatment of an overgrowth reported in the serious cause of antibiotic- US and UK. It also causes gas of clostridium species “beneficial/expected” associated diarrhea; produces gangrene. When present in food is not warranted. Also, toxins that can cause it is very easily killed by heating considering the potential role section of Doctor’s pseudomembranous colitis; to at least 74˚C; the bad news of plasmids in transferring • —can cause is that the spores can withstand various capacities, including Data’s standard C. perfringens a variety of symptoms and cooking temperatures and antibiotic resistance, from microbiology panel. illnesses ranging from food will germinate if cooked foods one organism to another, poisoning to gas gangrene; are left standing. Antibodies the use of antibiotics in the the organism that is used in to the toxins of food-borne C. treatment of clostridium place of yeast when making perfringens are common and overgrowth should be “salt rising” bread; and most infections are sub-clinical. considered cautiously. Some • C. tetani—found in soil and C. tetani is common in soil strains of probiotics such as wounds; produces toxins that and usually enters the host lactobacillus acidophilus may causes tetanus (tetanus is through a wound in the skin be useful in inhibiting such an the only vaccine-preventable where, once established, it overgrowth; sacharomyces illness that is infectious but produces the toxins that cause boulardi, a non-pathogenic not contagious from person tetanus. Eleven strains of C. yeast, may be of some to person). tetani have been identified and additional utility in this all strains that produce toxin same regard. For further C. botulinum produces seven produce the same neurotoxins information about the role of types of neurotoxins (A-G) with (by weight tetanospasmin is clostridium species in human most strains producing only one of the most potent toxins health and disease, or for one type but some strains known – only botulinum toxin complete citations on any of may produce several toxins. and the exotoxin produced by the publications mentioned Importantly, C. botulinum may Corynebacterium diphtheriae are in this article, please contact transfer neurotoxin genes to more toxic). the Scientific Support other clostridia—this is of Several other species are department at DDI.  concern because methods of interest especially to those designed to kill or inhibit C. clinicians treating patients with botulinum may not always work neurodevelopmental disorders well with other clostridium such as those on the autism species. spectrum. These species include

2 O dialog

OH Behind the Scenes at DDI— NH2 Meet Dean Bass, Assistant Laboratory Director

HO r. Dean Bass’ career has into the environment via feces. polyaromatic hydrocarbon focused on research Dr. Bass’ current projects at DDI compounds in coal using and operations in include improving laboratory high performance liquid Dthe areas of atomic accuracy and automation plus chromatography. He received spectroscopy, including trace developing reference ranges his Master’s and Doctorate at metal analysis, metal speciation, for trace metals in animals. He the University of Texas at Austin and neutron activation. He has has presented his findings in studying trace metal analysis used his expertise in a variety clinical journals and at numerous using thermal desorption of projects to improve the scientific meetings. In addition to mass spectrometry. As an analysis of hair, urine, blood and his role at Doctor’s Data, Dean applications scientist for Hitachi fecal testing for trace metals. also is part of the research team Instruments, he worked on trace He has developed a program at Argonne National Laboratory metal analysis applications to test water for the presence where his projects have been and contributed to the design of toxic metals which supports used to support the Department of trace metal analysis and enhances doctors’ clinical of Energy, the Environmental instruments. He also teaches findings related to metal toxicity. Protection Agency, the Army, the graduate and undergraduate Dean also has been involved with Navy, the Centers for Disease level chemistry at the Illinois the development of programs to Control and prevention, and Institute of Technology. He has test special samples, other than Homeland Security. 26 publications and has given in blood, urine or stool. He has Dr. Bass’ undergraduate over 60 presentations.  studied dental amalgams and research at the University the resultant release of mercury of Iowa looked at analyzing

What’s New at DDI— by David Quig, PhD DNA Oxidative Damage the damaged residues which a sensitive indicator of overall (urinary 8-hydroxy- are then excreted in urine. intracellular GSH status, cell Urinary 8-OH-d-G is a sensitive viability and the ability of cells 2’deoxyguanosine) biomarker associated with many to withstand toxic challenges. disease states including cancer GSH is important in many DNA OXIDATIVE DAMAGE— (e.g. prostate), and chronic biological processes including Oxidation of DNA results in diseases (e.g. cystic fibrosis, detoxification of xenobiotics, damage to guanosine residues atopic dermatitis, diabetes, removal of reactive oxygen and in the formation of rheumatoid arthritis and a species, regulation of the redox 8-hydroxy-2’-deoxyguanosine wide variety of neurological state of cells, the oxidative state (8-OH-d-G). Elevated levels of conditions including Parkinson’s, of protein sulfhydryl groups 8-OH-d-G in urine provide a Alzheimer’s and Huntington’s and the regulation of immune quantitative assessment of diseases). Moderately elevated function. Low levels of GSH have ongoing oxidative stress in the levels of urinary 8-OH-d-G are been reported in cardiovascular body. Chronic inflammatory associated with inadequate disease; cancer; AIDS; autism; processes and environmental intake of carotenoids, alcoholism; neurodegenerative factors such as toxic metals, antioxidant-rich foods and diseases; and chronic retention chemicals, ionizing radiation; supplemental antioxidants. of toxic metals, chemicals and life style choices such as some pharmaceutical agents. smoking and recreational Erythrocyte Consequences of insufficient drug use; and the use of some GSH include oxidative damage pharmaceuticals have been Glutathione (GSH) to proteins, membrane lipids associated with increases and DNA.  in the hydroxyl radical that ERYTHROCYTE has been implicated in the GLUTATHIONE (GSH)— oxidation of DNA. Enzymatic The level of total reduced and DNA repair mechanisms cut out oxidized GSH in erythrocytes is 3 SPRING 2010 DOCTOR’S DATA INC. dialog

Frequently Asked Questions

Q&A > On the DDI Comprehensive > I’ve heard that DDI is > Why does DDI always Stool Analysis please explain offering a new clostridium recommend ordering an the clinical significance of culture panel; what is this unprovoked urine toxic finding “no growth” of yeast and how is it different metals test prior to an in the Mycology (Yeast) from “Clostridium spp.” as initial provoked urine test? Culture section yet yeast reported under Bacteriology . . . DD, Raleigh, NC cells are reported in the Culture and from the Microscopy section? Clostridium difficile Toxins David Quig, PhD responds: . . . AS, Phoenix, AZ A and B test? The retention (body-burden) . . . SZ, Miami, FL of toxic metals can be best Barb Berta MS, RD responds: estimated by comparing the Fairly often no yeast may be Barb Berta MS, RD and levels of metals in urine before cultured yet we see yeast cells Chuck Masur MD respond: and after administration of a under microscopy in quantities Clostridia are common and metal binding agent/chelator. of “few” to “many”. These cells expected commensal bacteria The unprovoked specimen is could be dead organisms (e.g. in almost everyone. The new important because it reveals coming from a food source) or Comprehensive Clostridium current exposure to metals that they could be viable but very Culture reports all Clostridia are excreted by endogenous slow-growing and thus not species cultured from the stool detoxification processes. The showing up in the 3 to 5 day specimen including potential objective measure of metals culture period. Either of these pathogens such as C. botulinum, currently being excreted permits scenarios requires the clinician C. difficile, C. perfringens and calculation of the difference to ask more about diet and C. tetani. If C. difficile were to between the pre- and post- about symptoms that might be isolated on this panel, a C. provocation specimens, the relate to yeast overgrowth. A diff Toxins A and B test would estimate of bioaccumulation of clue that yeast might be an be done automatically and at no metals over time. For the most issue, even though there is no additional charge to the client. accurate estimation of metal evident growth, is a low level retention the unprovoked urine (2+ or less) of Lactobacillus or A report of Clostridium spp. specimen should be collected overall low levels of beneficial/ under Bacteriology Culture in very close temporal proximity expected bacteria. Typical simply indicates that one or to the provoked specimen. symptoms of yeast overgrowth more species of clostridia were Without both unprovoked and include headaches, sugar cultured from a stool sample; it provoked results, one is subject cravings, white coated tongue, does not specify any particular to a valid criticism that the diarrhea/constipation, rectal organism (pathogenic or not). provoked results might only itching, bloating, abdominal pain A clinician might “add on” a reflect a recent acute exposure and fatigue.  Clostridium difficile Toxins A to metals.  and B test if a patient were to present with unexplained profuse diarrhea, with mucous and We appreciate your continued sometimes blood, which often interest and urge you to CONTACT US is accompanied by fever and contact us should you have any abdominal pain. Patients at risk questions. Our Customer Service Phone: for contracting C. difficile are Department is available between 800-323-2784 those who have been on rounds 8:00 and 6:00 CST Monday or 630-377-8139 of antibiotic therapy, in contact through Friday. with animals, hospitalized, or Fax: visiting or working at a chronic 630-587-7860 care facility.  Email: [email protected]

3755 Illinois Avenue, St. Charles, IL 60174-2420 4 800.323.2784 (US & Canada) • 630.377.8139 (Elsewhere) • 44.(0)8712.180.052 (UK) 630.587.7860 (fax) • [email protected] • www.doctorsdata.com