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Advanced Stool Diagnos1cs

Presented by Chris.ne Stubbe, ND Medical Educa.on Specialist– Genova Diagnos.cs

GI Effects and the Evolu.on of Gut Health Assessment

•  Overview of GI Effects Comprehensive 2200

•  Microbial diversity discussion •  Individual biomarker review

•  Case Studies

Learning Objec.ves

© Genova Diagnos.cs

•  4 Func.onal Pillars •  2 Global Markers

Interpreta.on-‐at-‐a-‐Glance


•  Clinically ac.onable biomarkers highlighted in four key areas:

Interpreta.on-‐at-‐a-‐Glance: 4 Func.onal Pillars


–  Infec.on –  Inflamma.on

–  Insufficiency (Diges.ve)

–  Imbalance (Metabolic)

Interpreta.on-‐at-‐a-‐Glance: 4 Func.onal Pillars


–  Diversity Associa.on and Rela.ve Abundance graphics serve as broad markers of gut health.

Interpreta.on-‐at-‐a-‐Glance: Commensals

•  The new GI effects report assesses 24 clinically relevant microbial genera/species that map to 7 major phyla

•  Polymerase chain reac.on (PCR) amplifica.on technique is u.lized to iden.fy highly conserved 16S rRNA gene (i.e. DNA) sequences –  The method offers an enhanced DNA extrac.on method, updated

primer sequences, and op.mized thermal cycling for a wide reportable range

–  The reference ranges for individual commensals were derived from a healthy reference popula.on defined by specific clinical criteria

•  A computer algorithm maps the levels of selected commensals into the graphic representa.ons of Diversity Associa.on (DA) and Rela.ve Abundance (RA)

Commensal Bacteria

© Genova Diagnostics

!  The DAI and RA represent Genova’s initial implementation of this systems biology approach to gut microbiome diagnostics.

The Diversity Associa.on graphic represents the results of a proprietary algorithm that incorporates selected commensal targets that appear to correlate with gut health status, e.g. some genera/species have been associated with biodiversity in the literature (in the gut, higher diversity is associated with gut health).

•  A Diversity Associa.on in the lower half of the graphical bar indicates a very high likelihood that a pa.ent’s gut is not healthy

•  A Diversity Associa.on in the upper quar.le of the graphical bar indicates a high likelihood that a pa.ent’s gut is healthy

Diversity Associa.on


The Rela.ve Abundance graphic presents the propor.on of the pa.ent’s commensal targets rela.ve to the sum total of all commensal targets measured via a healthy cohort.

Significantly lower total RA may represent gut microbiota imbalance due to low beneficial commensal organisms.

Significantly higher total RA (compared to healthy cohort) may represent gut microbiota imbalance due to microbial overgrowth.

Rela.ve Abundance


Although research is evolving and the subject is complex, these same principles appear to apply to the microbial ecosystem in the human gut:

–  Greater microbial diversity is associated with the microbiome’s ability to deal with stressors such as opportunis.c pathogens or dietary perturba.ons;

–  Individuals with disease are more likely to have altera.ons in gut microbial communi.es when compared to healthy controls;

–  Mul.ple associa.ons between reduced microbial diversity and disease are being elucidated in the literature.

Source: Khanna S and Tosh PK. (2014). A clinician's primer on the role of the

microbiome in human health and disease. Mayo Clin Proc. 89(1): 107-‐114.

Biodiversity in the Human Gut


Mayo Clinical Proceedings: Diversity and Disease Associa.ons


Boeom Line: Markers of Diversity

•  ‘Diversity promotes self-‐organiza.on and healing within an ecosystem; it is a source of evolu.onary poten.al, innova.on, resilience, opportunity, and change.’

•  ‘When a system is out of balance… we find that measures such as stool culture, DNA probes, distribu.on of short chain faey acids, and β-‐glucuronidase ac.vity can give us a window into the richness/diversity and abundance of the en.re gut ecosystem.’

© Genova Diagnos.cs Source: Hanaway P. Diversity: From Diet to Flora to Life. Glob Adv Health Med. 2014 May;3(3):6-‐8.

D. I. G.

•  Pancrea.c Elastase 1 •  Products of Protein Breakdown •  Fecal Fats

D/Diges.on and Absorp.on


•  PE1 is a proteoly.c enzyme exclusively secreted by the pancreas, and serves as a noninvasive fecal biomarker of pancrea.c exocrine func.on.

•  Medical decision points are graphically displayed: –  201 µ/g to >500 µ/g normal exocrine pancrea.c func.on; –  100 µ/g to 200 µ/g moderate-‐to-‐mild exocrine pancrea.c insufficiency; –  <100 µ/g reflect severe exocrine pancrea.c insufficiency

•  Low levels of pancrea.c elastase are associated with chronic pancrea..s, gallstones, diabetes, celiac disease, cys.c fibrosis, IBD and excessive alcohol consump.on

•  If low, consider pancrea.c enzyme support with meals


Pancrea.c Elastase 1 (PE1)

•  Products of Protein Breakdown, which includes isovalerate, valerate and isobutyrate are produced by bacterial fermenta.on of proteinaceous material (polypep.des and amino acids) in the distal colon.

•  Elevated levels suggest increased protein material in the distal colon which may be due to: –  underlying gastrointes.nal condi.ons such as hypochlorhydria and

exocrine pancrea.c insufficiency (maldiges.on) –  bacterial overgrowth of the small intes.ne –  increased protein intake

Products of Protein Breakdown


•  Fecal fat measurement is an extrac.on method that provides a quan.ta.ve result of the amount of fat in the stool.

•  Fecal fat extrac.on methods have been found to correlate with degree of fat malabsorp.on.

•  Elevated fecal fat may indicate issue with diges.on or absorp.on of fat secondary to the following underlying condi.ons:

Fecal Fats


•  liver/gallbladder/bile issues •  low pancrea.c elastase 1 level •  increased transit .me •  food intolerances •  celiac disease

•  chronic use of NSAIDs •  dysbiosis •  infec.on •  inflammatory states (IBD) •  GI surgeries that limit absorp.ve

surface area

•  Calprotec.n and EPX are primary markers of inflamma.on

•  Fecal secretory IgA


I/Inflamma.on and Immunology

•  Fecal Calprotec.n is a marker of gastrointes.nal inflamma.on that is produced by neutrophils in the gastrointes.nal tract. –  Fecal Calprotec.n is FDA-‐cleared for differen.a.ng Irritable Bowel

Syndrome (IBS) from Inflammatory Bowel Disease (IBD) –  Fecal Calprotec.n is used to quan.fy degree of inflamma.on.

•  Medical decision points are graphically displayed. –  < 50 μg/g Normal, likely indica.ng no ac.ve GI inflamma.on. –  50 -‐ 120 μg/g Indicates some GI inflamma.on: IBD, infec.on, polyps,

neoplasia, NSAIDs. Treat inflamma.on and repeat in 4-‐6 weeks. –  >120 μg/g Abnormal: Determine source of inflamma.on and repeat as

clinically indicated. Referral may be indicated.

Calprotec.n


•  Fecal EPX is a marker of inflamma.on that is produced by eosinophils in the gastrointes.nal tract -‐ accumula.on of EPX in the GI tract is associated with inflamma.on and .ssue damage.

•  Clinical research indicates a significant correla.on between eosinophil mediators in stool, such as EPX, and the “gold standard” for assessing eosinophils in the gastrointes.nal tract (whole gut lavage fluids).

•  Clinically, eleva.ons in EPX indicate the presence of an IgE-‐mediated inflammatory process. Most common associa.ons are IgE-‐mediated food allergy, parasi.c infec.on, and IBD.

Eosinophil Protein X (EPX)


•  Produced across mucosal surfaces, sIgA is the first line of defense in protec.ng the gastrointes.nal epithelium from enteric toxins and pathogenic organisms.

•  Measurement of fecal sIgA may provide informa.on on the competence of mucosal immunity and the poten.al risk for epithelial barrier dysfunc.on.

•  Poten.al indica.ons for tes.ng include pa.ents with known or suspected disrup.ons of the GI epithelial barrier that may suffer from manifesta.ons of “leaky gut” such as excessive bacterial transloca.on (failure of immune exclusion) or food allergies (failures of immune inclusion and tolerance development).

Fecal sIgA


•  Metabolic –  SCFAs –  Beta-‐glucuronidase

•  Commensal Bacteria -‐ PCR –  Offers quan.ta.on of commensal bacteria –  24 bacterial genus/species targets –  Phylum F/B ra.o

•  Bacterial – culture + MALDI-‐TOF-‐MS •  Mycology – culture + MALDI-‐TOF-‐MS •  Parasitology – O&P and EIA •  Sensi.vi.es – Bacterial and Mycology •  Pathogens – Add-‐On via EIA

G/Gastrointes.nal Microbiome


•  The short chain faey acids (SCFAs) Acetate, n-‐Butyrate and Propionate are produced by anaerobic bacterial fermenta.on of indiges.ble carbohydrate (fiber)

–  SCFA (Total) reflects the sum total of these three primary SCFAs

•  Beta-‐glucuronidase is an enzyme induced by anaerobic bacteria (par.cularly E. coli, but also Peptostreptococcus, Bacteroides, and Clostridia)

G/Gastrointes.nal Microbiome-‐Metabolic


•  Total and individual SCFAs are produced by anaerobic bacterial fermenta.on of indiges.ble carbohydrate (fiber) –  Play an important role: in the salvage of unabsorbed carbohydrate; in

colonic absorp.on of electrolytes and water; as a fuel source for colonocytes; in maintenance of intes.nal barrier func.on; and support commensal bacteria

–  Altered levels of fecal SCFA can provide an indicator of an altered intes.nal microbial composi.on that has been associated with malignant transforma.on of colonic cells, inflammatory processes, and func.onal bowel disorders

Short Chain Faey Acids (SCFAs)


•  Beta-‐glucuronidase is a member of the glycosidase enzyme family that is an inducible, pH-‐dependent enzyme

•  Many toxins, hormones, and drugs are excreted from the body aver conjuga.on to a glucuronide molecule. Beta-‐glucuronidase uncouples these conjugates, freeing these poten.al carcinogens in the bowel and promo.ng recircula.on of these compounds through enterohepa.c pathways

•  Both high and low levels can be an issue: –  Limited research has noted that high levels may be related to colon cancer.

–  Low levels may decrease the release of polyphenols (like flavonoids), reducing their ability to do good

–  When high or low, consider probio.cs

–  When high, may consider calcium-‐d-‐glucarate to inhibit enzyme; or milk thistle

Beta-‐glucuronidase


•  24 Commensal molecular targets provide an assessment of the gut microbiome.

•  Diversity Associa.on and Rela.ve Abundance graphics serve as broad markers of gut health.


Commensal Bacteria

•  The gray-‐shaded por.on of a quin.le repor.ng bar represents the propor.on of the reference popula.on with results below detec.on limit.

Commensal Reference Range


•  The Firmicutes/Bacteroidetes (F/B ) Ra.o is a comparison of the two largest phyla that make up the gut microflora in human adults: Firmicutes and Bacteroidetes.

•  F/B ra.o has been associated with disrup.on of metabolic homeostasis (ie, higher extrac.on of caloric energy by the Firmicutes phyla bacteria); elevated BMI; elevated markers of inflamma.on; diabetes; non-‐alcoholic faey liver disease; and obesity.

Firmicutes/Bacteriodetes Ra.o



Tehrani A, et al. Obesity and its associated disease: A role for microbiota. Neurogastroenterol Mo.l. 2012; 24(4):305-‐311.


Culture: Bacteriology and Mycology

•  Bacterial and Mycology Culture with MALDI-‐TOF MS iden.fica.on

•  Vitek-‐MS (MALDI-‐TOF) technology is an FDA-‐cleared mass spectrometry plaxorm for the rapid iden.fica.on of bacteria and yeast from isolated culture colonies –  Matrix Assisted Laser Desorp.on Ioniza.on Time-‐of-‐Flight

•  MALDI-‐TOF MS examines paeerns of proteins detected directly from intact bacteria –  Technology ionizes chemical compounds (a combina.on

of the culture sample to be examined and an assay matrix) to generate charged molecules which provide a ‘tell-‐tale’ signature for iden.fica.on

MALDI-‐TOF MS: Bacteria and Yeast Iden.fica.on


•  Vitek-‐MS is the first FDA 510(k) cleared MALDI-‐TOF system for clinical use in the U.S. –  Covers most clinically relevant

organisms

–  Iden.fies about 200 different species –  Performance of MALDI-‐TOF MS

systems largely depends on size and valida.on of bioinforma.cs library


MALDI-‐TOF MS Primer

Bacterial and Mycology Sensi.vity


Parasitology


Add-‐Ons: EIA Pathogens & Lactoferrin



•  Fecal occult blood (FOB) iden.fies microscopic amounts of blood in the stool

–  Hemosure diagnos.c kit u.lizes immunochemical technology (FIT): has higher specificity due to its unique combina.on of monoclonal and polyclonal an.bodies that are specific to human hemoglobin.

–  FIT tes.ng is highly sensi.ve and specific-‐one posi.ve test warrants further inves.ga.on as to the source

•  Lactoferrin (LF) is an iron-‐binding glycoprotein secreted by most mucosal membranes; it is a major granular component of neutrophils (white blood cells). –  Liberated from the neutrophils in response to inflamma.on, LF binds

to iron, impeding microbial growth and facilita.ng genera.on of hydroxyl radicals.

•  Poten.al indica.ons for tes.ng are pa.ents exhibi.ng gastrointes.nal symptoms with suspected inflamma.on.

•  In studies of pa.ents undergoing colonoscopy for surveillance or for gastrointes.nal symptoms, fecal lactoferrin has been found to correlate with colorectal and intes.nal inflamma.on.

Lactoferrin Available as Add-‐On


•  Bacterial Overgrowth of the Small Intes.ne –  If pa.ent is symptoma.c for SIBO (bloa.ng, especially aver meals,

other IBS symptoms) –  If GIFx markers are elevated: products of protein breakdown, short

chain faey acids, n-‐butyrate concentra.on, fecal fats

•  IgG/IgE food An.body test –  If EPX or sIgA are elevated and there is no infec.on

•  Celiac and Gluten Sensi.vity –  If fecal fats are elevated or pancrea.c elastase 1 is low

•  NutrEval –  To assess nutri.onal status –  If not diges.ng/absorbing well, nutri.onal status may be compromised

Other tests that pair well with GI Effects


Clinical Applica.on Reviewing Case Studies


•  History of IgE food reac.ons since childhood-‐had 2 allergy aeacks requiring Benadryl last year aver ea.ng something; has never needed an epi pen, but face swelled like a balloon and O2 sats dropped; has always had nut allergies that cause vomi.ng; current IgE tes.ng showed mul.ple food and environmental allergies

•  IgG tes.ng showed mul.ple IgG food sensi.vi.es so Dr. ordered GI Effects stool test to iden.fy poten.al cause

•  Pa.ent also has eczema, .red, overweight •  Pa.ent filled out GI ques.onnaire and had liele to no

symptoms-‐ stool sample was harder/cons.pated •  She has been ea.ng more meat since elimina.ng so many

allergens, taking probio.cs with 3 strains Lactobacillus, 1 strain Bifidobacterium

Case 1: 13 year old female Allergies


Case 1: 13 year old female Allergies •  Clostridium difficile

•  Elevated EPX-‐ did have mul.ple food allergies on tes.ng

•  Elevated Fecal sIgA-‐ did have mul.ple food sensi.vi.es on tes.ng; or immune system responding to infec.on?

•  Other test abnormali.es may be resolved with addressing these above issues (will discuss on another slide)

•  Low beneficial bacteria, diversity and abundance-‐ consider broad spectrum probio.cs and prebio.cs

•  Low diversity = unhealthy gut •  Rela.ve Abundance slightly lower

than healthy popula.on


•  C difficile coloniza.on: pa.ent exhibits NO clinical symptoms (more common than C dif infec.on)

•  C dif infec.on: pa.ent exhibits clinical symptoms •  CDC recommenda.ons:

–  Tes.ng for C. difficile or its toxins should be performed only on diarrheal (unformed) stool, unless ileus due to C. difficile is suspected

–  Tes.ng of stool from asymptoma.c pa.ents is not clinically useful, including use as a test of cure. It is not recommended, except for epidemiological studies.

•  Do carriers need treatment? Perhaps if working in a healthcare facility where there is risk of infec.ng others

Cohen S, et. al. Clinical prac.ce Guidelines for Clostridium difficile infec.on in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the Infec.ous Diseases Society of Amrica (IDSA). Infec.on Control and Hospital Epidemiology May 2010: (31)5.

hep://www.cdc.gov/HAI/organisms/cdiff/Cdiff_faqs_HCP.html



Case 1: 13 year old female Allergies •  Protein products may be elevated

due to increased intake, maldiges.on or malabsorp.on-‐ pancrea.c elastase 1 is op.mal, so pancrea.c diges.on of protein is likely not the issue; may be insufficient Hydrochloric acid

•  Fecal fat total is the significant value in iden.fying malabsorp.on, individual fecal fats have minimal literature support

•  Beta glucuronidase recirculates fat soluble substances packaged for elimina.on; need for probio.cs, can be elevated in high faey meat diets, consider calcium d-‐glucarate to inhibit enzyme or milk thistle


•  Treatment approach: –  This pa.ent was treated with metronidazole for C difficile –  Gut gut healing protocol on board (glutamine, DGL, aloe, etc.) –  Probio.cs on board

•  Follow up IgG/IgE 4 months aver first food an.body test (and elimina.ng reac.ve foods) and 3 months aver GIFx showed: –  Leaky gut, but improvements in number of high food sensi.vi.es –  Total IgE remained elevated-‐ par.cularly to foods, pa.ent may not be

compliant-‐ pa.ent did go on vaca.on and ate freely –  Skin clearing up, not as itchy –  Aver ea.ng reac.ve foods on vaca.on no.ced upset stomach

•  Pa.ent adamant about not doing a follow up stool test (she’s 13)



•  Pa.ent diagnosed with collagenous coli.s (microscopic coli.s)

•  Pa.ent also has cirrhosis, smoker, history of an.bio.c use as well as food and environmental allergies

Case 2: 62 year old female Inflammatory Bowel Disease


Case 2: 62 year old female Inflammatory Bowel Disease

•  Inflamma.on significant with the red findings (yellow is considered a moderate finding)

•  Moderate Pancrea.c Insufficiency

•  Low beneficial bacteria, abundance and diversity-‐ probio.cs needed as well as prebio.cs and fiber (low SCFA)


Case 2: 62 year old female Inflammatory Bowel Disease •  Pancrea.c insufficiency-‐ watery

diarrhea can cause a low PE1 as well as other causes; pancrea.c enzymes with meals; correct underlying cause of pancrea.c insufficiency

•  Low protein breakdown and low fecal fats-‐ No clinical significance; since anaerobic bacteria make products of protein breakdown, an.bio.c use could produce a low finding

•  Calprotec.n-‐ significant inflamma.on; if this were prior to a diagnosis, referral for colonoscopy would be warranted

•  EPX can be seen with IBD as well as allergies (which she has both)

•  Low SCFA-‐ fiber and probio.cs; may consider butyrate with coli.s


Case 3: 41 year old female Celiac Disease

•  Confirmed Celiac disease

•  Not sure if avoiding gluten or not •  FOB •  Diversity in lower half likely gut is

not healthy


Case 3: 41 year old female Celiac Disease

•  PE1 can be low in Celiac

•  Fecal fats can be high in Celiac; also Celiac may have other food intolerances-‐ may check for those

•  Beta-‐glucuronidase upper end of normal, consider probio.cs, calcium-‐d-‐glucarate


•  Pa.ent is gluten intolerant-‐ feels beeer off gluten •  Experiences loose stools and fecal incon.nence-‐ on fiber

which helps

•  Prior stool test mostly unremarkable-‐ needed more fiber and probio.cs

•  Has been taking probio.cs last few years

Case 4: IBS-‐D 54 year old female



•  Fecal fats may be elevated due to rapid transit .me (looser stools)

•  Issue seems to be involving the microbiome-‐ poten.ally pathogenic yeast and low beneficial bacteria



•  Moderately elevated fecal fats with >500 PE1-‐ not an issue with pancrea.c diges.on of fats

•  Inflammatory markers look good

•  Low SCFA-‐ need fiber and probio.cs


•  Case 1: Food allergies and sensi.vi.es: address food allergies and sensi.vi.es-‐ heal leaky gut, address C dif?, probio.cs

•  Case 2: IBD: an.-‐inflammatory support, fiber, enzymes, probio.cs

•  Case 3: Celiac Disease: avoid gluten; r/o other causes of elevated FOB, probio.cs

•  Case 4: IBS-‐D: treat yeast, probio.cs •  “Clinical Cases Studies and Protocols for Difficult GI Cases”

with Jill Carnahan LiveGDX (BlastocysBs hominis, H pylori, SIBO, Celiac disease)

Case summary: Finding and trea.ng the root cause


Clinician Support

•  A literature-‐based Support Guide

•  A Gut Microbiota Clinical Associa.on Summary table

•  LearnGDX

•  LiveGDX-‐ 4th Wednesday of every month

•  Complimentary Medical Educa.on phone appointments for clinicians – 800.522.4762

•  All to support transla.on of innova.ve technology into clinically understandable informa.on


Q & A Session


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Advanced Stool Diagnos1cs

Presented by Chris.ne Stubbe, ND Medical Educa.on Specialist– Genova Diagnos.cs

GI Effects and the Evolu.on of Gut Health Assessment

advanced(stool(diagnos1cs - sync-opate.com · advanced(stool(diagnos1cs!...

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