Parasitology Stool DiagnosticsFrom Basics to Blastocystis

Christine Stubbe, ND FABNOGenova Medical Education Specialist

Lahnor Powell, ND, MPHMedical Education Specialist for Genova Diagnostics

Christine Stubbe, ND, FABNOMedical Education Specialist for Genova Diagnostics

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Parasitology Stool DiagnosticsFrom Basics to Blastocystis

Christine Stubbe, ND FABNOGenova Medical Education Specialist

• Learn about the most common parasites, risk factors, and clinical presentation

• Discuss Genova’s parasitology testing, including PCR detection

• Learn about the significance of Blastocystis subtyping

• Apply test results using patient case studies

Learning Objectives for This Presentation

• Basic definition:– A microorganism that causes, or can cause disease

• Pathogenic agents include:– Parasites

– Bacteria

– Yeast

– Viruses

• Ongoing debate – depends on organism virulence factors and host health status

What is a Pathogen?

• CDC definition:– A parasite is an organism that lives on or in a host organism and gets its food from or at the

expense of its host

• Are all parasites pathogens? No, not all parasites cause disease

• Are all parasites found in the intestines? No, many are, but some found on skin (lice), in blood (malaria parasites), etc…

• 3 types that can cause disease in humans:– Protozoa

– Helminths (worms)

– Ectoparasites (lice, bedbugs, etc.)

What is a Parasite?

https://www.cdc.gov/parasites/about.html

List of Stool Parasites

• Protozoa– Single celled organisms

– Microscopic

• Helminths – Multicellular organisms that can be seen

with the naked eye

– Ova (eggs) are microscopic

Risk Factors

• Patient living conditions and hygiene (crowded conditions, schools, drinking water, or hand-washing)

• Region of the world (GDX is an international company; majority of tests from USA and UK)

• Travel history (to endemic areas)

• Outbreaks (mainly through food)

• Use of feces as fertilizer

• Immune status (HIV or cancer)

• Age (infants or elderly)

• Childcare workers and children

• Exposure to animals

• Consumption of raw or undercooked pork, beef, or seafood

• Backpackers, hikers, campers, or swimmers drinking untreated water

• Exposure to feces through sexual contact

Symptoms associated with parasitic infections

• Diarrhea

• Abdominal pain or discomfort

• Bloating

• Nausea/vomiting

• Rash

• Anal itching

• Weight loss

• Loss of appetite

• May be asymptomatic

• The pre-test probability of having a parasite will vary from patient to patient due to risk factors

• Genova is an international laboratory, and receives samples mainly from developed countries; therefore pre-test probability of having a parasite is lower as compared to developing countries – Developed countries: protozoan parasites more common than helminths, but not prevalent due

to adequate public sanitation standards

– Developing countries: intestinal helminths and protozoan parasites are prevalent

Key Points

Genova’s Parasite Testing Methodologies

• Ova and Parasitology (O&P) Microscopy

• Macroscopic Exam for Worms

• Enzyme Immunoassay (EIA)

• Polymerase Chain Reaction (PCR) – qPCR and Next Generation Sequencing

Microscopic Ova & Parasite testing is the gold standard and currently is the only methodology capable of detecting any and all stool parasites

• Ideal for– Finding any and all parasites in stool

– Visualizing other findings including WBC, Charcot-Leyden crystals, etc…

• Limitations include– Difficult to visualize very small organisms that can be obscured

– Different organisms shed ova at different times (increased sensitivity with multiple stool samples – 3 day v 1 day)

– Training of the technician

Methodologies – O&P Microscopy

Methodologies – O&P Microscopy

GDX MicroscopyO&P MicroscopySample centrifuged

Slides prepared- Staining added- High powered fields

Looking for microscopic parasites (protozoa), ova of worms, RBCs, and/or Charcot-Leyden crystals

Microscopic O&P Results

• Reported as amount seen:

– Rare 1-2 per slide

– Few 1-2 per HPF

– Moderate 2-5 per HPF

– Many >5 per HPF

• Additional Findings

• Worms are primarily diagnosed by ova in the stool, which would be identified during the microscopic O&P exam

• The macroscopic add-on test is appropriate for patients who see worms in their stool; the patient must pull the worm out of the stool and make sure it is in the green-top container – it is preferred that they write ‘WORM’ on the container

• Macroscopic exam for worms must be marked on the requisition form

• The technician will perform a gross examination on the specimen to look for macroscopic evidence of parasitic infection prior to doing the microscopic examination

Macroscopic for Worms Test

• While pinworm eggs can be seen in a stool sample submitted for O&P exam, there is often a low yield

• The best way to diagnose is the “tape test,” or “Scotch tape test”

• The eggs stick to the tape and the tape can be placed on a glass slide and viewed microscopically

• Slides are obtained from the clinician, and Genova can analyze the slides sent in as a Direct Para evaluation

Pinworm Testing

Kucik CJ, et al. Am fam phy. 2004;69(5):1161-68.

CDC. https://www.cdc.gov/parasites/pinworm/. Updated 2013.

Huh S, et al. https://emedicine.medscape.com/article/225652-workup. Updated 2018.

Enzyme Immunoassay (EIA) detects a specific organism’s antigen

• Ideal for:– Can increase sensitivity of detecting some organisms

– FDA-cleared assays available

• Limitations include:– Limited to the individual organism being targeted

– May have higher false positive rates

– Commercial products only available for 3 parasites

Methodologies - EIA

EIA Test Results

• Results reported as negative (In Range) or positive (Out of Range)

• The diamond means FDA-cleared

Polymerase Chain Reaction (PCR) detects a specific organism’s DNA. Real time PCR (a.k.a. quantitative PCR, or qPCR) for detecting 6 organisms. Next-Generation DNA sequencing for Blastocystis subtypes.

• Ideal for– Those organisms that shed ova irregularly

– Can increase sensitivity of detecting some organisms

• Limitations include– Limited to the individual organism being targeted

– Limited number of FDA-cleared targets (4)

Methodologies – PCR

Garcia LS, et al. Clin Microbiol Rev. 2018;31(1).

• Laboratory Developed Test (LDT)

• Commercially available testing with FDA-cleared status– Cryptosporidium

– Cyclospora cayetanensis

– Entamoeba histolytica

– Giardia lamblia

PCR

PCR Amplification

• Parasite DNA isolated from the stool sample

• Strand of DNA is separated

• Fluorescent primer matches up to each of the 2 strands – now have 2 copies

• Each of the 2 copies is separated and process repeats x 40 cycles

• If DNA present, will light up/fluoresce

https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet

PCR Amplification

• Can PCR be too sensitive resulting in over-diagnosis?

– Increasing the number of amplification cycles can lead to false positives due to artifact

– There are standards for PCR usage; Genova follows these standards – Genova does not increase the number of amplification cycles

Kralik P, et al. Front Microbiol. 2017;8:108-17.

PCR Parasitology Results

• Results reported as detected or not detected

• PCR positive, O&P negative

• PCR negative, O&P positive

Genova offers a complete assessment for parasites on the following stool tests

Stool Tests with Parasitology

Included Add-on

GI Effects Comprehensive 2200

O&P, qPCR, Next GenerationSequencing

Macroscopic worms

GI Effects Microbial Ecology Profile 2205

O&P, qPCR, Next Generation Sequencing

Macroscopic worms

Gut Pathogen Profile 2207 O&P, qPCR, Next Generation Sequencing, Macroscopic worms

Comprehensive Digestive Stool Analysis 2.0 (CDSA2.0)

O&P, EIA Macroscopic worms

CDSA with parasitology O&P, EIA Macroscopic worms

• Currently, the only methodology capable of detecting any and all stool parasites remains the gold standard microscopic Ova and Parasite (O&P) exam

• A combination of methodologies is ideal to cover the breadth of organism detection – using a combination of assays results in a higher rate of positives

• The introduction of PCR parasite panels does not eliminate the need for O&P microscopy; there are no commercially available tests that are a complete replacement for O&P microscopy at this time

• Genova will continue to offer O&P microscopy, PCR, and EIA testing for parasites

Key Points

Blastocystis spp.

Blastocystis spp.

• Common; affects up to 30% in industrialized countries and up to 76% in developing countries

• Spread via the fecal-oral route

• Can live in the intestines for years

• Pathogenicity controversial

https://www.cdc.gov/parasites/blastocystis/

Barbosa CV, et al. PloS one. 2018;13(3):e0193860.

Popruk S, et al. J Trop Med Parasitol. 2013;36:88-97.

• Resides in the ileum and cecum, is adherent to the outer layer of mucus, and thrives in an anaerobic environment and in the presence of bacteria; eats bacteria as food source

• Associated with higher microbial diversity, richness, and microbial composition, and might be regarded as a commensal organism

• Others have regarded it as an indicator of dysbiosis

• Large-scale metagenomic analysis, the organism Methanobrevibacter smithii was strongly associated with the presence of Blastocystis Beghini F, et.al. ISME J. 2017;11(12):2848-63.

Cifre S, et. al. Med hypoth. 2018;116:4-9.

Nagel R, et. al. Microbiome. 2016;4(1):47-56.

Nieves-Ramirez ME, et al. mSystems. 2018;3(3):e00007-18.

Tan KS. Clin microbiol rev. 2008;21(4):639-65.

Tito RY, et al. Gut. 2019;68(7):1180-89.

Picture of Blastocystis under the microscope

Blastocystis spp.

• Blastocystis has a unique biomarker and microbiome profile on GI Effects 2200

• If only one methodology is positive for Blastocystis (PCR or O&P), the connection is much weaker

• The level of Blastocystis is the main driving force for the biomarker associations

• Biomarker pattern:– Significantly lower fecal inflammation markers including calprotectin, EPX, sIgA

– Correlation with Methanobrevibacter smithii

– Others

Stool Biomarker Analysis

Blastocystis Associated Symptoms and Conditions

• Nausea

• Loss of appetite

• Abdominal pain

• Flatulence

• Acute/chronic diarrhea

• Constipation

• Anal itching

• Fatigue

• Joint pain

• Urticaria (hives)

• Irritable Bowel Syndrome (IBS)

• Asymptomatic

• Subtypes are thought to be associated with different symptoms or clinical outcomes, although the literature is not yet clear

• The idea of subtypes in the literature is well established, but more research is needed for definitive answers regarding the clinical significance of each subtype

• Genova will gather data and provide more information over time

Why is it important to subtype Blastocystis?

Blastocystis subtypes (ST)1-9

Literature

• 17 STs identified; ST1-9 in humans – STs transmitted by different animals

(zoonotic transmission)

– STs found in different geographic regions

– ST1-4 make up 90% of findings

– ST3 most common at 60% of findings

• It is possible to have more than one ST

Genova’s Findings

Popruk S, et al. J Trop Med Parasitol. 2013;36:88-97.

Skotarczak B. AAEM. 2018;25(3):411-16.

Tito RY, et al. Gut. 2019;68(7):1180-89.

N/A = information not available

Cifre S, et. al. Med hypoth. 2018;116:4-9.

• Literature is mixed and preclinical

• Large-scale clinical outcomes studies regarding effective treatments for Blastocystis subtypes are lacking; however, this presents an opportunity for Genova and clinicians to correlate patient data as the literature evolves

• In the meantime, clinicians may decide to treat a symptomatic patient regardless of subtype until more definitive information is available

Treating subtypes

• Asymptomatic: no treatment recommended

• Symptomatic: often self-limited; metronidazole, TMP/SMX, iodoquinol, or nitazoxanide

• Difficult to eradicate; triple antibiotic therapy being explored versus monotherapy

• Certain subtypes may be more resistant to treatment

Blastocystis treatment

Coyle CM, et. al. Clin infect dis. 2012;54(1):105-10.

Mohapatra S, et. al. The Am j of gastroenterol. 2018;113(6):805-18.

Roberts T, et. al. Antimicr agents chemother. 2015;59(8):4417-23.

Sekar U, et al Trop parasitol. 2013;3(1):35-39.

• Saccharomyces boulardii had favorable cure rates, compared to metronidazole, in children infected with Blastocystis

• A high fiber and lactose-free diet may improve clinical signs and symptoms

• Botanicals such as Brucea javanica (Macassar Kernels), Coptis chinensis (Chinese goldthread), and garlic (in ST1) have some inhibitory activity in vitro

• A small study using oil of oregano showed eradication in eight out of 11 patients, and symptom improvement in seven out of 11 symptomatic adult patients, who had tested positive for Blastocystis

Blastocystis natural treatment

Coyle CM, et. al. Clin infect dis. 2012;54(1):105-10.

Dinleyici EC, et. al. Para res. 2011;108(3):541-45.

Force M, et al. Phytother Res. 2000;14(3):213-14.

Sekar U, Set al. Trop parasitol. 2013;3(1):35-39.

Case Study

• 26 year old male

• Acute onset, now chronic diarrhea x 4 months

• No recent travel

• Work-trade at a farm twice per month

• He thinks he may have eaten at a restaurant at onset

• GI Effects Gut Pathogen Profile 2207 ordered

Case Study #1

Case Study #1

Case Study #1

Case Study #1

Case Study #1 Treatment

• Patient requested natural treatments only

• Clinician selected a formula containing garlic, Coptis chinensis, and oregano

• Probiotic formula with Saccharomyces boulardii, Lactobacillus, Bifidobacterium

• Follow up – symptoms resolved

• 66 year old male

• Significant arthritis and pain

• Several rounds of metronidazole treatment for Blastocystis and each time joint pain resolved, then came back, along with another positive Blastocystis test

• Is this a resistant case of Blastocystis? Is he being reinfected between treatments?

• GI Effects® Comprehensive Profile 2200 ordered

Case Study #2

Case Study #2

Case Study #2

Case Study #2

Case Study #2

Case Study #2

Case Study #2

• Not a lot known about subtype 8

• TMP/SMX was chosen since metronidazole had failed previously; independent of subtype, TMP/SMX is suggested by the CDC as a possible treatment

• Does not travel, but works with people who have traveled

Case Study #2 Treatment

Sensitive Resistant

ST8TMP/SMX (most effective)MTZ (effective ↑ concentrations, but not total clearance)

Antifungals (fluconazole, nystatin, itraconazole)

Treatment for Organisms

• www.cdc.gov

• Sanford Guide to Antimicrobial Therapy

• Natural treatments – IFM or A4M training

– Emerson Ecologics or Natural Partners have consultants to answer supplement q’s

• Schedule a Medical Education consultation

• Genova performs a culture for bacteria and yeast; for those that are pathogenic or potentially pathogenic, sensitivities are performed using pharmaceutical and natural agents

• Genova cannot perform sensitivities on parasites because they arrive preserved

– The vial for O&P contains a preservative for accurate identification under the microscope

– The PCR test is looking for DNA and the PCR vial contains a fixative

– It is not standard for labs to perform sensitivities on parasites

FAQ: Does Genova offer sensitivities for parasites?

• Centers for Disease Control: https://www.cdc.gov/parasites/index.html

• PubMed: https://www.ncbi.nlm.nih.gov/pubmed/

• World Health Organization: http://www.who.int/

• Parasitic Organism Chart

• GI Effects Support Guide

Resources for Learning About Parasites

Questions?

Explore

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educational resources, including…

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Conferences – Schedule of events we attend

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Presenter:

Christine Stubbe, ND

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