The benefits of comprehensive stool testing

BY LUCY MAILING, MD/PHD STUDENT

This article was originally published in June 2018 and was updated in January 2019 to include the latest research and a more thorough analysis of available stool tests. I used to recommend Doctor’s Data CSAP, but now recommend a combination of GI-MAP, Doctor’s Data, and uBiome. In light of recent studies, I am also increasingly using comprehensive stool testing in combination with organic acids testing and breath testing to provide a better overall picture of the gut environment.

Let’s face it, collecting a stool sample in a special container is the last thing you want to do for your doctor. But what if I told you that your doctor’s office is likely only testing that sample for about one percent of what they could be testing your gut for?

Given how important gut health is to your overall health, you’d think that after all that effort, they’d at least provide you with some useful information. But here’s a typical result from a basic lab stool culture:

No visible ova or parasites

Occult blood: negative

Appearance: normal

Not only does this result provide little useful information, but it can also lead you to believe your gut is healthy, even if gut pathologies are the cause of your health struggles. I’ve had many clients that have had basic stool cultures come back normal, only to do comprehensive testing and find that they have a severe fungal infection or pathogenic overgrowth.

In this article, I’ll review the most commonly overlooked gut pathologies. I’ll also discuss the differences between stool testing methodologies and why choosing the right testing company is extremely important.

Who should get a comprehensive stool test?

More studies are published about the gut microbiome and its relevance to our health every day. The health of our gut is intricately linked to the health of just about every organ system, including the brain, liver, kidney, heart, skin, and bones. In many ways, testing the gut should be a part of routine part of preventive care, and I believe that anyone can benefit from comprehensive stool testing.

However, you might particularly benefit from the results of a comprehensive stool test if you have any of the following symptoms:

  • Digestive issues
  • Acid reflux (GERD)
  • Gas
  • Bloating
  • Abdominal pain
  • Constipation
  • Diarrhea

Those with other symptoms outside of the gut might also benefit from comprehensive stool testing:

  • Rashes or hives
  • Acne
  • Mood disturbances
  • Depression or anxiety
  • Fatigue
  • Insomnia
  • Brain fog
  • Joint pain
  • Chronic sinus and allergy issues
  • Rapid weight gain or weight loss

These symptoms can originate from several underlying causes, some of which are not related to gut health. However, an overlooked gut pathology will make it near impossible to truly address these symptoms, so it is essential to determine whether gut health is a potential cause or contributor.

Five commonly overlooked gut pathologies

There are five major gut pathologies that can be captured on a comprehensive stool test:

1) Gut dysbiosis / pathogenic overgrowth

Gut dysbiosis refers to an imbalance in the composition of the gut microbiota and is typically characterized by a lack of known beneficial bacteria and an overgrowth of potentially pathogenic microbes.

2) Parasitic infection

A parasite is an organism that lives in or on a host organism and gets its nourishment at the expense of the host. The two main classes of parasites in the human gut are protozoa and helminths. Protozoa are microscopic, single-celled organisms that are usually transmitted through the fecal-oral route. Helminths are large, multicellular organisms that are usually worm-like and visible to the naked eye.

3) Fungal overgrowth

Fungi are commonly detected in human fecal samples, but whether they actually colonize the gut of healthy individuals or simply pass through the healthy GI tract is still debated. In immunocompromised individuals, however, fungal colonization and overgrowth can occur quite rapidly. The most common fungal infections are Candida, Geotrichum, Microsporidium, and Rodoturula.

4) Macronutrient malabsorption

Malabsorption of protein, carbohydrates, and fats can result in abdominal pain, bloating, gas, and a variety of skin conditions. Most often, malabsorption is secondary to one of the GI pathologies mentioned above.

5) Immune dysregulation and inflammation

The gut microbiome is tightly regulated by the host immune system. Immune dysregulation and inflammation can contribute to gut dysbiosis or increase the risk of fungal or parasitic infection. Chronic gut inflammation can also lead to intestinal permeability (“leaky gut”) and food intolerances.

Which comprehensive stool testing method is best?

There are many different methods currently being used for stool testing. Unfortunately, not all of them are accurate or clinically useful. In this section, I’ll discuss the pros and cons of each method and note which companies are currently using them. If you don’t care for the details and just want to know which tests I recommend, feel free to skip to the next section.

Culture-based methodologies

For decades, scientific study of gut microbes relied on culture, staining, and microscopy. Growth media typically favored rapidly growing aerobic microbes like E. coli and Streptococcus, meaning that many anaerobic microbes could not be effectively cultured.

With the arrival of DNA sequencing, microbiology quickly moved away from culture towards sequencing-based approaches. High-throughput, next-generation sequencing allowed for massively parallel assessment of many microbes in a sample without any need to culture or find a suitable growth media.

In recent years, however, there has been a resurgence of interest in culture-based methodologies. Improvements in culture techniques and the introduction of special media that favors the growth of anaerobic bacteria significantly improved the range of microbes that could be cultured. Combined with proteomic-based mass spectrometry (also called MALDI-TOF), this high complexity stool culture allows for the rapid identification of over 1,400 species without having to purify or isolate them.

Several studies have validated the MALDI-TOF method, and shown that it has 99 percent accuracy in identification of normal gut flora, 100 percent accuracy in identification of common pathogenic species, and high reproducibility across laboratories.1,2 In fact, a 2016 study using high-complexity stool culture coupled with mass spectrometry was able to identify 513 new species in the human gut microbiome.3 However, MALDI-TOF still relies on successfully culturing the microbe first. While the study I just mentioned used a whopping 70 different culture conditions, most commercial laboratories that employ MALDI-TOF use only 3-5 culture conditions.

Moreover, culture-based methodologies tell you nothing about the relative abundance of microbes in a sample. A certain microbe could make up only 0.001% of a stool sample but grow rapidly when exposed to a particular culture media. The results might mislead you to believe you have an overgrowth of this microbe, and susceptibility testing performed by many of these laboratories may lead to poorly informed treatment choices.

Overall, culture-based methodologies can only identify a fraction of the microbes in the gut and often give you a false idea of what microbes are abundant in a stool sample. They may lead to inappropriate diagnoses and unnecessary antimicrobial or antibiotic treatments.

Popular companies that combine stool culture with MALDI-TOF: Doctor’s Data, Genova (GI Effects), BioHealth

Molecular techniques

In contrast to culture-based methodologies, molecular techniques give us a more accurate and more complete picture of the gut ecosystem. Several different molecular approaches are currently used in both research and commercial laboratory settings:

16S rRNA gene sequencing

In 1990, Carl Woese and George E. Fox discovered that the 16S ribosomal RNA gene had certain characteristics that meant it could be used to classify different microbes. Some regions of this gene evolve more slowly and are conserved across all bacterial species, while other regions are more hypervariable – they evolve rapidly and differ greatly from one species to the next. The conserved regions are used to design universal primers that allow for the amplification and sequencing of the hypervariable regions across a broad spectrum of microbes. These sequences can then be classified by comparing them to a curated database of fully sequenced 16S rRNA genes.

In other words, the 16S rRNA gene is like a bacterial fingerprint. By looking at just this fingerprint, we can do a pretty good job of determining which bacteria are present and in which relative abundance.

While this method can typically only get down to the genus level and is susceptible to primer bias, it is still the most widely used method to accurately and cost-effectively characterize the entire bacterial community in research settings. However, it’s important to note that it provides no information about fungi or parasites, so it is not truly a comprehensive stool test.

Popular companies: uBiome, Thryve, AmericanGut, Biohm, Atlas BioMed

Targeted PCR

Targeted PCR (polymerase chain reaction) requires a unique primer for each microbe that you want to screen for and involves amplification of a piece of DNA that has been captured by each primer. It is effective for asking whether a specific microbe is present and can accurately identify microbes to the genus or species level. The utility of targeted PCR for assessing the overall gut ecosystem depends on how many primers a specific laboratory uses. For example, a laboratory with specific primers for 20 of the most abundant microbes and the 15 most common pathogenic species in the human gut would be much more useful than a laboratory that only assesses five potential pathogens.

Popular companies: Genova (GI Effects)

Quantitative PCR

Also called qPCR or real-time PCR, this technique is similar to targeted PCR but amplifies and quantifies targeted microbial sequences in real-time, during the early phases of the reaction. This significantly improves the sensitivity, accuracy, and speed of the results. qPCR can detect as little as a two-fold change, while basic targeted PCR has only about ten-fold change resolution.

Popular companies: Diagnostic Solutions (GI-MAP)

Metagenomics

This method uses next-generation sequencing to assess the entire gene content of the gut microbiome. Metagenomics can identify microbes down to the species level and can determine their relative abundance with greater accuracy than 16S rRNA gene sequencing. It can also provide information about what functional capabilities these microbes have.

This is truly the cutting edge of microbiome science and will no doubt transform clinical practice in the next decade, as the cost of sequencing continues to drop and metagenomics becomes more widely available. In most cases, however, metagenomics does not yet provide sufficient additional, clinically actionable information to justify the difference in cost between metagenomics and 16S.

There are two cases where metagenomics might be worth the extra cost:

  • For individuals with diabetes or blood sugar dysregulation, the company DayTwo will run your metagenomic data through a machine learning algorithm and predict your blood sugar response to certain foods. Incredibly, the predictions have almost twice the accuracy of simple carb counting.
  • For those with a suspected acute or chronic infection that is not picked up by other stool testing methods, Aperiomics can utilize metagenomics to identify all of the potentially pathogenic bacteria, virus, parasites, and fungi in a sample.

Popular companies: DayTwo, Aperiomics, SmartDNA, Microba

Metabolomics and meta-transcriptomics

These high-throughput techniques assess the metabolic output of the gut microbiome and the sum total of all of the gene expression of the microbiome, respectively. While metagenomics asks the question “who is there?”, meta-transcriptomics and metabolomics ask the question “what are they doing?”. Like metagenomics, these techniques are widely used in research settings and are dramatically increasing our understanding of the gut microbiome. In the near future, metabolomics and meta-transcriptomics will likely complement other molecular approaches in a way that is accurate, affordable, clinically relevant, and actionable – but for now, they are impractical for clinical use.

While some companies are starting to employ these technologies, it’s important to note that metabolomics and meta-transcriptomics alone cannot accurately estimate bacterial abundance. Just because a particular species has higher metabolic activity does not necessarily mean that it is more abundant. I have now seen several bacterial abundance outputs from Viome that are simply incompatible with a human microbiome.

Popular companies: Viome

Example of culture vs molecular techniques for assessing bacterial abundance

Still not convinced that culture is inaccurate for assessing bacterial abundance? Hopefully, this example will help.

Here is a bacteriology culture report from Doctor’s Data:

Here is the exact same stool sample run through uBiome 16S sequencing. I downloaded the raw data and extracted only the taxa that were reported on Doctor’s Data:

You can see that the bacteria picked up by culture-based methods represents only 3.39% of the total bacteria present in the stool sample that can be detected by PCR-based methods. It also completely skews the relative abundance with the 0-4 scale, with both Bacteroides fragilis (2.26%) and Enterococcus (0.00%) registering as a 4+.

This is particularly concerning if you come back with a 4+ for pathogenic organisms like Pseudomonas aeruginosa and end up treating unnecessarily, when, in reality, this microbe may represent less than 0.00% of the ecosystem.

I’ve been aware of this issue for quite a while, but I still found Doctor’s Data CSAP useful for the parasitology, digestion, absorption, and inflammatory markers. Here’s an example of a CSAP that picked up Blastocystis hominis:

Nonetheless, I decided it was time to step back and objectively re-evaluate which comprehensive stool testing company I wanted to use going forward.

An independent analysis of popular stool tests

As someone who primarily consults on gut-related issues, it’s important to me that I am using the best and most evidence-based testing available. I decided to put together an independent analysis of four of the most widely used stool tests in clinical practice.

You can click here to download the full table.

There are essentially eight different categories to consider here, and the best stool test depends on exactly what you are looking for:

  • Bacterial abundances = uBiome
  • Bacterial pathogen detection = GI-MAP
  • Fungal detection = GI-MAP
  • Parasite detection = GI-MAP
  • Virus detection = GI-MAP
  • Digestion/absorption markers = Doctor’s Data or GI Effects
  • Inflammation/immunology markers = Doctor’s Data or GI Effects
  • Gut environment = Doctor’s Data, GI-MAP, or GI-Effects

Before I share what I’m currently using for gut testing, I want to answer a few questions relevant to the rankings above.

Is qPCR effective for detecting parasites?

Yes. Several recent studies and literature reviews suggest that qPCR detection of parasites is equivalent, and in many cases superior, to microscopy-based parasitology.4–9 Not only can qPCR detect the absence or presence of parasites, but it can also detect parasite load by providing an absolute number per gram of stool. Unlike traditional microscopy, this method does not require stool sampling on multiple days to ensure that parasites are detected and offers more rapid results.

Are fecal SCFAs reliable?

No. Short-chain fatty acids (SCFAs) are produced from the bacterial fermentation of dietary fiber and play important roles in the health of the gut and the rest of the body. However, quantifying them poses quite a challenge. Fecal excretion of SCFAs depends on the rate of absorption, cross-feeding interactions, and gut motility, so fecal SCFAs do not necessarily reflect SCFA production in the gut. For instance, ulcerative colitis patients have impaired absorption of butyrate, which means they will excrete a greater proportion of the butyrate they produce. One recent study also found that higher fecal SCFA excretion, which is typically thought to be indicative of good gut health, was associated with gut dysbiosis, obesity, hypertension, and cardiometabolic disease risk factors.10

Is zonulin a useful marker of gut permeability?

No. Blood or fecal zonulin has been shown to be elevated in depression, autism, diabetes, metabolic syndrome, PCOS, aging, and celiac disease. However, only a fraction of the patients in these studies have elevated zonulin.11,12 Moreover, many of the studies used to support the clinical use of zonulin testing were not measuring zonulin at all – they were measuring properdin, a zonulin analog.13

Furthermore, zonulin is only weakly correlated with other more validated measures of intestinal permeability, such as the differential sugar (lactulose-mannitol) absorption test and has been shown to fluctuate greatly throughout the day.11,12 Zonulin antibodies in the blood may be more stable and reflective of intestinal permeability, but these obviously will not be included on a stool test.

What I’m using in practice

Unfortunately, there is not currently a single stool test that provides all of the information I would be looking for in a test, so the best option is to combine comprehensive stool testing with 16S rRNA gene sequencing.

1) GI-MAP + Doctor’s Data CSAP + uBiome*

In a perfect world, this three-test combination is what I would run with all of my clients. GI-MAP provides the best pathogen detection, Doctor’s Data provides the most information about the gut environment, and uBiome provides the best assessment of bacterial abundance. uBiome is also cheap enough to run follow-up testing, so we can see how certain interventions alter bacterial abundance.

2) GI-MAP + uBiome*

Due to client financial constraints and the fact that I often want to run other tests, like Organic Acids or a SIBO breath test, I will often just order GI-MAP and uBiome. For clients with inflammatory bowel disease, where I want a better picture of their inflammatory markers, or for those with suspected carbohydrate malabsorption, I might choose to only run Doctor’s Data and uBiome.

3) Aperiomics

For extremely tough cases, where I suspect some sort of infection, but other testing does not identify the infectious agent, I might use Aperiomics. Aperiomics screens for every bacteria, virus, parasite, and fungus in a single test through metagenomic sequencing of fecal, blood, urine, oral swab, nasal swabs, and tissue samples. However, it only reports potential pathogens and does not provide any information about beneficial microbes. Aperiomics can be run through insurance, but often still costs anywhere between $500-$1000, depending on how many samples you submit.

*Note that other 16S rRNA gene sequencing companies could be used in place of uBiome, but it is important to use the same company to assess changes in the microbiome over time. Due to differences in DNA extraction processes, PCR primers, and downstream analysis, different 16S companies will often report slightly different results from the same sample.

But wait, isn’t the stool microbiome different from the gut microbiome?

You might recall a recent study that I reviewed in depth and discussed on Chris Kresser’s podcast, which suggests that stool samples don’t accurately reflect the luminal or mucosal gut microbiome. It’s true — stool samples tend to over or underrepresent different genera from even the most distal part of the colon.

In particular, stool tended to over-represent the abundance of Ruminococcus obeum, Coprococcus catus, Dorea longicatena, and two species of Eubacterium, and under-represent Parabacteroides and Bacteroides thetaiotamicron.14 In fact, Bacteroides represent approximately 50 percent of microbes associated with the colonic mucosa and 60 percent of all microbes in the colonic lumen yet constitute a mere 20 percent of the total bacteria in stool samples.

We also don’t know for sure whether someone who has a particularly high abundance of Bacteroides in the mucosa or lumen will necessarily have a high abundance in the stool. It is likely that there is a correlation here, but the exact strength of the correlation is unknown.

So, is stool testing useless? Maybe. But we have to consider the fact that the majority of published studies on the gut microbiome have measured – you guessed it – fecal samples. In fact, given the lack of studies on the gut mucosa, a stool sample is likely more clinically useful than an invasive mucosal biopsy.

If you think about it:

  • We know how certain interventions like diet, exercise, and antibiotics affect the fecal microbiome.
  • We know what fecal microbiome patterns are correlated with certain diseases
  • We have a general idea of which microbes should make up the bulk of a healthy fecal sample
  • We have a general idea of which microbes should not be present in large amounts in fecal samples.
  • We know that certain parasites and fungi can contribute to disease.

This means that while fecal samples are imperfect, they are still a useful proxy for the overall ecosystem and are currently one of the best ways to screen for various gut pathologies.

What do you make of all this research? Have you had comprehensive stool testing? Share your thoughts or experience in the comments below!

  1. Tan, K. E. et al. Prospective Evaluation of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System in a Hospital Clinical Microbiology Laboratory for Identification of Bacteria and Yeasts: a Bench-by-Bench Study for Assessing the Impact on Time to Identification and Cost-Effectiveness. J. Clin. Microbiol. 50, 3301–3308 (2012).
  2. Samb-Ba, B. et al. MALDI-TOF Identification of the Human Gut Microbiome in People with and without Diarrhea in Senegal. PLoS ONE 9, (2014).
  3. Lagier, J.-C. et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat. Microbiol. 1, 16203 (2016).
  4. Poirier, P. et al. Development and Evaluation of a Real-Time PCR Assay for Detection and Quantification of Blastocystis Parasites in Human Stool Samples: Prospective Study of Patients with Hematological Malignancies▿. J. Clin. Microbiol. 49, 975–983 (2011).
  5. Mejia, R. et al. A Novel, Multi-Parallel, Real-Time Polymerase Chain Reaction Approach for Eight Gastrointestinal Parasites Provides Improved Diagnostic Capabilities to Resource-Limited At-Risk Populations. Am. J. Trop. Med. Hyg. 88, 1041–1047 (2013).
  6. Verweij, J. J. et al. Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J. Clin. Microbiol. 42, 1220–1223 (2004).
  7. Llewellyn, S. et al. Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial. PLoS Negl. Trop. Dis. 10, e0004380 (2016).
  8. Cimino, R. O. et al. Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction. Parasit. Vectors 8, 380 (2015).
  9. Laude, A. et al. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis. 22, 190.e1-190.e8 (2016).
  10. de la Cuesta-Zuluaga, J. et al. Higher Fecal Short-Chain Fatty Acid Levels Are Associated with Gut Microbiome Dysbiosis, Obesity, Hypertension and Cardiometabolic Disease Risk Factors. Nutrients 11, (2018).
  11. Sapone, A. et al. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes 55, 1443–1449 (2006).
  12. Vojdani, A., Vojdani, E. & Kharrazian, D. Fluctuation of zonulin levels in blood vs stability of antibodies. World J. Gastroenterol. 23, 5669–5679 (2017).
  13. Scheffler, L. et al. Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family. Front. Endocrinol. 9, (2018).
  14. Zmora, N. et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell 174, 1388-1405.e21 (2018).
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The Best of DDW 2019

The Best of DDW 2019 BY LUCY MAILING, MD/PHD STUDENT A few weeks ago, I had the opportunity to attend Digestive Disease Week in San Diego, California! DDW [...]

The benefits of comprehensive stool testing

BY LUCY MAILING, MD/PHD STUDENT

This article was originally published in June 2018 and was updated in January 2019 to include the latest research and a more thorough analysis of available stool tests. I used to recommend Doctor’s Data CSAP, but now recommend a combination of GI-MAP, Doctor’s Data, and uBiome. In light of recent studies, I am also increasingly using comprehensive stool testing in combination with organic acids testing and breath testing to provide a better overall picture of the gut environment.

Let’s face it, collecting a stool sample in a special container is the last thing you want to do for your doctor. But what if I told you that your doctor’s office is likely only testing that sample for about one percent of what they could be testing your gut for?

Given how important gut health is to your overall health, you’d think that after all that effort, they’d at least provide you with some useful information. But here’s a typical result from a basic lab stool culture:

No visible ova or parasites

Occult blood: negative

Appearance: normal

Not only does this result provide little useful information, but it can also lead you to believe your gut is healthy, even if gut pathologies are the cause of your health struggles. I’ve had many clients that have had basic stool cultures come back normal, only to do comprehensive testing and find that they have a severe fungal infection or pathogenic overgrowth.

In this article, I’ll review the most commonly overlooked gut pathologies. I’ll also discuss the differences between stool testing methodologies and why choosing the right testing company is extremely important.

More studies are published about the gut microbiome and its relevance to our health every day. The health of our gut is intricately linked to the health of just about every organ system, including the brain, liver, kidney, heart, skin, and bones. In many ways, testing the gut should be a part of routine part of preventive care, and I believe that anyone can benefit from comprehensive stool testing.

However, you might particularly benefit from the results of a comprehensive stool test if you have any of the following symptoms:

  • Digestive issues
  • Acid reflux (GERD)
  • Gas
  • Bloating
  • Abdominal pain
  • Constipation
  • Diarrhea

Those with other symptoms outside of the gut might also benefit from comprehensive stool testing:

  • Rashes or hives
  • Acne
  • Mood disturbances
  • Depression or anxiety
  • Fatigue
  • Insomnia
  • Brain fog
  • Joint pain
  • Chronic sinus and allergy issues
  • Rapid weight gain or weight loss

These symptoms can originate from several underlying causes, some of which are not related to gut health. However, an overlooked gut pathology will make it near impossible to truly address these symptoms, so it is essential to determine whether gut health is a potential cause or contributor.

There are five major gut pathologies that can be captured on a comprehensive stool test:

1) Gut dysbiosis / pathogenic overgrowth

Gut dysbiosis refers to an imbalance in the composition of the gut microbiota and is typically characterized by a lack of known beneficial bacteria and an overgrowth of potentially pathogenic microbes.

2) Parasitic infection

A parasite is an organism that lives in or on a host organism and gets its nourishment at the expense of the host. The two main classes of parasites in the human gut are protozoa and helminths. Protozoa are microscopic, single-celled organisms that are usually transmitted through the fecal-oral route. Helminths are large, multicellular organisms that are usually worm-like and visible to the naked eye.

3) Fungal overgrowth

Fungi are commonly detected in human fecal samples, but whether they actually colonize the gut of healthy individuals or simply pass through the healthy GI tract is still debated. In immunocompromised individuals, however, fungal colonization and overgrowth can occur quite rapidly. The most common fungal infections are Candida, Geotrichum, Microsporidium, and Rodoturula.

4) Macronutrient malabsorption

Malabsorption of protein, carbohydrates, and fats can result in abdominal pain, bloating, gas, and a variety of skin conditions. Most often, malabsorption is secondary to one of the GI pathologies mentioned above.

5) Immune dysregulation and inflammation

The gut microbiome is tightly regulated by the host immune system. Immune dysregulation and inflammation can contribute to gut dysbiosis or increase the risk of fungal or parasitic infection. Chronic gut inflammation can also lead to intestinal permeability (“leaky gut”) and food intolerances.

There are many different methods currently being used for stool testing. Unfortunately, not all of them are accurate or clinically useful. In this section, I’ll discuss the pros and cons of each method and note which companies are currently using them. If you don’t care for the details and just want to know which tests I recommend, feel free to skip to the next section.

Culture-based methodologies

For decades, scientific study of gut microbes relied on culture, staining, and microscopy. Growth media typically favored rapidly growing aerobic microbes like E. coli and Streptococcus, meaning that many anaerobic microbes could not be effectively cultured.

With the arrival of DNA sequencing, microbiology quickly moved away from culture towards sequencing-based approaches. High-throughput, next-generation sequencing allowed for massively parallel assessment of many microbes in a sample without any need to culture or find a suitable growth media.

In recent years, however, there has been a resurgence of interest in culture-based methodologies. Improvements in culture techniques and the introduction of special media that favors the growth of anaerobic bacteria significantly improved the range of microbes that could be cultured. Combined with proteomic-based mass spectrometry (also called MALDI-TOF), this high complexity stool culture allows for the rapid identification of over 1,400 species without having to purify or isolate them.

Several studies have validated the MALDI-TOF method, and shown that it has 99 percent accuracy in identification of normal gut flora, 100 percent accuracy in identification of common pathogenic species, and high reproducibility across laboratories.1,2 In fact, a 2016 study using high-complexity stool culture coupled with mass spectrometry was able to identify 513 new species in the human gut microbiome.3 However, MALDI-TOF still relies on successfully culturing the microbe first. While the study I just mentioned used a whopping 70 different culture conditions, most commercial laboratories that employ MALDI-TOF use only 3-5 culture conditions.

Moreover, culture-based methodologies tell you nothing about the relative abundance of microbes in a sample. A certain microbe could make up only 0.001% of a stool sample but grow rapidly when exposed to a particular culture media. The results might mislead you to believe you have an overgrowth of this microbe, and susceptibility testing performed by many of these laboratories may lead to poorly informed treatment choices.

Overall, culture-based methodologies can only identify a fraction of the microbes in the gut and often give you a false idea of what microbes are abundant in a stool sample. They may lead to inappropriate diagnoses and unnecessary antimicrobial or antibiotic treatments.

Popular companies that combine stool culture with MALDI-TOF: Doctor’s Data, Genova (GI Effects), BioHealth

Molecular techniques

In contrast to culture-based methodologies, molecular techniques give us a more accurate and more complete picture of the gut ecosystem. Several different molecular approaches are currently used in both research and commercial laboratory settings:

16S rRNA gene sequencing

In 1990, Carl Woese and George E. Fox discovered that the 16S ribosomal RNA gene had certain characteristics that meant it could be used to classify different microbes. Some regions of this gene evolve more slowly and are conserved across all bacterial species, while other regions are more hypervariable – they evolve rapidly and differ greatly from one species to the next. The conserved regions are used to design universal primers that allow for the amplification and sequencing of the hypervariable regions across a broad spectrum of microbes. These sequences can then be classified by comparing them to a curated database of fully sequenced 16S rRNA genes.

In other words, the 16S rRNA gene is like a bacterial fingerprint. By looking at just this fingerprint, we can do a pretty good job of determining which bacteria are present and in which relative abundance.

While this method can typically only get down to the genus level and is susceptible to primer bias, it is still the most widely used method to accurately and cost-effectively characterize the entire bacterial community in research settings. However, it’s important to note that it provides no information about fungi or parasites, so it is not truly a comprehensive stool test.

Popular companies: uBiome, Thryve, AmericanGut, Biohm, Atlas BioMed

Targeted PCR

Targeted PCR (polymerase chain reaction) requires a unique primer for each microbe that you want to screen for and involves amplification of a piece of DNA that has been captured by each primer. It is effective for asking whether a specific microbe is present and can accurately identify microbes to the genus or species level. The utility of targeted PCR for assessing the overall gut ecosystem depends on how many primers a specific laboratory uses. For example, a laboratory with specific primers for 20 of the most abundant microbes and the 15 most common pathogenic species in the human gut would be much more useful than a laboratory that only assesses five potential pathogens.

Popular companies: Genova (GI Effects)

Quantitative PCR

Also called qPCR or real-time PCR, this technique is similar to targeted PCR but amplifies and quantifies targeted microbial sequences in real-time, during the early phases of the reaction. This significantly improves the sensitivity, accuracy, and speed of the results. qPCR can detect as little as a two-fold change, while basic targeted PCR has only about ten-fold change resolution.

Popular companies: Diagnostic Solutions (GI-MAP)

Metagenomics

This method uses next-generation sequencing to assess the entire gene content of the gut microbiome. Metagenomics can identify microbes down to the species level and can determine their relative abundance with greater accuracy than 16S rRNA gene sequencing. It can also provide information about what functional capabilities these microbes have.

This is truly the cutting edge of microbiome science and will no doubt transform clinical practice in the next decade, as the cost of sequencing continues to drop and metagenomics becomes more widely available. In most cases, however, metagenomics does not yet provide sufficient additional, clinically actionable information to justify the difference in cost between metagenomics and 16S.

There are two cases where metagenomics might be worth the extra cost:

  • For individuals with diabetes or blood sugar dysregulation, the company DayTwo will run your metagenomic data through a machine learning algorithm and predict your blood sugar response to certain foods. Incredibly, the predictions have almost twice the accuracy of simple carb counting.
  • For those with a suspected acute or chronic infection that is not picked up by other stool testing methods, Aperiomics can utilize metagenomics to identify all of the potentially pathogenic bacteria, virus, parasites, and fungi in a sample.

Popular companies: DayTwo, Aperiomics, SmartDNA, Microba

Metabolomics and meta-transcriptomics

These high-throughput techniques assess the metabolic output of the gut microbiome and the sum total of all of the gene expression of the microbiome, respectively. While metagenomics asks the question “who is there?”, meta-transcriptomics and metabolomics ask the question “what are they doing?”. Like metagenomics, these techniques are widely used in research settings and are dramatically increasing our understanding of the gut microbiome. In the near future, metabolomics and meta-transcriptomics will likely complement other molecular approaches in a way that is accurate, affordable, clinically relevant, and actionable – but for now, they are impractical for clinical use.

While some companies are starting to employ these technologies, it’s important to note that metabolomics and meta-transcriptomics alone cannot accurately estimate bacterial abundance. Just because a particular species has higher metabolic activity does not necessarily mean that it is more abundant. I have now seen several bacterial abundance outputs from Viome that are simply incompatible with a human microbiome.

Popular companies: Viome

Still not convinced that culture is inaccurate for assessing bacterial abundance? Hopefully, this example will help.

Here is a bacteriology culture report from Doctor’s Data:

Here is the exact same stool sample run through uBiome 16S sequencing. I downloaded the raw data and extracted only the taxa that were reported on Doctor’s Data:

You can see that the bacteria picked up by culture-based methods represents only 3.39% of the total bacteria present in the stool sample that can be detected by PCR-based methods. It also completely skews the relative abundance with the 0-4 scale, with both Bacteroides fragilis (2.26%) and Enterococcus (0.00%) registering as a 4+.

This is particularly concerning if you come back with a 4+ for pathogenic organisms like Pseudomonas aeruginosa and end up treating unnecessarily, when, in reality, this microbe may represent less than 0.00% of the ecosystem.

I’ve been aware of this issue for quite a while, but I still found Doctor’s Data CSAP useful for the parasitology, digestion, absorption, and inflammatory markers. Here’s an example of a CSAP that picked up Blastocystis hominis:

Nonetheless, I decided it was time to step back and objectively re-evaluate which comprehensive stool testing company I wanted to use going forward.

As someone who primarily consults on gut-related issues, it’s important to me that I am using the best and most evidence-based testing available. I decided to put together an independent analysis of four of the most widely used stool tests in clinical practice.

You can click here to download the full table.

There are essentially eight different categories to consider here, and the best stool test depends on exactly what you are looking for:

  • Bacterial abundances = uBiome
  • Bacterial pathogen detection = GI-MAP
  • Fungal detection = GI-MAP
  • Parasite detection = GI-MAP
  • Virus detection = GI-MAP
  • Digestion/absorption markers = Doctor’s Data or GI Effects
  • Inflammation/immunology markers = Doctor’s Data or GI Effects
  • Gut environment = Doctor’s Data, GI-MAP, or GI-Effects

Before I share what I’m currently using for gut testing, I want to answer a few questions relevant to the rankings above.

Is qPCR effective for detecting parasites?

Yes. Several recent studies and literature reviews suggest that qPCR detection of parasites is equivalent, and in many cases superior, to microscopy-based parasitology.4–9 Not only can qPCR detect the absence or presence of parasites, but it can also detect parasite load by providing an absolute number per gram of stool. Unlike traditional microscopy, this method does not require stool sampling on multiple days to ensure that parasites are detected and offers more rapid results.

Are fecal SCFAs reliable?

No. Short-chain fatty acids (SCFAs) are produced from the bacterial fermentation of dietary fiber and play important roles in the health of the gut and the rest of the body. However, quantifying them poses quite a challenge. Fecal excretion of SCFAs depends on the rate of absorption, cross-feeding interactions, and gut motility, so fecal SCFAs do not necessarily reflect SCFA production in the gut. For instance, ulcerative colitis patients have impaired absorption of butyrate, which means they will excrete a greater proportion of the butyrate they produce. One recent study also found that higher fecal SCFA excretion, which is typically thought to be indicative of good gut health, was associated with gut dysbiosis, obesity, hypertension, and cardiometabolic disease risk factors.10

Is zonulin a useful marker of gut permeability?

No. Blood or fecal zonulin has been shown to be elevated in depression, autism, diabetes, metabolic syndrome, PCOS, aging, and celiac disease. However, only a fraction of the patients in these studies have elevated zonulin.11,12 Moreover, many of the studies used to support the clinical use of zonulin testing were not measuring zonulin at all – they were measuring properdin, a zonulin analog.13

Furthermore, zonulin is only weakly correlated with other more validated measures of intestinal permeability, such as the differential sugar (lactulose-mannitol) absorption test and has been shown to fluctuate greatly throughout the day.11,12 Zonulin antibodies in the blood may be more stable and reflective of intestinal permeability, but these obviously will not be included on a stool test.

Unfortunately, there is not currently a single stool test that provides all of the information I would be looking for in a test, so the best option is to combine comprehensive stool testing with 16S rRNA gene sequencing.

1) GI-MAP + Doctor’s Data CSAP + uBiome*

In a perfect world, this three-test combination is what I would run with all of my clients. GI-MAP provides the best pathogen detection, Doctor’s Data provides the most information about the gut environment, and uBiome provides the best assessment of bacterial abundance. uBiome is also cheap enough to run follow-up testing, so we can see how certain interventions alter bacterial abundance.

2) GI-MAP + uBiome*

Due to client financial constraints and the fact that I often want to run other tests, like Organic Acids or a SIBO breath test, I will often just order GI-MAP and uBiome. For clients with inflammatory bowel disease, where I want a better picture of their inflammatory markers, or for those with suspected carbohydrate malabsorption, I might choose to only run Doctor’s Data and uBiome.

3) Aperiomics

For extremely tough cases, where I suspect some sort of infection, but other testing does not identify the infectious agent, I might use Aperiomics. Aperiomics screens for every bacteria, virus, parasite, and fungus in a single test through metagenomic sequencing of fecal, blood, urine, oral swab, nasal swabs, and tissue samples. However, it only reports potential pathogens and does not provide any information about beneficial microbes. Aperiomics can be run through insurance, but often still costs anywhere between $500-$1000, depending on how many samples you submit.

*Note that other 16S rRNA gene sequencing companies could be used in place of uBiome, but it is important to use the same company to assess changes in the microbiome over time. Due to differences in DNA extraction processes, PCR primers, and downstream analysis, different 16S companies will often report slightly different results from the same sample.

You might recall a recent study that I reviewed in depth and discussed on Chris Kresser’s podcast, which suggests that stool samples don’t accurately reflect the luminal or mucosal gut microbiome. It’s true — stool samples tend to over or underrepresent different genera from even the most distal part of the colon.

In particular, stool tended to over-represent the abundance of Ruminococcus obeum, Coprococcus catus, Dorea longicatena, and two species of Eubacterium, and under-represent Parabacteroides and Bacteroides thetaiotamicron.14 In fact, Bacteroides represent approximately 50 percent of microbes associated with the colonic mucosa and 60 percent of all microbes in the colonic lumen yet constitute a mere 20 percent of the total bacteria in stool samples.

We also don’t know for sure whether someone who has a particularly high abundance of Bacteroides in the mucosa or lumen will necessarily have a high abundance in the stool. It is likely that there is a correlation here, but the exact strength of the correlation is unknown.

So, is stool testing useless? Maybe. But we have to consider the fact that the majority of published studies on the gut microbiome have measured – you guessed it – fecal samples. In fact, given the lack of studies on the gut mucosa, a stool sample is likely more clinically useful than an invasive mucosal biopsy.

If you think about it:

  • We know how certain interventions like diet, exercise, and antibiotics affect the fecal microbiome.
  • We know what fecal microbiome patterns are correlated with certain diseases
  • We have a general idea of which microbes should make up the bulk of a healthy fecal sample
  • We have a general idea of which microbes should not be present in large amounts in fecal samples.
  • We know that certain parasites and fungi can contribute to disease.

This means that while fecal samples are imperfect, they are still a useful proxy for the overall ecosystem and are currently one of the best ways to screen for various gut pathologies.

What do you make of all this research? Have you had comprehensive stool testing? Share your thoughts or experience in the comments below!

  1. Tan, K. E. et al. Prospective Evaluation of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System in a Hospital Clinical Microbiology Laboratory for Identification of Bacteria and Yeasts: a Bench-by-Bench Study for Assessing the Impact on Time to Identification and Cost-Effectiveness. J. Clin. Microbiol. 50, 3301–3308 (2012).
  2. Samb-Ba, B. et al. MALDI-TOF Identification of the Human Gut Microbiome in People with and without Diarrhea in Senegal. PLoS ONE 9, (2014).
  3. Lagier, J.-C. et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat. Microbiol. 1, 16203 (2016).
  4. Poirier, P. et al. Development and Evaluation of a Real-Time PCR Assay for Detection and Quantification of Blastocystis Parasites in Human Stool Samples: Prospective Study of Patients with Hematological Malignancies▿. J. Clin. Microbiol. 49, 975–983 (2011).
  5. Mejia, R. et al. A Novel, Multi-Parallel, Real-Time Polymerase Chain Reaction Approach for Eight Gastrointestinal Parasites Provides Improved Diagnostic Capabilities to Resource-Limited At-Risk Populations. Am. J. Trop. Med. Hyg. 88, 1041–1047 (2013).
  6. Verweij, J. J. et al. Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J. Clin. Microbiol. 42, 1220–1223 (2004).
  7. Llewellyn, S. et al. Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial. PLoS Negl. Trop. Dis. 10, e0004380 (2016).
  8. Cimino, R. O. et al. Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction. Parasit. Vectors 8, 380 (2015).
  9. Laude, A. et al. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis. 22, 190.e1-190.e8 (2016).
  10. de la Cuesta-Zuluaga, J. et al. Higher Fecal Short-Chain Fatty Acid Levels Are Associated with Gut Microbiome Dysbiosis, Obesity, Hypertension and Cardiometabolic Disease Risk Factors. Nutrients 11, (2018).
  11. Sapone, A. et al. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes 55, 1443–1449 (2006).
  12. Vojdani, A., Vojdani, E. & Kharrazian, D. Fluctuation of zonulin levels in blood vs stability of antibodies. World J. Gastroenterol. 23, 5669–5679 (2017).
  13. Scheffler, L. et al. Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family. Front. Endocrinol. 9, (2018).
  14. Zmora, N. et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell 174, 1388-1405.e21 (2018).
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