Laboratory Assessment of GI Terrain and Function

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Culture-based methods offer overall superiority to molecular diagnostic testing

Dr Chuck Masur MD from Drs Data laboratory in the USA contributed this article to their in house e news letter in spring 2011. Chuck has kindly agreed that we may reproduce here.

The terrain of the gastrointestinal (GI) tract is vast and varied.  Villi and microvilli in the small intestine along with the absorptive cells of the large intestine contribute an effective absorptive surface area of about 200 m2 (equivalent to about 10 times the surface area of the human body or an area the size of a singles’ tennis court playing surface).  This terrain is occupied by about 10 x 1013 micro-organisms (the microbiota) – by comparison the microbiota exceed by 10-fold the total number of cells comprising the human body.  More than 1000 different species and sub-species contribute to the population of human GI micro-organisms; about 500 of these species are bacteria.

The microbiome is the totality of these micro-organisms, their genetic elements (genomes) and all of their interactions in their defined environment.  Any one person may have about 160 different micro-organisms colonizing their own particular microbiome, and thus about 80 of these would be bacterial species.  Approximately 99% of the total bacterial load is contributed by only 40 to 50 species; these fecal bacteria account for about 60% of the dry weight of stool.

The microbiota, generally, are imagined as homogeneously populating stool in the gut lumen such that one might envision taking a swab of a stool sample and simply plating it out on culture media and “analysing” it.  In truth, this simple construct is far from reality.  There are a multitude of distinct sub-populations in many micro-ecosystems throughout the GI tract from mouth to anus.  The environment in the small intestine is highly anaerobic and stool is a loose slurry-like mixture, while in the large intestine the environment becomes progressively more aerobic and, as water is reabsorbed, stool becomes more and more formed.  The microbiota can be free-living in the lumen (“planktonic”) or can be adherent in biofilms on food particles, on implanted devices and on the mucosal surfaces of the gut wall.  The composition of the sub-microbiota in these micro-ecosystems can change dynamically; this is especially true in the small intestine where changes can occur hour-to-hour depending on the availability of food substrates.

Imagining a simple co-existence between gut bacteria and the human host also is far from what takes place in reality.  Beneficial gut organisms perform a multitude of different functions upon which their human hosts depend, such as:

  • Inhibiting the growth of microbial pathogens (lactocidin, lactobicillin, acidolin, H2O2),
  • Helping to maintain appropriate pH in the GI tract (lactic acid; short chain fatty acids),
  • Competing for adhesion sites on epithelial surfaces (biofilms; “competitive exclusion”),
  • Defending against opportunistic fungi,
  • Fermenting unused energy substrates (SCFA),
  • Training the immune system to defend against pathogens,
  • Producing important vitamins (e.g. B7 (biotin), K),
  • Helping to break down and rebuild hormones,
  • Facilitating the absorption of minerals (Ca, Mg, Fe),
  • Helping to regulate gut development, and
  • Affecting cellular changes that prevent increased intestinal permeability (“leaky gut”).

Molecular diagnostic technology has contributed immensely to the identification of the vast majority of micro-organisms inhabiting the GI tract.  Many sub-populations of the GI microbiota reside primarily in biofilms colonising the mucosa of the gut wall and the make-up of these colonies is distinctly different from those living a planktonic existence in the gut lumen or adherent to food particles in stool.  It must be understood that gut wall biofilms are not generally accessible except by destructive methods (e.g. biopsy).  Therefore, an accurate depiction by molecular diagnostic techniques of the totality of the GI microbiota in any individual stool sample would necessitate numerous mucosal biopsies and the use of at least 40 to 50 distinct molecular diagnostic probes.  At a cost of $150 to $200 per commercially available, FDA-approved probe, the cost of the probes alone would price such a depiction at a minimum of $6,000 to $7,500 – a very expensive stool analysis indeed!

Some commercial laboratories have developed their own low cost “in house” molecular diagnostic (DNA/PCR-based) probes but their reliability, especially with regard to clinical sensitivity and specificity (even if one were to grant that they have acceptable levels of analytical sensitivity and specificity) is questionable.  Therefore their use must be accompanied by a disclaimer that such tests are not to be used for diagnostic purposes.  Further, regarding “sensitivity”, high analytical sensitivity does not guarantee high clinical sensitivity, for example, as many as 15 % of known HIV-1 seropositive individuals may be “negative” on the exquisitely [analytically] sensitive PCR test for presence of the DNA of that virus.

Marketing materials for “in house” molecular diagnostic technology have claimed that their technology is “more sensitive than classic laboratory methods”.  Although not clearly stated, they are probably referring to “analytical sensitivity”; the ability to identify the fewest number of organisms.  This is not the same as “clinical sensitivity”; the ability to state, with a reasonable degree of certainty, that an organism is present when the technology reports to have found it (i.e. few false positives).

Similar materials have claimed that their molecular diagnostic technology is “specific and accurate”.  Again, although not clearly stated, they are probably referring to “analytical specificity”; the ability of a test to clearly differentiate one organism from another [and accurately report the findings].  This is not the same as “clinical specificity”; the ability to state, with a reasonable degree of certainty, that an organism is not present when the technology didn’t find it (i.e. few false negatives).

Marketing PCR/DNA-based stool testing as the only reliable way to get an “accurate” enumeration of total GI microbial load, is misleading and especially so given the fact that many important organisms are known to reside primarily in biofilms adherent to mucosal surfaces (not in the lumen) and thus would not be enumerated accurately by molecular testing of lumen contents.  For PCR/DNA-based enumeration to be sufficiently “accurate” for diagnostic purposes would require, at the very least, the aforementioned biopsies plus an analysis of lumenal contents not to mention the use of peer-reviewed, FDA approved molecular diagnostic technology.  Even with accurate enumeration, molecular diagnostic technology would include non-viable (dead) organisms and so could not give an accurate picture of the “balance” amongst living, metabolically active organisms in the GI tract.

In reality, the only non-destructive way to accurately and reliably (excellent clinical sensitivity and specificity) assess the degree of “balance” among the microbiota within the GI ecosystem is with culture-based stool analysis.  For this reason, culture-based stool analysis remains the Cornerstone to the Standard of Care for the assessment of gastrointestinal ecological balance and function: it is not “old school” or “old technology”it is the “gold standard” against which the performance of all other technology is measured world-wide!

In summary, Comprehensive Stool Analysis expands on the standard of care for any complete assessment of gastrointestinal terrain and function:

  • Permits positive ID of specific living pathogens;
  • Clearly portrays the balance (or imbalance) of beneficial, commensal and dysbiotic bacteria and yeast and definitively IDs  pathogens including parasites;
  • Provides phenotypic susceptibility testing results; plus
  • Offers valuable insight into absorption, digestion, inflammation, humoral immune function and other important markers of GI health and disease.

State-of-the-art culture-based technology is the cornerstone of the International Standard of Practice for any comprehensive assessment of GI function.

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2 Comments. Leave new

  • I disagree with this opinion for certain GI problems. Cultures need live bacteria, and many bacteria are strict anaerobes and cannot be cultured. Additionally, if the patient has parasites, some cannot be easily detected by the common stool tests, since many parasites neither live nor lay eggs into the stool itself.
    If there is a big digestive problem with high impact on the quality of life for the patient, and all common tests were negative, a PCR based test might be able to pick something up.

    Reply
    • I have asked Charles Masur MD to respond to your comments in order that a more substantive view may be added.

      “I DISAGREE WITH THIS OPINION FOR CERTAIN GI PROBLEMS.” The poster was not clear as to what, exactly, those “certain GI problems” might be. However, I was happy to note that the writer did not disagree outright with the over-riding utility of culture-based stool assessment. After all, culture-based technology is recognised as the world standard for bacteriology and mycology assessments and it is the standard to which all other stool analysis technologies are compared. One major problem with other technologies, including molecular-based technology, is that all have lower clinical sensitivity and/or clinical specificity compared to culture-based stool analysis. Another is that, even if molecular technology (e.g. with PCR) were to correctly identify the presence of a particular organism, it would still be necessary to culture, isolate and test the specific phenotype that was isolated in order to determine its sensitivity to any natural and/or pharmaceutical agent(s).

      “CULTURES NEED LIVE BACTERIA, AND MANY BACTERIA ARE STRICT ANAEROBES AND CANNOT BE CULTURED.” This would be a fair comment for traditional aerobic technologies however, with the use of more up-to-date methods, it is possible to identify, collect and culture (under anerobic conditions) the spores of anerobic organisms.

      “ADDITIONALLY, IF THE PATIENT HAS PARASITES, SOME CANNOT BE EASILY DETECTED BY THE COMMON STOOL TESTS, SINCE MANY PARASITES NEITHER LIVE NOR LAY EGGS INTO THE STOOL ITSELF.” Intestinal parasites by definition must spend at least some part of their lifecycle in the lumen of the gut (i.e. in the stool) so some part of their life-cycle (e.g. larval forms, adult worms, gravid proglotids, eggs, etc.) will be found therein. This being said. it is true that some parasites are difficult to detect and may not be found in each and every stool sample. For this reason at least three samples, taken at least 24 hours apart, should be collected and analyzed to maximize the detection and identification of any parasites if they are indeed present. While it is true that a few parasites do not lay eggs in the stool itself (e.g. strongyloides) either immature or mature forms of most of those organism can be found and identified by examination of stool.

      “IF THERE IS A BIG DIGESTIVE PROBLEM WITH HIGH IMPACT ON THE QUALITY OF LIFE FOR THE PATIENT, AND ALL COMMON TESTS WERE NEGATIVE, A PCR BASED TEST MIGHT BE ABLE TO PICK SOMETHING UP.” I would agree with this statement to a certain extent, not as an initial “fishing expedition” but as a last resort and after fully utilising culture-based aerobic and anaerobic technology as well as traditional microscopic techniques as performed by a highly experienced and reputable laboratory.

      Reply

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