GI360 FAQs

GI360™ Frequently Asked Questions

Q: What is the significance of the microbiome Diversity Score, and how should it be interpreted in conjunction with the Dysbiosis Index?

A: Diversity of species is an important component of health for any ecosystem, and the gastrointestinal microbiome is no exception. Recent research clearly indicates that greater diversity is associated with a robust and healthy microbiome. Greater diversity is important for redundancy with respect to important functional guilds, i.e. butyrate production. For the GI360™ the Diversity Score is calculated from the microbiota abundance data (PCR), using the well-established Shannon's diversity index. Data from DDI indicates that the Dysbiosis Index and Diversity Score are inversely correlated- the higher the Dysbiosis Index, the lower the Diversity Score. As such, a Diversity Score would be expected to improve with successful clinical intervention to restore normobiosis.

Q: Why does DDI continue to use culturomics now that you are using PCR?

A: Culture and PCR are extremely comprehensive and complimentary methods to investigate clinically important issues in the gastrointestinal microbiome. There isn't a single perfect method for addressing all clinical questions regarding the gastrointestinal microbiota. PCR is awesome, but it is not the be-all end-all with respect to comprehensive stool analysis. Culture with proteomic identification via MALDI-TOF MS remains clinically significant and is here to stay. PCR addresses the question- Is it there? PCR is limited by the relatively small number of probes in hand. Culture addresses the question of what is there, with the limitation that most obligate anaerobic bacteria are not routinely cultured. That being said, Doctor's Data has the ability to identify >1,600 genera and species of bacteria and yeast; for the vast majority there aren't PCR probes. Culture and PCR are extremely comprehensive and complimentary methods to investigate clinically important issues regarding the gastrointestinal microbiota.

Further, clinically applicable susceptibility testing requires cultured isolates of live bacteria and yeast.

Q: Does the GI360™ include susceptibility testing?

A: Yes, GI360™ includes direct susceptibility testing for cultured pathogenic and dysbiotic bacteria, and all cultured yeast. Susceptibilities to antibiotics and natural/botanical agents are provided for each individual patient's pure isolates- no "wall charts."

Q: Can you talk about susceptibility testing (standardized) vs. detection of resistance factors?

A: Hundreds of resistance markers can be detected in a "stool pool" of well patients. Resistance markers may be present in the microbiome as a result of previous exposure to antibiotics and environmental exposures; elemental mercury is a classic example. Resistance mechanisms may very well be associated with non-pathogenic, commensal organisms. Simply evaluating the presence of resistance markers in a total stool DNA extract, without direct connection to a specific pathogenic bacteria, has no value in clinical microbiology or patient care.

Direct susceptibility testing is performed with highly standardized and validated techniques with regards to natural antibacterial and prescriptive agents. Bona fide susceptibility testing provides actionable results that aid in targeted clinical intervention, and is especially helpful when avoidance of pharmaceutical antibiotics is a first-line preference.

Q: Some stool tests include bacterial ratios like Firmicutes: Bacteriodetes (F:B) - does the GI360™?

A: One certainly can get an idea about imbalance among those predominant phyla by looking at the web diagram on the first page of the report (Abundance and Diversity). Defining a robust, clinically meaningful F:B ratio was not an objective of the incorporated dysbiosis model. Some labs offer a ratio based on very limited probe sets. An expanded and clinically validated PCR probe set that characterizes a microbial profile associated with obesity, Metabolic syndrome and T2DM beyond an F:B ratio is highly desirable, but yet to be conclusively elucidated.

Q: Other labs report some different bacteria by PCR - how were the bacteria that GI360™ reports selected?

A: The microbiota abundance and diversity section of the report is a novel test within the GI360™ test. Using a unique approach, the specific probes were selected to cover consensus observations from published literature regarding the most clinically significant bacteria with respect to dysbiosis associated with IBD and the different classifications of IBS. A scientific process was applied to select DNA probes targeting ≥300 bacterial species, based on ability to confidently discern dysbiosis in that clinical setting. The specific set of probes and the model has been rigorously tested and validated to identify and characterize dysbiosis. Doctor's Data also utilizes additional DNA probes for pathogenic bacteria, viruses and parasites, separate from the dysbiosis test. The latter probe set includes the probes that are used in an FDA cleared panel for the detection of microbes most commonly associated with diarrheagenic disease.

Q: What are the "requirements" for validation of molecular testing?

A: Great question as not all PCR platforms are equal. There are currently no specific requirements for molecular testing in the clinical laboratory so clinicians should consider published performance characteristics for a given PCR platform. Very strict assay performance evaluation is required for FDA/EU (CE-marked) clearance. The performance characteristics of the primary PCR platform that DDI uses (CE-marked) has been published in a peer-reviewed journal (doi:10.1111/apt.13236), and confirmed in-house. The PCR testing at Doctor's Data has been evaluated against Illumina deep sequencing.

Q: Comparison with other DDI stool tests. When to choose this over the comprehensive stool analysis.

A: Both the GI360™ and CSAP are excellent tests providing actionable information. The CSAP utilizes growth-based culture and ID by MALDI-TOF, PCR for specific pathogenic microbes, important stool chemistries, and microscopy to detect and assess the status of commensals, dysbiotic and pathogenic bacteria, yeast and parasites. The GI360™ built on the CSAP with addition of an additional PCR platform. The GI360™ includes indexes of Dysbiosis and Diversity, which are calculated from the results of the Microbiome Profile (PCR). Both the GI360™ and the CSAP include PCR analysis for a total of 14 pathogenic bacteria, viruses and parasites.

Q: Does this test include H. pylori and beta-glucuronidase?

A: Beta-glucuronidase activity is included in the GI360™. Many clients do not want H. Pylori, and it is appreciated that many clinicians and patients do not want to pay for something that they don't want or need. H. pylori testing is available as a discounted add-on. Doctor's Data uses the best stool test for H. pylori; the FDA cleared stool antigen immunoassay.

Q: PCR vs culture, and microscopy - advantages and disadvantages.

A: PCR is a well-established method for detection of microbe specific, nucleotide sequences. It is very sensitive and specific. But, it can only detect what one has probes for. Many more bacteria, yeast and parasites can be detected via standardized, high quality culturomics and O&P. The combined methodologies are the paragon of complementation.

Q: Is there something in the results of these tests that would point to a strong probability of SIBO presence?

A: There isn't a stool test that permits direct diagnosis of SIBO, and breath testing is unreliable with poor positive predictive value. Although not practical, gavage sampling from the upper bowel followed by culture or PCR has been used in the research setting.

Q: In terms of clinical indications, how does the selection of GI360™ build upon/differ significantly from the CSA w/parasitology?

A: Key term is builds upon, an already powerful clinical test. Difference? Improved sensitivity for detection and characterization of dysbiosis (not just insufficiency dysbiosis), poor diversity, and detection of pathogenic bacteria, viruses and parasites. The sensitivity and reproducibility associated the highly validated PCR is downright phenomenal.

Q: I've heard PCR testing can pick up bacteria that were present in the past but are no longer present and contributing to dysbiosis (whereas culture picks up only things present currently). Is this accurate?

A: PCR can be so sensitive that it is recommended that test of cure by repeat PCR testing is performed about 3 weeks after treatment. Case history and patient presentation should always be taken into account when considering results for any laboratory test.

Q: Is stool testing a consideration in pediatric populations?

A: GI360 and CSAP are testing options for pediatric populations. The CSAP may be more appropriate for those under 2 years of age. The Microbiome Abundance and Diversity portion of the GI360 is best utilized for ages 2 and above due to variations in the microbiome ages 0-2.

There are clinical nuances when interpreting stool reports in pediatric populations. For example, breast fed infant stool contains higher levels of inflammatory markers, calprotectin and lactoferrin. Pediatric patients are more frequent carriers of C. difficile than adults. Cultures of stool "normal" and "abnormal" organisms and quantities differ in pediatric patients vs. adults and require careful interpretation before treatment. GI pathogens and parasites, however, are significant findings, irrespective of age.

Pediatric reference ranges have not been established for stool testing, including the GI360, CSAP and all add-ons.