FAQ’s on the GI 360 Test

1. What is the reason/significance in continuing culture methods now that DDI is using PCR?

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 to 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 >1,400 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 in the gastrointestinal microbiome.

Direct, clinically applicable susceptibility testing requires the phenotypic expression provided by pure isolates of live bacteria and yeast. Susceptibilities to antibiotics and natural/botanical agents are provided.

Culture and PCR are extremely comprehensive and complimentary methods to investigate clinically important issues in the gastrointestinal microbiome.

2. Does the GI360™ include sensitivity testing?

Yes, GI360™ includes sensitivity testing for cultured pathogenic and dysbiotic bacteria, and all cultured yeast.

3. Some stool tests include bacterial ratios like Firmicutes:Bacteriodetes (F:B) – does the GI360?

One certainly can get an idea about undesirable predominance of Firmicutes by looking at the web diagram on the first page of the report (Abundance and Diversity). Defining a robust F:B ratio was not an objective of the incorporated dysbiosis model. More Bacteroidetes probes would be required to offer a robust F:B ratio. 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.

4. Other labs report some different bacteria by PCR – how were the bacteria that GI360™ reports selected?

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 major bacterial observations from published literature regarding the most clinically significant bacteria with respect to dysbiosis associated with IBS classifications and IBD. A scientific process was applied to select DNA probes targeting ≥300 bacteria, based on ability to confidently discern dysbiosis in the 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.

5. What are the “requirements” for validation of molecular testing?

Great question as not all “PCR” is the same. There are currently no specific requirements for molecular testing in the clinical laboratory so clinicians should consider performance characteristics, most importantly reproducibility. Very strict assay performance evaluation is required for FDA/EU (CE-marked) clearance. All of the PCR testing at Doctor’s Data has been evaluated against Illumina sequencing. If one questions PCR-based laboratory results with respect to discordant patient presentation, consider split sampling (same specimen, different names)… that’s a good test for reproducibility.

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

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 extract, without direct connection with a pathogenic bacteria, has no value in clinical microbiology or patient care.

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

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

Both the GI360™ and CSAP are excellent tests providing actionable information.  The CSAP utilizes growth-based culture and ID by MALDI-TOF, 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 remarkably sensitive and reproducible PCR technologies.  The GI360™ includes The Dysbiosis Index, a calculation based on the overall bacterial abundance and profile within the patient’s sample, as well as PCR detection of pathogenic bacteria, viruses and parasites.

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

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 an add-on for an affordable price. Doctor’s Data uses the best stool test for H. pylori; the FDA cleared stool antigen immunoassay.

9. PCR vs culture, and microscopy – advantages and disadvantages.

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

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

There is not a stool test available that can detect the presence of SIBO. Although not practical, gavage sampling from the upper bowel followed by culture or PCR has been used in the research setting. Analysis of expiratory gasses is riddled with poor quality sample collection.

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

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

12. 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?

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.