What is Next-Gen Sequencing?
MicroGen DX is a Molecular Laboratory that utilizes Next-Gen DNA Sequencing to accurately identify all microbes within each sample. The extracted DNA from each sample is matched to the DNA sequence codes of the National data base of over 25,000+ known microbes of bacterial and fungal species with 99.9% accuracy. Conversely, we now know that culture methods can only grow 1% of all known microbes and have an alarming rate of negative or no growth results leaving physicians guessing what microbes are causing the infection and impacting treatment decisions.*
*Bjarnsholt T., “The role of bacterial biofilms in chronic infections.” US National Library of Medicine, APMIS Suppl. 2013 May;(136):1-51. doi: 10.1111/apm.12099., PubMed.gov, web.
Chronic Infections, Biofilms and Next-Gen Sequencing
All planktonic or single cell microorganisms will eventually move to a biofilm state, making diagnosing the causative microbes by way of culture methods nearly impossible and leading to chronic and ongoing infections to remain.
“The CDC and the NIH have estimated that biofilm infections now constitute 65 - 80 % (respectively) of bacterial infection.”
CDC/ NIH research shows that 80% of infections are caused by Bio-Films. Cultures fail to identify the microorganisms in a biofilm phenotype.”*
*Orthopaedic biofilm infections - Paul Stoodley,a Garth D. Ehrlich,b Parish P. Sedghizadeh,c Luanne Hall-Stoodley,d Mark E. Baratz,e Daniel T. Altman,e Nicholas G. Sotereanos,e John William Costerton,e and Patrick DeMeoe
To understand WHY cultures fail in correctly identifying the causitive microbes we need to understand how biofilms form and work together to survive. Microbes have switched from an acute frontal attack by planktonic cells, to a strategy of biofilm growth and chronic attack on infected tissues, and the most serious long-term effect of this tactical change is that they evade detection by the classic methods of Medical Microbiology (culture method).
Patients will be treated more effectively when the identity of any and all bacteria in their tissues can be accurately determined.
Next-Gen Sequencing (NGS)
We live in the era of precise DNA-based forensics, and of whole genome sequencing, and patients will be best served when we mobilize these resources for the prompt and accurate diagnoses of bacterial disease. ONLY Next-Gen Sequencing (NGS) can detect all of the microbes who are living at the site of the infection.
Molecular Diagnostics versus and my Microlab
Q: But if these microbes are really there, why doesn’t my microlab grow the same microorganisms that NGS can detect?
A: There are many reasons NGS is a far superior tool in identifying microbes, NOT having to grow anything is just one reason why.
Reasons the microlab did not find or grow microbes that we detect with DNA sequencing:
- Science has proven that less than 1% of all microbes can be detected by traditional culture.
- Pradeep Singh, MD has recently shown (Singh P Copenhagen biofilm meeting 2011-site this) “that cells of Pseudomonas aeruginosa undergo a structural change in the lipopolysaccharide (LPS) of their outer membranes that makes them unable to grow on the surface of agar plates, even if the medium is otherwise ideal.
- There are National Guidelines for Microbiology (cultures). These guidelines are established by the IDSA and ASM. They require your sample for culture to be at the microlab in 2 hours and kept at room temperature. Why does the IDSA and ASM have strict sample management criteria ? They know that in order for the sample to “grow” it has to be at the lab in a condition that allows the lab to have a chance to successfully grow the specimen on the plate. Look at the lab boxes sitting outside medical offices and ask yourself, “Is that swab or urine specimen going to be at the lab in 2 hours?”
- PCR and NGS do not have the same limitations of time and temperature as culture!
- The microbes were part of a biofilm and have changed their genotype and phenotype and “refuse” to grow.
a. The microbes created a team to successfully fool traditional and archaic testing methods.
- Wrong media was used: If the microbes are anaerobes, they are extremely difficult to grow in desired conditions and will not grow in “normal” agar environments.
- Fungi takes weeks to grow, if at all.
- MicroGen DX laboratory uses Molecular Diagnostics of PCR and NGS to identify the microbes by extracting the microbial DNA within each sample. In the PCR Test Level I we detect a precise number of microbes typically known to cause the specific infection. We also identify Antibiotic resistance gene markers and deliver the overall bacteria load within 24 hours. We then take the extracted DNA and use this on our NGS Test or Level II and match the DNA sequence codes to a national data base of 25,000 known microbes to accurately deliver the microbial data of what is really inside the sample collected from the infection site.
Q: But, could any of the microbes detected be contaminants?
A: Yes, there may be contaminants listed on your report.
How could contaminants appear on my lab report?
Contamination could happen during the sampling process. For example, if proper skin prep is not done correctly, normal flora on the skin may attach itself to the needle while penetrating the skin for an aspiration sample of say a knee or shoulder. For example, this should be considered if Staph Epi or P Acne are listed on your lab report. Also we know that lactobacillus is a normal flora in the vaginal area. Normal flora may be on the list but not part of the problem. Remember, these microbes are there for a reason and if balanced, live in harmony with the host. The area of the sample collection needs to be considered when reviewing your report to determine which microbes are to be treated.
Sites with Normal Healthy Microbiome and sites with No Microbiome
Sites that have a Normal Microbiome?
Sinus Cavities, for example have a microbiome of bacteria that work in harmony to keep the host healthy. When taking a sample it is crucial that the sample is taken from the site of the infection. The sample should be collected from the area of inflamation.
Sites that Do Not have Normal Microbiome:
When the sample is taken from host areas that do not have a microbiome such as joints, wounds, urine or lungs, in other words, bacteria should not be colonizing those host spaces. In these cases our report is not as difficult to interpret. NGS is detecting the bacteria and fungus that have been found at the site of collection. If the patient is not showing any other markers of infection than the host is dealing with the species that have found their way to that part of the body. If you have any other laboratory tests, WBC, CRP, LE and clinical signs of inflammation and infection, then our report is giving you the make up of the microorganisms found at the site.
What do I do with all the information on my lab report?
Part I is the PCR Test Result
qPCR Rapid Screening will detect 8-16+ microbes traditionally found or determined to cause a certain type of infection. Within 24hrs qPCR will identify the “usual suspects” at the scene of the infection but can not tell you if any other species are there.
We also detect and deliver Antibiotic Resistance Genes to identify what antibiotics may not work to treat detected microbes.
But the limitation of PCR stops at those 8-16+ identified microbes. Within all chronic infections the story doesn’t stop with 16 or even 45 species of microbes. In human samples alone our NGS technology has detected over 6,500 species of microorganisms! This is why NGS is so crucial to provide a comprehensive analysis of exactly which microbial species are at the site of the infection.
Part II Next-Gen Sequencing
To date, we have found over 6,500 microbial species in human samples. This level of detail is crucial in assisting physicians treat the actual cause of the infection. We are talking about those chronic infections that some physicians have stopped treating due to “zero growth culture reports” and/or “no signs of improvement”. But with our test we have given physicians and patients the answers they could not get before that made the differnce in clearing a 20 year sinus infection, years of UTI pain and suffering and put a stop to amputations to see patients walk out on their own two feet. This is not magic or a miracle. This is the most comprehensive diagnostic tool you will ever need to assist with delivering you accurate data on the microbes causing the infection. Furthermore, if you stop with a culture report or even PCR report there is a great possiblity you may be only seeing a very, very small part of the whole microbial picture. BUT now you have the option for Next-Gen Sequencing.
Making a treatment decision based on your NGS Report
When you receive your lab report you will see a list of microbes detected by their DNA by percentage detected. This seems great...What do I do and what do I treat here?
First School of Thought
One School of Thought to treat infection is to target the dominant species detected. By destroying the strongest and most numbered microbes the biofilm “house” or community can collapse and the rest will be taken out under host pressure. Microlabs using traditional culture are also looking for “dominant species” based on what will grow. NGS is telling you with DNA evidence what the true dominant species is at the host site.
Similar to going to battle with 300 front line men with 30 cavalry. Once you destroy the 300 it will make wiping the rest of the army out easier.
Second School of Thought
Another School of Thought is to target all the species detected not just the dominant species. This strategy has to take into consideration the use of multiple antimicrobials and the impact to the host. We know that multiple systemic antibiotics have consequences to the microbiome of the gut. We also know that these agents have side effects to take into consideration. For certain infections ( sinus & wound, UTI ) a topical application of antimicrobials can provide a route of administration that provides the benefit of avoiding the impact to the host microbiome in the gut. Topical application of antimicrobials can avoid the toxicity and side effects of systemic delivery while delivering 1K to 5K times the concentration of antimicrobials directly on top of the site of infection. This high concentration would never have been achieved with oral or IV delivery and would provide greater penetration of biofilm EPS matrix.
Do I have to follow the recommendations in the report?
Cultures were designed to grow single cell / planktonic bacteria otherwise known as “professional pathogens”. Molecular with PCR and NGS give you the complete picture. You make the decision as to what you will treat. The recommended antibiotics and antifungals are only offered as suggestions based on a research data base where antimicrobials were shown to be effective against the species detected. It is up to the physician to make use of the information in the report, along with other information gathered in the treatment of the patient, to decide on the best course of action. But the most significant factor we deliver is accurate data of what is living inside your patient’s infection so you can be confident in your treatment decisions for their recovery and overall health.