Next Generation Sequencing (NGS)

Bacteria can be classified using conventional microbiology methods, such as microscopy or being grown on specific media in a laboratory and using antibiotic sensitivity assessments. In recent decades, molecular microbiology methods have revolutionized bacterial identification. A popular method is 16S ribosomal RNA (rRNA) gene sequencing. This method is not only faster and more accurate than conventional methods, but also allows identification of strains that are difficult to grow in standard laboratory conditions.

The 16S rRNA gene is present in all bacteria. This makes it an ideal genetic fragment to be used in identification and comparison of bacteria. NGS is a process which uses this 16S rRNA gene identification to analyse for quality. Only the highest quality bacteria, anaerobes or fungi identified will be interpreted and reported. The data for each detected bacterial or fungal species is then reported as a percentage of specimens identified.

Deep NGS or Shotgun sequencing goes further, it creates a genetic fingerprint of everything in your urine sample and is able to identify tens of thousands of microorganisms in one sample. This can include not only bacteria but parasites and viruses something that PCR and simple 16S NGS testing cannot offer.

Benefits of NGS testing

  • NGS tests are not affected by temperature or time delays unlike traditional urine cultures where the urine sample must be delivered to the laboratory within two hours to prevent sample degradation. Often standard cultures will return a no growth result when samples have not arrived at the laboratory within the time frame required for successful culturing.
  • NGS testing does not grow microbes, they are instead extracting the microbial DNA from a sample and can identify multiple bacteria, anaerobes or fungi. They require just need one sample.
  • These reports will be comprehensive in nature covering all micro-organisms in your sample rather than focusing on a single pathogen which the standard laboratory culture is directed towards.

Issues with NGS testing

There are some uncertainties about NGS testing in a clinical setting:

  • All testing is reliant on the sample containing the presence of biofilm pieces or infected bladder lining cells.  If these are not present in the sample you submit, no laboratory test offered by these companies will be able to detect the pathogens contained within them. Dormant, embedded bacterial DNA will not be detected.
  • At present, it isn’t possible for a DNA sequencing test to tell you whether any micro-organisms identified were part of a biofilm or intracellular community or not.
  • It is currently unknown how the urinary microbiome may differ from the microbiome of the bladder wall and in particular those bacteria embedded into bladder wall cells or those affected by biofilm coverage. To understand the differences further will likely require more invasive sampling procedures such as biopsies.
  • The DNA extraction technique chosen will significantly affect how faithfully the bacterial composition of the original sample is represented by the DNA extracted from it. In some bacteria, such as Gram-positive bacteria and Mycobacteria, it can be more difficult to break down the cell membrane of the bacteria (known as lysis) for study than others in a microbial community and these bacteria may be less represented in a report. On the other hand, if an extraction method is too harsh, the DNA from the easily lysed species may become sheared. Currently, there is no standard technique that works equally well for lysing all bacteria in a given sample.
  • Another limitation of sequencing-based approaches is that they only yield information about the microbial DNA in a sample. While this allows for identification of the types of bacteria present in a biological sample, it does not distinguish between bacteria that are live from those that are dead.
  • NGS reports give percentages on the bacteria/fungi or microbes found in your urine. But the understanding of the urinary microbiome is still in development. Are all those identified responsible for your urine infection? Does a higher percentage equate to the causative agent behind your infection?
  • The testing methods are based on individual, unique, patented methods used by each laboratory.  They test against a panel of microbes/fungi/anaerobes established by that company.  If the infection causing micro-organism is not found against that panel, how are you to be treated?
  • There is also the issue of cost. Treatment of a chronic UTI may involve several NGS tests over a period of time and at present, these are extremely expensive. Some patients in the US are able to offset their costs through health insurance but others are unable to do so. International patients cannot offset against health insurance.
  • It can be difficult to find a practitioner who can interpret the laboratory findings and treat accordingly. Treatment guidelines used by GPs and consultants worldwide are based on standard testing protocols. Some patients have found their results have been dismissed or ignored by their doctor or consultant.
  • Finally, as yet, no research studies have been published using NGS testing in a clinical setting to determine success outcomes for patients using this diagnostic method.
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