Category Archives: Future of Microbiology

“Metagenomic disruption of the diagnostic microbiology lab”

Future of Microbiology, ,

I am 50 now, so I only have another 25-30 years of my working career left. So much to do in so little time!

I often wonder what the microbiology lab will look like on my last day at work. (I have posted on this before). Possibly a more interesting question is how I will look on my last day at work…

I am interested in how metagenomic approaches are going to disrupt the diagnostic microbiology lab and whether this will eventually become a mainstay of microbiological diagnosis, and eventually consign the agar plate to the museum.

The strangely named “shotgun metagenomic sequencing” involves sequencing all genes present in a clinical sample thus allowing identification of pathogens, and (as a bonus) their associated resistance genes and pathogenicity factors.

It sounds relatively straightforward, but there are certain challenges that one needs to be aware of.

  • Sensitivity:- Unlike PCR, the process does not involve an amplification step, therefore it may not be as sensitive as other currently existing methodology. In addition, if the sample contains a lot of human DNA, or DNA from non-pathogens, then the pathogen can be “drowned out” in the testing process. (stoichiometric ratios). Methods to enhance pathogen and suppress non-pathogen/host nucleic acid during the sequencing process are in development to mitigate this issue, but it is a work in progress.
  • Turnaround Time:- Traditional sequencing can take 1-2 weeks when you add the time for the various steps; extraction, library preparation, sequencing and bioinformatic analysis. This is still slow compared to current culture-based and PCR methodology. Newer Real-time sequencing techniques such as Oxford Nanopore can potentially reduce this turnaround down to a couple of days.
  • Cost:- Cost is coming down, and depending on what sequencing platform you use, can be anything from a hundred dollars to a few hundred dollars per sample. The cost will almost certainly come down further but we are still some way from the cost of a couple of agar plates.
  • Bioinformatic analysis and validation thereof:- The bioinformatic analysis of genetic sequences remains somewhat foreign to most microbiologists. Slowly but surely automated bio-informatic pipelines are being developed which automates this step for an increasing number of pathogens. However, validation of these pipelines is laborious and difficult and requires the input of specialist bioinformaticians.

There are now metagenomic assays commercially available in several areas, the most promising possibly being metagenomic analysis of CSF samples for infective causes of meningo-encephalitis. But it is still only a small niche area of the market, and it has a long way to go before becoming mainstream.

If you look at MALDI-TOF, it took approximately 25 years from the technology becoming available, until being widely adopted in clinical microbiology labs. The reason it has been so successful is because it is fast, accurate, cost-efficient and scalable. I think metagenomic sequencing will take just as long. Operationalisation of exciting technology is a protracted and somewhat painful process…

So, on my last day at work, around about 2050, I think metagenomics will be commonplace in most reasonable sized diagnostic microbiology laboratories. But I have a feeling that the tried and trusted agar plate will still be around… 

Michael

“The Molecular Revolution”

Confessions of a Microbiologist, Future of Microbiology, , ,

Time to get back to some writing “post” COVID!

When I started at the laboratory I currently work at in New Zealand in 2007, we only did one molecular assay, a chlamydia PCR, and we did this with separate extraction and amplification platforms on an open bench, with all sorts of potential for contamination. And we were/are not a small lab, a sizeable regional centre, processing well over 1000 microbiology samples a day.

2007, it’s actually not that long ago…

Fast forward 15 years and everything has changed. We now have a very sizeable menu of molecular assays performed on a range of different platforms. CSF, respiratory virus and GI panels, gonorrhoea, trichomonas, HSV/VZV,  HIV, HBV &HCV viral loads, Legionella spp., Mycoplasma pneumoniae, C. pneumoniae, C. difficile to name just a few. We even have a Mpox PCR!

A lot of these assays are now on commercial platforms that perform both the extraction and amplification steps in an automated fashion in a closed environment, essentially allowing placement of the platform anywhere, and can be run by most of our staff. The results are often available within a few hours of the sample being received in the laboratory.

In summary, the clinical service we can now offer is vastly improved from 15 years ago. I suspect it is much the same in many diagnostic labs throughout the world.

The big question is what will happen in the next 15 years? Will high volume sample types such as throat swabs, vaginal swabs, sputum samples, all still culture based at my lab, succumb to the revolution and go molecular? It is entirely possible that this will be the case. It will probably come down to cost first and foremost. Personally I see throat swabs switching to molecular very soon.

And what place will there be for whole genome sequencing in the diagnostic lab? That is a whole other question in itself but there are quite a few labs now in NZ who have acquired Nanopore Minions and are now “playing” with them in the areas of Infection Control and metagenomics.

My prediction is by 2030, for most diagnostic microbiology labs, their molecular department will be bigger than their traditional culture-based bacteriology department…

What do you think?

Michael

“Will Nanopore sequencing be the next big disruptor in clinical microbiology laboratories?”

Future of Microbiology, , , ,

Will the next big “disruption” in clinical microbiology be Nanopore sequencing technology? I believe this is entirely possible, but there is still work to do…

The last big “disruption” to take place in clinical microbiology laboratories was the introduction of MALDI-TOF for organism identification. From proof of concept to commercial introduction of this technology took a couple of decades. Major disruptions actually take a lot of fine tuning and polishing…

For a platform to be successful in a routine diagnostic microbiology laboratory it needs to have several key characteristics. It needs to be fast, it needs to be cost-effective (compared with existing methodology), it needs to be scalable, and it needs to perform well (good sensitivity and specificity). MALDI-TOF has achieved each of these key goals. That’s why it has been adopted, almost universally in clinical microbiology laboratories. Lots of other innovative technologies come close, but don’t quite get there…

So what about Nanopore sequencing?

Championed by Oxford Nanotech , nanopore sequencing is a unique, scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein “nanopore”. The different bases give a specific change in ionic current. The resulting signal can thus be decoded to provide the specific DNA or RNA sequence. Nanopore sequencing enables direct, real-time long-read analysis of DNA or RNA fragments.

Nanopore sequencing has several potential applications in the clinical microbiology laboratory, such as:

  • Organism detection directly from clinical samples, either by 16S/18S rRNA or by a metagenomic approach
  • Detection of genotypic resistance determinants
  • Detection of genotypic virulence determinants
  • Typing of microorganisms for infection control or public health reasons

How does Nanopore sequencing weigh up on each of the key features required to break into a routine diagnostic microbiology laboratory.

  • Speed: The technology allows both base reading and bioinformatic analysis of the sequences to be performed in real-time. Depending on what is being sequenced, it is possible to get useful information from sequencing in a matter of minutes. Potentially the sequencing process can be “stopped” when the necessary information has been obtained, saving on both time and flow cell.
  • Cost-effectiveness: Compared to other sequencing platforms, the start up costs are relatively low. For just a few thousand dollars, it is possible to get hold of a MinION, a few flow cells, and start “sequencing”. However other costs to consider include the bioinformatic software, and hardware to assess DNA/RNA quality and quantity. The flow cells containing the nanopores are still expensive at the moment, but the cost is decreasing. Flow cells can be washed and re-used to a certain extent, which will reduce costs. In addition, a smaller & cheaper flow cell called a “flongle” has just been released.


    “Flongle”
  • Scalability: The ability to “barcode” the nucleic acid extracts going into the flow cell allows the processing of multiple samples simultaneously. Platforms which include multiple flow cells such as the GridION and PromethION can thus process literally hundreds and thousands of samples in the one day. The recently released LamPORE testifies to this.
  • Good performance: There is a lot of validation work currently going on for Nanopore sequencing for various clinical applications, both microbiological and non-microbiological. A lot of the bioinformatic pipelines that would facilitate commercialisation of Nanopore sequencing are still in development. This will take time. Metagenomic approaches to organism identification from clinical samples using Nanopore sequencing are potentially very attractive. The issues of filtering pathogenic DNA out from the human DNA are currently being addressed.

The exciting thing about this technology is that it seems to be improving very quickly. One of the main issues historically with nanopore sequencing was the fidelity/accuracy of the base calling. However recent improvements in the nanopore design and the reading software have improved this dramatically.

I suspect over the next few years, routine clinical microbiology laboratories, like my own, will start looking closely at this technology to see whether it is ready for implementation in diagnostic clinical microbiology. I suspect it will have an initial role in sterile site samples and resistance genotyping, but may well extend to more routine samples in due course.

I think it is just a matter of time…

Michael

I am keen to hear from clinical microbiologists who have Nanopore sequencing in their laboratory, so I can learn from their experiences!