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

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!

“Avoiding Burnout in the COVID Marathon”

I have seen a lot of tired looking colleagues around. I have spoken to people that are tetchy who are not normally tetchy. I know of colleagues who have been working far in excess of their normal working hours…

Is this the new normal?

It has made me wonder about the sustainability of it all, and how many people are going to “fall over” next year when they come to realise that this pandemic is not going to go away anytime soon, even with the introduction of a vaccine.

It became obvious quite quickly that COVID-19 was going to be a marathon, not a sprint. Being a marathon runner myself, I am well used to pacing and knowing what happens if one sets off too quickly! There will be few people working in microbiology who have not been affected by the extra work pressures that this pandemic has recently caused. In my opinion 2021 will be more of a risk for burnout than 2020. Continued pressure leads to burnout…

Here are a few of my own personal tips for avoiding burnout, in the hope that I will be able to get through the next year relatively unscathed.

  1. Be very selective about voluntary work:- We may not realise it, but a lot of work we do is to a large extent “voluntary” in nature. Whether it is giving presentations, reviewing research papers, attending webinars, participating in advisory groups. In a pandemic situation all this type of work just adds to the stress, and eats away at our working hours. Please, be ruthless with this type of work, and just focus on your core work, even if some onlookers think you are being lazy…
  2. Don’t get caught up in the “work” hype:- In the pandemic situation, people lose perspective, and have a tendency to get caught up in the work frenzy, feeding off each other’s nervous tension, reading and responding to every work request. Don’t do this, keep perspective and be aware that you only have so many working hours each week to be as productive as possible.
  3. Take leave:- Mix this up between intermittent “treat” days off and the occasional period of longer leave (1 week plus). When you are on leave, you must be completely detached from work, no email contact, no texts, no Whatsapp groups, nothing. Switch off all your notifications. Ideally have a dedicated work phone and leave it there. I find that the people that adhere to these leave rules are the ones who have the best work-life balance.
  4. Don’t do backlogged work on return from leave:-  Taking leave is one thing. Returning from it is quite another! The last several times I have been on leave I have left an Out of Office message saying. I will be on leave between dates X and Y. Emails received during this period will not be read. If you have an important personal message for me then please resend it after date Y. I  then have an empty inbox within 1 minute of returning to work because all the emails received during leave get dumped immediately and unceremoniously into an archive folder without review. Trust me, the sky does not fall!, and I do not return from leave with an impending sense of doom. Rarely does anyone actually resend an email on my return, which goes to show how “impersonal” or trivial most email is…
  5. Don’t go to every meeting you are invited to:- I get invited to approximately 15 meetings a week. If I went to all of them, half my work hours would be gone in one fell swoop, just like that. I therefore am fairly ruthless about which meetings I go to. Are they meetings where I can influence the outcome, or where I need agreement from others for the agenda I am following? Otherwise it is just another hour down the drain.
  6. Be ignorant:- Nobody can be expected to be up to date with all the COVID research. Even if you are up to date, you will almost certainly be out of date within weeks, such is the pace of knowledge acquisition. Accept that you will be a bit ignorant! Pick a topic within COVID that particularly interests you and keep abreast of developments in that field. Then try and contribute in that area.
  7. Leave work at work:- Working out of hours should very much be the exception as opposed to the norm. When you finish work, go home to your family and the rest of your life and your other passions outside of work. You can start thinking about work again the next day.
  8. Connect and be kind:-. It is very easy to become short-tempered when you have a lot of work on your plate. However, before you have an argument, remember that you need to see and work with this person the next day, and the day after that. You can connect and be kind without having to be best friends.
  9. Share the blame:- I am a big believer in the philosphophy that a problem shared is a problem halved. There is nothing more stressful than trying to sort out a complex issue single handedly. 
  10. Build teams rather than being a linchpin:- From an individual point of view, it is nice to be seen as a linchpin, to be viewed as indispensable in your particular department or area of expertise. However from an institutional point of view and particularly during a simmering pandemic like this one, linchpins can be detrimental, because of an over-reliance on the linchpin. When the linchpin eventually falls over, because nobody can keep going forever, then the whole system runs into trouble. What you need is an effective team, where one person can go off on leave (or off sick) and there will be little difference to the productivity. This pandemic has made me realise how critical it is to function as a team, rather than putting all your efforts into being a linchpin.

I currently have 2 jobs and a family of 8 at home. If anyone is going to burn out, it will be me! I am very aware of the need to listen to my own advice and follow the rules above. Sometimes I lapse and then need to discipline myself in order to optimise work-life balance. 

Even with a vaccine on the horizon, the COVID-19 pandemic will keep us all occupied for at least the next year, and most likely a bit longer than that. We each need to have a personal plan of how to navigate through it from a work point of view.

Look after yourself, and look after each other.

Michael

“Between the devil and the deep blue pool…”

Pooling of COVID-19/SARS-CoV-2 samples has been an important and integral part of the NZ laboratory response to COVID-19.

Two weeks ago, following the appearance of COVID-19 cases in the community following a 100 day hiatus, test volumes surged  nationally from 4000 samples to 27000 samples a day, literally overnight…

It goes without saying that without widespread pooling of samples, we would have had testing backlogs of several days if not weeks, completely devaluing the usefulness of the results in terms of contact tracing and significantly increasing the risk of exponential growth in the outbreak.

Microbiologists, by nature, are purists. They understandably want their laboratory to produce the “perfect” result. Accreditation agencies may have similar views, with a narrow focus on the quality of the results produced. That’s their job after all…

But the world, and in particular the COVID world that we now live in, is far from perfect, and we need to keep looking at the big picture.

Pooling of clinical samples for a PCR assay has a small effect on sensitivity. Because we measure virus counts on a logarithmic scale this effect is almost, but not quite negligible, if a small number of samples are pooled. We have the potential to miss “positive” samples with very low viral loads, likely coming from patients who are almost certainly non-infectious. In my anecdotal experience, most of the results produced at the limit of detection are in patients who are recovering from infection, in the recent or not so recent past. Our experience shows that the loss of sensitivity by pooling samples is probably less than using a throat swab instead of a nasopharyngeal swab.

The other potential drawback of pooling is that if you get a positive pool, you then need to test all the samples in the pool individually. If positivity rates are high then pooling becomes self-defeating, creating even more work! However positivity rates in NZ have up until now been very low, so this has not been an issue for us.

As far as I am aware, NZ diagnostic laboratories that have utilised pooling (most of them) have validated the methodology over different platforms to the best of their ability, within the considerable time and resource constraints they have had to work within. In addition they have implemented IT solutions to facilitate the pooling of samples from a pre-analytical point of view.

Registration and molecular staff all over the world have been under the pump recently due to COVID-19 testing. Long, long hours, validation of new assays and platforms, pressure to get results out quickly… It is tough and I am in utmost admiration of our molecular team. Pooling is one of several ways to reduce this pressure on staff and try and prevent burnout. COVID-19 and the associated high testing volumes are not going to go away. This was always going to be a marathon effort, not a sprint, so testing processes need to be sustainable in the long term.

COVID-19 is a new disease but pooling of laboratory samples is not. The thing that has become very clear with regards to this infection, is that effective control depends to a large degree on testing large numbers of people and getting the results out quickly so that appropriate isolation and contact tracing can be performed. We should be embracing policies that allow us to achieve this goal.

Up until now at my own lab, our largest volume molecular test was Neisseria gonorrhoeae/Chlamydia trachomatis PCR, approximately 60,000 tests per annum. SARS-CoV-2 test numbers are going to completely and utterly dwarf this!

We need to adapt, in a pragmatic and realistic fashion, to the situation that we are currently faced with.

Michael

There are plenty of examples of SARS-CoV-2 pooling studies out there. Here is one for starters!