Category Archives: The Art of Microbiology

“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!

“Taking the crap out of enteric microbiology”

Just because a stool sample turns up at your microbiology laboratory, it doesn’t mean you have to test it… This is old style microbiology reasoning, testing for everything in the hope that you will find something!

There are many different microbiology tests that one can do on a stool sample. Here is a sample list of what is offered at the lab I work at:

  • PCR for common bacterial pathogens, e.g. salmonella, campylobacter, shigella, VTEC, yersinia.
  • Culture for more opportunistic bacterial pathogens such as Aeromonas
  • EIA for cryptosporidium and giardia
  • GDH/PCR for C. difficile toxin
  • Faecal concentration and trichrome stain for ova, cysts and parasites
  • Immunochromatographic assay for rotavirus
  • Multiplex PCR for other enteric viruses (e.g. noro, astro, sapo)
  • Faecal antigen test for H. pylori.

With appropriate clinical details present, we can then choose objectively from the list above which tests are appropriate to perform for a specific sample.

However, without clinical details, it would be utterly unreasonable for the lab to do all of these tests, and without clinical details there is no way of deciding which tests we should be doing.

Yet so many microbiology labs still take this approach. Receive a stool sample and test it for something! This is blindfold microbiology.

Extending this philosophy further, clinical details of “diarrhoea” doesn’t really cut the mustard either. That is to some extent stating the obvious!

Fit healthy adults who present with a short history of diarrhoea in general do not require laboratory testing. Personally I get 2 or 3 episodes of loose stools every year. I am sure the rest of the world has a similar experience! I do not need laboratory testing. So clinical details simply of “diarrhoea” or “loose stools” is insufficient to justify testing. There needs to be more than that…

The lab I work at will only test stool samples if one of the following applies, even when clinical details of “diarrhoea” or something similar is on the form:

  • Something to indicate an illness on the more severe end of the spectrum, such as prolonged diarrhoea, bloody diarrhoea, hospitalised, systemic symptoms, etc.
  • Or something that suggests there might be a public health issue, e.g. food handler, group meal, overseas travel, farm worker, etc.

“Carte blanche” approaches to enteric microbiology are hideously costly, and also give rise to quality issues such as overdiagnosis and overtreatment.

If you test every stool sample you receive for putative pathogens such as Blastocystis hominis or Dientamoeba fragilis, you are going to end up overdiagnosing and overtreating a whole heap of people. Don’t go there!

By taking a considered and objective approach to microbiology testing of stool samples you can dramatically reduce the amount of testing that you perform, and increase the quality of results at the same time.

Michael

 

“Workflow trumps Fancy Tests”

I happened to be visiting a microbiology lab in a large teaching hospital last year. We were shown all the assays they used to rapidly identify a pathogen from positive blood cultures: PCR assays, FIuorescent In-Situ Hybridisation (FISH). They had the works!

The range of tests available was very impressive, and would be the envy of most diagnostic microbiology laboratories.

But there was a catch… At 8pm in the evening, the microbiology department shut up shop and everybody went home. The blood culture analyser stood there completely untouched until 8am the next morning, including any bottles that flagged positive during this time.

So a blood culture that went positive at 9pm would be sitting in the analyser for at least 11 hours before any attempt was made to identify the pathogen.

This got me thinking!

It actually doesn’t matter that much how many fancy assays you have, or how much money your laboratory has. If you can’t get your workflow right then it all becomes a bit academic.

I am a big proponent of 24/7 staffing of microbiology laboratories, or at the very least the processing of positive blood cultures being done 24/7. It is after all one of the most important samples in the microbiology department. We have plenty of lesser importance!

Turnaround times generally don’t just include the actual analysis of the sample. More often than not, it includes storage time, transport/courier time, registration time, verification time, etc.

And then the final result has to be both received and acted on by a clinician. This communication step is also vitally important. There are so many steps, pre-analytical, analytical and post-analytical that contribute to the total turnaround time.

It is useful to do intermittent vertical timeline audits of your critical samples, to see where the delays are occurring, and then sort these out first before you consider fancy assays. And often such delays can be sorted without having to spend a lot of money. It might just be a case of relocating a blood culture analyser, or adding an extra courier run…

I am not against fancy assays, they have their place, but only as part of the whole process…

Michael