Tag Archives: malditof

“Metagenomic disruption of the diagnostic microbiology lab”

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… 


“The dying art of traditional microbiology”


I had the opportunity to visit a food microbiology laboratory a few weeks ago. It was clearly a good laboratory with excellent quality assurance measures, but it was interesting to visit a microbiology lab that did not have a MALDI-TOF platform in place. The lab still relied very much on traditional identification techniques for identification such as colonial morphology, Gram stain, oxidase, catalase, and of course the API biochemical strips!

This made me reminisce about my training days in microbiology in Glasgow back at the turn of the century, when we still used Kohn’s tubes for Salmonella & Shigella identification, X and V discs were used for Haemophilus influenzae, and the CAMP and Reverse CAMP test were used for Streptococcus agalactiae and Clostridium perfringens respectively.

Nowadays, in most clinical diagnostic microbiology labs, everything just gets “thrown” on to the MALDI-TOF platform and an ID is usually forthcoming. In the rare occasions that a “Maldi” ID is not obtainable, one can always send the isolate for 16s/18s RNA sequencing.

It is a changed world, and all the mystery has disappeared..

There are of course very good reasons for clinical microbiology labs to move to MALDI-TOF identification. It is fast, mostly accurate and cost-efficient when utilised in a mid to large sized laboratory. It is also probably safer in terms of risk of laboratory-acquired infections.

But it is a little bit dull, tedious even. I do miss the old-fashioned methods, their variety and their idiosyncrasies. It also gave staff a good grounding in the basic principles of microbiology and bacterial identification. Most new staff that start in medical microbiology laboratories these days will be unfamiliar with anything else apart from MALDI-TOF.

Traditional diagnostic microbiology is a dying art, and the labs that still practice it are becoming fewer by the year. 

I am by no means a luddite, and I am all for progress, but I don’t think the recent technological advances have necessarily made us better microbiologists, or made the job more enjoyable…



“Nothing is perfect..”

Apologies for the paucity of posts recently. I have had an unwell child.

MALDI-TOF (Matrix Assisted Laser Desorption & Ionisation- Time Of Flight) has revolutionised microrganism identification in the past decade, and in particular bacterial identification in the microbiology laboratory. It will continue to evolve into fields such as filamentous fungi, mycobacteria, antimicrobial resistance etc.

Rarely has such an innovation been so rapidly and comprehensively adopted by the laboratory community. And you can see why. It’s quick, it’s easy, it’s cheap (the consumables are anyway) and it’s reliable.

But it’s not perfect….

Some well known identification limitations are listed below where MALDI-TOF struggles to give the microbe a label, or at least one which fits in with our pre-defined beliefs.

Take the following well known, and not so well known examples…

  • E. coli and Shigella species
  • Streptococcus pneumoniae and Streptococcus mitis
  • Bordetella pertussis and Bordetella bronchiseptica
  • Klebsiella oxytoca and Raoultella ornitholytica
  • Shewanella putrifaciens and Shewanella algae
  • Proteus vulgaris and Proteus penneri
  • Klebsiella pneumoniae and Klebsiella variicola
  • Haemophilus haemolyticus and Haemophilus influenzae
  • Serratia ureilytica and Serratia marcescens

There are many more. Please feel free to suggest others that you have come across. There will be even be differences between different MALDI-TOF platforms, e.g. Bruker and VITEK MS.

Most of the time it does not matter too much from a clinical point of view. Some of the time, it does, and we need to try and clarify with molecular or biochemical means.

It shouldn’t surprise us however that the identification is not perfect. All identification systems are essentially typing systems in disguise, and they all have databases or libraries at the core of their function..

  • “APIs” are a typing system based on biochemical reactions.
  • MALDI-TOF is a typing system based on proteonomics.
  • 16SRNA sequencing is a typing system based on nucleic acid sequences.

Identification systems therefore suffer from the inherent problems of all typing systems. No typing system can be matched exactly against any other typing system.

For all typing systems the ability to group isolates and the ability to discriminate them is a trade-off of sorts. For MALDI-TOF, the bigger the databases get, the more the system can discriminate, and thus the more it can potentially differ from the original (textbook) identification.

So no system is perfect, including MALDI-TOF.

But it’s very, very good….