3.3 Advanced testing

Some testing methods are beyond the scope or resources of the hospital laboratory and are more likely to be found in public health and reference laboratories. MALDI-TOF and automated systems are two methods found increasingly in larger clinical laboratories. Most molecular methods (e.g. whole genome sequencing, 16S rDNA PCR, molecular typing) are currently only used widely for research and in reference laboratories. We describe some of these methods below.

3.3.1 MALDI-TOF

Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (Croxatto et al., 2012) gives a result faster than most standard biochemical test methods and at a lower cost per sample. It is both accurate and reliable, and gives identification to species level from a pure culture within minutes. In recent years, MALDI-TOF has revolutionised microbiology practice in laboratories where it is used, having largely replaced most biochemical identification methods.

Disadvantages of MALDI-TOF are that the equipment is expensive and often requires expensive maintenance contracts. It is also not effective for all organisms. For example, it cannot distinguish well between E. coli and Shigella, nor between S.pneumoniae and viridans type Streptococci. For these, additional biochemical testing is still needed for confirmation.

The rapid throughput means that hundreds of isolates can be processed daily using very little technologist time, meaning that it is most cost-effective for laboratories with large sample throughputs (identifying several thousand isolates per year). For these laboratories, the cost of the instrument and the maintenance contracts is very rapidly offset in savings on reagents and staff time.

It is possible to use MALDI-TOF to identify a small number of resistance mechanisms (e.g. for MRSA) but it is still necessary to perform separate antibiotic susceptibility tests (AST).

If you are not familiar with MALDI-TOF, watch the following short video about this method (Theory of MALDI-TOF Mass Spectometry, 2016).

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3.3.2 Automated systems

Automated systems, such as Vitek II (Biomerieux), BD Phoenix (Beckton Dickinson) and Beckman-Coulter-MicroScan are used in some clinical laboratories. Effectively these are an automated version of the biochemical test strips, with more reagents and analytic software. The cost of such systems is high, but less work is needed to complete the tests. A further advantage is that AST can be done at the same time.

3.3.3 Commercial PCR

Commercial PCR is often used for example, with enteric pathogens or for Methicillin-resistant Staphylococcus aureus (MRSA) detection. Advantages are the reduced work involved, as PCR tests can run on an automated ‘plug-in and play’ platform, and the rapid turnaround time for results – the same day. However, commercial PCR tests are expensive, and isolates must still be cultured if AST is needed. Most kits are specific for one or two bacterial species so are more useful for screening or confirmatory testing for specific target organisms, rather than testing for all the clinical pathogens that might be in a sample.

Two commercial PCR systems are:

  • Cepheid’s GeneXpert® System, a family of workstations that can run 2–80 PCR tests on a single, consolidated workstation.
  • The BioFire® FilmArray® System, a scalable system with multiplex PCR testing for infectious disease diagnostics.

3.3.4 Whole genome sequencing (WGS)

Whole genome sequencing (WGS) is a method for rapid sequencing of the chromosomal DNA and mitochondrial DNA (and chloroplasts DNA in plants). WGS is carried out using next-generation sequencing (NGS) technologies, which allow the rapid generation of large quantities of genomic data.

WGS has the potential to become an important tool in pathogen surveillance. However, the challenge in AMR is the timely processing of the large volumes of data, particularly during emergency pathogen outbreaks (Bogaerts, 2021 and Jauneikaite, 2023). As a result, the Surveillance and Epidemiology of Drug-resistant Infections Consortium (SEDRIC, n.d.) working group are working on a series of recommendations on the use of genomic surveillance via WGS for AMR. The working group has identified various advantages and barriers to the use of WGS in AMR:

  • Advantages

    • Health-care associated infections in hospitalised patients pose a significant challenge globally. Genomic AMR surveillance of pathogens using WGS from health-care associated infections can provide much better identification of the causative agent, such as genomic subtypes. It can also help to identify facility-level trends.
    • One particular advantage of WGS is its use to investigate outbreaks and to improve support for infection prevention and control in AMR surveillance in health-care facilities. It can help to identify complex epidemiological patterns, such as the emergence of new strains and expansion of new multidrug-resistant strains.

  • Barriers

    • Despite many advantages, there are still many barriers to WGS in AMR surveillance. The initial set up and running costs can be prohibitive or the equipment difficult to obtain due to poor distribution networks and supply chains.
    • There are also significant challenges in the analysis and interpretation of the genomic data, which typically requires bioinformaticians. Quality assurance processes for both laboratory sequencing and bioinformatic analysis have still yet to be clearly defined.

You will learn more about WGS in the Whole genome sequencing in AMR surveillance course.

3.2 Chromogenic media

4 Identifying the key pathogens in global AMR surveillance