MICROBIAL TECHNOLOGY


compared with $12.23 for standard testing.


The first installation was completed in the National Reference Laboratory in Uganda in February 2020. Then the COVID-19 pandemic struck causing logistical problems and a shift in priorities. Dr Gordon described other non- COVID-associated logistical challenges associated with transporting MALDI-ToF instruments, which do not tolerate tilting into airports such as that in Bhutan that has a steep approach or needing to plan flood resilience by installing a benchtop MALDI-ToF rather than a floor-standing equivalent.


An important consideration for such a


programme is ensuring the sustainability of the solution. Applying MALDI-ToF methods for use in other investigations, such as mosquito identification (Kenya), research and public health applications (Timor Leste) and investigating deaths in fish (Ghana), could lead to additional funding and support for long-term service provision.


While this ambitious project has been additionally challenging due to the COVID-19 pandemic, lessons have been learned for rollout of future programmes. In addition to the logistical learning points, although local doctors were supportive of laboratory development, early engagement with clinicians in the grant application and implementation stages is also key if programmes in LMICs are to be successful.


Detecting resistance mechanisms Dr Mandy Wooton, Scientific Lead at The Specialist Antimicrobial Chemotherapy Unit, Public Health Wales Cardiff, discussed how the routine diagnostic laboratory detects AMR


focused on Gram-negative resistance mechanisms including extended- spectrum β-lactamases (ESBLs), AmpC cephalosporinases and carbapenemases. Detection of these enzymes remains a challenge because of the diversity of emerging resistance, which is continuously evolving with newer targets being increasingly identified, with currently 5522 core AMR genes in the reference gene catalogue. Dr Wooton discussed the complexity of each of the molecular classes of β-lactamases and their different resistance profiles and mechanisms, and how these can be manipulated in phenotypic tests to aid their detection. The β-lactamases are the most significant group of enzymes involved in conferring resistance to β-lactam antibiotics in Gram-negative bacteria, which work by hydrolysing the β-lactam bond of substrates thus rendering the antibiotic ineffective. While standard antibiotic susceptibility testing detects most resistances, Dr Wooton described the current methods and phenotypic tests as the preferred methods in routine diagnostic laboratories for the ‘difficult to test’ resistances. In general, isolates are screened by


either chromogenic/differential agar, disc diffusion or a variety of automated methods. Phenotypic confirmatory tests include combination disc testing and gradient strips, while MALDI-ToF colorimetric tests and automated methods have so far proved less popular. Genotypic confirmatory tests include PCR/real-time PCR, rapid testing direct from sample, whole-genome sequencing (WGS) and microarrays. Dr Wooton discussed how inhibitor- based tests with an indicator antibiotic, including combination disc testing, are relatively cheap, easy to perform


and extensively validated for definitive identification and differentiation of enzyme types. Although there are some limitations due to the complex nature of resistance, for example, the detection of OXA-48 resistance, and difficulty for detection in non-fermenters, they have a good overall sensitivity and specificity. While rapid technologies have a clinical benefit, in that they are rapid and easy to use, and are particularly useful in outbreak situations, they are expensive and the probes and primers may not be updated regularly, leading to limitations in the detection of novel or rare targets and an increase in false negatives. Similarly, WGS is currently used for special circumstances (eg tuberculosis detection), and in the future could be implemented in a routine setting; howeve, challenges in cost and timelines could be a problem.


Next-generation sequencing After the morning break, Dr Nathaniel Storey from the Department of Bioinformatics, Great Ormond Street Hospital (GOSH) London reviewed advances in next-generation sequencing (NGS) for the detection of antimicrobial resistance and how this could be used in diagnostics and infection prevention control (IPC). The majority of existing NGS services use short read Illumina sequencing technologies. These are highly accurate and use short DNA reads (typically 100 bp) but generate enormous amounts of data. However, library preparations can be slow, taking one to four days, and sequencing can be expensive. These problems are compounded by the need to batch process samples to keep down costs as far as possible.


In contrast, Dr Storey described the


Delegates to the conference contributed to the success of the conference, and it is planned to make the presentations available to all who attended.


WWW.PATHOLOGYINPRACTICE.COM JUNE 2023


approach used by Oxford Nanopore for NGS. This is becoming increasingly popular and may supplant Illumina-based sequencing technology if sequencing is to be implemented in smaller laboratories. Nanopore sequencing offers a number of benefits including decreased library preparation time yields very long reads of up to several megabytes of sequence and reduced hardware and consumable costs. Hardware costs for NGS are a fraction of Illumina-based technology costs with a starter kit costing less than £1000. The space required is also low and the cost of consumables reasonable. The accuracy of this method of sequencing was much lower than that of Illumina initially but has improved hugely over the last few years. Dr Storey took us through the use of bacterial WGS at GOSH using software from Aries Genetics – an Austrian company – enabling cloud-based data analysis that facilitates the identification and typing of bacterial pathogens from


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