MICROBIAL TECHNOLOGY
In particular, multiplex testing presents certain challenges to molecular QC and QA, particularly when balancing the need for a clinically relevant and cost-effective EQA service in line with the relevant regulatory requirements (ISO 17043 and ISO 15189). Supporting diagnosis based on clinical presentation, multiplex assays often comprise a combination of bacterial, viral and fungal genes (± antibiotic resistance genes). To support a decreased turnaround time, multiplex assays may report preliminary findings at an early stage with a final identification report later.
Whole-genome sequencing is being applied to M. tuberculosis (pictured) and other mycobacterial infections (scanning electron micrograph)
and next-generation sequencing (NGS). Dr McCulloch described how an
external quality assessment (EQA) scheme needs to be fit for purpose, meeting regulatory requirements (ISO 17043:2010 as an EQA / PT provider) and the requirements of a clinical laboratory (ISO 15189:2012 or equivalent). A scheme needs to be practical and cost-effective, and be driven from a regulatory perspective, but also include educational elements. External quality assessment samples that are as close to a clinical sample as possible ensure that the scheme is as clinically relevant as practicable. An EQA scheme for molecular diagnostics needs to keep up with current clinical and technological developments that are occurring in ‘real-life’ clinical and laboratory settings. In terms of QC requirements for accreditation, QC materials should be different from calibration materials, and the use of independent third party controls should be considered. Dr McCulloch stressed the importance
of internal QC (IQC), in particular for monitoring variation in test results. Variation is expected and may indicate different issues according to its nature. For instance, random variation is represented by individual spikes observed in the data. These are easy to detect and are likely to be caused by unpredictable fluctuations. Conversely, systematic variation is represented by trends or drift
in the data. This type of variation is harder to detect in a single dataset and may lead to hidden, persistent and/or growing errors.
While the majority (86%) of molecular
assays remain qualitative, there is a need for viral quantitation in certain infectious diseases (eg cytomegalovirus [CMV], Epstein-Barr virus [EBV] and bloodborne viruses) and certain patient populations (eg transplant recipients). Currently, ‘Official Standards’ are only available for less than 8% of all the QCMD EQA schemes and pathogen/strain level coverage is less than 0.5%. In the absence of an international standard, an EQA provider needs to find suitable reference material, and QCMD chose to use an alternative approach and developed International Reference Materials (IRMs). Once an international standard becomes available, it will be possible and necessary to back calibrate IRMs. With over 60% of laboratories
reporting the use of commercial molecular assays, the diverse nature of molecular assays available can complicate the quality assessment (QA) process.
In the scenario where more than one pathogen has been identified from a single sample, identifying the causative agent of infection rather than colonisation can be challenging as it is possible that the pathogens present may represent clinically relevant dual infections. From an EQA sample perspective, combinations of multiple pathogens in a single tube are not considered appropriate, although such ‘mixtures’ can be used as control material. QCMD supports the rotation of different pathogens through the annual EQA challenges to be more relevant than providing all pathogens in a single tube/ challenge.
The diagnostic landscape is changing
and there is an increased interest in the provision of near-patient testing in community settings, which presents another set of challenges to molecular QC and QA. In the near patient setting, the location and personnel completing the testing can be varied, ranging from testing being completed in a different laboratory by personnel not employed by your laboratory (eg by satellite laboratories), in a primary care facility (eg GP surgery), in an accident and emergency department, or in an ambulance or other ‘in the field’ setting. In addition, there are many different point-of-care (POC) devices/platforms on the market with different specimen requirements and testing algorithms, all of which need to be covered by an EQA scheme. Whatever the setting, personnel and device used, according to ISO 15189:2012 near-patient testing comes under the responsibility of an accredited laboratory.
Next-generation sequencing (NGS) is an emerging area of diagnostic microbiology as we heard in the previous
One great advantage of NGS in clinical microbiology is that it is ‘agnostic’ and should allow for an unbiased approach to the detection of pathogens
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