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Contemporary molecular approaches in the clinical microbiology laboratory: progress and pitfalls.

Over the past two decades, the detection, identification and classification of pathogens has changed considerably, with a major shift from phenotypic methodologies to those utilising molecular techniques. For some pathogens, this has dramatically changed aspects of the diagnosis and epidemiology of clinical syndromes caused by these pathogens. For example, molecular assays for some slow-growing pathogens (e.g., Mycobacterium tuberculosis), have considerably decreased the time to diagnosis, and for other pathogens, particularly those which are unable to be routinely cultured (e.g., Chlamydia trachomatis, Mycoplasma genitalium), the increased diagnostic sensitivity has cast new light on the incidence, demographics and types of infection. In addition, the ability to detect genes that confer antimicrobial resistance in bacteria, viruses and fungi, and to quantitate levels of pathogens (e.g., HIV) has allowed improved and educated monitoring of therapeutic interventions. Advances in ‘user-friendly’ commercially available molecular technology, including so called ‘black box’ assays, coupled with a number of high profile and disruptive outbreaks (e.g., pandemic influenza H1N1, Clostridium difficile 027) have greatly facilitated the implementation of molecular methods into the routine clinical microbiology workflow. Many commercial systems have greatly simplified technologies for nucleic acid extraction, obviating the requirement for specialised extraction facilities. In particular, the use of multiplex, real time polymerase chain reaction (PCR) assays has enabled the rapid detection of several pathogens from clinical samples whilst retaining excellent analytical sensitivity, and allowed diagnostic testing to become more ‘syndromic’, rather than testing for individual pathogens in multiple assays. More recently, the introduction of next-generation sequencing technology has heralded a potential ‘game changer’ in diagnostic and molecular typing methods. Although mainly confined to the research environment at present, the use of whole genome sequencing (WGS) of bacterial, viral and fungal pathogens promises a paradigm shift in our understanding of the pathogenesis, evolution and transmission of infectious agents. Recent work has attempted to better ‘interface’ this technology with the clinical microbiology sector, with advances being made in DNA extraction methods, and correlation of in silico genotypic and phenotypic antimicrobial susceptibility results. Despite their transformational nature, there remain a number of challenges surrounding the universal adoption of molecular methods into the clinical microbiology setting, predominantly relating to cost, workflow and diagnostic interpretation. In particular, the clinical significance of low levels of detection for some pathogens may cause more confusion than clarity, and