Molecular methods are based on the detection of genetic material in a sample. These methods can detect infectious pathogens and their associated drug resistance directly from a patient’s sample, by detecting the microorganism’s DNA and genes associated with antimicrobial resistance. Therefore, the method relies on having a comprehensive database of these genes and their antimicrobial resistance and cannot detect any resistance due to new, uncharacterised mechanisms. Even in the case where a genetic marker is identified, the method cannot distinguish whether it will be active or not to confirm that resistance will take place phenotypically.
Polymerase Chain Reaction (PCR) and DNA Microarrays
Molecular based tests developed to detect certain infectious diseases are called molecular based assays. The most common molecular assays are based on the Polymerase Chain Reaction (PCR). When a specific segment of DNA of interest (e.g. part of a virus’ DNA) is present in the sample it gets amplified to return a measurable signal to confirm its presence. DNA-Microarrays is a similar method where a microarray chip is used to simultaneously detect different specific pathogens and vast numbers of different resistance genes in a single test.
A big advantage of molecular based assays is that they are generally faster and more sensitive than culture-based methods, returning results within a few hours. In contrast to LFTs they are easy to multiplex, allowing screening for tens of pathogens/genes in a single test. They are, however, more expensive than both the other methods and require specialised equipment and trained staff to operate them, making them unavailable at the point of prescribing. Compact, user-friendly lab-on-a-bench-like systems such as the VERIGENE System (Luminex), the BioFire System (Biomerieux) and T2Dx System (T2 Biosystems) have the potential to enable clinicians to rapidly identify pathogens directly from a patient sample, all within two hours.
Whilst molecular assays are excellent for answering the “what is it?” question, the method does not distinguish infection from asymptomatic colonisation, making the interpretation of results a challenge particularly when many pathogens are detected at once.
Genome sequencing and metagenomics
Whereas conventional molecular diagnostics can identify tens of common pathogens and resistant markers at a time, metagenomics allows the identification of the entire community of microorganism DNA within a sample (microbiome) using methods common to whole genome sequencing (WGS).
In WGS, the whole genome of an organism is determined in one process, by detecting in a sequential order each nucleotide (the basic building block of DNA) present in a sample. The derived sequences are then checked against known databases of unique pathogen sequences and genetic resistance markers, to identify the microorganism and its antimicrobial resistance. Metagenomics offers the capacity to detect all potential pathogens —bacteria, viruses, fungi and parasites — and all genes involved in AMR in a single sample. It therefore has the potential to be a great clinical diagnostic tool as well as a research tool for unraveling new pathogens and emerging resistance factors.
Another powerful capability of metagenomics is the capacity to study human host immune responses to infection (transcriptomics) making it possible to answer all three key diagnostic questions in one test: “what is it?”, “how can we kill it?” and “how is the patient doing?”.
Despite these advantages, metagenomics is not routinely performed in clinical practice, as turn-around times for sequencing are too long (the sequencing run alone takes >18 hours). It also requires specialised labs and staff, has a higher cost compared to all the other methods and has the same limitations as molecular based assays.
Genomics providers are, however, increasingly expanding their infectious diseases portfolio, aiming towards making their solutions more comprehensive, portable, cheaper, and faster.
For example, Illumina has established a collaboration with IDbyDNA to deliver sample-to-answer solutions, which have been used by Synergy Laboratories to offer the first comprehensive NGS-based test for urinary tract infections (UTI). The test includes ~100 uropathogens and 371 select genetic markers of antibiotic resistance.
Oxford Nanopore Technologies has also recently announced collaborations with BioMérieux and leading China-based diagnostic companies, towards the development of in-vitro diagnostic (IVD) tests for metagenomics and infectious disease.
Another method, beyond the traditional phenotypic and molecular pathogen detection methods but still relevant for infectious diseases and AMR diagnostics, is biomarker testing. Biomarkers are biological molecules found in blood or other body fluids that are associated with a condition or a disease – and measuring them is a way of characterising the patient’s response to the disease. The two most common biomarkers relevant to infectious diseases are C-reactive protein (CRP) and procalcitonin (PCT).
CRP/PCT tests are often based on the ELISA method, mentioned previously. The level of CRP and PCT biomarkers in the blood increases during an infection and is often higher during a bacterial infection than a viral one. The level of biomarkers and speed at which they are being produced can be used to differentiate bacterial from most viral infections. However, this is not always the case. Some viruses (such as COVID-19) or autoimmune diseases can result in a similar biomarker increase as bacterial infections, making the interpretation of these tests very challenging. As a result, biomarker testing has been more useful as a complementary test , interpreted alongside clinical symptoms and the experience of healthcare professionals rather than a standalone sample-to-answer diagnostic.