Editor Note: Dr. Graham F. Cope and Bruce Savage are two individuals participating in the Longitude Prize Challenge. The Longitude Prize is a competition that brings together innovators from different fields from around the world to create a new diagnostic technology that combats the developing antimicrobial resistance epidemic.
Abstract
The spread of methicillin-resistant Staphylococcus aureus (MRSA) in the community is a major concern and is associated with death and severe morbidity. Rapid detection can reduce the time to begin appropriate therapy and reduce further the spread of infection. Early detection is complicated by the presence of wild type S. aureus and with other Staphylococcus species. Detection of MRSA requires detection of specific genes that provide antimicrobial resistance, namely the mecA, nuc, or femA genes. Several commercially available molecular kits are available using molecular techniques that have significant advantages over traditional culture-based techniques. However, they are mostly based on the polymerase chain reaction (PCR) technique, which require specialize equipment and expertise and therefore are expensive. A new rapid, easy-to-use technique has been developed based on the established technology of DNA hybridisation. This has resulted in a simpler method that does not require expensive equipment and can be performed by people with no prior molecular biology experience. This makes it suitable for rapid screening and more attractive for smaller laboratories and use in developing countries.
Main Article
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of both hospital and community-acquired infection, causing significant morbidity and mortality through skin and soft tissue infections and necrotizing pneumonia (Moran et al., 2006). Delays in giving patients appropriate antibiotic therapy is a risk factor for a longer hospital stay, hospital-acquired infection, and premature mortality (Paul et al., 2010).
Targeted therapy is currently based on the well established, conventional culture and susceptibility testing which takes at least 24–48 hours. In the last few years, various rapid tests have been developed for use in clinical laboratories that detect MRSA directly from nasal swabs and blood cultures. These new methodologies have the advantage of faster turnaround time and further reduce the cost of healthcare.
Rapid detection of MRSA from swabs can identify infected individuals and enable clinicians to quickly select appropriate antibiotic therapy and initiate additional infection control if necessary. A common clinical complication in patients who presents with sepsis is the presence of Gram-positive cocci clusters in the blood. These clusters could be wild type S. aureus, a potentially serious pathogenic organism that is susceptible to methicillin (MSSA), or coagulase-negative Staphylococcus, which are other species of the same genus and account for the majority of post-surgical infections (Hall and Lyman, 2006). Therefore, it is essential that rapid tests can distinguish between the different strains of Staphylococci with a high degree of accuracy (Bakthavatchalam et al, 2017).
Following diagnosis of Staphylococci, patients are usually treated with broad-spectrum antibiotics until the susceptibility of the organism has fully been established. This can take up to 24-48 hours. However, if clinicians give appropriate antibiotics for MSSA to a severely unwell patient with MRSA, that patient has an increased risk of mortality. The reverse is also true with MRSA targeted antimicrobials such as vancomycin, which results in prolonged bacterial infection and higher mortality rates (Gentry et al., 1997). The use of rapid diagnostics for MRSA has been shown to result in a more effective therapy that significantly reduces the length of hospital stay and cost (Brown et al., 2010).
An early integrated PCR detection assay was published which targeted mecA. In this assay, one of the primers was labelled with a biotin group and the other primer labelled with a dinitrophenol group. Following PCR, the product was captured on a 96-well microtitre plate coated with streptavidin. After capture and washing, an anti-dinitrophenol antibody conjugated to horseradish peroxidase (HRP) was added, followed by the substrate. The sensitivity of the assay was regarded as good and initial trials were successful. Further developments were achieved in 2004 with a novel PCR for rapid identification of MRSA (Huletsky et al., 2004,), which was then followed by several modifications and improvements that led to a range of commercial assays detecting the same target.
These tests, particularly the early manifestations, had two limitations. First, they were not specific to MRSA and gave false positives for the other strains. Secondly, they did not directly detect the mecA gene, which is responsible for methicillin resistance, but rather depended on the integration of the group of genes in which the mecA gene lies. This was regarded as a surrogate marker of resistance (Bakthavatchalam et al., 2017).
Nevertheless, these tests had the major advantage of being easy to perform with a rapid turnaround time of less than 1 hour. As PCR based techniques are continually being developed, there is still a persistent problem in that these assays are complex, require sophisticated technology with many operator-dependent steps, and therefore require highly trained staff. A number of completely integrated, easy-to-use systems have been developed (e.g. Cepheid), but these require very sophisticated, dedicated instrumentation and are very expensive.
An alternative to PCR is the old technique of DNA hybridisation, which is now making a return. This technique provides a simpler approach without the need for complex instrumentation of highly trained staff (Khodakov et al., 2016). Hybridisation is based on the fact that cytosine forms base pairs with guanine and adenine forms base pairs with either thymidine (in DNA) or uracil (in RNA). Early hybridisations were performed with target DNA immobilized on a nitrocellulose membrane, but nowadays, a variety of different solid supports are used (Tenover, 1993).
DNA hydridisation with enzyme amplification
Our lab has developed a technique known as DNA hydridisation with enzyme amplification. This involves coating an ELISA plate or plastic tube with streptavidin which forms a substrate for the support of two uniquely designed oligonucleotides. The oligonucleotide sequences specifically complement the target gene in the antibiotic resistant bacteria. The unique quality of this assay is that it can be used directly on samples from nasal swabs since the very small oligonucleotides are able to permeate through the bacterial cell wall. Most molecular biology based techniques require bacterial DNA to be extracted and purified before use in a PCR-based reaction. This takes time and adds a further complication to the test procedure. In the assay, one oligo is linked to biotin which forms a strong link to streptavidin on the solid support. The other oligo is liked to HRP. The presence of specific antibiotic resistance genes, such as mecA from MRSA, will hybridise to the bound oligonucleotides. The introduction of 3,3′,5,5′-Tetramethylbenzidine (TMB) reacts with the HRP to develop a soluble blue reaction product that may be read at 370 or 655 nm. This is a unique approach based on previously published methods (Chen et a.l, 2015, Fang et al., 2014), but has been extensively modified and re-engineered. The total assay time from receipt of sample is 30 minutes, which is substantially faster than any PCR-based technique on the market.
Conclusion
DNA hydridisation with enzyme amplification provides an alternative method to PCR to rapidly identify antimicrobial resistant strains of bacteria in less than 30 minutes. Furthermore, it does not require expensive equipment or highly trained staff. In the clinical microbiology laboratory, it can be used when rapid results are required, such as pre-operative assessment of MRSA prior to emergency surgery. In addition, this technique may ultimately lend itself to point of care applications to address the challenge of the Longitude Prize, which is to create a cost-effective, accurate, rapid, and easy-to-use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time.
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