A Plexor®-based multiplex real-time PCR assay was designed to simultaneously detect the ESBL genes CTX-M, TEM, OXA and SHV, using amplicon melting temperatures, without the use of melting curve analysis. Figure 2 shows the results of the development of the real-time PCR method Table 1.

179 samples were collected from the Leicester Royal Infirmary and tested by RT-PCR. The result of the multiplex assay were mostly in agreement with the results of the multiplex end-point PCR. The assay correctly identified 91.7% isolates tested. A summary of the statistical results can be found in Table 3. Seven very major errors and seven major errors were identified. Very major errors were defined as samples that showed as negative for ESBL genes by the RT-PCR assay, but positive by end-point PCR. Major errors were defined as samples that showed as positive for ESBL genes by the RT-PCR assay, but negative by end-point PCR.

Interestingly, the real-time PCR assay picked up additional TEM genes in two of the isolates, which the end-point PCR had failed to. The NTC was assigned correctly as predicted.

In comparison, the disk diffusion method correctly identified ESBL production in 87.36% of tested isolates. Whilst the sensitivity was slightly higher than the RT-PCR, the specificity was significantly lower (see Table 3). Eighteen very major errors and five major errors were identified.

Although other methods have been described for the detection of ESBLs, to our knowledge, this is the first study to accurately and rapidly describe a multiplex real-time PCR assay to detect ESBLs, based on amplicon melting temperatures, without the need for a high-resolution melting curve analysis. Whilst there are other chemistries applied in real-time PCR that do not require high-resolution melt-curve analysis for detection, such as Taqman, these tend to be far more expensive and require a higher level of user experience. 13 In this assay, the detection of products is based upon the melting temperature of amplicons, within each cycle. In the present study, 179 samples previously identified as ESBL-producers by disk diffusion methods, were tested using a Plexor®-based multiplex real-time PCR assay for the detection of 149 variants of the CTX-M, TEM, OXA and SHV genes in one amplification. The assay was able to correctly detect genes in 91.7% of the isolates in under 3 hours. In comparison, disk diffusion methods were able to correctly detect ESBL production in 87.4% of isolates. Whilst in this study, the disk diffusion method showed a slightly higher sensitivity than the RT-PCR method, this has not been seen in other studies. 1617 It has also been reported that clinical failures may occur, even when an isolate is negative phenotypically. 18 This may have been seen in this study, as there were eighteen cases where ESBL genes were detected by end-point PCR, but the isolate was negative phenotypically. This further advocates the use of genotypic detection.

ESBL-producing Enterobacteriaceae are a global growing concern, especially when it comes to UTIs. Current susceptibility testing requires at least 24 hr., therefore it is common practice to treat with empirical antibiotic therapy, without full pathogen information. Treating in this way can lead to ineffective therapy, causing an increase in clinical symptoms, including the possibility of urosepsis and ascending infection. It is well known that empirical treatment with antibiotics increases the population of resistant pathogens. 19 Therefore, it is crucial that reliable, accurate and rapid detection methods of antibiotic resistance are available. In addition, prevalence studies are highly important for monitoring the spread and evolution of antibiotic resistance genes.

Not only can the assay detect ESBL production in isolates in under 3 hours, it can also differentiate between the major classes of ESBL, aiding in surveillance studies. In addition, this assay did not require any laborious primer modifications and the results are easy to interpret, with no further analysis required. The results from this assay could lead to treatment escalation much more quickly than current methods. However, due to the low number of negative samples tested in this study, it is difficult to determine if treatment de-escalation could be concluded from the results of this assay. Nevertheless, due to the increased expense of PCR-based methods compared to current susceptibility testing methods, it is likely that only those patients who are suspected of having an ESBL producing UTI, such as patients who suffer from recurrent UTIs or those that are at higher risk (pregnant women, children, immunocompromised) would be tested. Therefore, the patient population tested in this study, represents the samples that would most likely be tested in practice. Until the negative likelihood ratio of this test can be more reliably proven, it is suggested that this test be used as an add-on to current testing in any patients that do not fall into the above criteria.

In the future, more targets could be added to this multiplex assay, as long as they have a different amplicon melting temperature. This is achieved through primer design, as amplicon melting temperature is related to length and the ratio of CG/AT content. As technology improves, the melting temperature difference required between amplicons can decrease, further increasing the number of targets possible in the assay. We recommend that primers for other Carbapenamases and ampCs (other major antibiotic resistance genes) are added, to give this assay full breadth across the antibiotic resistance spectrum seen in UTIs.

A multiplex Plexor®-based real-time PCR assay could provide a rapid, more sensitive, easy to interpret detection method, thereby helping to prevent the inappropriate use of antibiotics. It was found that this method could aid antibiotic susceptibility testing, if ESBL production is suspected. With further investigation this assay could form the basis of a high throughput kit that health services could use to detect ESBLS or other antibiotic resistance genes.