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Molecular Macrolide Resistance Assay Shows Promise for Point-of-Care Use

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NEW YORK (GenomeWeb) – Researchers at American University have developed an isothermal amplification assay to detect a resistance gene that is passed among bacterial species.

The gene, called mef(A), is common in Streptococcus bacteria, and the test could potentially be adapted for point-of-care use to detect resistance in specific diagnosed infections and to aid surveillance and tracking of antimicrobial resistance.

Researchers John Bracht and Megan Nelson at AU chose the recombinase polymerase amplification (RPA) chemistries from TwistDx for their assay. As described recently in BMC Infectious Diseases, they were able to detect mef(A) from raw lysates of Streptococcus pyogenes, S. pneumoniae, S. salivarius, and Enterococcus faecium in less than 10 minutes. The assay also correlated with antimicrobial resistance as determined by culture methods, and was not affected by SNPs within the gene.

Resistance conferred by macrolide efflux mechanisms in bacteria was described in 1990s. The mef(A) gene allows bacteria to pump out antibacterial drugs, specifically two extremely common drugs, erythromycin and azithromycin, also called Z-pack.

But, "Bacteria transfer this gene around, so lots of species have it," Bracht, a genomics researcher at AU, said.

Bracht speculated that bacteria have such a mechanism because the antibacterials themselves were developed essentially using the chemical weapons already deployed in the eons-old warfare among microbes.

However, the increasing inappropriate use of antibacterials in the past decades has led to an evolutionary advantage for strains with the efflux pump, such that they are essentially being selected for. One study, for example, noted that mass azithromycin distributions among communities in Ethiopia to combat trachoma — a type of Chlamydia trachomatis infection of the eyes that can cause blindness — had the side-effect of increasing mef(A)-induced resistance.

And, research in the early 2000's suggested mef(A) was among the most common resistance genes that led to up to 30 percent of S. pneumonia in the US being macrolide resistant.

The most immediate use for the AU test might be in a point-of-care diagnostics workflow for Strep A, Bracht said.

RPA has been used by other groups for resistance gene detection. For example, researchers in China have used it to detect the plasmid-mediated mcr-1 gene that confers colistin resistance. It has also been used by researchers at the University of Southampton in the UK as part of a microfluidic device to detect genes encoding CTX-M β-lactamases, as well as in a multiplex test for CTX-M-15, KPC and NDM-1 genes conferring resistance to extended spectrum β-lactam and carbapenem antibiotics. The Southampton team recently developed the method to incorporate bead-based detection and also paired CTX-M detection using RPA with thin film transistor sensors for detection. That device measured amplification of the resistance gene through an electrochemical detection of pH changes rather than pH-based dye technique or a more standard fluorescence methods, enabling a positive test to be read in a few minutes.

RPA is also being used by a company called mFluiDx in a microfluidic device to detect methicillin-resistant Staphylococcus aureus genes. And, researchers at Rice University have been pioneering paper-based HIV resistance detection using RPA as well as methods to make RPA quantitative and able to be run using body heat.

While RPA is a popular isothermal method being applied to resistance detection, others are using technologies such as loop mediated isothermal amplification (LAMP). For example, Talis Biomedical is commercializing a LAMP-based SlipChip method and recently won funding for an assay to detect antimicrobial resistance in gonorrhea infections.

Bracht found LAMP to be slower and more complex, however, while the RPA method had an added advantage of support from TwistDx, an Abbott subsidiary that has recently relocated from the UK to San Diego.

"We did consult with the TwistDx team, and they helped us design the probe and made sure the primers were designed correctly," Nelson said. The final assay was robust enough that the sample prep step simply involved lysis with three minutes of boiling followed by dilution.

Now, the AU team plans to develop additional assays and spin the technology out into a company to commercialize it. The group is in the process of filing a patent and is also undertaking research using clinical samples.

In the long run, Bracht said the test could "pair quite nicely" with rapid tests or point-of-care molecular tests for Step A detection. The Infectious Disease Society of America guidelines for Strep A testing have not been updated recently, so there is not yet clear guidance for clinicians on using rapid molecular testing, although Roche, Abbot, and Quidel, all have tests available.

Bracht said the AU test has been adapted for use with lateral flow strips, but it is currently a lab-based test due using qPCR fluorescence readout for the proof-of-concept experiments.

Bracht and Nelson also see that the mef(A) test, or a resistance panel the lab is also striving for, could be used for surveillance in the future.

For example, the team found that S. salivarius commensal bacteria in some of the negative control saliva samples in the study carried the mef(A) gene. A future application might be testing to determine a person's status as a resistance gene carrier essentially determine their potential to generate de novo "superbugs," and then avoiding certain antibiotics in resistance gene carriers whenever possible.

"We may not need to wait until a person is sick; it might be just as important to know if a person has these genes when they are healthy," Bracht said.

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