A NOVEL AIR-DRIED MULTIPLEX HIGH RESOLUTION MELT ASSAY FOR THE DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASE AND CARBAPENEMASE GENES

Here we describe the development and evaluation of a novel an air-dried high-resolution melt (HRM) assay to detect eight major extended spectrum beta-Lactamase (ESBL) (SHV and CTXM groups 1 and 9) and Carbapenemase (NDM, IMP, KPC, VIM and OXA-48) genes that cause antimicrobial resistance. The assay was evaluated using 440 DNA samples extracted from bacterial isolates from Nepal, Malawi and UK and 390 clinical Enterobacteriaceae isolates with known resistance phenotypes from Nepal. The sensitivity and specificity for detecting the ESBL and Carbapenemase genes in comparison to the reference gel-base PCR and sequencing was 94.7% (95%CI: 92.5%-96.5%) and 99.2% (95%CI: 98.8%-99.5%) and 98.5% (95%CI: 97.0%-99.4%) and 98.5% (95%CI: 98.0%-98.9%) when compared to the original wet format. The overall phenotypic agreement was 91.1% (95%CI: 90.0%-92.9%) on predicting resistance to cefotaxime and carbapenems. We observed good inter-machine reproducibility of the air-dried HRM assay using the Rotor-Gene Q, QuantStudioTM 5, CFX96, LightCycler 480 and MIC. Assay stability upon storage in the fridge, room temperature and oven were assessed at six time points for eight months and no loss of sensitivity occurred under all conditions. We present here a ready-to-use air-dried HRM-PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and Carbapenamase genes to improve AMR diagnosis and treatment.


INTRODUCTION
Antimicrobial resistance (AMR) is a major global cause of death and the development of new antibiotics is considered a public health priority. 1 AMR causes an estimated 700,000 deaths globally each year, and this number is predicted to rise to 10 million by 2050. 2 Identification of AMR is typically by culture-based phenotypic antimicrobial susceptibility testing (AST) which require incubation, from primary sample, for 48 to 96 hours. As clinical management decisions are often taken rapidly, the lack of timeliness of AST leads to an inaccurate diagnosis and inappropriate treatment. 3,4 First line or broad-spectrum antibiotics are often used in large doses to ensure their efficacy on the suspected but unknown aetiological pathogens. 3,5 Empirical treatment facilitates the emergence of AMR, increases the duration of hospitalisation, damages the patient microbiota and increases the cost of therapy. 6-8 Rapid diagnosis of AMR can enable targeted usage of antibiotics, improved patient outcomes and antimicrobial stewardship. 3,5 Improved use of antibiotics through the development of rapid diagnostics is an important approach to tackle AMR. 9 The most common mechanism of drug resistance in Gram-negative bacteria is mediated by the production of -lactamases, including the extended-spectrum -lactamases (ESBLs) and Carbapenemases, 10 which provide resistance to the -lactam antibiotics. Polymerase Chain Reaction (PCR) based detection of ESBLs and Carbapenemase genes may provide a faster diagnosis of AMR than phenotypic methods, which might in turn generate more timely information for treatment decisions. 11,12 Whist molecular methods for the detection and characterisation of microbial resistance genes are becoming increasingly established, with good agreement with phenotypic methods, producing faster results, 13-15 their use in clinical settings is, however, hampered by the high degree of multiplexing needed due to the many genes involved in resistance. In addition, PCR requires a cold chain to maintain the integrity of reagents, equipment, and trained staff, which are often unavailable in low-resource . CC-BY 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.21251950 doi: medRxiv preprint settings, especially in low-and middle-income countries (LMICs). One approach that could facilitate the implementation of PCR assays in LMICs would be to provide the PCR primers, Taq enzyme and buffer components dry in the PCR plastics. This process would eliminate the need for the cold chain, ensure their biological integrity and simplify preparation, as only nuclease-free water and the DNA template would need to be added to resuspend the PCR reagents. [16][17][18] Typically, this process would be done by lyophilisation of the reagents.
Lyophilisation, also called freeze-drying, is the process of the removal of water from a product by volatilization and desorption to increase the lifespan of a product. However, lyophilisation process is costly and requires the addition of excipients, such as cryoprotectants and bulking agents. 17,19 We report here the development and validation of an air dried HRM-PCR mix to detect the most frequent ESBLs and Carbapenemase enzymes based on a previously validated in-house AMR HRM-PCR assay. 20

Air-dried HRM assay optimisation
We adapted an in-house 9-plex HRM PCR that detects nine major ESBL genes (TEM, SHV and CTXM groups 1 and 9) and Carbapenemase genes (NDM, IMP, KPC, VIM and OXA-48) developed in our laboratory 20 to a dry format. For the dry-out process, AmpDRY™ (Biofortuna, UK) was used, which is a PCR reaction mix that allows direct air drying of the whole reaction including primers and reporter molecules and removes the need for lyophilisation systems and reagents. The composition of each HRM reaction included a mixture of 1x EvaGreen® dye (Biotium, Canada), primers for detecting ESBL groups and Carbapenemase genes, 20 the proprietary air-drying PCR buffer AmpDRY™ (Biofortuna, UK) and PCR grade water to a final volume of 6.25l. The reaction mixture was added into each of the wells of a 96-well PCR plate (Starlab, Germany) and was dried in an oven-drier . CC-BY 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint and 500 mM Betaine (Sigma Aldrich, UK) in PCR grade water to each PCR well containing the dried reagents for a final reaction volume of 12.5l. PCR plates were briefly centrifugated before PCR amplification and, when plates were not compatible with the thermocycler used, the mixture was transferred to the appropriate reaction vessels. The optimised PCR amplification protocol consisted of an initial incubation step at 80°C for 15 minutes, followed by 30 cycles of denaturation for 10 seconds at 95°C, annealing for 60 seconds at 66°C and elongation for 10 seconds at 72°C monitoring the fluorescence in the FAM/SYBR channel.
HRM analysis was carried out over a temperature range of 75°C to 95°C taking a reading in the HRM/SYBR channel every 0.1°C, with a 2 second stabilisation between each step. Positivity was indicated by a peak at the predictive melting temperature (Tm) of the target  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  DNA from the Nepal and Malawi isolates was extracted using the boilate 21 method and isolates from the UK were extracted using the DNeasy Blood and Tissue kit (Qiagen). The isolates sourced in the UK and Nepal were screened for ESBL and Carbapenemase markers using reference PCR published protocols 11,12 with some modifications and the air-dried HRM assay. The reference PCR reaction mix was performed using DreamTaq PCR reaction mix (Thermo Fisher, UK), 2.5l of DNA and nuclease free water to a final volume of 12.5l.
PCR amplification was visualised with PicoGreen TM (Life Technologies, USA) staining on a 1% TBE (Tris-borate-EDTA) gel with 1% to 2% of agarose depending on the fragment size to resolve. This reference gel-based PCR was not performed with the Malawian isolates as Next Generation sequencing data was available from previous studies. 7,20 In addition, the 440 isolates were screened using the in-house 9-plex HRM PCR assay originally developed in our laboratory 20 using the commercially available Type-it® HRM kit (Qiagen). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

Bacterial strains for phenotype prediction evaluation from Nepal
The copyright holder for this this version posted February 19, 2021. Isolates were resuscitated on MacConkey or nutrient agar and DNA extracted by a boiling lysis method as described elsewhere. 21

Limit of detection
Limit of detection (LOD) of the air-dried assay was evaluated for the ESBL genes CTXM-1 and SHV, one positive for CTXM-1 (isolate 1), one positive for SHV (isolate 2), and a positive isolate for both genes (isolate 3) to estimate the LOD in isolates coproducing multiple genes. Two aliquots of 200l of each of the suspensions were taken and processed following two extraction methodologies: DNeasy Blood and Tissue kit (Qiagen) and the boilate technique. DNA samples for each dilution series were tested in triplicate using the HRM assay. The LOD was defined as the lowest concentration at which the AMR genes were detected in all three replicates.

Cross-platform validation
To evaluate the compatibility of the air-dried HRM assay in a wide range of platforms, a set of 94 samples comprising all the resistance genes were tested using different qPCR systems including the Rotor-Gene Q, QuantStudio TM 5 (Thermofisher, USA), CFX96 (BioRad, USA), LightCycler ® 480 (Roche Life Sciences, Germany) and MIC (Bio Molecular Systems, Australia). Amplification of the markers was assessed together with changes in Tms between platforms.

Evaluation of the stability upon storage at different temperatures
Stability of the dried-HRM assay was evaluated over time under different storge temperatures. A set of 89 samples comprising all the markers and isolates 1-3 at the dilution of the LOD and previous dilution were tested with plates stored at different conditions. One PCR plate with the dried reaction mix was stored for each of the following periods of time; . CC-BY 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 19, 2021. respectively. 22 Statistical significance of differences in Tms between platforms was measured using One-Way-ANOVA and differences of peak height between different storage conditions using One-Way-ANOVA with Tukey's test for Post-Hoc analysis. Statistical significance was set at a p-value < 0.05.

Air-dried HRM assay evaluation using banked DNA
The air-dried HRM assay was capable of identifying the eight markers, each of which was characterised by the presence of a single peak at the expected Tm (Fig. 1a). The assay was also able to identify isolates co-producers of four AMR markers (Fig. 1b). There was no overlap between adjacent peaks with a minimum separation of peak Tm of 0.8 °C allowing easy identification of multiple genes within the same sample.
Measures of diagnostic accuracy and agreement of the air-dried HRM assay for detecting individual genes compared to the reference tests are detailed in Table 1 (PCR and WGS) and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint and Carbapenemase markers and moderate agreement ( = 0.79) for SHV. SHV was often found in coproducers of multiple genes and the sensitivity of SHV was lower in isolates carrying two (76.7%) and three genes (59.3%) than single producers of SHV (92.6%).

Cross-platform validation
A good reproducibility was obtained on all instruments. Peak calling was performed by visual observation by the presence of a peak at the expected Tm and cut-off was established for each instrument by evaluating five threshold values set as 20%, 10%, 7.5%, 5% and 3% of the fluorescence of the highest peak. The optimal cut-off for the Rotor-Gene Q, QuantStudio and MIC was 5% of the fluoresce of the highest peak and for CFX96 and LightCycler® 480 it was 10%. These cut-offs produced almost perfect agreement with the reference tests (ĸ =0.935). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The amplicon Tm (°C) shifted across platforms (Fig. 2) and ranged from ± 0.013°C to ± 0.99°C for CTXM-1, ± 0.07-1.09°C for CTXM-9, ± 0.08-1.15°C for IMP, ±0.02-1.26°C for KPC, ±0.01-1.38°C for NDM, ±0.19-1.5°C for OXA-48, ±0.08-0.94°C for SHV and ±0.12-1.27°C depending on the platform used. The Tm differences within the same peak and neighbouring peaks is shown in Tables 3a and 3b for each of the platforms. The Tm difference was not statistically significant for any of the platforms for either the type of peak, peaks within the same cluster (p=0.318) and neighbouring clusters (p=1.00).

Limit of detection
The limit of detection was 11.5, 102 and 960 cfu/reaction using DNeasy kit and 2.3, 20.4 and 192 cfu/reaction by the boilate method for isolates carrying the CTXM-1, SHV and both CTXM-1 and SHV genes, respectively.

Stability upon different storage conditions
The effect of storage time and temperature was assessed on the AmpDry mix by analysing the plate mean fluorescence peak height and amplification of isolates, including isolates at LOD dilutions. The average temperature for room storage, fridge and oven was 20.35°C ± 0.7, 6.2°C ± 0.9 and 29.7°C ± 1.4 respectively, the humidity of the room was at 36.5% ± 9.34. Overall, room temperature was the best storage condition compared to fridge and oven.
The difference of mean fluoresce peak hight was not statistically significant within the same time point but was statistically significant between different time points (Fig. 3). The peak height started decreasing after storage time T3 for room and oven storage, and at T2 for fridge storage (Fig. 3). Nonetheless, the difference of mean peak height produced with the AmpDry mix stored at time T3 (one month) was not statistically significant to the produced at T0, T1 and T2 at all storage conditions. The AmpDry mix recovered at T4 and T5 (fridge only) produced significantly lower peak heights when compared to T1, T2 and T3 (room temperature only). The mean peak height produced with the AmpDry mix stored at time T5 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.21251950 doi: medRxiv preprint at room temperature, was comparable to all time points at all storage conditions and timepoints except at T1 for fridge storage (Fig. 3).
Isolate 1 was negative at the LOD dilution at T3 under oven storage; isolate 2 was negative at the LOD dilution at T3 under room temperature and oven storage, and isolate 3 was positive in all runs tested (Fig 4). Of the 89 isolates tested, 100% were positive for all markers at all storage times and conditions, except for one sample that had one of three marker peaks below the cut-off (NDM) at T4 fridge storage (data not shown).

Discussion
In this study, we evaluated the performance of an 8-plex HRM PCR assay in dry format to detect ESBL and Carbapenemase genes. The assay showed high sensitivity, specificity and measures of agreement for all markers when compared to the reference tests. In addition, the drying process did not result in loss of performance, with all the resistance genes of the 89 clinical isolates correctly classified after 6 months of storage.
The dry format of the assay overcomes key real-world challenges relating to transport, storage, and freezing/thawing issues, which can substantially lower the sensitivity of PCR. 23,24 This HRM assay presents several major advantages over fresh qPCR mixes as its resistant to long periods of storage at relatively warm temperatures (30 °C) and its stability during handling in warm conditions enables easy storage and transportation of the assay at ambient temperature for long periods of time. This would be of particular importance in LMICs where laboratories face insufficient and suboptimal cold chain capacity. 25 As this assay is easy to set-up and interpretation of results with analysis of the melting data can be automated, it may be straight-forward to implement in laboratories with access to qPCR facilities, but otherwise moderate resources, as all that is required is to reconstitute the mix and add template DNA. The assay has good performance using the boilate extraction method, which is fast, simple and easy to implement with minimal resources. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Cross-platform validation illustrates a remarkably good performance on all 5 q-PCR systems (Rotor-Gene Q, QuantStudio TM 5, CFX96, LightCycler ® 480 and MIC) evaluated, with minimal variation on the peak Tms, which was not statistically significant. The cut-offs however required slight adjustment (5% or 10% of the highest peak) to achieve the best performance, nevertheless this is straightforward correction that can be applied with simple instructions.
The protocol has some constraints as a 24h incubation from primary sample to grow the isolates is still required prior DNA extraction. The assay has not been evaluated using direct clinical samples but the LOD obtained here indicates sensitivity to be insufficient to detect the low CFU/ml (>1/ml) possible in bacterial bloodstream infections. 27,28 The overall agreement to predict bacterial phenotypes was strong ( = 0.82) amongst Enterobacteriaceae isolates but weak in non-Enterobacterial isolates. Thus, we do not recommend the use of the assay in non-Enterobacterial isolates. The high discrepancy among non-Enterobacteriaceae isolates can be explained as Acinetobacter spp. and Pseudomas spp. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Other reasons for phenotype-genotype mismatches include enzyme modifications that change the spectrum of activity and susceptibility profile 33 , and also isolates with MICs close to the breakpoint being incorrectly classified during phenotypic susceptibility testing. 34 To summarise, the air-dried HRM assay detected ESBL and Carbapenemese genes fast, effectively and with high specificity and sensitivity and maintained performance after six months of storage at room temperatures. This 8-plex dry HRM assay was also successfully transferred to 5 different PCR platforms indicating that can be reliably implemented in many laboratories. The assay can become a useful tool for AMR diagnosis and surveillance.

Funding
The study was funded through the MRC Proximity to Discovery (P2D) award number MC_PC_17196. The funders had no role in the design of the study, data collection, analysis, or preparation of the manuscript.

Acknowledgments
We would like to thank the staff at Patan Hospital in Kathmandu for their assistance with the isolates used in this study and Biofortuna for advice.  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 19, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 19, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint      is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.21251950 doi: medRxiv preprint