PathCrisp: an innovative molecular diagnostic tool for early detection of NDM-resistant infections

Abstract

The rapid and early detection of infections and antibiotic resistance markers is a critical challenge in healthcare. Currently, most commercial diagnostic tools for analyzing antimicrobial resistance patterns of pathogens require elaborate culture-based testing. Our study aims to develop a rapid, accurate molecular detection system that can be used directly from culture, thereby introducing molecular testing in conjunction with culture tests to reduce turnaround time and guide therapy. PathCrisp assay, a combination of loop-mediated isothermal amplification and CRISPR-based detection, maintained at a single temperature, was designed and tested on clinical isolates. The specificity and sensitivity of the assay was analyzed, post which the assay was compared with the polymerase chain reaction (PCR) method to detect the New Delhi metallo-beta-lactamase (NDM) gene in carbapenem-resistant enterobacteriaceae clinical samples. Our PathCrisp assay demonstrated the ability to detect as few as 700 copies of the NDM gene from clinical isolates. Our assay demonstrated 100% concordance with the PCR-Sanger sequencing method, more commonly used. Additionally, the lack of the need for a kit-based DNA purification step, rather a crude extraction via heating, enables the direct use of culture samples. The PathCrisp assay is precise, specific and rapid, providing results in approximately 2 h, and operates at a constant temperature, reducing the need for complex equipment handling. In the near future, we hope that this assay can be further optimized and designed as a point-of-care test kit, facilitating its use in various healthcare settings and aiding clinicians in the choice of antibiotics for therapy.

Fig. 1

Similarity of NDM-1 variant with different variants: primer and guide design. (a) Conserved regions across deposited NDM variants: partial NDM gene sequences from NCBI and CARD were aligned to study conserved regions across NDM variants (NDM-1 to NDM-43, except NDM-32). The green-coloured histogram at the beginning of the alignment depicts the conserved region. The Cas12a guide target is marked in pink, and is conserved across all variants. SNPs in the sequences are marked with block colours: dATP (red), dGTP (yellow), dCTP (blue), dTTP (green). (b) Conservation analysis in Indian patient samples: The NDM gene sequence of 101 NDM-positive clinical samples from different priority pathogens, such as E. coli, K. pneumoniae, and A. baumannii, from Indian patients, were screened to check for the conservation of the designed oligos. Whole genome sequences of these samples were used to extract the NDM gene sequences (793–813 base pairs in length). The NDM gene regions were screened to detect homology with the designed Cas12a guide and LAMP PCR primer sequences. The Figure shows conservation of the regions used for LAMP primer design (F1, B1, F2,B2, B3, LF and LB regions). Only the F3 region, shown in dark blue, shows SNPs at the 12th bp (A/C). All the other regions, including the Cas12a guide (red) are conserved across the recent Indian patient samples sequenced. Abbreviations: EC—E. coli, KP—K. pneumoniae, AB—A. baumannii, RC—reverse complementary sequence, F-Forward, B-Backward, L-Loop. (c) Map of LAMP primers designed with Cas12a guide: The layout of the designed LAMP primers with the sgRNA.

Fig. 2

Validation of designed oligos for NDM variants: (a) testing with genomic DNA of K. pneumoniae: the genomic DNA of K. pneumoniae, positive for NDM-1 (obtained from Vircell), is being used to test the designed LAMP primers and sgRNA. Our designed guide and primers can detect NDM-1. (b) specificity testing with negative controls: different known negative genomic DNAs are being used to test the specificity of the PathCrisp assay, with previously used K. pneumoniae (NDM-1) as assay control. The assay was repeated twice: once with Bst1.0 (without UDG) depicted as circles in the graph, and a second time with BST2.0 (with UDG) depicted as diamonds in the graph. The average value of each sample is represented as a bar graph, while individual values are marked either as circles or diamonds. Grey dotted lines represent the cutoff to consider if the sample is positive, which is the average of RFU of LAMP-NTC plus 3 times the standard deviation. (c) Testing with carbapenem-resistant E. coli isolates: sixteen carbapenem-resistant E. coli isolates from patient samples were tested with designed LAMP primers and sgRNA. VIR-5, VIR-8 and VIR-9 were negative for NDM, consistent with the previous report. The NDM as per previous sequencing data is mentioned for the samples. In each assay, the negative control for LAMP is no-template control, depicted as LAMP-NTC. For detection controls, negative control, with no LAMP product, is shown as Det. negative in the graph. For positive control, pJET1.2_NDM was used, depicted as Det. positive.

Fig. 3

Limit of detection of NDM-PathCrisp assay. Purified pJET1.2_NDM plasmid was serially dilated up to 2.86E−05 picogram (which roughly corresponds to 7 copies) and then used as a template for Pathcrisp. The assay can consistently detect up to 2.86E−03 picogram (~ 700 copies). The mean value for each sample is represented as a bar graph while individual values are marked either as circles The Grey dotted line represents the cutoff to consider if the sample is positive, which is the sum of the average LAMP-NTC RFU and 3 times its Standard deviation. The negative control for LAMP is no-template control, depicted as LAMP-NTC. For detection controls, negative control, with no LAMP product, is shown as Det. negative in the graph. For positive control, pJET1.2_NDM was used, depicted as Det. positive.

Fig. 4

Tested PathCrisp assay on unknown CRE samples 49 carbapenem-resistant enterobacteriaceae (CRE) isolates obtained from patient samples were tested for the presence of NDM using PathCrisp assay. The obtained RFU values are normalized to the detection negative of each batch and plotted here. For a few samples, the assay was repeated twice, once with Bst 1.0 (without UDG) depicted as circles in the graph and a second time with BST 2.0 (with UDG) depicted as diamonds in the graph. The average value of each sample in case of repetition is represented as a bar graph, while individual values are marked either as circles or diamonds. Grey dotted lines represent the cutoff to consider if the sample is positive, which is the average of RFU of LAMP-NTC plus 3 times Std. deviation. The 49 samples obtained were either K. pneumoniae (striped bars) or E. coli (solid coloured bar). In each batch, the negative control for LAMP is no-template control, depicted as LAMP-NTC. For detection controls, negative control, with no LAMP product, is shown as Det. negative in the graph. For positive control, pJET1.2_NDM was used, depicted as Det. positive.

Fig. 5

PathCrisp test on crude extract pJET1.2_NDM transformed DH5⍺ E. coli cells were used to test the PathCrisp assay, after crude extraction. The assay was repeated twice, once with Bst1.0 (without UDG) depicted as circles in the graph and the second time with BST2.0 (with UDG) depicted as diamond shapes in the graph. The average value of each sample is represented as a bar graph while individual values are marked either as a circle or a diamond. The grey dotted lines represent the cutoff to consider if the sample is positive, which is the average of RFU of LAMP-NTC plus 3 times standard deviation. Negative control for media was also included for each type of culture: LB Agar plate and LB broth, respectively. The negative control for LAMP is no-template control, depicted as LAMP-NTC. For detection controls, negative control, with no LAMP product, is shown as Det. negative in the graph. For positive control, 0.3 nM pJET1.2_NDM was used, depicted as Det. positive.