Research Article
Lateral Flow Immunoassay and the EDTA-Colistin Broth Disk Elution for mcr-1 gene detection in colistin-resistant E. coli and K. pneumoniae
Inmunoensayo de flujo lateral y elución de disco en caldo con colistina-EDTA para detectar el gen mcr-1 en E. coli y K. pneumoniae resistentes a la colistina
Phu Truong Thien1,2 https://orcid.org/0000-0003-0868-2811
Ngoc Tran Bich1* https://orcid.org/0000-0002-2255-6398
Thao Nguyen Thi Ngoc1,2 https://orcid.org/0009-0004-1295-401X
Van Hung Pham3,4 https://orcid.org/0000-0002-1672-3292
Vinh Tran Thanh Hua1 https://orcid.org/0009-0008-8064-782X
Van Tran Thi Hue1 https://orcid.org/0009-0007-0677-2553
Dat Ngo Quoc1 https://orcid.org/0000-0003-1461-0216
1University of Medicine and Pharmacy. Ho Chi Minh City, Vietnam.
2Cho Ray Hospital. Ho Chi Minh City, Vietnam.
3Nam Khoa CO., Ltd., Ho Chi Minh City, Vietnam.
4Vietnam Institute of Research and Development Clinical Microbiology. Ho Chi Minh City, Vietnam.
*Author for correspondence. Email: bichngoctran@ump.edu.vn
Introduction: The global emergence of colistin-resistant Enterobacterales, especially strains carrying plasmid-mediated mcr genes, poses a significant threat to public health. Rapid detection of mcr-mediated resistance is critical for timely clinical management and infection control, particularly in resource-limited settings such as Southeast Asia.
Objectives: To evaluate the performance of the lateral flow immunoassay (NG-Test MCR-1) in comparison with the combined reference method of EDTA-Colistin Broth Disk Elution (CBDE/EDTA) and polymerase chain reaction (PCR) for detecting mcr-mediated colistin.
Methods: A cross-sectional study was conducted at Cho Ray Hospital, Ho Chi Minh City, Vietnam. A total of 160 clinical isolates of Klebsiella pneumoniae (n = 128) and Escherichia coli (n = 32), along with 2 additional E. coli strains carrying the mcr-1 gene, were tested for colistin resistance using Broth Microdilution (BMD) combined with the NG-Test MCR-1 in comparison with the CBDE/EDTA combined with PCR detection of mcr-1 to mcr-10.
Results: Among 32/160 colistin-resistant isolates, only 2 E. coli strains were confirmed to carry mcr-1 by both NG-Test MCR-1 and PCR, while no K. pneumoniae isolates tested positive for mcr genes. The NG-Test MCR-1 showed perfect agreement with the CBDE/EDTA method combined with PCR. Furthermore, it achieved 100% PPA and NPA, with wide 95% confidence intervals due to the limited number of mcr-positive isolates.
Conclusions: NG Test MCR-1 provides rapid, specific detection of mcr-1, while CBDE/EDTA is a cost-effective screening tool but less definitive. Combining these methods is recommended to improve mcr-mediated colistin resistance detection.
Keywords: Edetic Acid; Enterobacteriaceae; Genes; Immunoassay; Plasmids.
RESUMEN
Introducción: La propagación de Enterobacterales resistentes a la colistina, especialmente cepas portadoras de genes mcr mediados por plásmidos, representa una amenaza para la salud pública y limita las opciones terapéuticas. La detección rápida y precisa de la resistencia mediada por mcr es esencial para el manejo clínico y controlar infecciones, sobre todo en entornos con recursos limitados.
Objetivos: Evaluar el rendimiento diagnóstico del inmunoensayo de flujo lateral NG-Test MCR-1 en comparación con el método de referencia EDTA-Colistin Broth Disk Elution (CBDE/EDTA) combinado con reacción en cadena de la polimerasa (PCR) para detectar genes mcr.
Métodos: Estudio transversal en el Hospital Cho Ray, Vietnam, con 160 aislamientos clínicos (Klebsiella pneumoniae = 128; Escherichia coli = 32) y dos cepas adicionales de E. coli portadoras de mcr-1. La resistencia a colistina se determinó mediante microdilución en caldo (BMD). Las cepas resistentes (CMI ≥ 2 µg/mL) se analizaron con NG-Test MCR-1 y CBDE/EDTA, con confirmación por PCR (mcr-1 a mcr-10).
Resultados: De los 34 aislamientos resistentes, solo 2 de E. coli fueron positivos para mcr-1 por NG-Test MCR-1 y PCR. Ninguna K. pneumoniae presentó genes mcr. NG-Test MCR-1 mostró concordancia perfecta con CBDE/EDTA + PCR (κ = 1,00; PPA = 100 % [IC95 %: 56–100 %]; NPA= 100 % [IC95 %: 92–100 %]).
Conclusiones: NG-Test MCR-1 permite una detección rápida y específica de mcr-1, mientras que CBDE/EDTA es útil como herramienta de cribado. Se recomienda un enfoque combinado para optimizar la detección y apoyar la vigilancia de la resistencia a colistina.
Palabras clave: Ácido edético; Enterobacteriaceae; Genes; Inmunoensayo; Plásmidos.
Received: 16/05/2025
Approved: 20/08/2025
INTRODUCTION
The global emergence of carbapenem-resistant Enterobacterales (CRE), particularly Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae), poses a significant threat to public health.(1) An even greater concern is the increasing prevalence of multidrug-resistant and extensively drug-resistant strains that exhibit resistance to both carbapenems and colistin, further complicating treatment options.(1,2) In developing regions such as South and Southeast Asia, antimicrobial resistance has escalated significantly, exacerbated by the limited availability of novel antibiotics. In these settings, colistin remains a last-resort therapeutic option for multidrug-resistant infections, underscoring the critical need to detect colistin-resistant pathogens to guide effective clinical decisions.(3)
Colistin resistance in Enterobacterales is primarily mediated through mechanisms such as chromosomal mutations affecting lipid A and the presence of plasmid-mediated mcr genes. Previously, the combination of EDTA-Colistin Broth Disk Elution (CBDE/EDTA) and PCR has been widely supported and utilized for detecting mcr genes. As an enzyme-targeted screening tool, CBDE/EDTA identifies the functional presence of MCR enzymes associated with mcr genes. In contrast, PCR offers molecular confirmation.(4,5) However, this combined method requires technical expertise and longer turnaround times. In contrast, the NG-Test MCR-1 test by the Lateral Flow Immunoassay method (LFIA) offers a simpler and faster alternative, with high sensitivity and specificity, making it more user-friendly for routine laboratory use.
This study was conducted at Cho Ray Hospital, a major referral center in Vietnam, which serves as a representative site for studying antimicrobial resistance patterns in Southeast Asia. By assessing the performance of this rapid diagnostic tool. It is intended to provide valuable information insights into its potential application in clinical microbiology laboratories across Vietnam and the broader Southeast Asian region.
This study aims to evaluate the performance of the NG-Test MCR-1 in comparison with the combined reference method of EDTA-Colistin Broth Disk Elution (CBDE/EDTA) and polymerase chain reaction (PCR) for detecting mcr-mediated colistin resistance.
METHOD
Study design and participants
A prospective, cross-sectional study was conducted at Cho Ray Hospital in Ho Chi Minh City, Vietnam, from October to December 2023.
During this period, 160 non-duplicate clinical isolates were collected (128 Klebsiella pneumoniae and 32 Escherichia coli strains), along with 2 additional E. coli strains carrying the mcr-1 gene, as confirmed by whole-genome sequencing using the Illumina platform. The isolates were obtained from inpatients across various clinical departments using a convenience sampling method, based on the availability of eligible specimens during the study period
Study variables
For each bacterial isolate, the following variables were recorded: The bacterial species (Klebsiella pneumoniae or Escherichia coli), the clinical specimen source (blood, urine, sputum, pus, or body fluids), and the colistin minimum inhibitory concentration (MIC) determined by the broth microdilution (BMD) method by CLSI M100-S34 guidelines. In addition, results from the NG-Test MCR-1 assay (positive or negative) for MCR-1 protein detection, findings from the CBDE and CBDE/EDTA methods (including any MIC changes in the presence of EDTA), and PCR outcomes for mcr genes (mcr-1 to mcr-10) were documented. Metadata such as the date of collection and hospital department were also noted, without including any patient-identifiable information.
Data collection
Broth Microdilution (BMD) method, combined with the LFIA: NG Test MCR-1
Colistin's minimum inhibitory concentrations (MICs) were initially determined using the BMD method, following the Clinical and Laboratory Standards Institute (CLSI M100S34) guidelines. In brief, bacterial isolates were cultured in Mueller-Hinton broth and subjected to serial dilutions of colistin. The MIC was defined as the lowest concentration at which no visible bacterial growth was observed after 18–24 hours of incubation at 35°C.(6)
Subsequently, isolates exhibiting colistin resistance (MIC ≥ 2 µg/mL) underwent further testing with the NG Test MCR-1, a lateral flow immunoassay designed to detect MCR-1-producing strains rapidly. Bacterial colonies were re-suspended in an extraction buffer, and the test strip was immersed in the solution. The appearance of a test line indicated a positive result, confirming the presence of the MCR-1 protein.
EDTA-Colistin Broth Disk Elution(CBDE/EDTA) method, combined with the PCR mcr1-10 test
Initially, colistin resistance was assessed using the CBDE method, following the Clinical and Laboratory Standards Institute (CLSI M100 S34) guidelines. In brief, bacterial isolates were inoculated into cation-adjusted Mueller-Hinton broth, containing a colistin disk (10 µg) and incubated at 35°C for 16–20 hours. The minimum inhibitory concentration (MIC) was determined based on the turbidity of the bacterial suspension, with an MIC ≥ 2 µg/mL indicating colistin resistance.(6)
To further differentiate mcr-mediated colistin resistance from other resistance mechanisms, we performed the CBDE/EDTA test, which involves adding ethylenediaminetetraacetic acid (EDTA) to the CBDE assay.(4) The presence of metalloenzyme activity, which EDTA inhibits, was evaluated by comparing the MIC results between the CBDE and CBDE/EDTA tests. A presumptive positive result for plasmid-mediated colistin resistance (PMCR) by the EDTA-CBDE screening method was defined as any observed reduction in MIC in the presence of EDTA. These screening results were subsequently compared to the corresponding molecular findings for confirmation.
For isolates testing positive in the CBDE/EDTA assay, PCR detection of mcr genes (mcr-1 to mcr‑10) was conducted at an external reference laboratory using standardized protocols.
Statistical analysis
The agreement between the NG-Test MCR-1 combined with the Broth Microdilution (BMD) method and the CBDE/EDTA method combined with PCR (mcr 1–10) was evaluated using Cohen's Kappa coefficient (κ), NPA, and PPA with 95% confidence intervals. Due to the small number of mcr-positive isolates, confidence intervals for PPA and NPA were calculated using the Jeffreys method, a Bayesian approach suitable for small sample sizes or when observed proportions are close to 0% or 100%. The association between bacterial species, specimen type, and presence of mcr genes using Fisher’s exact test. These statistical measures assessed the inter-method concordance for detecting mcr-mediated colistin resistance among Enterobacterales isolates.
Ethical considerations
The study collected frozen bacterial strains that met the selection criteria without patient intervention. The Ethics Committee of the University of Medicine and Pharmacy in Ho Chi Minh City approved this research under contract number 142 /HĐĐĐ-ĐHYD.
RESULTS
Characteristics of the study strains
Among the 160 bacterial isolates, the most common specimen sources were phlegm (30.3%), body fluids (28.0%), and blood (19.3%). The dominant bacterial species identified were Klebsiella pneumoniae (80.0%) and Escherichia coli (20.0%) (table 1).
Broth Microdilution (BMD) in combination with the NG Test MCR-1 method
Using the broth microdilution (BMD) method, colistin resistance was detected in 2/32 (6.25%) E. coli and 30/128 (23.4%) K. pneumoniae isolates. Among the 34 colistin-resistant isolates, only two E. coli strains were positive for mcr-1 by NG-Test MCR-1, while no K. pneumoniae isolate carried mcr genes. All mcr-1-positive isolates were confirmed colistin-resistant by BMD, with significant differences observed between species for both colistin resistance (p = 0.039) and mcr-1 positivity (p = 0.045).
Using the CBDE/EDTA method, potential mcr-mediated resistance was detected in 2/32 E. coli and 1/30 K. pneumoniae isolates. PCR confirmation identified mcr-1 only in the two E. coli strains, with no detection in K. pneumoniae. Discrepancies between CBDE/EDTA and PCR results highlight limitations of phenotypic screening, particularly in K. pneumoniae (p = 0.002) (table 2).
EDTA-Colistin Broth Disk Elution (CBDE/ EDTA) method in combination with PCR
By the CBDE method, colistin resistance was found in 2/32 E. coli isolates and 30/128 K. pneumoniae isolates. CBDE/EDTA results were positive in 2/2 colistin-resistant E. coli and 1/30 colistin-resistant K. pneumoniae isolates. PCR detection of mcr-1 to mcr-10 genes confirmed positivity in 2/2 E. coli isolates and 0/30 K. pneumoniae isolates. Overall, among the 34 colistin-resistant isolates, 5 were positive by CBDE/EDTA and 4 were confirmed by PCR. The p-values for CBDE/EDTA positivity and PCR confirmation were 0.045 and 0.002, respectively (table 3).
Interpretation result of BMD with the NG Test MCR-1 to the CBDE/ EDTA
There were 4 isolates confirmed to carry mcr genes by PCR were also detected by both the NG Test MCR-1. No false-positive or false-negative results were observed in this small subset, suggesting high concordance between genetic and phenotypic methods for detecting mcr-mediated resistance in E. coli (table 4).
The diagnostic agreement between BMD combined with NG-Test MCR-1 and CBDE/EDTA combined with PCR showed a Kappa (κ) value of 1.00 with a 95% confidence interval of [1.00–1.00]. Both Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were 100%, with 95% confidence intervals of [56%–100%] and [92%–100%], respectively. Confidence intervals were calculated using the Jeffreys method (table 5).
DISCUSSION
This study provides valuable insights into the characteristics of bacterial isolates and the detection of mcr-mediated colistin resistance using different methodologies. Among the 160 clinical isolates analyzed, K. pneumoniae accounted for the majority (80%), while E. coli represented 20% of cases. The observed distribution of colistin resistance in this study, where 32 out of 160 isolates were resistant to colistin, comprising 30 Klebsiella pneumoniae strains without the mcr gene and 2 Escherichia coli strains both harboring the mcr-1 gene, aligns with global patterns in colistin resistance mechanisms.(7)
In K. pneumoniae, colistin resistance is predominantly associated with chromosomal mutations rather than plasmid-mediated mcr genes. Mutations in genes such as mgrB, pmrA, and pmrB lead to modifications in the bacterial outer membrane, reducing colistin binding and efficacy. A study highlighted that colistin resistance in K. pneumoniae is often due to the inactivation of the mgrB gene, a negative regulator of the PhoPQ signaling system, resulting in lipid A modifications that confer resistance.(8) Conversely, in E. coli, colistin resistance is frequently linked to plasmid-mediated mcr genes, particularly mcr-1. The mcr-1 gene encodes a phosphoethanolamine transferase that modifies lipid A, diminishing colistin's binding affinity. A comprehensive review reported a global prevalence of mcr-mediated colistin-resistant E. coli at approximately 6.51%, with significant variations across regions and sources.(9)
In this study, the absence of mcr genes in colistin-resistant K. pneumoniae isolates and their presence in all colistin-resistant E. coli isolates underscores the distinct resistance mechanisms between these species. This distinction is crucial for developing targeted diagnostic and treatment strategies, as plasmid-mediated resistance can disseminate more rapidly across bacterial populations than chromosomal mutations.(7)
The NG Test MCR-1 demonstrated high accuracy in detecting the mcr-1 gene among colistin-resistant E. coli isolates, with perfect concordance with PCR results. This LFIA test offers a rapid, specific, and user-friendly approach to identifying mcr-1 in microbiology laboratories. The speed and ease of NG Test MCR-1 make it a promising tool for early detection, allowing clinicians to adjust antimicrobial therapy promptly. Although mcr-1 has emerged as the most prevalent variant globally, including in Southeast Asian settings, its major limitation is the potential failure to detect other mcr variants (mcr-2 to mcr-10), which may lead to false-negative results in strains carrying alternative resistance mechanisms.(10)
In contrast, the CBDE/EDTA method provides a phenotypic approach to detecting colistin resistance and can identify transferable mcr-mediated resistance. This method is considered cost-effective and practical for routine laboratory use. However, its accuracy remains limited, as observed in the study where CBDE/EDTA detected mcr genes in one K. pneumoniae isolate that later tested negative by PCR. This discrepancy suggests that CBDE/EDTA may not differentiate between mcr-mediated resistance and other mechanisms of colistin resistance, such as mutations in the PmrAB and PhoPQ regulatory systems. PCR confirmation is still essential when CBDE/EDTA results are favorable to determine the exact mcr variant and avoid misinterpretation.(7)
The comparison between BMD with NG Test MCR-1 and CBDE/EDTA with PCR revealed a perfect agreement (κ = 1.00), Furthermore, it achieved 100% PPA and NPA, with wide 95% confidence intervals due to the limited number of mcr-positive isolates, reinforcing the reliability of NG Test MCR-1 for detecting mcr-1 in E. coli. However, the absence of mcr gene detection in colistin-resistant K. pneumoniae suggests that resistance in this species may be attributed to non-mcr mechanisms, further supporting the need for a combined approach using phenotypic and molecular tests.
These results are consistent with global studies evaluating the performance of the NG-Test MCR-1. A study published in the Journal of Clinical Microbiology assessed the NG-Test MCR-1 LFA across 238 Gram-negative bacillus isolates, including 126 Enterobacterales, 50 Pseudomonas aeruginosa, and 50 Acinetobacter species. Before repeat testing, the assay demonstrated a positive percent agreement (PPA) of 100% and a negative percent agreement (NPA) of 96.1%. Upon retesting, only one false-positive result remained, attributed to an MCR-2 producer, indicating the high specificity of the test.(5) As the current study was conducted in Vietnam, where data on NG Test MCR-1 performance remains limited, it provides valuable insights into the effectiveness of this assay for detecting mcr-1 in Southeast Asia. This research not only expands the application of NG Test MCR-1 but also provides essential data for antimicrobial resistance surveillance and control strategies in the region.
Clinical and Laboratory Implications: The findings of this study highlight the value of incorporating the NG-Test MCR-1 into routine diagnostic workflows for the rapid detection of mcr-1, especially in resource-limited settings with restricted access to molecular diagnostics. Its high specificity for mcr-1 renders it a valuable point-of-care tool, significantly reducing turnaround time and strengthening infection control strategies. A stepwise diagnostic approach, utilizing CBDE/EDTA for initial screening followed by NG-Test MCR-1 or multiplex real-time PCR to identify mcr variants (mcr-1 to mcr-10) comprehensively, could optimize laboratory efficiency and enhance diagnostic accuracy.
Study Limitations and Future Directions: This study has several limitations. First, the number of mcr-positive isolates was very small (n = 4), which resulted in wide 95% confidence intervals for both PPA and NPA estimates, despite point estimates being 100%. This statistical limitation reduces the precision of diagnostic performance measurements and warrants cautious interpretation. Second, the research was conducted in a single tertiary-care hospital (Cho Ray Hospital), which may limit the generalizability of the findings to other healthcare settings in Vietnam or Southeast Asia. Third, the study focused exclusively on mcr-1 detection, and other plasmid-mediated mcr variants (mcr-2 to mcr-10) could not be identified, potentially underestimating the overall prevalence of mcr-mediated colistin resistance. Finally, only two species (Klebsiella pneumoniae and Escherichia coli) were examined, which does not capture the full diversity of Enterobacterales in clinical practice.
Future research should include larger sample sizes from multiple hospitals across different regions, incorporate a broader range of Enterobacterales species, and utilize multiplex PCR or whole genome sequencing to detect all mcr variants comprehensively. These improvements would enhance the robustness of prevalence estimates, narrow confidence intervals, and strengthen the evidence base for diagnostic and infection control strategies.
The NG-Test MCR-1 demonstrated perfect agreement with the reference method (κ = 1.00), with 100% PPA and NPA, although wide 95% confidence intervals reflected the limited number of mcr-positive isolates. The assay provided rapid and specific detection of mcr-1, while CBDE/EDTA served as a cost-effective screening tool. These findings support the combined use of NG-Test MCR-1 and CBDE/EDTA to optimize the detection of mcr-mediated colistin resistance, enabling timely clinical decision-making and strengthening antimicrobial stewardship, particularly in resource-limited settings.
NG Test MCR-1 provides rapid, specific detection of mcr-1, while CBDE/EDTA is a cost-effective screening tool but less definitive. Combining these methods is recommended to improve mcr-mediated colistin resistance detection.
Acknowledgments
We would like to extend our special thanks to Cho Ray Hospital for providing the facilities for our research. Furthermore, we sincerely appreciate the Microbiology Department, Cho Ray Hospital, for kindly providing the bacterial strains essential for this study. We also thank Nam Khoa CO., Ltd., for supporting the genotyping of mcr-1 to mcr-10 in colistin-resistant strains.
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Conflict of interest
The authors declare that they have no competing interests.
Financial information
This study did not receive any specific funding from public, commercial, or non-profit organizations.
Authors' Contributions
Conceptualization: Tran Bich Ngoc, Truong Thien Phu.
Data curation: Tran Bich Ngoc, Truong Thien Phu, Thao Nguyen Thi Ngoc, Vinh Tran Thanh Hua.
Formal analysis: Tran Bich Ngoc, Truong Thien Phu,Thao Nguyen Thi Ngoc.
Research: Tran Bich Ngoc, Truong Thien Phu, Thao Nguyen Thi Ngoc.
Methodology: Tran Bich Ngoc, Truong Thien Phu,Thao Nguyen Thi Ngoc, Van Hung Pham, Vinh Tran Thanh Hua.
Supervision: Truong Thien Phu, Dat Ngo Quoc.
Validation: Van Tran Thi Hue, Van Hung Pham.
Writing – original draft: Tran Bich Ngoc, Thao Nguyen Thi Ngoc, Truong Thien Phu.
Writing – review and editing: Tran Bich Ngoc, Truong Thien Phu.
Data availability
This research data is confidential according to the applicable confidentiality agreements and regulations and, therefore, cannot be publicly displayed or shared. The data are securely stored at the Integrated Planning Department at Cho Ray Hospital. Access to these data requires proper authorization. If you have any questions or need further information, please contact Ngoc Bich Tran at bichngoctran@ump.edu.vn
