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Recombinant polymerase amplification combined with lateral flow strips for the detection of deep-seated Candida
Mengdi Zhao1, Xizhen Wang2, Kun Wang2
1Department of Materials Science and Engineering, Suzhou University of Science and Technology, Suzhou, China.
Abstract:
The incidence of Candida infections in intensive care units (ICU) has significantly increased in recent years, and these infections have become one of the most serious complications threatening the lives of ICU patients. The proportion of non-Candida albicans infections, such as Candida krusei and Candida glabrata infections, which are resistant to fluconazole, is increasing each year. Early identification of the strains causing Candida infections is important for the timely implementation of targeted treatments to save patients' lives. However, the current methods of direct microscopy, culture, and histopathology, as well as other diagnostic methods, have many shortcomings, such as their low sensitivity and long assay times; therefore, they cannot meet the needs for early clinical diagnosis. Recombinant polymerase amplification (RPA) is a promising isothermal amplification technique that can be performed without sophisticated instruments and equipment, and is suitable for use in resource-poor areas. RPA combined with lateral flow strips (LFS) can be used to rapidly amplify and visualize target genes within 20 min. In this study, RPA-LFS was used to amplify the internal transcribed spacer 2 (ITS2) region of C. krusei. The primer-probe design was optimized by introduction of base mismatches (probe modification of five bases) to obtain a specific and sensitive primer-probe combination for the detection of clinical specimens. Thirty-five common clinical pathogens were tested with RPA-LFS to determine the specificity of the detection system. The RPA-LFS system specifically detected C. krusei without cross-reaction with other fungi or bacteria. A gradient dilution of the template was tested to explore the lower limit of detection and sensitivity of the assay. The sensitivity was 10 CFU/50 µL per reaction, without interference from genomic DNA of other species. The RPA-LFS and qPCR assays were performed on 189 clinical specimens to evaluate the detection performance of the RPA-LFS system. Seventy-six specimens were identified as C. krusei, indicating a detection rate of 40.2%. The results were consistent with those of qPCR and conventional culture methods. The RPA-LFS system established in our study provides a reliable molecular diagnostic method for the detection of C. krusei, thus meeting the urgent need for rapid, specific, sensitive, and portable clinical field testing.
Insights
A new Recombinant Polymerase Amplification (RPA) combined with Lateral Flow Strips (LFS) assay rapidly and accurately detects Candida krusei infections. This molecular diagnostic method offers a sensitive, specific, and portable solution for early clinical diagnosis in intensive care units (ICUs).
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Intensive care unit (ICU) patients face increasing risks from Candida infections, particularly non-albicans strains like Candida krusei, which exhibit antifungal resistance.
- Current diagnostic methods for Candida infections lack the sensitivity and speed required for timely clinical intervention, leading to poor patient outcomes.
- Emerging molecular techniques offer potential for rapid and accurate pathogen identification, crucial for effective treatment strategies.
Purpose of the Study:
- To develop and validate a rapid, sensitive, and specific molecular diagnostic assay for the early detection of Candida krusei.
- To evaluate the performance of a Recombinant Polymerase Amplification (RPA) combined with Lateral Flow Strips (LFS) assay for identifying Candida krusei in clinical specimens.
Main Methods:
- The study optimized a Recombinant Polymerase Amplification (RPA) assay targeting the internal transcribed spacer 2 (ITS2) region of Candida krusei.
- The RPA assay was coupled with Lateral Flow Strips (LFS) for rapid visual detection of amplified DNA within 20 minutes.
- Specificity was confirmed against 35 common clinical pathogens, and sensitivity was determined using serial dilutions, reaching a limit of 10 CFU/50 µL.
Main Results:
- The developed RPA-LFS assay demonstrated high specificity for Candida krusei, with no cross-reactivity observed with other fungi or bacteria.
- The assay achieved a sensitivity of 10 CFU/50 µL, proving effective without interference from other genomic DNA.
- In clinical evaluation, the RPA-LFS assay identified Candida krusei in 76 out of 189 specimens (40.2% detection rate), showing consistent results with qPCR and culture methods.
Conclusions:
- The established RPA-LFS system provides a reliable, rapid, specific, and sensitive molecular diagnostic tool for Candida krusei detection.
- This assay meets the urgent clinical need for portable and efficient field testing, facilitating early diagnosis and targeted treatment in ICUs.
- The RPA-LFS method holds significant promise for improving the management of invasive fungal infections, especially in resource-limited settings.
