Development of reverse transcription loop-mediated isothermal amplification-based assay for rapid and specific detection of human fungal pathogen, Candida auris

Abstract

Background and purpose: Due to the ability of Candida auris, a multidrug-resistant human fungal pathogen, to colonize the skin and hospital surfaces, it is pertinent to control its nosocomial outbreaks through rapid diagnosis. Delayed and improper diagnosis of C. auris due to misidentification becomes a major hurdle in the prevention of employment of efficient therapeutics leading to the development of drug resistance. The culture-based methods are slow and less sensitive while PCR-based methods are costly. Loop-mediated amplification (LAMP) is a feasible alternative, but it fails to differentiate between live and dead cells. Therefore, this study aimed to evaluate the diagnostic efficiency of the reverse transcription (RT) LAMP approach and compare it with that of the LAMP assay for the detection of C. auris.

Materials and methods: RT-LAMP method was developed for the detection of C. auris and its clinical isolates. The limit of detection (LOD), sensitivity, and specificity were evaluated for the developed method using culture RNA. The RT-LAMP reaction for C. auris detection was standardized using the primers of a specific 869-bp DNA segment (accession no. XM_018317007), encoding a pyruvate: ferredoxin oxidoreductase domain, from the genome of C. auris.

Results: The LOD for the RT-LAMP method was 1ag contrary to 10fg for LAMP method using DNA. Specificity was 100% as determined using a gram-negative bacteria and several other Candida species. The RT-LAMP method was intraspecific and displayed no cross reaction even with closely related Candida species. The RT-LAMP method was validated on 10 clinical isolates of C. auris and showed 100% concordance with a culture-based method.

Conclusion: The RT-LAMP-based method in the present study offered a proof of concept that warrants clinical validation on a large number of samples. Therefore, its diagnostic potential for the rapid, sensitive, and specific detection of C. auris could be further exploited in resource-limited regions.

Keywords: Loop-mediated isothermal amplification; RT-LAMP; Sensitivity; Specificity; Candida auris.

Figure 1

Optimization reverse transcription loop-mediated amplification (RT-LAMP) (a) RT-LAMP reaction with Candida auris RNA and negative template control visualized by color change from pink to yellow after 40 min at 65 ℃. (b) Agarose gel image of the amplified product obtained from LAMP reaction.

Figure 2

Limit of detection of reverse transcription loop-mediated amplification (RT-LAMP) reaction (a) RT-LAMP reaction in tubes 1-14 with RNA concentrations: Lane 1)1000 ng, 2)100 ng, 3)10 ng, 4)1ng, 5)100 pg, 6)10 pg, 7)1 pg (left panel) 8)1000 fg, 9)100 fg, 10)10 fg, 11)1 fg, 12)100 ag, 13)10 ag, 14)1 ag (right panel), visualised by color change from pink to yellow after 40 min at 65 ℃. (b) Agarose gel images of amplified products obtained from RT-LAMP reactions.

Figure 3

Lane 1)1000 pg, 2)100 pg, 3)10 pg, 4)1pg, 5)100 fg, 6)10 fg, (left panel), 7)10 fg, 8)1 fg, 9)100 ag, 10)10 ag, 11) 1 ag (right panel), visualized by colour change from pink to yellow after 40 minutes at 65℃. (b) Agarose gel images of amplified products obtained from LAMP reactions.

Figure 4

Interspecies specificity of reverse transcription loop-mediated amplification (RT-LAMP) reaction (a) RT-LAMP reaction of culture RNA extracted from Lane 1) C. auris, 2) C. albicans, 3) Mycobacterium smegmatis and 4) E coli, visualized by color change from pink to yellow after 40 minutes at 65℃. (b) Agarose gel image of the amplified product obtained from RT-LAMP reaction.

Figure 5

Intraspecies specificity reverse transcription loop-mediated amplification (RT-LAMP) reaction (a) RT-LAMP reaction of culture RNA extracted from lane 1) Candida auris, 2) Candida albicans, 3) Candida glabrata, 4) Candida tropicalis, 5) Candida parapsilosis, and 6) Candida krusei., visualized by color change from pink to yellow after 40 min at 65 °C. (b) Agarose gel image of the amplified product obtained from RT-LAMP reaction.

Figure 6

Clinical validation of reverse transcription loop-mediated amplification (RT-LAMP) for Candida auris (a) RT-LAMP reaction of culture RNA extracted from clinical isolates of C. auris in lanes 1) 1622, 2) 218834, 3) 1251, 4) C22/6089, 5) 37229, 6) 202916, 7) 202982, 8) P1188, 9) C-33, 10) C-34, 11) CBS10913T, and 12) NTC. (b) Agarose gel image of the amplified product obtained from RT-LAMP reaction.