A simple and sensitive test for Candida auris colonization, surveillance, and infection control suitable for near patient use

ABSTRACT

Candida auris is a multidrug-resistant fungal pathogen with a propensity to colonize humans and persist on environmental surfaces. C. auris invasive fungal disease is being increasingly identified in acute and long-term care settings. We have developed a prototype cartridge-based C. auris surveillance assay (CaurisSurV cartridge; “research use only”) that includes integrated sample processing and nucleic acid amplification to detect C. auris from surveillance skin swabs in the GeneXpert instrument and is designed for point-of-care use. The assay limit of detection (LoD) in the skin swab matrix was 10.5 and 14.8 CFU/mL for non-aggregative (AR0388) and aggregative (AR0382) strains of C. auris, respectively. All five known clades of C. auris were detected at 2-3-5× (31.5–52.5 CFU/mL) the LoD. The assay was validated using a total of 85 clinical swab samples banked at two different institutions (University of California Los Angeles, CA and Wadsworth Center, NY). Compared to culture, sensitivity was 96.8% (30/31) and 100% (10/10) in the UCLA and Wadsworth cohorts, respectively, providing a combined sensitivity of 97.5% (40/41), and compared to PCR, the combined sensitivity was 92% (46/50). Specificity was 100% with both clinical (C. auris negative matrix, N = 31) and analytical (non-C. auris strains, N = 32) samples. An additional blinded study with N = 60 samples from Wadsworth Center, NY yielded 97% (29/30) sensitivity and 100% (28/28) specificity. We have developed a completely integrated, sensitive, specific, and 58-min prototype test, which can be used for routine surveillance of C. auris and might help prevent colonization and outbreaks in acute and chronic healthcare settings.

IMPORTANCE

This study has the potential to offer a better solution to healthcare providers at hospitals and long-term care facilities in their ongoing efforts for effective and timely control of Candida auris infection and hence quicker response for any potential future outbreaks.

INTRODUCTION

The United States Centers for Disease Control and Prevention (CDC) has reported increasing incidences of invasive Candida auris infections in healthcare facilities in recent years (1, 2). Although C. auris is primarily a skin colonizer, this often multi-drug-resistant (MDR) fungal pathogen can cause bloodstream infections (3, 4) that lead to a crude mortality rate of 30%–40% (5, 6). Consequently, C. auris is the first fungal pathogen that the CDC has classified as an “urgent threat” (6–8). The World Health Organization declared antibiotic-resistant (AR) fungal pathogens as a major public health threat and has designated it in the “critical priority group” for public health and research (7–9). The COVID-19 pandemic has also been associated with a further surge in the number of C. auris cases in the United States (10) and worldwide (11–14), with clinical cases increasing from 478 (2019) to 2,377 cases in 2022 in the United States alone (1, 13, 15).

There are five different clades of C. auris, which were originally classified based on their geographical distribution (4, 16–19): the South Asian clade (clade I), the East Asian clade (clade II), the South African clade (clade III), the South American clade (clade IV), and Iranian clade (clade V) (6, 16). However, C. auris clades are now reported on all continents except Antarctica (18, 20, 21). Clade I isolates have the highest reported rate of multi-drug resistance including isolates resistant to both azoles and amphotericin B (22) and pan-resistant isolates (23); Clade II isolates are often drug susceptible (24, 25), whereas Clade III isolates are most often resistant to azoles (24); Clade IV isolates carry the highest percentage of echinocandin resistance (24). Clades I, III, and IV isolates include the MDR strains most often associated with hospital infections and outbreaks (24, 26) and are the clades responsible for most cases of candidemia and associated mortality (19, 27). Strains belonging to Clades II (25) and V commonly cause ear infections and rarely cause bloodborne infections and outbreaks (22, 28). The majority of these clades include both aggregating and non-aggregating phenotypes (29, 30), which differ from each other in terms of resistance to disinfectants and antifungals, biofilm formation, host immune response evasion, and colonizing ability on biotic and abiotic surfaces (29–32).

Given the high potential of C. auris for colonization and nosocomial spread, a simple and rapid test to detect this pathogen on human and environmental surfaces could provide critical protection against C. auris outbreaks especially in Intensive Care Unit settings (33) and in long-term care facilities (34). Routine testing for C. auris could help guide proper treatment as well as the implementation of effective infection control practices (1, 2, 35). Indeed, PCR-based tests have become the preferred choice for identifying C. auris from clinical and surveillance samples by many laboratories (1, 36–43). Here, we present a highly specific, sensitive, and a <1 h test to enable the detection of C. auris.

MATERIALS AND METHODS

Sample matrix, media, and pathogens

All bacterial and fungal strains used in this study are listed in Table 1. Assay optimization and limit of detection (LoD) studies were performed with clade I C. auris strains AR0388 (non-aggregative) and AR0382 (aggregative). All strains were obtained from the CDC and Food and Drug Administration (FDA) Antimicrobial Resistance Isolate Bank, Atlanta, GA (CAU, 2022) and used for all analytical studies. Initial inoculum was prepared by culture in Sabouraud dextrose (SD) broth for 16–18 hours at 37°C in a shaker incubator. Tenfold serial dilutions were made in SD broth and plated on SD agar to enumerate CFU/mL.

TABLE 1.

Microbial strains^a

		Microorganism	Strain no.	Source	Clade	CaurisSurV test result
Inclusivity panel		C. auris	AR 0381	CDC-FDA AR	II	+
			AR 0383	CDC-FDA AR	III	+
			AR 0384	CDC-FDA AR	III	+
			AR 0385	CDC-FDA AR	IV	+
			AR 0386	CDC-FDA AR	IV	+
			AR 0387	CDC-FDA AR	I	+
			AR 0389	CDC-FDA AR	I	+
			AR 0390	CDC-FDA AR	I	+
			AR 0931	CDC-FDA AR	IV	+
			AR 1097	CDC-FDA AR	V	+
			AR 1099	CDC-FDA AR	NK	+
			AR 1100	CDC-FDA AR	NK	+
			AR 1101	CDC-FDA AR	II	+
			AR 1102	CDC-FDA AR	III	+
			AR 1103	CDC-FDA AR	III	+
			AR 1104	CDC-FDA AR	IV	+
			AR 1105	CDC-FDA AR	NK	+
Exclusivity panel	Non-auris Yeast and other fungi	Candida duobushaemulonii	AR 0391	CDC-FDA AR		−
			AR 0392	CDC-FDA AR		−
			AR 0394	CDC-FDA AR		−
		Candida haemulonii	AR 0393	CDC-FDA AR		−
			AR 0395	CDC-FDA AR		−
			AR 0932	CDC-FDA AR		−
		Candida lusitaniae	AR 0398	CDC-FDA AR		−
		Candida albicans	470547	ATCC		−
		Candida tropicalis	66029	ATCC		−
			CAB54-6763-3, HM-1124	BEI		−
		Candida parapsilosis	22019	ATCC		−
		Candida glabrata	15126	ATCC		−
		Candida krusei	6258	ATCC		−
			AR 0397	CDC-FDA AR		−
		Kodameae ohmeri	AR 0396	CDC-FDA AR		−
		Cryptococcus neoformans	China Isolate 5, NR-41295	BEI		−
			NIH398, NR-50333	BEI		−
		Cryptococcus gattii	CBS1930, NR-50185	BEI		−
			MIC64-C1, NR-50421	BEI		−
		Saccharomyces cerevisiae	AR 0399	CDC-FDA AR		−
			AR 0400	CDC-FDA AR		−
		Aspergillus fumigatus	B6081, NR-50386	BEI		−
	Gram-positive bacteria	Staphylococcus aureus	Clinical isolate	UH		−
		Staphylococcus epidermidis	SK135, HM-118	BEI		−
		Streptococcus pyogenes	ABC020063118, NR-48702	BEI		−
		Corynebacterium amycolatum	SK46, HM-109	BEI		−
		Enterococcus faecium	TX1330, HM-204	BEI		−
		Propionibacterium acnes	HL030PA2, HM-505	BEI		−
			HL005PA1, HM-519	BEI		−
	Gram-negative bacteria	Proteus mirabilis	WGLW4, HM-752	BEI		−
		Klebsiella oxytoca	MIT 10–5244, HM-625	BEI		−
		Pseudomonas aeruginosa	Shr42, NR-48982	BEI		−

^a NK, not known; UH, University Hospital; CDC-FDA-AR, The United States Center for Disease Control and FDA Antimicrobial Resistance Bank; ATCC, American Type Culture Collection; BEI, BEI resources.

PCR (BD Max System) confirmed C. auris-negative skin swab matrix (skin swabs collected in liquid Amies transport media) collected at the Department of Pathology and Laboratory Medicine, the University of California Los Angeles (UCLA), Los Angeles, CA, were used as a negative matrix in the analytical dynamic range and LoD experiments. The negative samples were banked at −20°C or −80°C and were aliquoted and shipped to our lab at Public Health Research Institute (PHRI), Rutgers New Jersey Medical School, Newark, NJ where they were stored at −20°C until further use.

Cartridge-based real-time PCR assay

The assay was optimized in a GeneXpert filter-based cartridge system (44–46) controlled by a GeneXpert instrument. It is a real-time molecular beacon-based PCR assay targeting the ITS2 gene of C. auris (proprietary). An internal control (IC) assay (proprietary) was also included in the cartridge, serving as a positive control for both sample processing and PCR amplification. After optimization of the reagents and the sample preparation, robotically built, reagent -filled, ready-to-use filter-based cartridges for C. auris surveillance assay (CaurisSurV cartridge) were made by Cepheid for this study. All the experiments presented in this manuscript were performed using CaurisSurV cartridges.

Sample processing

We developed a simple point-of-care applicable method that involves a single manual step of adding the sample to the CaurisSurV cartridge, producing results within an hour (Fig. 1). The cartridge-based system in the GeneXpert instruments offers the advantage of completely automating the assay after the sample is added as described earlier (44–48). We tested the performance of CaurisSurV cartridge by spiking C. auris at various concentrations into Amies transport medium (Thomas Scientific, Swedesboro, NJ) and/or C. auris negative skin swab matrix. Five hundred microliters of this spiked sample was then added to the CaurisSurV cartridges and loaded into the GeneXpert system (Fig. 1). During automated processing in the CaurisSurV cartridge, the programmed microfluidic movements resuspended the internal control assay target with 200 µL of 50 mM Tris–0.1 mM EDTA (0.1 mM)–0.1% Tween (TET) buffer (pH 8) which was then added to the sample matrix. The sample was then mixed with 1.5 mL of TET buffer and passed through the internal cartridge filter. The filter-captured fungal cells were extensively washed with both TET and guanidine hydrochloride buffers. Glass beads present in the filter were then agitated by an ultrasonic horn to lyse the captured fungal cells. Finally, approximately 80% of the total eluted DNA was moved into the PCR tube that is integrated into the assay cartridge for PCR amplification and detection. The test was considered positive when real-time cycles vs fluorescence units for the C. auris-specific molecular beacon reached a value above 20. The cycle threshold (Ct) was calculated by the GeneXpert software. A negative test required a positive internal control (IC) reaction. All negative tests with negative IC reactions were considered invalid.

Fig 1.

A simple three-step sample to result in testing protocol.

Analytical dynamic range and LoD

The dynamic range of the assay was evaluated in C. auris-negative skin swab matrix by spiking C. auris AR0388 (non-aggregative strain) at 10⁷ CFU/mL through 1 CFU/mL concentrations (N = 4). Five hundred microliters of the spiked matrix was then added to the sample chamber of the CaurisSurV cartridge. The analytical sensitivity of the CaurisSurV cartridge was assessed using a representative strain of both the aggregative (AR0382) and non-aggregative (AR0388) phenotype of C. auris. The analytical LoD was determined by testing 10-fold serial dilutions of respective C. auris CFU spiked into PCR-negative skin swab matrix (Fig. 2). A total of 20 technical replicates were tested at each concentration including negatives. For this study, the LoD was defined as the lowest concentration of CFU at which 19/20 or 95% of the replicates tested were positive.

Fig 2.

C. auris surveillance assay cartridge (CaurisSurV cartridge) performance is demonstrated analytically showing wide dynamic range of C. auris spiked from 1 to 10⁷ CFU/mL in skin swab matrix (A, R² = 0.9911); LoD with non-aggregative strain (AR0388, B) and an aggregative strain (AR0382, C) of C. auris spiked in clinical skin swab matrix.

Assay inclusivity and exclusivity

Assay inclusivity was carried out in Amies transport media at 3× (31.5 CFU/mL) or 5× (52.5 CFU/mL) the LOD (of AR0388) for 17 different strains of C. auris belonging to all five clades (Table 1 inclusivity). All these isolates were obtained from the CDC and FDA Antimicrobial Resistance (AR) Isolate Bank, Atlanta, GA (CAU 2022). The specificity of the CaurisSurV cartridge was determined by spiking 10⁶ to 10⁸ CFU/mL of different bacteria or non-C. auris yeast/fungal species or genomic DNA where quantified cells were not possible, into Amies transport media (Table 1 exclusivity). Test isolates included non-C. auris yeast species (N = 21), other related fungi (N = 1), representative gram-positive (N = 7), and gram-negative bacteria (N = 3).

Clinical evaluation

A clinical evaluation was performed in two phases: (i) an initial validation phase and (ii) a blinded study where the tester was blinded to the sample PCR or culture results. For the initial validation study, a total of 85 PCR-confirmed C. auris positive and negative skin swab samples (Table S1) were collected from the sample banks of Dr. Garner, UCLA, Los Angeles, CA (N = 72) and Dr. Chaturvedi at Public Health Laboratory, Wadsworth Center, NY (N = 13). UCLA C. auris PCR-positive skin swab samples (N = 41) were initially identified using BD Max PCR using BioGx C. auris reagents (Cat. No.450–043), and 31 were further confirmed for C. auris and clade identity by culture (HardyCHROM Candida) and whole-genome sequencing (Illumina). C. auris positive samples (N = 10) from the Wadsworth Center, NY were identified both by BD Max using PCR established at Dr. Chaturvedi’s lab (42) and culture using CHROMagar Candida plus agar media (49). At both sites, PCR-negative samples were not further confirmed by culture. Our primary analysis used culture positivity and culture negativity as reference for positive and negative standards, respectively. We also performed a secondary analysis against PCR positivity regardless of culture result as the reference positive standard and both culture and PCR positivity as the reference negative standard.

For the blinded study, 60 skin swab samples were coded and shipped to our lab at PHRI, Rutgers New Jersey Medical School, Newark, NJ from Wadsworth Center, NY (Table S2). The tester at the PHRI laboratory was blinded to the sample identification at the time of testing. Once the testing was completed for all 60 samples, the resulting output from the CaurisSurV cartridge was matched with the answer key.

For the UCLA samples, a final volume of 0.5 mL of the skin swab matrix was added to the sample chamber of the CaurisSurV cartridge and then loaded into the GeneXpert instrument. Only 0.25 mL of the matrix was available for the Wadsworth samples which were supplemented with liquid Amies transport media to make the final test volume up to 0.5 mL and then tested in CaurisSurV cartridges. All samples used for the clinical evaluation study were aliquoted and kept frozen (−80°C/−20°C) or refrigerated (4°C) for different lengths of time (Tables S1 and S2), before shipping to Rutgers and were tested at the PHRI laboratory within a week of receiving the samples.

Statistical analysis

Standard statistical analyses (average, SD, and correlation) and graphing were performed using Microsoft Excel (ver 2102) and GraphPad Prism 8.4.3 for Windows. Pearson’s and Spearman’s correlation coefficient r and R² (coefficient of determination and square of the regression line) values were interpreted based on the literature (50), where r value of >0.7 indicates a strong to very strong correlation.

RESULTS

Dynamic range and analytical LoD

C. auris surveillance assay dynamic range was tested to determine its logarithmic range of detection and linearity as measured by Ct vs target concentration. C. auris AR0388 was spiked at 1 to 10⁷ CFU/mL in the skin swab matrix and tested in the CaurisSurV cartridge. As shown in Fig. 2A, the assay demonstrated an excellent dynamic range in the skin swab matrix as well as linearity across this range (R² = 0.9911). The CaurisSurV cartridge LoD was then evaluated by spiking C. auris AR0388 (non-aggregative strain) or AR0382 (aggregative strain) in skin swab matrix at 1, 5, 10, 20, and 50 CFU/mL (e.g., 0.5, 2.5, 5, 10, and 25 CFU/test) and then tested in replicates of 20. In this study, the LoD was defined as the CFU/mL concentration that was positive at a 95% rate. Microsoft Excel-based probit analysis determined the assay LoD to be 10.5 CFU/mL (Ct = 34.8) for the non-aggregative strain AR0388 (Fig. 2B) and 14.8 CFU/mL (Ct ~ 34) for the aggregative strain AR0382 (Fig. 2C) of C. auris.

Inclusivity and exclusivity

The assay inclusivity was tested on 17 different strains of C. auris belonging to all five clades reported until now (Table 1). All tests were performed in Amies transport medium at 3× (31.5 CFU/mL) or 5× (52.5 CFU/mL) the LOD of C. auris AR0388. The assay detected C. auris in all samples tested regardless of their clades yielding an analytic assay inclusivity of 100%. The assay exclusivity (specificity) was evaluated by testing 10⁶ to 10⁸ CFU/mL of different bacteria and non-C. auris yeast/fungal species spiked into Amies transport medium (Table 1). The assay did not detect any of the non-target pathogens tested, indicating an analytic specificity of 100%.

Clinical evaluation

The initial clinical sample validation was performed using banked skin swab specimens stored at the UCLA (N = 72) and Wadsworth (N = 13) laboratories. As summarized in Fig. 3, out of 41 BDMAX PCR-positive samples identified at the UCLA lab, 31 were both PCR and culture positive, and 10 were positive by PCR but negative by culture. The remaining 31 out of 72 samples were PCR negative (which were not culture confirmed). The Wadsworth Center provided 10 PCR and culture-positive samples and 3 PCR-negative samples (which were not culture confirmed) for validation (Fig. 3). All samples were tested with the CaurisSurV cartridge. Considering culture as the positive reference standard (Table 2), we detected 30/31 (96.8%) of the positive samples from the UCLA site and 10/10 (100%) of the Wadsworth samples, for a combined sensitivity of 97.5%. Considering PCR-negative samples as the negative reference standard, we confirmed 32/32 samples as negative for a combined specificity of 100% and two invalids. In a secondary analysis, we considered PCR positivity as the secondary positive reference standard regardless of the culture results. Using this analysis, we determined a combined sensitivity of 92% (46/50) from both sites. Of the 10 culture-negative PCR-positive samples from UCLA, we detected six as C. auris, three as C. auris negative, with one invalid sample. A total of three invalid results due to failure of the internal control were seen out of 85 samples (3.5%), which could not be repeated due to lack of sample volume. Although PCR testing at the sites and CaurisSurV testing were not concurrently done, a correlation plot of the Ct values from our CaurisSurV cartridge to the respective PCR Ct values from both sites showed a correlation of r = 0.86/R² = 0.74 for UCLA (Fig. S1A) and R² = 0.98 (Fig. S1B) r = 0.99/for Wadsworth validation sample sets.

Fig 3.

Flow chart of the clinical evaluation study performed with C. auris surveillance assay cartridge (CaurisSurV cartridge).

TABLE 2.

C. auris surveillance assay cartridge (CaurisSurV cartridge) performance against the sample sets from two different sites^e

		Culture positives PCRa positives		Culture negatives PCR positives		Combined PCR Positivesb	PCR negativesc
		UCLA (N = 31)	WW (N = 10)	UCLA (N = 10)	WW	UCLA + WW (N = 51)	UCLA (N = 31)	WW (N = 3)
CaurisSurV Cartridge	Positives	30	10	6	0	46	0	0
	Negatives	1	0	3	0	4	29	3
	Invalidsd	0	0	1	0	1	2	0
	Sensitivity (%)	96.8	100.0	66.7	NA		NA
	Combined sensitivity (%)	97.5				92
	Specificity (%)						100.0	100.0

^a BDMax PCR assay.

^b Irrespective of culture positivity.

^c Culture not done.

^d Invalids were excluded from sensitivity/specificity calculations.

^e WW, Wadsworth Center, NY; NA, not applicable.

In the blinded study, out of 60 samples, 29 tested positive, 29 negative, and 2 were invalid. Referencing against the PCR and culture positivity key from Wadsworth Center, CaurisSurV cartridge had correctly identified 29/30 (96.6%) positives and 28/28 (100%) negatives (Table 3). Although the tests were not concurrently done at both sites, we evaluated the Ct correlation between the BDMax PCR performed at Wadsworth Center vs CaurisSurV cartridge (Fig. S1C) and found a Pearson’s correlation coefficient r = 0.94/R² = 0.89, indicating strong correlation (50). The sample WW19, the CaurisSurV cartridge missed detecting, was further investigated and found to have BDMax PCR Ct value of 31.4. This pauci fungal nature of the sample might have resulted in aliquot-to-aliquot variation resulting in CFU quantities below the LoD in the aliquot tested in the CaurisSurV cartridge. Therefore, we obtained the culture isolate from this sample (WW19) and tested in the CaurisSurV cartridge, which correctly identified the isolate further asserting our reasoning of Poisson distribution.

TABLE 3.

C. auris surveillance assay cartridge (CaurisSurV cartridge) performance against the skin swab sample set (N = 60) from Wadsworth Center, NY in a blinded study

		Culture positives PCRa positives	PCR negativesb
CaurisSurV Cartridge	Positives	29	0
	Negatives	1	28
	Invalidsc	0	2
	Sensitivity (%)	96.6
	Specificity (%)	100.0

^a BDMax PCR assay.

^b Culture not done.

^c Invalids were excluded from sensitivity and specificity calculations.

As shown in Table S1, the UCLA C. auris positive sample set (N = 41) had been in storage at −20°C or −80°C from 6 to 66 weeks (Av 147.5 ± 121.4 days). The Wadsworth validation sample set (N = 10) was stored in refrigerated condition (2°C–8°C) for <8 weeks.

DISCUSSION

The CaurisSurV prototype cartridge is a completely integrated sample processing-DNA extraction-PCR approach that detects C. auris from skin swabs within 1 h (Fig. 1). Users are only required to add 0.5 mL of the sample to the cartridge and load the cartridge into the GeneXpert instrument for C. auris testing. Results are produced in less than an hour. The test is recommended to be used for composite skin swab samples (bilateral axilla, groin) collected in Amies transport media as per CDC recommendations (https://www.cdc.gov/candida-auris/hcp/screening-hcp/?CDC_AAref_Val=https://www.cdc.gov/fungal/candida-auris/c-auris-screening.html). To the best of our knowledge, there are no commercially available point of care capable PCR-based tests for C. auris surveillance testing similar to CaurisSurV test. However, the CDC and other public health laboratories use fully automated PCR-based tests such as the T2Cauris test that runs on the T2Dx instrument (51) and the partially automated BDMax PCR-based test, for detection of C. auris from patient skin samples (41, 52). The CDC also recommends a lab-designed traditional PCR test for use in ABI 7500 platform (https://www.cdc.gov/candida-auris/hcp/laboratories/real-time-pcr-identification.html). However, these assays are not rapid, include many manual steps and are not suitable for point of care or near patient testing which this assay is expected to offer.

The CaurisSurV cartridge demonstrated an analytical LoD of 10.5–14.8 CFU/mL for both non-aggregative and aggregative strains of clade 1 C. auris and confirmatory tests of the four other C. auris clades at 3×–5× this LOD were positive, confirming a high sensitivity for all C. auris clades. Although this test can detect all five clades of C. auris, it does not cross-react with other closely related Candida species or any other fungal or bacterial pathogens, demonstrating high specificity for C. auris. Further validation of the CaurisSurV cartridge assay performed using banked specimens showed the test to be highly sensitive (97%) and 100% specific compared to the culture as primary reference standard. Our test missed detecting four PCR-positive samples from the UCLA laboratory. Three of these four samples were culture negative indicating a low pathogen load (BDMax PCR Ct values of 33.7, 34.8, 33.8, and 27.8). This low load could have resulted in CFU levels below the assay LoD in the aliquots we received. We analyzed these four samples on their length, temperature of storage, and comparison to Ct values from the BD max PCR assay (Table S1), noting that these samples had all been stored frozen for long periods (70–461 days) without any cryo-preservatives before testing in CaurisSurV cartridges. It should also be noted that the samples were not concurrently tested in both systems and were freeze thawed at least two times before testing in CaurisSurV cartridge. Other than aliquot variation and long-term storage effect on sample stability, we could not conclusively establish a reason for the CaurisSurV-negative PCR, for these culture negatives samples. The blinded study further confirmed the assay sensitivity of 96.6% (29/30) and specificity of 100% (28/28). The single sample that was missed had a Ct value of 31.4 by BDMax PCR, but a culture isolate from this sample tested in CaurisSurV cartridge was positive. Due to similar reasons as explained above, this could be likely due to aliquot-to-aliquot variation or reduced sample stability in this stored (4°C–8°C for 4–8 weeks) sample containing a very low CFU titer (Table S2). Overall, our assay had a low invalid rate at 3.4% (5/145) as indicated by the failed internal control, which was likely caused either by the presence of PCR inhibitors, the absence of PCR reagents in the cartridge, or other in-cartridge assay errors. We could not repeat these samples due to low available sample volume. Future work will focus on improving the assay to be more sensitive and resilient to the inhibitors and other assay errors thereby reducing the invalid rate.

In summary, we have developed a completely integrated, rapid, low-complexity C. auris surveillance assay that is highly sensitive and specific. This assay offers a better solution to healthcare providers at hospitals and long-term care facilities in their ongoing efforts for effective and timely control of C. auris infection and hence quicker response for any potential future outbreaks.

ETHICS APPROVAL

The study was approved by the Rutgers-Newark Institutional Review Board (IRB) under protocol number Pro20170000818.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jcm.00525-24.

Supplemental material. jcm.00525-24-s0001.docx.

Fig. S1; Tables S1 and S2.

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