Ultrasensitive candida auris detection

Abstract

Kits, compositions, and methods for detection of Candida auris make use of nucleic acid molecules that specifically bind a target region of 18S rRNA or 28S rRNA of Candida auris. The ultrasensitive detection of Candida auris 18S rRNA at concentrations at least 100-fold higher than the coding genomic DNA means this RT-PCR approach is capable of detecting a single colony forming unit of C. auris. The assay is also specific for C. auris and does not detect other Candida species. This approach leverages the biologically amplified nature of C. auris 18S and 28S rRNA as compared to the coding DNA and provides a highly sensitive screening assay for C. auris in healthcare or other settings. The assay is amongst the most sensitive and specific approaches for C. auris screening, and can be adapted in high-throughput laboratory approaches.

Description

ULTRASENSITIVE CANDIDA AURIS DETECTION

[0001] This application claims benefit of United States provisional patent application number 63 / 562,415, filed March 7, 2024, the entire contents of which are incorporated by reference into this application. REFERENCE TO A SEQUENCE LISTING

[0002] The content of the XML file of the sequence listing named “UW084_seq”, which is 29 kb in size, created on March 5, 2025, and electronically submitted herewith the application, is incorporated herein by reference in its entirety. BACKGROUND

[0003] Candida auris is an increasingly important, multidrug-resistant fungal pathogen that was first identified in Japan in 2009 (1). Since that time, C. auris has spread globally, including in the United States. Most of the outbreaks from C. auris have occurred in adult healthcare settings. C. auris is well adapted as a nosocomial pathogen and is also often resistant to multiple antifungal drugs (2). This drug resistance profile makes it difficult to prophylactically and therapeutically control C. auris infections. People who are most vulnerable to C. auris are older and immunocompromised. Healthcare facilities that work with such patients are beginning to develop and deploy screening strategies to detect C. auris colonized persons and to implement infection control procedures to minimize the threat of C. auris to hospitalized patients and those in skilled nursing facilities.

[0004] However, identification of C. auris is difficult because there are many other Candida yeast species that look very similar and because Candida and other yeasts are relatively ubiquitous in the environment. Testing for C. auris can occur at the screening stage (e.g., usually before the patient is sick from C. auris) or to inform acute care needs (e.g., because the patient is sick with C. auris fungemia). For screening, culture-based methods are usually employed, often starting from an axillary or groin swab to test for colonization. Chromogenic media that was long used to screen for and identify other medically-important Candida spp. like C. albicans fail to reliably differentiate C. auris so newer chromogenic media have been developed for C. auris-specific screening (3, 4). Culture-based methods are relatively inexpensive but require time for the organism to grow. Blood samples collected from sick patients can also be subjected to similar culture-based methods and organisms that grow canbe identified by a variety of biochemical and mass spectrometry methods that are routinely used in clinical microbiology laboratories (5).

[0005] Beyond cultures, molecular diagnostics are also used for C. auris detection. Several DNA-based methods have been developed, with most relying on the C. auris internal transcribed spacer 2 (ITS2) gene, which is present as a spacer region amongst the 18S, 5.8S, and 28S rRNA subunit genes. Whereas culture-based methods are considered the gold standard and can detect even a single colony forming unit (cfu) of C. auris, molecular methods are somewhat limited in this regard because most of the tools cannot detect C. auris at such ultra-low densities. Molecular methods that can improve analytical sensitivity while accelerating and simplifying detection could have a positive impact on the effectiveness of C. auris screening.

[0006] There remains a need for materials and methods that enable sensitive and specific detection of C. auris that are not hindered by the disadvantages of culture-based methods or DNA-based methods. SUMMARY

[0007] The methods described herein provide molecules, materials, compositions, and methods for detection of Candida auris. Described herein, in one embodiment, is a kit or composition that comprises nucleic acid molecules that specifically bind a target region of 18S rRNA or 28S rRNA of Candida auris.

[0008] In some embodiments, the nucleic acid molecules comprise primers useful for amplification of 18S rRNA or 28S rRNA. In some embodiments, the nucleic acid molecules comprise a probe. In some embodiments, the molecules are optimized for amplification-based detection methods. In some embodiments, the molecules are optimized for non-amplification- based detection methods.

[0009] In some embodiments, the kit or composition comprises a first nucleic acid primer having at least 90% identity with SEQ ID NO: 1; a second nucleic acid primer having at least 90% identity with SEQ ID NO: 2; and a nucleic acid probe having at least 90% identity with SEQ ID NO: 3. The first and second nucleic acid primers amplify 18S ribosomal RNA (rRNA) of Candida auris, and the nucleic acid probe specifically binds to 18S rRNA of Candida auris. In some embodiments, the nucleic acid probe is labeled with a detectable marker.

[0010] Also described is a method of detecting Candida auris in a specimen. In some embodiments, the method comprises contacting the specimen with a first nucleic acid molecule that specifically binds to SEQ ID NO: 5 (target region of 18S rRNA sequence) or SEQ ID NO: 7 / 8 (target region of 28S rRNA sequence); and detecting specific binding of the first nucleic acid molecule to Candida auris 18S rRNA or to 28S rRNA. Specific binding of the first nucleic acid molecule is indicative of the presence of Candida auris in the specimen. The first nucleic acid molecule can be a primer that amplifies the 18S rRNA or 28S rRNA, and its binding is detected indirectly through use of a probe. In other embodiments, the first nucleic acid molecule is a probe whose binding is detected directly, for example, through use of a molecular beacon that emits a detectable signal upon binding to the target.

[0011] In some embodiments, the first nucleic acid molecule is 15 to 30 nucleotides in length. In some embodiments, the first nucleic acid molecule has at least 12 contiguous nucleotides of that are 100% complementary to a corresponding region of SEQ ID NO: 5.

[0012] In some embodiments, the method further comprises amplifying the 18S rRNA or 28S rRNA with the first nucleic acid molecule and with a second nucleic acid molecule that specifically binds to SEQ ID NO: 5 (target region of 18S rRNA sequence) or SEQ ID NO: 7 / 8 (target region of 28S rRNA sequence). In such embodiments, the first and second nucleic acid molecules act as primers, and the method further comprises contacting the amplified 18S or 28S rRNA with a nucleic acid probe that specifically binds to SEQ ID NO: 5 or SEQ ID NO: 7 / 8.

[0013] In other embodiments, wherein a molecular beacon is employed, the first nucleic acid molecule emits a detectable signal upon binding to SEQ ID NO: 5 or SEQ ID NO: 7 / 8.

[0014] Additionally described is a method of detecting Candida auris in a specimen. In some embodiments, the method comprises amplifying 18S rRNA in the specimen with a first nucleic acid primer having at least 90% identity with 5’-TCACGGTGAGTACTTCCATATC-3’ (SEQ ID NO: 1), and with a second nucleic acid primer having at least 90% identity with 5’- GCTAACAGATTCAAGCGTGC-3’ (SEQ ID NO: 2). The method further comprises contacting the amplified 18S rRNA with a nucleic acid probe having at least 90% identity with 5’- ACCTTTCCTCTGCTTCCTCGCAAG-3’ (SEQ ID NO: 3) under conditions permitting binding of the nucleic acid probe to Candida auris 18S rRNA. Detecting specific binding of the nucleic acid probe to Candida auris 18S rRNA is indicative of the presence of Candida auris in the specimen.

[0015] In some embodiments of the above methods, the nucleic acid probe is labeled with a detectable marker. In some embodiments, the detectable marker is a fluorescent marker. In some embodiments, the amplifying comprises reverse transcription polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA), or other molecular diagnostic methods.

[0016] Also described is a method of screening for Candida auris in a sample. In some embodiments, the method comprises performing one of the methods described herein on a sample obtained from a subject or from an environmental source. The method further comprises identifying the sample as positive for Candida auris when specific binding of the nucleic acid probe is detected in the sample. In some embodiments, the sample is obtained from a swab of the subject’s axilla, groin, nares, ear canal, oropharynx, vagina, rectum, wound site, catheter exit site, from a urine or blood sample, from a swab of an environmental source, and / or from pooled samples.

[0017] With respect to the kits, methods, and compositions described herein, in some embodiments, the first and second nucleic acid molecules have at least 95% identity with SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In some embodiments, the nucleic acid probe has at least 95% identity with SEQ ID NO: 3. In some embodiments, the identity between these molecules and the corresponding sequence is 96%, 97%, 98%, or 99%. In some embodiments, the first nucleic acid primer is SEQ ID NO: 1 and the second nucleic acid primer is SEQ ID NO: 2. In some embodiments, the nucleic acid probe is SEQ ID NO: 3.

[0018] Shown in FIG.1 is an alignment of C. auris and other Candida spp 18S rRNA, revealing the C. auris specific diagnostic target region. The C. auris specific diagnostic region to be targeted can be from the 18S rRNA, such as SEQ ID NO: 5, which corresponds to the bold text in FIG.1. In some embodiments, the C. auris specific diagnostic region to be targeted can be from the 28S rRNA, such as SEQ ID NO: 7 or SEQ ID NO: 8, which corresponds to the bold text in Figures 2 and 3. Reference to “SEQ ID NO: 7 / 8” means SEQ ID NO: 7 and / or SEQ ID NO: 8. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG.1: Alignment of C. auris and other Candida spp 18S rRNA to identify C. auris specific diagnostic target region. Bold italicized text = target region of interest (SEQ ID NO: 5); underlined text = homology to C. auris. C. auris (MN658534; SEQ ID NO: 9); C. albicans (XR_002086442.1; SEQ ID NO: 10); C. glabrata (AY218893.1; SEQ ID NO: 11); C. duobushaemuloni (MK394153.1; SEQ ID NO: 12), C. (Clavispora) lusitaniae (MH545926.1;SEQ ID NO: 13); C. haemuloni (MW281621.1; SEQ ID NO: 14). Asterisks indicate homology across all species shown.

[0020] FIG.2. Alignment of C. auris and other Candida spp 28S rRNA Region 1 of interest (URS000216E5F7_498019 Nucleotides 375-572). Bold italicized text, area of species-specific interest (SEQ ID NO: 7); underlined text, homology to C. auris. Sequences are shown for C. auris 28S rRNA (SEQ ID NO: 15); C. albicans 28S rRNA (SEQ ID NO: 16); C. haemuloni 28S rRNA (SEQ ID NO: 17); and C. duobushaemuloni 28S rRNA (SEQ ID NO: 18).

[0021] FIG.3. Alignment of C. auris and other Candida spp 28S rRNA Region 3 of interest (URS000216E5F7_498019 Nucleotides 664-961). Bold italicized text, area of species-specific interest (SEQ ID NO: 8); underlined text, homology to C. auris. Sequences are shown for C. auris 28S rRNA (SEQ ID NO: 19); C. albicans 28S rRNA (SEQ ID NO: 20); C. haemuloni 28S rRNA (SEQ ID NO: 21); and C. duobushaemuloni 28S rRNA (SEQ ID NO: 22).

[0022] FIG.4. Species-specific detection of C. auris 18S rRNA using novel RT-PCR approach. Fluorescence detection by RT-PCR shown for C. auris as compared to other Candida spp (C. albicans, C. glabrata, C. parapsilosis shown). Additional runs showed no cross-reactivity against C. haemuloni and C. duobushaemuloni.

[0023] FIG.5. Significant enrichment of C. auris 18S rRNA compared to coding DNA that enhances assay analytical sensitivity by the novel RT-PCR approach compared to PCR. Fluorescence detection after RT-PCR versus PCR for C. auris.

[0024] FIG.6. Linearity of C. auris 18S rRNA RT-PCR. Fluorescence detection after RT-PCR across a 6-log dilution series of C. auris-containing samples. Top panel shows cycle thresholds (y-axis) plotted against log10dilutions to derive the slope (efficiency) of the RT-PCR. The bottom panel shows the actual fluorescence amplification curves for the constituent data. DETAILED DESCRIPTION

[0025] Candida auris is an emerging, drug-resistant fungal pathogen, and screening assays are needed to identify this organism to protect patients and healthcare facilities. Conventional screening relies on either culture-based methods or on DNA PCR-based diagnostics. The invention described herein is based on the surprising discovery of high levels of expression of C. auris 18S rRNA using a Reverse Transcription PCR (RT-PCR)-based approach for C. auris detection.

[0026] The ultrasensitive detection of Candida auris 18S rRNA at concentrations at least 100- fold higher than the coding genomic DNA means this RT-PCR approach is capable of detecting a single colony forming unit of C. auris. The assay is also specific for C. auris and does not detect other Candida species. This approach leverages the biologically amplified nature of C. auris 18S rRNA as compared to the coding DNA and provides a highly sensitive screening assay for C. auris in healthcare or other settings. The assay is amongst the most sensitive and specific approaches for C. auris screening, and can be adapted in high-throughput laboratory approaches.

[0027] Definitions

[0028] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0029] “Nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of expressing the encoded protein.

[0030] The term "primer," as used herein, means an oligonucleotide designed to flank a region of rRNA or DNA to be amplified. In a primer pair, one primer is complementary to nucleotides present on the sense strand at one end of a polynucleotide fragment to be amplified and another primer is complementary to nucleotides present on the antisense strand at the other end of the polynucleotide fragment to be amplified. A primer can have at least about 11 nucleotides, and preferably, at least about 16 nucleotides and no more than about 35 nucleotides. Typically, a primer has at least about 80% sequence identity, preferably at least about 90% sequence identity with the complement of a target polynucleotide to which the primer hybridizes.

[0031] As used herein, the term “probe” refers to an oligonucleotide, naturally or synthetically produced, via recombinant methods or by PCR amplification, that hybridizes to at least part of another oligonucleotide of interest. A probe can be single-stranded or double-stranded. In some embodiments, the probe is a single-stranded bi-labeled fluorescent molecular beacon that takes a hairpin / loop conformation when complementary arm sequences on either side of the probe anneal to one another, and then emit fluorescence upon hybridization of the probe to its target.When not hybridized to its target, no fluorescence is emitted due to quenching of the fluorophore. In some embodiments, the probe portion of the molecular beacon is 15-30 bp in length, which portion forms a loop, and a complementary stem portion comprises 5-7 bp on each side of the probe portion. A fluorescent molecule at the 5’ end is quenched by a non- fluorescent quencher located at the 3’ end, and activated when the stem / loop structure is dissociated upon binding of the probe to its target.

[0032] As used herein, the term “active fragment” refers to a substantial portion of an oligonucleotide that is capable of performing the same function of specifically hybridizing to a target polynucleotide.

[0033] As used herein, "hybridizes," "hybridizing," and "hybridization" means that the oligonucleotide forms a noncovalent interaction with the target DNA and / or rRNA molecule under standard conditions. Standard hybridizing conditions are those conditions that allow an oligonucleotide probe or primer to hybridize to a target DNA and / or rRNA molecule. Such conditions are readily determined for an oligonucleotide probe or primer and the target DNA and / or rRNA molecule using techniques well known to those skilled in the art. The nucleotide sequence of a target polynucleotide is generally a sequence complementary to the oligonucleotide primer or probe. The hybridizing oligonucleotide may contain nonhybridizing nucleotides that do not interfere with forming the noncovalent interaction. The nonhybridizing nucleotides of an oligonucleotide primer or probe may be located at an end of the hybridizing oligonucleotide or within the hybridizing oligonucleotide. Thus, an oligonucleotide probe or primer does not have to be complementary to all the nucleotides of the target sequence as long as there is hybridization under standard hybridization conditions.

[0034] The term "complement" and "complementary" as used herein, refers to the ability of two nucleic acid molecules to base pair with each other. For example, in DNA, adenine (A) is complementary to thymine (T). In RNA, adenine (A) is complementary to uracil (U). Typically, two DNA molecules are complementary if they hybridize under the standard conditions referred to above. Typically, two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity.

[0035] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non- human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In a typical embodiment, the subject is a human.

[0036] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.

[0037] Nucleic Acid Molecules, Kits & Compositions

[0038] The molecules, materials, compositions, and kits described herein can be used for detection of Candida auris. In one embodiment, provided is a kit or composition that comprises nucleic acid molecules that specifically bind a target region of 18S rRNA or 28S rRNA of Candida auris. By directing the detection at rRNA, in these target regions, superior sensitivity is achieved by taking advantage of the high biological expression of rRNA.

[0039] In some embodiments, the nucleic acid molecules comprise primers useful for amplification of 18S rRNA or 28S rRNA. In some embodiments, the nucleic acid molecules comprise a probe. In some embodiments, the molecules are optimized for amplification-based detection methods. In some embodiments, the molecules are optimized for non-amplification- based detection methods.

[0040] In some embodiments, the kit or composition comprises a first nucleic acid primer having at least 90% identity with SEQ ID NO: 1; a second nucleic acid primer having at least 90% identity with SEQ ID NO: 2; and a nucleic acid probe having at least 90% identity with SEQ ID NO: 3. The first and second nucleic acid primers amplify 18S ribosomal RNA (rRNA) of Candida auris, and the nucleic acid probe specifically binds to 18S rRNA of Candida auris. In some embodiments, the nucleic acid probe is labeled with a detectable marker.

[0041] Additional primers and probes can be directed against the target regions of 18S rRNA or 28S rRNA of Candida auris shown with bold text in Figures 1-3, or SEQ ID NOs: 5, 7, or 8. Those skilled in the art understand that such primers and probes would be complementary to and capable of specifically binding to these target regions that are species-specific.

[0042] Methods

[0043] Provided is a method of detecting Candida auris in a specimen. In some embodiments, the method comprises contacting the specimen with a first nucleic acid molecule that specifically binds to SEQ ID NO: 5 (target region of 18S rRNA sequence) or SEQ ID NO: 7 / 8 (target region of 28S rRNA sequence); and detecting specific binding of the first nucleic acid molecule to Candida auris 18S rRNA or to 28S rRNA. Specific binding of the first nucleic acid molecule is indicative of the presence of Candida auris in the specimen. The first nucleic acid molecule can be a primer that amplifies the 18S rRNA or 28S rRNA, and its binding is detected indirectlythrough use of a probe. In other embodiments, the first nucleic acid molecule is a probe whose binding is detected directly, for example, through use of a molecular beacon that emits a detectable signal upon binding to the target.

[0044] In some embodiments, the first nucleic acid molecule is 15 to 30 nucleotides in length. In some embodiments, the first nucleic acid molecule has at least 12 contiguous nucleotides of that are 100% complementary to a corresponding region of SEQ ID NO: 5.

[0045] In some embodiments, the method further comprises amplifying the 18S rRNA or 28S rRNA with the first nucleic acid molecule and with a second nucleic acid molecule that specifically binds to SEQ ID NO: 5 (target region of 18S rRNA sequence) or SEQ ID NO: 7 / 8 (target region of 28S rRNA sequence). In such embodiments, the first and second nucleic acid molecules act as primers, and the method further comprises contacting the amplified 18S or 28S rRNA with a nucleic acid probe that specifically binds to SEQ ID NO: 5 or SEQ ID NO: 7 / 8.

[0046] In other embodiments, wherein a molecular beacon is employed, the first nucleic acid molecule emits a detectable signal upon binding to SEQ ID NO: 5 or SEQ ID NO: 7 / 8.

[0047] In some embodiments, the method comprises amplifying 18S rRNA in the specimen with a first nucleic acid primer having at least 90% identity with 5’-TCACGGTGAGTACTTCCATATC- 3’ (SEQ ID NO: 1), and with a second nucleic acid primer having at least 90% identity with 5’- GCTAACAGATTCAAGCGTGC-3’ (SEQ ID NO: 2). The method further comprises contacting the amplified 18S rRNA with a nucleic acid probe having at least 90% identity with 5’- ACCTTTCCTCTGCTTCCTCGCAAG-3’ (SEQ ID NO: 3) under conditions permitting binding of the nucleic acid probe to Candida auris 18S rRNA. Detecting specific binding of the nucleic acid probe to Candida auris 18S rRNA is indicative of the presence of Candida auris in the specimen.

[0048] In some embodiments of the above methods, the nucleic acid probe is labeled with a detectable marker. In some embodiments, the detectable marker is a fluorescent marker. In some embodiments, the amplifying comprises reverse transcription polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA), or other molecular diagnostic methods.

[0049] Also described is a method of screening for Candida auris in a sample. In some embodiments, the method comprises performing one of the methods described herein on a sample obtained from a subject or from an environmental source. The method further comprisesidentifying the sample as positive for Candida auris when specific binding of the nucleic acid probe is detected in the sample.

[0050] In some embodiments, the specimen or sample is obtained from a swab of the subject’s axilla, groin, nares, ear canal, oropharynx, vagina, rectum, wound site, catheter exit site, from a urine or blood sample, from a swab of an environmental source, and / or from pooled samples.

[0051] In some embodiments, the first and second nucleic acid molecules have at least 95% identity with SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In some embodiments, the nucleic acid probe has at least 95% identity with SEQ ID NO: 3. In some embodiments, the identity between these molecules and the corresponding sequence is 96%, 97%, 98%, or 99%. In some embodiments, the first nucleic acid primer is SEQ ID NO: 1 and the second nucleic acid primer is SEQ ID NO: 2. In some embodiments, the nucleic acid probe is SEQ ID NO: 3.

[0052] Shown in FIG.1 is an alignment of C. auris and other Candida spp 18S rRNA, revealing the C. auris specific diagnostic target region. Bold text highlights the region of interest to be targeted for a C. auris specific assay (SEQ ID NO: 5); underlining indicates homology to C. auris. MN658534: C. auris 18S rRNA (SEQ ID NO: 9); XR_002086442.1: C. albicans 18S rRNA (SEQ ID NO: 10); AY218893.1: C. glabrata 18S rRNA (SEQ ID NO: 11); MW281621.1: C. haemuloni 18S rRNA (SEQ ID NO: 12); MK394153.1: C. duobushaemuloni 18S rRNA (SEQ ID NO: 13); MH545926.1: C. (Clavispora) lusitaniae 18S rRNA (SEQ ID NO: 14).

[0053] The C. auris specific diagnostic region to be targeted can be from the 18S rRNA, such as SEQ ID NO: 5, which corresponds to the bold text in FIG.1. In some embodiments, the C. auris specific diagnostic region to be targeted can be from the 28S rRNA, such as SEQ ID NO: 7 or SEQ ID NO: 8, which corresponds to the bold text in Figures 2 and 3. Reference to “SEQ ID NO: 7 / 8” means SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0054] The methods described herein can be applied in settings in which it is essential to screen for patients who might be carrying C. auris. Some examples of such settings include screening patients upon hospital or care center admission, pooling samples in order to monitor a population, e.g., in a nursing home or retirement center.EXAMPLES

[0055] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention. Example 1: C. auris 18S ribosomal RNA for ultrasensitive Candida auris detection

[0056] This Example describes the identification of high levels of expression of C. auris 18S rRNA and the development of a Reverse Transcription PCR (RT-PCR)-based approach for C. auris detection. Specific primers and a probe were used to detect C. auris 18S rRNA at concentrations at least >100-fold higher than the coding genomic DNA. As such, the C. auris RT-PCR approach is capable of detecting a single colony forming unit of C. auris. The assay is also specific for C. auris and does not detect other Candida species. This approach leverages the biologically amplified nature of C. auris 18S rRNA as compared to the coding DNA and may serve as a highly sensitive screening assay for C. auris in healthcare or other settings. The assay offers a most sensitive and specific approach for C. auris screening and could be adapted in high-throughput laboratory approaches.

[0057] Candida auris is an increasingly important, multidrug-resistant fungal pathogen that was first identified in Japan in 2009 (1). Since that time, C. auris has spread globally, including in the United States. Most of the outbreaks from C. auris have occurred in adult healthcare settings. C. auris is well adapted as a nosocomial pathogen and is also often resistant to multiple antifungal drugs (2). This drug resistance profile makes it difficult to prophylactically and therapeutically control C. auris infections. People who are most vulnerable to C. auris are older and immunocompromised. Healthcare facilities that work with such patients are beginning to develop and deploy screening strategies to detect C. auris colonized persons and to implement infection control procedures to minimize the threat of C. auris to hospitalized patients and those in skilled nursing facilities.

[0058] However, identification of C. auris is difficult because there are many other Candida yeast species that look very similar and because Candida and other yeasts are relatively ubiquitous in the environment. Testing for C. auris can occur at the screening stage (e.g., usually before the patient is sick from C. auris) or to inform acute care needs (e.g., because the patient is sick with C. auris fungemia). For screening, culture-based methods are usually employed, often starting from an axillary or groin swab to test for colonization. Chromogenic media that was long used to screen for and identify other medically important Candida spp. likeC. albicans fail to reliably differentiate C. auris so newer chromogenic media have been developed for C. auris-specific screening (3, 4). Culture-based methods are relatively inexpensive but require time for the organism to grow. Blood samples collected from sick patients can also be subjected to similar culture-based methods and organisms that grow can be identified by a variety of biochemical and mass spectrometry methods that are routinely used in clinical microbiology laboratories (5).

[0059] Beyond cultures, molecular diagnostics are also used for C. auris detection. Several DNA-based methods have been developed, with most relying on the C. auris internal transcribed spacer 2 (ITS2) gene, which is present as a spacer region amongst the 18S, 5.8S, and 28S rRNA subunit genes. Whereas culture-based methods are considered the gold standard and can detect even a single colony forming unit (cfu) of C. auris, molecular methods are somewhat limited in this regard because most of the tools cannot detect C. auris at this ultra-low density. Molecular methods that can improve analytical sensitivity while accelerating and simplifying detection could have a positive impact on the effectiveness of C. auris screening. To our knowledge, one group has performed RT-PCR on this ITS2 region to leverage it as a viability marker (6) because the transcribed ITS2 region is rapidly degraded after transcription (7). Because we previously studied 18S rRNA to improve diagnostics in other fungi (8), here we sought to develop such an assay after identifying high levels of expression of C. auris 18S rRNA. This Example describes the development of a Reverse Transcription PCR (RT-PCR)-based approach for C. auris detection with favorable performance characteristics for a next-generation C. auris molecular screening tool.

[0060] MATERIALS AND METHODS

[0061] C. auris strains included ATCC strains 0382, 0384, 0913, 1099, 1101 as well as standard isolates of C.albicans, C. glabrata, C. parapsilosis, C. haemuloni and C. duobushaemuloni.

[0062] C. auris cultures were prepared using standard media and were serially diluted in phosphate buffered saline (PBS) before being aliquoted into either bioMerieux NucliSENS lysis buffer or being plated on additional fungal culture media. Cultures were performed in the Seattle Children’s Hospital Clinical Microbiology Laboratory. Molecular testing on lysed and decontaminated nucleic acids was performed at the University of Washington.

[0063] Total nucleic acids were extracted from 1 mL volumes of lysis buffer using Specific Protocol B on the bioMerieux EasyMAG automated nucleic acid extraction system. RT-PCRwas subsequently performed using a Bioline SensiFAST Probe Lo-ROX mastermix kit.20-uL reactions were prepared as follows: 10 μL 2X master mix, 0.4 μL ribosafe RNase inhibitor, 0.2 μL reverse transcriptase (RT), 3.6 μL molecular-grade water, forward and reverse probes at 0.4 μM final, and C. auris FAM probe as 0.1 μM final, 4 μL extracted total nucleic acid sample). Primers / probes were from IDT DNA (Coralville, IA). RT-PCR was performed on a QuantStudio 5 (QS5) real-time PCR instrument (Applied Biosystems) using the following cycling conditions: 10 min at 45°C for reverse transcription followed by 2 min at 95°C for polymerase activation followed by 40 cycles of 5 sec at 95°C and 25 sec at 60°C. Where indicated, the assay was run with or without RT to examine the enrichment of the 18S rRNA molecule relative to coding DNA.

[0064] RESULTS

[0065] Reference sequences of Candida were examined in silico to identify regions conserved in C. auris, but divergent in other even closely-associated Candida spp. This comparison identified a 117-bp C. auris-specific region that we predicted could be specifically targeted by RT-PCR methods (Figure 1). Although this assay development work was focused on C. auris 18S rRNA, this approach may also be applied to the C. auris 28S rRNA, which also offers C. auris-specific sequences that could be targeted by specific RT-PCR or other molecular methods (Figures 2-3).

[0066] From this alignment, forward and reverse primers and a specific probe were designed as follows. The forward primer was: 5’-TCACGGTGAGTACTTCCATATC-3’ (SEQ ID NO: 1); the FAM-labeled probe was 5’-[FAM]-ACCTTTCCTCTGCTTCCTCGCAAG-3’ (SEQ ID NO: 3); and the reverse primer was 5’-GCTAACAGATTCAAGCGTGC-3’ (SEQ ID NO: 2) for a 117-bp amplicon in most C. auris 18S rRNA sequences. These reagents showed 100% homology to all clades of C. auris tested in silico and lower homology with other species (Table 1).

[0067] TABLE 1. Conservation of C. auris primer / probe reagents with Candida spp.

[0068] RT-PCR on total nucleic acids extracted from C. auris or other Candida spp showed specific amplification of C. auris 18S rRNA target and no detection of 18S rRNA from other species of Candida (Figure 4).

[0069] Based on the specific performance of the C. auris 18S rRNA RT-PCR assay, we next sought to determine how much the diagnostic signal was enhanced by the RT-PCR approach compared to PCR-only approaches. C. auris samples were tested with or without RT (Figure 5). These data show at least >100-fold higher concentration of 18S rRNA in C. auris compared to the coding genomic DNA sequences. As such, the C. auris RT-PCR approach is capable of detecting a single colony forming unit of C. auris.

[0070] The linearity of the C. auris 18S rRNA RT-PCR was also assessed (Figure 6) and was found to provide linear performance over a six-log range. Additional clinical performance characteristics and further validation can be performed to assess the assay in other matrices and under actual use conditions.

[0071] The RT-PCR assay described herein is sensitive and specific for highly expressed C. auris 18S rRNA. This assay does not become positive in the presence of high amounts of 18S rRNA from other Candida species. This approach leverages the biologically amplified nature of C. auris 18S rRNA as compared to the coding DNA and provides a highly sensitive screening assay for C. auris in healthcare or other settings. The assay has the potential to be amongst the most sensitive and specific approaches for C. auris screening and could be adapted to high- throughput laboratory approaches and to rapid molecular diagnostics to improve healthcare screening. This strategy may also be applied to the mature 28S rRNA of C. auris.

[0072] REFERENCES

[0073] 1. Spivak ES, Hanson KE. Candida auris: an Emerging Fungal Pathogen. J Clin Microbiol.2018;56(2).

[0074] 2. Jacobs SE, Jacobs JL, Dennis EK, Taimur S, Rana M, Patel D, et al. Candida auris Pan-Drug-Resistant to Four Classes of Antifungal Agents. Antimicrob Agents Chemother. 2022;66(7):e0005322.

[0075] 3. Mulet Bayona JV, Salvador Garcia C, Tormo Palop N, Gimeno Cardona C. Evaluation of a novel chromogenic medium for Candida spp. identification and comparison with CHROMagar Candida for the detection of Candida auris in surveillance samples. Diagn Microbiol Infect Dis.2020;98(4):115168.

[0076] 4. Tamura T, Alshahni MM, Makimura K. Evaluation of CHROMagar Candida Plus chromogenic agar for the presumptive identification of Candida auris. Microbiol Immunol. 2022;66(6):292-8.

[0077] 5. Lockhart SR, Lyman MM, Sexton DJ. Tools for Detecting a "Superbug": Updates on Candida auris Testing. J Clin Microbiol.2022;60(5):e0080821.

[0078] 6. Freitas BL, Leach L, Chaturvedi V, Chaturvedi S. Reverse Transcription-Quantitative Real-Time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris Cells from the Health Care Environment. J Clin Microbiol.2022;60(2):e0077921.

[0079] 7. Milligan L, Decourty L, Saveanu C, Rappsilber J, Ceulemans H, Jacquier A, et al. A yeast exosome cofactor, Mpp6, functions in RNA surveillance and in the degradation of noncoding RNA transcripts. Mol Cell Biol.2008;28(17):5446-57.

[0080] 8. Glasgow HL, Cruz K, Murphy SC. Reverse-transcription PCR increases sensitivity of broad-range fungal detection in bronchoalveolar lavage fluid. Med Mycol.2021;60(1).

[0081] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.

[0082] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is:

1. A method of detecting Candida auris in a specimen, the method comprising: (a) contacting the specimen with a first nucleic acid molecule that specifically binds to SEQ ID NO: 5 (target region of 18S rRNA sequence) or SEQ ID NO: 7 / 8 (target region of 28S rRNA sequence); and (b) detecting specific binding of the first nucleic acid molecule to Candida auris 18S rRNA; wherein specific binding of the first nucleic acid molecule is indicative of the presence of Candida auris in the specimen.

2. The method of claim 1, wherein the first nucleic acid molecule is 15 to 30 nucleotides in length.

3. The method of claim 1 or 2, wherein the first nucleic acid molecule has at least 12 contiguous nucleotides of that are 100% complementary to a corresponding region of SEQ ID NO:

5.

4. The method of claim 1, further comprising amplifying the 18S rRNA or 28S rRNA with the first nucleic acid molecule and with a second nucleic acid molecule that specifically binds to SEQ ID NO: 5 (target region of 18S rRNA sequence) or SEQ ID NO: 7 / 8 (target region of 28S rRNA sequence), wherein the first and second nucleic acid molecules act as primers, and further comprising contacting the amplified 18S rRNA or 28S rRNA with a nucleic acid probe that specifically binds to SEQ ID NO: 5 or SEQ ID NO: 7 / 8.

5. The method of claim 1 or 2, wherein the first nucleic acid molecule emits a detectable signal upon binding to SEQ ID NO: 5 or SEQ ID NO: 7 / 8.

6. A method of detecting Candida auris in a specimen, the method comprising: (a) amplifying 18S rRNA in the specimen with a first nucleic acid primer having at least 90% identity with 5’-TCACGGTGAGTACTTCCATATC-3’ (SEQ ID NO: 1), and with a second nucleic acid primer having at least 90% identity with 5’-GCTAACAGATTCAAGCGTGC-3’ (SEQ ID NO: 2); (b) contacting the amplified 18S rRNA with a nucleic acid probe having at least 90% identity with 5’-ACCTTTCCTCTGCTTCCTCGCAAG-3’ (SEQ ID NO: 3) under conditions permitting binding of the nucleic acid probe to Candida auris 18S rRNA; and(c) detecting specific binding of the nucleic acid probe to Candida auris 18S rRNA; wherein specific binding of the nucleic acid probe is indicative of the presence of Candida auris in the specimen.

7. The method of claim 4 or 6, wherein the nucleic acid probe is labeled with a detectable marker.

8. The method of claim 7, wherein the detectable marker is a fluorescent marker.

9. The method of claim 4 or 6, wherein the amplifying comprises reverse transcription polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), or transcription mediated amplification (TMA), or other molecular diagnostic techniques.

10. A method of screening for Candida auris in a sample, the method comprising: (a) performing the method of one of the preceding claims on a sample obtained from a subject or from an environmental source; and (b) identifying the sample as positive for Candida auris when specific binding of the nucleic acid probe is detected in the sample.

11. The method of claim 10, wherein the sample is obtained from a swab of the subject’s axilla, groin, nares, ear canal, oropharynx, vagina, rectum, wound site, catheter exit site, from a urine or blood sample, from a swab of an environmental source, and / or from pooled samples.

12. A kit comprising: (a) a first nucleic acid primer having at least 90% identity with SEQ ID NO: 1; (b) a second nucleic acid primer having at least 90% identity with SEQ ID NO: 2; and (c) a nucleic acid probe having at least 90% identity with SEQ ID NO: 3; wherein the first and second nucleic acid primers amplify 18S ribosomal RNA (rRNA) of Candida auris, and wherein the nucleic acid probe specifically binds to 18S rRNA of Candida auris.

13. The kit of claim 12, wherein the nucleic acid probe is labeled with a detectable marker.

14. The kit or method of any of the preceding claims, wherein the first and second nucleic acid molecules have at least 95% identity with SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

15. The kit or method of any of the preceding claims, wherein the nucleic acid probe has at least 95% identity with SEQ ID NO: 3.

16. The kit or method of any of the preceding claims, wherein the first nucleic acid primer is SEQ ID NO: 1 and the second nucleic acid primer is SEQ ID NO:

2.

17. The kit or method of any of the preceding claims, wherein the nucleic acid probe is SEQ ID NO: 3.

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