The use of readily available laboratory tests for the identification of the emerging yeast Candida auris in Mexico

doi:10.21203/rs.3.rs-1516575/v1

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The use of readily available laboratory tests for the identification of the emerging yeast Candida auris in Mexico

https://doi.org/10.21203/rs.3.rs-1516575/v1

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Identification of the emerging multidrug resistant yeast Candida auris is challenging. Here we described the role of the Mexico national reference laboratory Instituto de Diagnóstico y Referencia Epidemiológicos Dr. Manuel Martínez Báez (InDRE) and the Mexican national laboratory network in the identification of C. auris. Reference identification was performed based on phenotypic and molecular laboratory methods, including growth in special media, evaluation of isolate micromorphology, and species-specific PCR and pan-fungal PCR and sequencing. C. auris isolates were able to grow on modified Sabouraud agar with 10% NaCl incubated at 40°C. With one exception, isolates of C. auris were spherical to ovoid yeast-like cells and blastoconidia, with no hyphae or pseudohyphae on cornmeal agar. C. auris isolates were resistant to fluconazole. Species-specific and pan-fungal PCR confirmed isolates as C. auris. Sequence analysis revealed the presence of two different C. auris clades in Mexico, clade I (South Asia) and clade IV (South America).

Candida auris

laboratory identification

antifungal resistance

Candida auris is an emerging multidrug resistance yeast able to cause invasive fungal infections and hospital outbreaks. This species of Candida differs from other species in that it more readily spreads from patient to patient in healthcare settings through contact with contaminated environmental surfaces or equipment (1, 2). In addition, the laboratory identification of C. auris using commercially available methods for yeast identification is challenging because most of the systems used for yeast identification, such as Phoenix BD, API 20 and MicroScan, misidentify C. auris as other Candida species (3), mainly species of the C. haemulonii species complex (2, 4), but also as C. famata, C. guilliermondii, C. lusitaniae, C. glabrata, C. parapsilosis and C. catenulata (5). Currently, the most reliable methods for identification of C. auris include the VITEK® 2 with the most current software update, MALDI-TOF, molecular methods using specific PCR assays (4, 6, 7), or by the amplification and sequencing of the ITS1-ITS2 or the D1/D2 region of the ribosomal cistron (7–10).

In Mexico, C. auris was reported for the first time in May 2020 in a woman with severe endometriosis (11). Three months later the same hospital reported a large outbreak of C. auris associated with high mortality in patients with severe COVID-19 (12). The impact of the current COVID-19 pandemic resulted in overcrowded hospitals, with larger numbers of critically ill patients at increased risk of invasive candidiasis. These overcrowded conditions set the stage for an increase in hospital-associated infections, including those caused by C. auris (13).

Here, we describe the role of the Mexico national reference laboratory, Instituto de Diagnóstico y Referencia Epidemiológicos Dr. Manuel Martínez Báez (InDRE), and the Mexican national laboratory network in the detection of emerging problems, and the identification of the emerging yeast C. auris. This report presents a summary of the microbiological characteristics, focused on the phenotypic and molecular characteristics of Mexican isolates proven to be C. auris.

Phenotypic identification. Suspected isolates were grown on Sabouraud dextrose agar (SDA) incubated at 30°C for 120 hours. In parallel, isolates were grown on a selective modified Sabouraud agar (1), supplemented with 10% NaCl (Sab-10% NaCl) and dulcitol instead of dextrose as carbon source. Isolates on this media were incubated at 40°C for 72 hours. In addition, morphological characteristics of isolates were evaluated, including growth on Cornmeal agar.

Isolates were also tested using the VITEK® 2 system. Identification was performed using VITEK® 2 YST cards and results were analyzed using the software version 8.01 (bioMérieux, Marcy, L'Etoile, France). Additionally, assimilation of the following carbohydrates was evaluated: glucose, galactose, cellobiose, raffinose, melezitose, rhamnose, trehalose, dulcitol and xylose.

Molecular identification. Genomic DNA (gDNA) extraction was performed using the FastDNA™ kit (MP Biomedicals, USA) and the FastPrep® equipment as suggested by the manufacturer. Lysed material was further purified using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany).

Two PCR amplifications were used for molecular identification. The first was a pan-fungal PCR targeting the internal transcribed spacers (ITS1-ITS2) of the ribosomal cistron using primers ITS1F (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4R (5´-TCCTCCGCTTATTGATATGC-3´). The second amplification was a species-specific PCR for C. auris using primers ITS1F and CaurisR (5'-CCACCGCGAAGATTGGTG-3'). The PCR conditions have been previously described (14), and PCR products were sequenced by the Sanger method (15). Sequence analysis was done using the nBLAST tool at the NCBI database (http://www.ncbi.nlm.nih.gov/blast). A phylogenetic tree of our data and the ITS regions of 20 non-redundant sequences extracted from the GenBank database were constructed using MEGA7.0 software and Jukes-Cantor nucleotide substitution model with 1000 bootstraps (16).

Fluconazole susceptibility testing

fluconazole (FLU) susceptibility testing was performed using the agar dilution method, as previously described (17). Minimum inhibitory concentration (MIC) was assessed in concentrations ranging from 2 to 256 µg/ml. After 48h incubation, the MIC was determined as the lowest concentration that inhibits yeast growth (18). For susceptibility testing, Candida parapsilosis (ATCC 22019) was used as a susceptible strain, and Candida krusei (PAHO-ARG13) as a resistant strain (CLSI-M60). Candida auris MIC values for were interpretated using the tentative cutoff values proposed by the CDC (MIC ≥ 32 µg/mL) (19).

Six isolates suspected to be C. auris were submitted to InDRE for species identification. One blood and two urine isolates came from a private hospital at the state of Nuevo León (labeled MYC-28, MYC-29, and MYC-30). The other three isolates were referred from public hospitals: a sputum isolate from Tabasco state (MYC-43), a blood isolate from Querétaro state (MYC-121), and a urine isolate from Morelos state (MYC-221). Before shipment, MYC-28, MYC-29 and MYC-30 were identified as C. auris using MALDI-TOF MS Bruker, and MYC-43 as C. auris, MYC-121 as C. krusei, and MYC-221 as C. parapsilosis using the VITEK® 2 system, v8.01 software. Four isolates were from men and two from women, with a median age of 59 years (range: 54–78 years). All patients were diagnosed with COVID-19 and were hospitalized in the intensive care unit (ICU). Median ICU stay was 35 days (range: 23–71 days). Five of the six patients died.

Using phenotypic methods, we found that isolates originally identified as C. auris (MYC-28, MYC-29, MYC-30 and MYC-43) grew on SDA at 37°C and Sab-10% NaCl at 40°C, Microscopic examination revealed spherical and ovoid yeast-like cells and blastoconidia, but clamydoconidia and hyphae or pseudohyphae were not observed on cornmeal agar for isolates MYC-28, MYC-29, MYC-30 (Fig. 1A). Isolate MYC-43 displayed clamydoconidia and hyphae or pseudohyphae on cornmeal agar (Fig. 1B). Isolates originally identified as C. parapsilosis and C. krusei did not grow in Sab-10% NaCl at 40°C and produced pseudohyphae and chlamydoconidia on cornmeal agar (Fig. 1C and 1D).

Assimilation of carbohydrates testing shows that isolates originally identified as C. auris (MYC-28, MYC-29, MYC-30 and MYC-43) assimilated five of the nine carbohydrates tested: glucose, raffinose, melezitose, trehalose and dulcitol. Final species identification was performed using the species-specific C. auris PCR. An approximately 279 bp band was amplified for MYC-28, MYC-29, MYC-30 and MYC-43 isolates while no amplification was observed for isolates MYC-121 and MYC-221 (Fig. 2). Using pan-fungal PCR sequencing, C. auris isolates had 99–100% identity with other C. auris sequences within GenBank. Phylogenetic analysis of the sequence of ITS1-ITS2 regions showed that Mexican C. auris isolates were clustered with the C. auris South American clade (clade IV) for isolates MYC-28, MYC-29 and MYC-30, and isolate MYC-43 grouped with C. auris South Asian clade (clade I) (10) (Fig. 3). Sequences were deposited at the GenBank database under accession numbers MW358012 (MYC-28), MW358013 (MYC-29), MW358014 (MYC-30), MZ648436 (MYC-43), MZ020643 (MYC-121) and MZ020644 (MYC-221).

Fluconazole susceptibility testing of the four C. auris isolates showed high MIC values. Isolates MYC-28, MYC-43, MYC-29 and MYC-30 had MICs of 128 µg/mL, based on proposed breakpoints (MIC ≥ 32) these isolates were consider resistant to fluconazole (19).

The national laboratory surveillance confirmed four cases of C. auris in Mexico belonging to two different clades, all cases were identified in patients in the ICU with severe COVID-19. With the identification of C. auris in several Mexican states and the increase in the number of patients hospitalized in ICUs as a result of the COVID-19 pandemic, the mycology reference service of the InDRE implemented national laboratory surveillance for C. auris. Several challenges had to be addressed during the implementation of this surveillance, especially the limited availability of reference methods, such as MALDI-TOF, for the confirmation of C. auris.

In combination with several other low-cost tests, we were able to successfully use species-specific PCR for definitive identification of C. auris in a resource-limited setting, obtaining reliable and comparable results as those obtained with the reference methods. Candida auris-specific PCR tests have been used for the identification of colonized patients by the direct detection of C. auris DNA on skin swabs (20). This type of non-culture based methodology has shown relevant utility in the early detection and control of C. auris outbreaks (21). Here, we have used this test to definitively identify isolates of C. auris using simple PCR and gel electrophoresis rather than real time PCR. We have shown that this test could be employed in combination with other simple tests when MALDI-TOF and DNA sequencing are not available.

Without specialized Candida chromogenic media, the use of basic mycology methods, such as the evaluation of growth under different culture conditions, such as high salt and high temperature, were key to identifying potential isolates of C. auris. On cornmeal agar, C. auris generally has homogeneous size and shaped blastoconida and no hyphae and pseudohyphae. However, one of the four isolates did present pseudohyphae, showing that is not a definitive characteristic of C. auris.

The evaluation of the profile of carbohydrates assimilation it is not widely used for identification of Candida spp, mainly because it is a complex and laborious laboratory method. We confirmed that dulcitol assimilation is key to identify C. auris to other closely related species of Candida (1–3). Despite the close genetic relationship between C. auris and the C. haemolunii complex, the biochemical assimilation profile between them is distinct, so that a four carbohydrate assimilation combination (galactose, raffinose, rhamnose and dulcitol) is decisive for the differentiation of C. auris from the C. haemulonii species complex (2).

Using the VITEK® 2 system, we found that all four isolates were correctly identified as C. auris. As reported previously, VITEK® 2 system using the software version 8.01 is able to identify C. auris, especially isolates from the South American clade (clade IV) (22).

Here we observed all isolates were resistant to fluconazole, with high MIC values (≥ 128 µg/mL). This high resistance to fluconazole is similar to others reports around the world. Unfortunately, our information about antifungal susceptibly testing (AFST) had the limitation of not being a gold standard methodology, but it is important to highlight that the method used is a viable alternative in settings where access to AFST using broth microdilution or gradient diffusion is not available.

Phylogenetic analysis of sequences showed that the three Mexican C. auris isolates from the north of the country belonged to the South American clade, (clade IV). Surprisingly, the Mexican C. auris isolate from the south of the country clustered with the South Asian clade (Clade I), indicating that there have been multiple introductions of C. auris into Mexico and several clades are now circulating through the population.

With limited resources and limited access to reference methods for Candida species identification such as MALDI-TOF we were able to confirm the species identification of isolates suspected to be C. auris. Here we describe how conventional mycology laboratory methods, such as microscopy, growth on differential media and growth at elevated temperatures were key aspects to tentative identification of C. auris. The use of a simple species-specific PCR test confirmed the tentative identification. These methods can all be easily implemented in laboratories in countries with limited resources, or with logistical difficulties in accessing reference methods for Candida identification.

Acknowledgements: We would like to thank the State Laboratory Network of Public Health (LESP) and the Support Laboratory in Epidemiological Surveillance (LAVE-IMSS) to contribution for the epidemiological surveillance of C. auris and other species.

Compliance with Ethical Standards

Conflict of interest: The authors declare that they have no conflict of interest. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the Instituto de Diagnóstico y Referencia Epidemiológicos Dr. Manuel Martínez Báez (InDRE).

Ethical approval: This article does not contain any studies with human participants performed by any of the authors.

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The use of readily available laboratory tests for the identification of the emerging yeast Candida auris in Mexico

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Introduction

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