Emerging multidrug-resistant Candida duobushaemulonii infections in. Panama hospitals: importance of laboratory surveillance and accurate

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1 JCM Accepted Manuscript Posted Online 25 April 2018 J. Clin. Microbiol. doi: /jcm Copyright 2018 American Society for Microbiology. All Rights Reserved Emerging multidrug-resistant Candida duobushaemulonii infections in Panama hospitals: importance of laboratory surveillance and accurate identification Running Title: Candida duobushaemulonii infections in Panama. Authors Ruben Ramos BSc 1*, Diego H. Caceres MSc 2,3*, Marilyn Perez BSc 1, Nicole Garcia BSc 1, Wendy Castillo BSc 1, Erika Santiago BSc 4, Jovanna Borace BSc 4, Shawn R. Lockhart PhD 2, Elizabeth L. Berkow PhD 2, Lizbeth Hayer BSc 5, Andres Espinosa-Bode MD 2, Jose Moreno BSc 1, Brendan R. Jackson MD 2, Jackeline Moran BSc 1, Tom Chiller MD 2, Gloriela de Villarreal BSc 1, Nestor Sosa MD 1, the Red Nacional de Vigilancia Epidemiologica en Microbiologia Clinica 1,5, and Snigdha Vallabhaneni MD 2 (*) These authors contributed equally Affilitations: 1 Instituto Conmemorativo Gorgas de Estudios de la Salud, Panama City, Panama 2 Centers for Disease Control and Prevention, Atlanta, GA, United States of America. 3 Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, Tennessee, United States of America. 4 Hospital Santo Tomas, Panama City, Panama. 5 Ministerio de Salud de Panama, Panama City, Panama. Corresponding authors: Ruben Ramos, rramos@gorgas.gob.pa, and Diego H. Caceres, diegocaceres84@gmail.com. Key words: Candida duobushaemulonii, laboratory surveillance, invasive candidiasis, mutidrug-resistance, emerging yeast 1

2 ABSTRACT Candida duobushaemulonii, a yeast closely related to Candida auris, is thought to rarely cause infections, and is often misidentified. In October 2016, the Panamanian Ministry of Health implemented laboratory surveillance for C. auris Suspected C. auris isolates were forwarded to the national reference laboratory for identification by Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry and antifungal susceptibility testing. During November 2016 May 2017, 17 of 36 (47%) isolates suspected to be C. auris were identified as C. duobushaemulonii. These 17 isolates were obtained from 14 patients at six hospitals. Ten patients, including three children, had bloodstream infections, MICs for fluconazole, voriconazole, and amphotericin B were elevated. No resistance to echinocandins was observed. C. duobushaemulonii causes more invasive infections than previously appreciated, and poses a substantial problem given it is resistant to multiple antifungals. Expanded laboratory surveillance is an important step in the detection and control of such emerging pathogens. 2

3 INTRODUCTION Candida duobushaemulonii is a yeast that belongs to the Candida haemulonii species complex along with Candida haemulonii and C. haemulonii var. vulnera. Yeasts in this complex are closely related to Candida pseudohaemulonii and Candida auris, the latter of which is an emerging global health threat because it is often multidrug-resistant (MDR) and can cause outbreaks in the healthcare setting (1,2,3). Little is known about species in the C. haemulonii complex because infections are rare and these organisms are difficult to identify without advanced laboratory identification methods (4). C. haemulonii has been the species best studied; like C. auris, C. haemulonii tends to be multidrug-resistant, but unlike C. auris, which often causes invasive bloodstream infection, most reported C. haemulonii infections have involved skin wounds in patients with diabetes or peripheral vascular disease (5-8). C. duobushaemulonii was previously classified as C. haemulonii group II. Using phenotypic and molecular methods, C. duobushaemulonii was recategorized as its own species in 2012 (1). Since then, a small number of C. duobushaemulonii infections have been reported from China (5), India (7) and Brazil (6,8). It has been documented to cause vulvovaginal candidiasis and chronic lower extremity wound infections (8,9), but also invasive infections (5,6). Like C. auris, C. duobushaemulonii is frequently multidrug resistant, typically with high-level resistance to amphotericin B and azoles (5,6,10). Most commercially available yeast identification methods, such as VITEK 2 (software version before 8.01), BD Phoenix, and API 20C AUX, cannot distinguish between C. duobushaemulonii, C. haemulonii var. vulnera, C. 3

4 pseudohaemulonii, and C. auris. These systems typically yield an identification of C. haemulonii for all of the above species, although Candida famata, or no identification are sometimes reported (4,11). Given the differences in types of infection caused and antifungal susceptibility patterns, accurate species identification is necessary but can only be be performed with sequence analysis of internal transcribed spacer (ITS) region of the ribosomal DNA or mass patterns by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) (4,11). After the first cases of C. auris were identified in Panama in 2016 (12), the Panamanian Ministry of Health initiated prospective laboratory-based surveillance for C. auris. Clinical laboratories were asked to forward all clinical isolates suspected of being C. auris to the Central Reference Laboratory of Public Health at the Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES) for further identification. Suspected C. auris isolates included all identified as C. haemulonii, C. famata, or Candida spp. by VITEK 2, the only yeast identification instrument available in laboratories within Panama s national network. This report describes 14 patients with C. duobushaemulonii infection or colonization in Panama, Central America, identified through the surveillance system set up to detect C. auris and highlights the importance of the laboratory surveillance and accurate species identification. RESULTS During November 2016 May 2017, the ICGES s national microbiology reference laboratory received 36 isolates suspected to be C. auris from 31 patients. Most isolates were from blood (n=17) and central venous catheter (CVC) 4

5 tip (n=3), and the remaining 16 were from other sites, including urine (n=4), skin and nails (n=5), respiratory samples (n=3), surgical sites (n=2), bone (n=1), and vaginal discharge (n=1). All isolates were initially identified as C. haemulonii by VITEK 2 at local participating laboratories. Of the 36 isolates suspected of being C. auris, 17 (47%) were identified by both biomérieux and Bruker MALDI-TOF, and confirmed by sequencing of the D1/D2 region of the 28S as C. duobushaemulonii. Eight (22%) were identified as C. haemulonii, five (14%) as C. auris, and six (16%) as other Candida species (four C. parapsilosis, one C. tropicalis and one C. inconspicua). Thirteen (76%) of 17 isolates from the bloodstream were C. duoboshaemulonii, two were C. haemulonii, one was C. auris, and one was C. parapsilosis. All three isolated from CVC tip were confirmed as C. duobushaemulonii. Four of sixteen (25%) of isolates from other sites were C. duobushaemulonii. The 17 C. duobushaemulonii isolates were obtained from 14 patients who were hospitalized at six medical institutions in four geographical regions of Panama (Figure 1A). Half of these patients were from one hospital in Panama City, and most isolates came from the end of 2016 (Figure 1B). Of the 14 patients with C. duobushaemulonii infection or colonization, 10 had bloodstream infection, including three who concurrently had a CVC tip culture that yielded C. duobushaemulonii. In the remaining four of fourteen patients, C. duobushaemulonii was recovered from endotracheal secretions (n=2), likely to represent colonization, surgical mesh (n=1) and exposed bone (n=1). Nine (64%) patients were male, with a mean age of 47 years (range: 0 77). Three cases were identified in children (two were 2 years old and one was <30 days old), and all involved bloodstream infections. Information on 5

6 underlying conditions, treatment, outcomes, and any epidemiologic links between patients was not available. Based on Centers for Disease Control and Prevention (CDC) s tentative breakpoints for C. auris, antifungal susceptibility testing (AFST) by VITEK 2 revealed elevated MICs for fluconazole ( 32 µg/ml; n=11; 65%), voriconazole ( 4 µg/ml; n=8; 47%) and amphotericin B ( 2 µg/ml; n=14; 82%). The overall MIC 50 was 32 µg/ml for fluconazole, 1 µg/ml for voriconazole, and >16 µg/ml for amphotericin B. No resistance to caspofungin or micafungin was observed (Table 1). AFST for four isolates was conducted at CDC using broth microdilution. All four isolates had MICs for fluconazole of 32 by VITEK 2 ; however two had fluconazole MICs below the breakpoint (4 and 8 µg/ml) by broth microdilution. Results for amphotericin B showed consistently high MICs, all >8 by both VITEK 2 and Etest. Although individual MIC values for micafungin varied slightly between AFST methodologies, the interpretation of susceptible remained regardless of method used. We were unable to perform AFST by broth microdilution and Etest at CDC for the remaining 13 of 17 C. duobushaemulonii isolates because growth of these strains under test conditions was insufficient to obtain valid MICs. DISCUSSION Our report documents the presence in Panama of invasive infections with C. duobushaemulonii, a multidrug-resistant Candida species. Notably, in a laboratory surveillance system established in Panama to detect C. auris infections, nearly half of the isolates suspected to be C. auris were in fact C. duobushaemulonii. Given that C. duobushaemulonii was detected only through this laboratory-based surveillance and not through clinical testing, we are unable to determine whether 6

7 this species is a relatively new cause of infection in Panama or a longstanding but unrecognized one. Regardless, detection of this organism is of importance since it causes serious, invasive infections. Most C. duobushaemulonii isolates were cultured from the bloodstream (and most bloodstream isolates sent for confirmatory testing turned out to be C. duobushaemulonii), confirming that, like C. auris, C. duobushaemulonii, can also cause invasive infections. Despite belonging to the same species complex, this organism may be quite different from C. haemulonii, which is more often seen in superficial wound infections, although as found in this surveillance system, infections can also occur in the bloodstream. Older reports of C. haemulonii infections in bloodstream may in fact have represented infections with other species, such as C. auris, C. duobushaemulonii, or C. pseudohaemulonii, because advanced identification to distinguish between these species was not routinely performed or because these species were not yet named (5-10). The high levels of azole and amphotericin B resistance observed for C. duobushaemulonii using broth microdilution and Etest, as well as VITEK 2, suggest that echinocandins, when available, may be a better choice for treatment of these infections (14). These drugs are also recommended as first-line treatment for C. auris. More work is needed to determine the relationship between C. duobushaemulonii MICs and clinical outcomes. Additionally, MICs for individual strains likely vary, and broth microdilution and Etest should be used when possible to evaluate for resistance. When these methods are unavailable, VITEK 2 may be used, as it accurately identified elevated MICs to amphotericin B and low MICs to 7

8 echinocandins; however, in a limited sample it yielded falsely high results for azoles. Although several outbreaks of Candida parapsilosis have been documented (15-18), transmission of Candida between patients in healthcare settings has rarely been an infection control concern. Candida infections were thought to primarily originate from translocation of host flora to normally sterile sites such as the bloodstream or, in the case of oral or vulvovaginal candidiasis, overgrowth of normal flora. The emergence of C. auris has called this paradigm into question, as ample evidence suggests that C. auris is transmitted in healthcare settings (2,19-21). However, data on whether closely related C. haemulonii complex species can be transmitted are limited. One report suggests that C. haemulonii may have been transmitted between patients receiving care in a peripheral vascular disease clinic (22). Whether C. duobushaemulonii can be similarly transmitted remains to be seen. We found fourteen patients infected or colonized with this relatively rare Candida species in a period of seven months, about half from a single medical institution in Panama City, suggesting the possibility of healthcare transmission, although we did not have information on epidemiologic links between patients. Whole-genome sequencing of isolates from Panama and other parts of the world along with more detailed epidemiologic information may help answer this question. If in fact C. duobushaemulonii can also be transmitted in healthcare settings and continues to cause multidrug-resistant invasive infections, it may require the same infection control measures as those recommended for C. auris (23,24). Surveillance established to detect C. auris has helped identify another highly resistant yeast causing invasive infections in Panama. This finding highlights the 8

9 crucial role laboratory surveillance has in identifying emerging pathogens. Clinicians, microbiologists, and laboratory technicians should pay special attention to fungal isolates identified by automated tests as uncommon or unidentified species or that have unexpected characteristics for the identified species (e.g., morphology, susceptibility patterns and other additional taxonomy keys) (25). In such cases, confirmatory testing with MALDI-TOF or DNA sequencing is recommended (11). Our report is limited by the lack of information on clinical conditions, treatments, and outcomes. This information was not available because the surveillance was primarily laboratory based. Such information would add to the knowledge about invasive disease caused by C. duobushaemulonii.because some clinical labs may not perform species level identification of isolates from noninvasive sites, we may not know the true spectrum of infections caused by C. duobushaemulonii.. In recent years, we have seen the emergence of invasive infections caused by highly drug-resistant Candida, such as C. auris and now C. duobushaemulonii. These species are often misidentified using commonly used yeast identification methods, and much remains to be understood about their transmission, pathogenesis, and reservoirs. Integrated epidemiologic and laboratory surveillance is a fundamental component of global health security necessary for the detection and control of such emerging pathogens. METHODS Isolates received through the surveillance program underwent confirmatory identification with MALDI-TOF using the VITEK MS (biomérieux) (Research Use 9

10 Only data base) available at the microbiology laboratory of Hospital Santo Tomas (Panama City, Panama). AFST was conducted at each local hospital using VITEK 2 automated systems (VITEK 2 susceptibility card for yeast. Reference: AST- YS07). For further species confirmation and AFST, isolates were sent to the Mycotic Diseases Branch Laboratory at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, United States. At CDC, isolates were tested by MALDI-TOF (Microflex, Bruker) and sequencing of the D1/D2 region of the 28S. AFST was performed at CDC on four isolates using broth microdilution with custom-made frozen panels for azoles and echinocandins (TREK Diagnostic Systems) and Etest for amphotericin B. There are no published minimum inhibitory concentrations (MIC) cutoff for C. duobushaemulonii (13); therefore we used tentative breakpoints for C. auris proposed by CDC (11). A case was defined as C. duobushaemulonii isolated from any body site in a patient hospitalized in Panama during the surveillance period (14). The local ethics committee reviewed this project and deemed it non-research public health response. ACKNOWLEDGMENTS: We thank Reina Turcios-Ruiz, Loren Cadena, Susan Kaydos-Daniels, Paige Armstrong, Alex Bandea, Ngoc Le and Colleen Lysen at Centers for Disease Control and Prevention. Itza Barahona de Mosca, Lourdes Garcia and Felicia Tulloch at Ministerio de Salud de Panama. The members of the Red Nacional de Vigilancia Epidemiologica en Microbiologia Clinica: Alexis Solis, Osvaldo Cisterna, Mellisa Herrera, Nayarith Rojas, Ana Samudio, Lisbeth Wong. 10

11 CONFLICTS OF INTEREST: All other authors not report potential conflicts 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. REFERENCES 1. Cendejas-Bueno E, Kolecka A, Alastruey-Izquierdo A, Theelen B, Groenewald M, Kostrzewa M, Cuenca-Estrella M, Gómez-López A, Boekhout T. Reclassification of the Candida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant human pathogenic yeasts. J Clin Microbiol Nov;50(11): doi: /JCM Epub 2012 Sep Chowdhary A, Sharma C, Meis JF. Candida auris: A rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog. 2017; 18;13(5):e Lockhart SR, Etienne KA, Vallabhaneni S, Farooqi J, Chowdhary A, Govender NP Colombo AL, Calvo B, Cuomo CA, Desjardins CA, Berkow EL, Castanheira M, Magobo RE, Jabeen K, Asghar RJ, Meis JF, Jackson B, Chiller T, Litvintseva AP. Simultaneous Emergence of Multidrug-Resistant Candida auris on 3 Continents Confirmed by Whole-Genome Sequencing and Epidemiological Analyses. Clin Infect Dis. 2017;64: Kathuria S, Singh PK, Sharma C, Prakash A, Masih A, Kumar A, Meis JF, Chowdhary A. Multidrug-Resistant Candida auris Misidentified as Candida 11

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16 Figure 1. Confirmed isolates of the emerging pathogen Candida duobushaemulonii in Panama, November 2016 May 2017: (A) Geographical distribution of C duobushaemulonii 14 cases in Panama by provinces, (B) Timeline of cases of C. duobushaemulonii in Panama by medical institution. 16

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18 1 2 Table 1. Antifungal susceptibility results of Candida duobushaemulonii isolates from Panama, Isolated FCZ VCZ CSF MCF AmpB MIC µg/ml 1 µg/ml 0.25 µg/ml µg/ml >16 µg/ml MIC 90 >64 µg/ml >8 µg/ml 0.25 µg/ml 0.25 µg/ml >16 µg/ml Testing performed by by VITEK 2 automated system. (FCZ) fluconazole, (VCZ) voriconazole, (CSF) caspofungin, (MCF) micafungin (AmpB) amphotericin B Downloaded from on December 19, 2018 by guest 1