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Molecular characterization and antifungal susceptibility testing of Candida nivariensis from blood samples – an Iranian multicentre study and a review of the literature

Molecular characterization and antifungal susceptibility testing of Candida nivariensis from... Purpose. Identification of the emerging yeast species Candida nivariensis among presumptively identified Iranian Candida glabrata isolates. Methodology. Clinical C. glabrata species complex isolates from blood (n=100; 46.9%), vaginal swabs (n=20; 9.4%), bronchoal- veolar lavage (n=10; 4.7%) and sputum (n=12; 5.6%) from 68 patients from Iran were investigated. Isolates were characterized by CHROMagar, multiplex PCRs, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), amplified fragment length polymorphism (AFLP) fingerprinting, internal transcribed spacer (ITS)/large subunit (LSU) rDNA and FKS1/FKS2 sequencing, and the European Committee on Antimicrobial Susceptibility Testing broth microdilution method. A comprehensive literature review was conducted and all the relevant clinical and microbiological data were collected. Results. Four C. nivariensis isolates were recovered from blood samples of three subjects and were all consistently identified by nine-plex PCR, Bruker MALDI-TOF MS, and LSU and ITS rDNA sequencing. AFLP genotyping clustered the isolates into two groups. Sequencing of the FKS1 and FKS2 hotspots showed no accountable amino acid substitutions. All isolates were suscep- tible to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin and micafungin. Conclusion. In total, 4 out of 213 clinical C. glabrata species complex candidemia isolates were C. nivariensis. Improvement of the BioMerieux Vitek MS database is required to accurately identify C. nivariensis and it is advised to alternatively use CHROMagar and/or PCR-based techniques. As other species within the Nakaseomyces clade may cause infection and showed high MIC values for antifungals, inclusion of their spectra into the MALDI-TOF MS database seems relevant. Due to developing resistance to fluconazole and insufficient efficacy of caspofungin, the combination of catheter removal plus treatment with caspofungin, or voriconazole, or micafungin might be effective for patients. such as CHROMagar, can easily distinguish these species InTR oduCTIon from C. glabrata, molecular assays increased the accuracy Candida glabrata is the second most common cause of and shortened the identification turn-around time [9]. candidemia in the USA [1] and its prevalence is increasing Moreover, not only is little known about their virulence, worldwide [2, 3]. Acquisition of resistance to azoles, antifungal susceptibility and prevalence, but also rarity of echinocandins, amphotericin B and multidrug resistant spectra of these species has hampered the accuracy of Vitek (MDR) traits are alarming features of C. glabrata [3–5]. MS to correctly identify clinical isolates of C. bracarensis For a decade C. glabrata has been recognized as a cryptic and C. nivariensis [10]. Accordingly, identification and species complex containing the emerging opportunistic sharing data on nationwide and worldwide scales could yeast species C. nivariensis [6] and Candida bracarensis enrich our knowledge about various aspects of these species [7]. These species belong to the Nakeseomyces clade of and will contribute to diagnostic and even therapeutic the Saccharomycotina [8]. Comparative genomic studies improvement. showed that C. bracarensis and C. nivariensis are more related to the non-pathogenic yeast species Nakaseomyces Here, we retrospectively studied a large collection of clinical delphensis than to C. glabrata [8]. C. glabrata isolates recovered from Iranian patients. Subse- quently, amplified fragment length polymorphism (AFLP) Although some studies showed that time-consuming, fingerprinting [11], antifungal susceptibility patterning, using but relative simple phenotypic identification techniques, 000963 © 2019 The Authors This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 770 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 European Committee on Antimicrobial Susceptibility Testing diagnosis, some masses were visible around the mitral valve (EUCAST) broth microdilution, and sequencing of the FKS1 on his Trans esophageal echocardiography (TEE). With being and FKS2 hotspot 1 (HS1) regions were performed. Lastly, a suspicious of mitral valve endocarditis, a blood sample was comprehensive literature review was performed to collect taken and incubated in Bactec device (Bactec 9420, Becton relevant clinical and microbiological data of C. nivariensis cases. Dickinson, Franklin Lakes, NJ, USA). Broad spectrum anti- biotics, including cefepime 2 g/IV/TDS, gentamicin 80 mg/ IV/TDS, rifampin 300 mg/PO/BD and vancomycin 1 g/IV/ METHodS BD were prescribed aer b ft lood culture sampling. Aer 36 h, ft Retrieving data of published cases the blood culture was positive for Enterobacter spp. Vanco- mycin was discontinued and other antibiotics continued All published studies containing C. nivariensis cases from and his fever completely resolved aer 7 d ft ays. On 18 April 2005 (year of description of the species C. nivariensis) to fever started again, hence, the blood sample was taken and 1 October 2018 were retrieved. The keyword ‘Candida aer 48 h i ft t was positive for Candida spp. Caspofungin nivariensis’ without any limitations in languages and dates (70 mg/IV/stat and 50 mg/IV/daily) was added to the previous was searched in PubMed and Google search engines. antibiotic regimen and this treatment was continued for Clinical aspects, including year of publication and location, 10 days. His follow-up blood culture was negative aer 3 d ft ays. age, sex, sample type, risk factors and underlying conditions, Despite being partially recovered, due to his family member's antifungals used and duration of therapy, outcome and request, he was discharged while he was under treatment with microbiological data, including number of isolates, pheno- uco fl nazole (400 mg daily/PO) 22 days post admission. type on CHROMagar, antifungal susceptibility testing (AFST) protocol, and the MIC values for the antifungals fluconazole Case 2 (isolate 5n) (FLC), voriconazole (VRC), itraconazole (ITC), posaconazole e s Th ame patient (case 1) aer 30 d ft ays with fever complaint (PSC), caspofungin (CSP), micafungin (MCF), anidulafungin was referred to Ghaem hospital and admitted to the infection (ANF), amphotericin B (AMB), and 5-fluorocytosine (5FC) ward on 26 May 2015. He described his previous frequency were recorded. In order to assure the accuracy of collected of dysuria 5 days before admission and he was evaluated for clinical and microbiological data, they were checked and urosepsis. Urine analysis revealed the presence of many white recorded by two individuals to reach consensus. 5 −1 blood cells (leukocyturia) and yeast cells (>10 c.f.u. ml ) and accordingly AMB deoxycholate (75 mg/IV/ daily) was Patient information prescribed. A blood sample was taken and aer 6 d ft ays incu- Case 1 (isolate 2n) bation yielded yeast cells. Although, not fully recovered and A 67-year-old man, who underwent mitral and aortic valves' his general health status was not satisfactory for the medical repair, two months aer c ft ardiac surgery and with clinical team, the patient left the hospital and no information was neurological deficits compatible with cerebrovascular acci - available on his follow-up. dent (CVA) was admitted in Ghaem hospital, Mashad, Iran, on 5 April 2015. Brain MRI (T1 with contrast view) revealed Case 3 (isolate 35n) two ring enhancement masses on right frontal and temporal A 14-year-old girl with severe burnings was hospitalized lobes that suggested brain abscesses. Aer t ft his primary in the burn intensive care unit (ICU), Ghaem Hospital, Received 15 December 2018; Accepted 19 February 2019; Published 29 March 2019 1 2 Author affiliations: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; Department of Infectious Diseases and Tropical Medicine, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, 4 5 Iran; Department of Medical Mycology and Parasitology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Medical Mycology and Parasitology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; Department of Medical Mycology and Parasitology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Basic Sciences in Infectious Diseases Research Center, and Department of Medical Mycology and Parasitology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Medical Parasitology and Mycology, School of Medicine, Isfahan 10 11 University of Medical Sciences, Isfahan, Iran; BioAware Life Sciences Data Management Software, Belgium; CBMR Scientific Inc, Edmonton, Canada; Department of Dermatology, Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Medical Mycology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, PR China; Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands. *Correspondence: Weihua Pan, panweihua@ smmu. edu. cn; Ferry Hagen, f. hagen@ westerdijkinstitute. nl Keywords: Candida glabrata complex; multicenter study; Candida nivariensis; multi-plex PCR; MALDI-TOF MS; ribosomal DNA sequencing. Abbreviations: AFLP, amplified fragmentlength polymorphism; AMB, amphotericin B; AND, anidulafungin; BAL, Bronchoalveolar lavage; EUCAST, European Committee of Antifungal Susceptibility Testing; 5-FC, 5-fluorocytosine; FLC, fluconazole; ITC, itraconazole; ITS, internal transcribed spacer; LSU, large subunit; MALDI-TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry; MCF, micafungn; PSC, posaconazole; VRC, voriconazole. Four isolates of C. nivariensis were deposited in the culture collection of Westerdijk Fungal Biodiversity Institute (accession numbers CBS 15642– 15645). Sequences of the large subunit and internal transcribed sequences of rDNA and hotspot 1 of FKS1 and FKS2 were deposited in GenBank under the accession numbers, MK503934-MK503937, MK503955-MK503958, and MK509355-MK509362. †These authors contributed equally to this work Two supplementary tables are available with the online version of this article. 771 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 Mashhad, Iran, on 26 October 2015. The whole upper body Isfahan and Mashhad, were retrospectively collected. Isolates part, neck and face were involved with second to third degree were obtained from blood (n=71; 33.3%), urine (n=100; burns. In the beginning of her admission, she was alert and in 46.9%), vaginal swabs (n=20; 9.4%), bronchoalveolar lavage order to relieve the pain, fentanyl (50 µg/IV/infusion/hour) (BAL) fluid (n=10; 4.7%) and sputum (n=12; 5.6%) samples. Isolates were streaked on Saboraud dextrose agar at 37 C was prescribed for a few days and daily washing and dressing of wounds were performed. The first episode of fever was for 24–48 h. Colonies that showed white and pink colonies on CHROMagar were selected and subjected to a compre- noticed 7 days post admission. Physical examinations showed that there was a significant infection of the burn wounds. hensive multiplex 21-plex PCR [12]. Further analysis by a nine-plex PCR [9], Bruker MALDI-TOF MS (Microflex LT, Hence, wound drainage and blood were subjected to culture, and imipenem (500 mg/IV/QID) and vancomycin (1 g/IV/ Bruker Daltonics, Bremen, Germany), large subunit (LSU) and internal transcribed spacer (ITS) rDNA sequencing, BID) treatments were started. The blood and wound drainage culture yielded both Acinetobacter baumannii, vanco- antifungal susceptibility testing (EUCAST v9.0) and AFLP was performed. Identification by MALDI-TOF MS was mycin was replaced by colistin (9 mu/IV/loading and then 4.5 mu/IV/BID) and she was treated with both colistin and performed using a full extraction method [13]. In order to evaluate the accuracy of MALDI-TOF MS and the nine-plex imipenem. Aer ft 2 days, fever signs were eliminated and first debridement surgery was performed. On the fourteenth PCR, sequencing of LSU of ribosomal DNA (rDNA) using primers of LROR (5′-ACCCGCTGAACTTAAGC-3′) and admission day, the ae ff cted site was repaired with allogra ft skin surgery. en 3 Th days aer ft the surgery (seventeenth day LR5 (5′-TCCTGAGGGAAACTTCG-3′), and ITS rDNA using primers of ITS1 (5′-TCC GTA GGT GAA CCT GCG of admission), the patient became febrile and discharges were G-3′) and ITS4 (5′-TCC GTA GGT GAA CCT GCG G-3′) observed around the skin graft. A blood sample yielded an was performed [14]. imipenem and amikacin resistant strain of Pseudomonas aeruginosa and Candida spp., while the wound culture C. glabrata increasingly shows resistance to echinocandins remained negative. Imipenem treatment was stopped and that is mainly associated with acquisition of mutations in the meropenem (2 g/TDS/IV) and fluconazole (200 mg/daily/ hotspot 1 (HS1) of FKS1 and FKS2 genes [15]. C. nivariensis PO) were added to colistin. On the twenty-first day of admis- is phylogenetically closely related to C. glabrata, therefore, sion, blood cultures remained positive with Pseudomonas we sequenced HS1 of these two genes for the isolates of aeruginosa and Candida spp. On 26 November 2015, in spite C. bracarensis and C. nivariensis. Primers targeting HS1 of of the extensive debridement of the graft and the replace - FKS1 (389 bp) and FKS2 (718 bp) of C. glabrata were used ment of fluconazole with amphotericin B deoxycholate for this purpose (unpublished data). (50 mg/daily/IV) the patient succumbed. Genotyping of isolates of C. nivariensis using AFLP Case 4 (isolate 81) In order to investigate the genetic relationship and genotypic A 62-year-old female with left limb sarcoma was admitted with diversity of our isolates, AFLP was followed as described necrotizing fasciitis and severe sepsis in the emergency ward in before [11]. Besides our clinical isolates of C. nivariensis Imam Khomeini complex hospital, Tehran, Iran, on 23 July 2018. (n=4), CBS reference and type strains of C. nivariensis (CBS Treatment with meropenem (1 gr/TDS/IV) and vancomycin 9984, CBS 9985 and CBS 10161), C. bracarensis (CBS 10154) (1 g/IV/BID) was started and amputation of the left limb was and a clinical isolate of C. bracarensis (H111) generously immediately performed. During 24–26 July, despite antibiotic provided by Professor W. Liao (Shanghai, China), C. glabrata therapy and amputation, the patient was suffering from fever and (CBS 138), C. uthaithanina (CBS 10932), C. kungkrabaensis sepsis. Hence, being suspicious for an infection caused by other (CBS 10927), N. delphensis (CBS 2170) and N. bacillisporus micro-organisms, blood samples were taken and incubated in (CBS 7720) were included in the AFLP experiment. Bactec 9420 device (Becton Dickinson). On 26 July, following the isolation of Pseudomonas aeruginosa from a wound culture Antifungal susceptibility testing of the amputated leg discharges, colistin (9 mu/IV/Loading and To define the MIC of our isolates, antifungal susceptibility then 4.5 mu/IV/BID) and gentamycin (240 mg/IV/TDS) were testing was performed using broth microdilution procedure administered and treatment with vancomycin was stopped. of EUCAST version 9.0 ( www. eucast. org/ fileadmin/ src/ On 27 July, blood cultures yielded yeast cells and subsequent m e di a/ P D Fs/ EUCA ST_ fi les/ AFST/ C linic a l_ bre a kp oints/ PCR reported them as C. glabrata and antifungal therapy with Antifungal_ breakpoints_ v_ 9. 0_ 180212. pdf ). Amphotericin 300 mg of liposomal amphotericin B was started the following B (AMB), 5-flucytosine (5FC; Sigma Chemical Corporation, day. On 29 July, liposomal AMB was replaced with 50 mg of St. Louis, MO, USA), fluconazole (FLC), voriconazole (VCZ) caspofungin. On 8 August 2018, the patient died due to septic and anidulafungin (AND; Pfizer, New York, NY, USA), itra- shock and cardiac arrest. conazole (ITC; Santa Cruz Biotech, Dallas, TX, USA), posa- conazole (PSC; MSD, Kenilworth, NJ, USA), and micafungin Isolates, identification, and part sequencing of FKS1 (MCF; Astellas Pharma, Tokio, Japan) were included. As and FKS2 genes caspofungin is associated with interlaboratory variations Preserved clinical isolates of C. glabrata (2015–2018) from and obtained MIC values of this drug cannot differentiate four metropolitan cities of Iran, including, Tehran, Shiraz, wild-type and mutated strains of C. glabrata [16], we did not 772 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 include this drug in our antifungal susceptibility assessment. (n=2), sputum (n=2), CSF (n=2), pleural uid fl , urorstomy It is known that MIC values obtained for anidulafungin uid fl , lung biopsy, pelvic collection, nasal secretion, exit site and micafungin better predict the presence of mutations in swab, nail with each one sample (for three samples no source FKS1 and FKS2, and thus both echinocandins were included was mentioned). Candidemia (24%), vaginitis (22.66%) and [16]. AMB, AND, FLC and MCF were interpreted based on candiduria (8%) were among the most prevalent clinical mani- clinical breakpoints, while ITC, PSC, VRC and 5FC were festations caused by C. nivariensis. Broad-spectrum antibiotic interpreted based on epidemiological cut-off values. For anti - therapy and catheter insertion were the most encountered risk fungal susceptibility testing, apart from our clinical isolates factors for development of candidiasis due to C. nivariensis. of C. nivariensis (n=4), four reference and type strains of Among 18 patients with candidemia due to C. nivariensis, C. nivariensis (CBS 9983, CBS 9984, CBS 9985 and CBS treatment option and clinical outcome (death/survival) were 10161) were included. reported for only three and four patients, respectively. These three patients were primarily treated with fluconazole and during the course of infection, fluconazole was changed to RESuLTS voriconazole and micafungin followed by micafungin alone data obtained from published cases of (n=1), or catheter removal and CSP (n=1), or CSP treatment C. nivariensis (n=1). From these three patients two were treated with VCZ All clinically and microbiologically relevant data of published plus MCF or CSP plus catheter removal survived, while the literature and our cases are presented in Tables S1 and S2 one treated with CSP died. With respect to cases with vagi- (available in the online version of this article). In total, 75 nitis, FLC was the most frequently used antifungal (n=10/16), C. nivariensis isolates were found in 13 countries. Isolates of followed by miconazole (n=3/16), boric acid in combination C. nivariensis were recovered from four continents and with ITC and chlorhexidine (n=2/16), and FLC in combina- Europe with 39 isolates had the highest number of isolates, tion with nystatin (n=1/16). From these 16 vaginitis cases only followed by Asia with 30 isolates, America with 4 isolates and 12 mentioned clinical oucome, among them four out of eight Australia with only 1 isolate. Country-wise, China contained individuals administered with FLC, two out of three patients the highest number of isolates of C. nivariensis (n=20), treated with miconazole, and one individual treated with the followed by the UK (n=16), Poland (n=14), Spain (n=8), India combination of FLC and nystatin showed recurrent vaginitis. (n=6), Argentina (n=3), Malaysia (n=2), and Australia, Brazil, France, Indonesia, Italy and Japan reported only one isolate. Comparison of identification systems and e m Th ajority of infections caused by C. nivariensis were sequencing of HS1 FKS1 and FKS2 found in adults aged >20 years (89.65%) with a median age of Using 21-plex PCR, 213 clinical isolates were identified as 62 years. Two-thirds of cases of infections (n=29) (with C. glabrata. In contrast, the nine-plex PCR identified four known data of age and sex) were acquired by women and isolates of C. nivariensis and no C. bracarensis in agreement the rest by men (n=10). C. nivariensis isolates were recov- ered from a wide ranges of clinical samples, including blood with MALDI-TOF MS (green scores>2) and sequencing of LSU rDNA (Fig. 1). In total, 4 out of the 213 Iranian (n=18), vagina (n=17), urine (n=6), BAL and tracheal aspirate (n=4), peritoneal uid fl (n=6), oral samples (n=3), abscess C. glabrata species complex isolates were C. nivariensis and the (n=2), urine and renal catheter (n=2), vascular tip catheter rest were C. glabrata. Interestingly, all isolates of C. nivariensis Fig. 1. Successful identification of C. nivariensis isolates using nine-plex PCR. 773 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 Fig. 2. AFLP fingerprinting profiles generated for C. nivariensis isolates. were from blood samples and were obtained from Mashhad in 9983–9985 (Fig. 2). The isolates 2 N and 5 N that were isolated 2015 (n=3) and Tehran in 2018 (n=1) that comprised 4.41% – with an interval of >30 days – from the same patient were of the Iranian candidemia cases caused by C. glabrata. found to share the same AFLP genotype. Despite the fact that primers targeting HS1 of FKS1 and FKS2 Antifungal susceptibility testing were based on the corresponding sequences of C. glabrata, FKS2 was successfully amplified for all four isolates of C. nivariensis, Based on clinical breakpoints, all of our isolates showed a −1 while FKS1 showed only faint bands. However, subjecting these susceptible phenotype for FLC (0.0625–1 µg ml ), AND and −1 −1 amplicons to PCR sequencing yielded sequences with decent MCF (≤0.016 µg ml ) and AMB (0.5–1 µg ml ). Based on qualities. No mutations were observed in the HS1 for FKS2 and epidemiological cut-off value, our isolates were susceptible −1 −1 in FKS1 two of the isolates contained silent mutations. The first to ITC (≤0.016–0.0625 µg ml ), VRC (≤0.016–0.03 µg ml ), −1 −1 isolate carried a homozygote and a heterozygote mutations in PSC (≤0.016–0.125 µg ml ) and 5-FC (0.0625–0.125 µg ml ) the residues of 628 (A1884T) and 630 (C1888Y), and the second (Table 1). isolate harboured a mutation in the residue of 630 (C1888T). All these mutations were silent and did not ae ff ct the amino acid sequence of the HS1 of FKS1. dISCuSSIon Despite an increasing number of reported C. nivariensis cases Genotyping by AFLP from different countries, limited data is presented with regards AFLP analysis clustered our isolates with the CBS reference to antifungals' susceptibility pattern, virulence factors, genuine and type strains of C. nivariensis and they were grouped in distribution and epidemiology, and biological niches of this two discrete clusters. Three of our isolates (2 N, 5 N and 100B) species. Here, we have systematically screened clinical isolates of from Mashhad and Tehran clustered with CBS 10161, while C. glabrata collected from 2015 to 2018 to assess the nation- the other isolate from Mashhad (35 N) clustered with CBS wide epidemiology of C. nivariensis in Iran. Table 1. MIC of our isolates of C. nivariensis and CBS reference and type strains of C. bracarensis and C. nivariensis determined by EUCAST methodology −1 Strains MIC values (µg ml ) Fluconazole Itraconazole Voriconazole Posaconazole 5FC AMB Anidulafungin Micafungin CBS9983 1 0.06 0.6 0.125 0.06 0.5 ≤0.016 ≤0.016 CBS9984 1 0.125 0.6 0.125 0.06 1 ≤0.016 ≤0.016 CBS9985 1 0.06 0.03 0.125 0.06 0.5 ≤0.016 ≤0.016 CBS10161 0.125 ≤0.016 ≤0.016 ≤0.016 0.125 1 ≤0.016 ≤0.016 2N 0.06 ≤0.016 ≤0.016 ≤0.016 0.125 1 ≤0.016 ≤0.016 5N 0.125 ≤0.016 ≤0.016 ≤0.016 0.125 1 ≤0.016 ≤0.016 35 N 0.125 ≤0.016 ≤0.016 ≤0.016 0.125 0.5 ≤0.016 ≤0.016 81–7 1 0.06 0.03 0.125 0.06 1 ≤0.016 ≤0.016 774 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 the importance of using highly resolutive genotyping tech- C. nivariensis was exclusively found in blood niques, such as AFLP, in microbiological investigations. Using samples AFLP, our isolates clustered into two distinct genotypes of Isolates of C. nivariensis were only obtained from blood C. nivariensis, where three of our isolates (one from Tehran samples, and we did not find this species from other clinical and two from Mashhad from the same patient) clustered with samples, such as urine, vaginal swabs, BAL, sputum and stool. CBS 10161 and the fourth isolate (from Mashhad) clustered es Th e four isolates were recovered from three patients (two with CBS 9983, CBS 9984 and CBS 9985. females and one male) and for one of them within 30 days two isolates were recovered, which represented two episodes. Variability in antifungal susceptibility patterns and es Th e four cases were reported from two different hospitals. clinical outcomes e m Th edian age of infected patients was 62 years and it seems that C. glabrata and C. nivariensis tend to cause infection in Our C. nivariensis isolates were susceptible to all tested the elderlies [17]. All of our patients had received broad spec- drugs, including AMB, AND, FLC, ITC, MCF, PSC, VRC trum antibiotics, which is one of the highlighted risk factors and 5FC (Table  1). On the other hand, some clinical for development of candidemia [18]. isolates of C. nivariensis recovered from blood [23] and vaginal swabs [24] showed high MIC values for FLC and infected patients were not responsive to this drug. Data Appropriate identification tool is a prerequisite derived from the literature review revealed that there is a As a preliminary screening tool, all of the presumptive isolates strain and geographical variability for antifungal suscep- of C. glabrata were cultured on CHROMagar and isolates of tibility patterns of isolates of C. nivariensis and it seems C. glabrata and C. nivariensis showed pink and white colora- that this phenomenon is common for clinical isolates of tion, respectively, while a 21-plex PCR [12] identified them all C. glabrata, as well [15, 25]. Although, one of our patients as C. glabrata species complex. MALDI-TOF MS and nine-plex that was treated with CSP plus FLC and AMB deoxy- PCR, unequivocally identified four isolates of C. nivariensis in cholate survived, unfortunately no data was available on agreement with rDNA sequencing. In contrast, Vitek MS does his follow-up to ensure that he is alive. Moreover, two of not have the ability to identify isolates of C. nivariensis and our patients died despite treatment with FLC plus AMB C. bracarensis [19]. Hence, it is recommended that laborato- deoxycholate and AMB plus CSP. Considering that there ries equipped with this device reidentify isolates of C. glabrata is limited information with regards to treatment options with inexpensive alternative means of identification such as and outcomes of patients infected with C. nivariensis, it CHROMagar and/or PCR-based approaches. In our study, appears that the sole reliance on FLZ and CSP does not 4.41% (3/68) of candidemia cases caused by members of the seem enough to resolve candidemia and vaginitis caused cryptic complex species of C. glabrata were attributable to by this species [24, 26, 27], while a combination of VRC C. nivariensis. Although, most of studies have shown a low clinical plus MCF followed by micafungin alone [23] or CSP plus prevalence of C. bracarensis and C. nivariensis [10], the preva- catheter removal [27] for candidemia cases and oral VCZ lence we reported here is in agreement with the study conducted [24] for recurrent vaginitis cases showed successful treat- in Poland, where they did not find C. bracarensis, while they ment outcomes. A higher efficacy of MCF for clearance of showed that C. nivariensis constituted 6% of clinical isolates of infections caused by C. glabrata might be behind the fact C. glabrata [20]. The lower clinical prevalence of C. nivariensis that this drug causes mutations in HS1 of FKS1 and FKS2 and C. bracarensis is partially explained by genomic studies, approximately 11 times less than CSP and five times AND where it was shown that these two species are more closely [28]. Possible misidentification cases of C. nivariensis and related to the non-pathogenic members of the Nakaeseomyces C. bracarensis as C. glabrata and high MIC values for azole clades than to C. glabrata [8]. However, accelerated evolution of drugs might prompt clinicians to use echinocandins for their genome enable them to become more virulent [8] and with treatment of candidemia cases caused by C. nivariensis. On an increase in the population of immunocompromised patients the other hand, mutations in HS1 of FKS1 and FKS2 are a might lead to a higher prevalence of infections pertained to these predictor of therapeutic failure of echinocandins against two species. Moreover, all pathogenic attributes of C. glabrata C. glabrata [29], hence, screening of mutations in HS1 of are present even in environmental (non-pathogenic) species of FKS1 and FKS2 could be an imperative initiative to detect Nakaseomyces clade, for example N. delphensis [8]. Furthermore, resistance isolates of C. nivariensis and C. bracarensis to N. delphensis compared to clinical isolates of C. bracarensis was echninocandins. Lack of amino acid substitution in the HS1 found to be pan-azole-resistant (FLC=128, ITC>16, PSC>8, of FKS1 and FKS2 was in agreement with low MIC values −1 VRC>4 µg ml ) and its MIC values for echinocandins and AMB obtained for aniulafungin and micafungin. were higher or equal to that of C. bracarensis [21]. As a result, expanding appropriate and accurate means of identifications Where is the original biological niche of will result in a more comprehensive view on the distribution C. nivariensis? and epidemiology, antifungal susceptibility patterns, and even Comparative genomic studies have disclosed that C. glabrata pathogenicity of species within the Nakaseomyces clade. is well-adapted to the human gut as its main biological niche, Various genotypes of C. glabrata proved to differ in their while C. nivariensis might have adapted to other environmental antifungal susceptibility and mortality rates [22], highlighting niches [8]. This is in line with the findings of environmental 775 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 were signed by patients and in order to prevent exposing their informa- sampling studies from Thailand that isolated C. nivariensis tion, clinical data and isolates derived from them were designated with from leaves of sugarcane [30], barks and soil [10], while specific codes. C. glabrata was not cultured. So far, all C. bracarensis isolates are from clinical sources, and the majority of C. nivariensis References 1. Lockhart SR, Iqbal N, Cleveland AA, Farley MM, Harrison LH isolates has a clinical source. This might raise the question if et  al. Species identification and antifungal susceptibility testing these two species are part of human mycobiota and they will of Candida bloodstream isolates from population-based surveil- invade the bloodstream, once there is an opportunity. This lance studies in two U.S. cities from 2008 to 2011. J Clin Microbiol might be further reinforced by the fact that one of the patients 2012;50:3435–3442. that had a duodenal perforation [26] developed C. nivariensis 2. Pham CD, Iqbal N, Bolden CB, Kuykendall RJ, Harrison LH et  al. Role of FKS Mutations in Candida glabrata: MIC values, echino- candidemia. Hence, it could be speculated that this species candin resistance, and multidrug resistance. Antimicrob Agents occur in the environment as the main biological niche and Chemother 2014;58:4690–4696. subsequently it undergoes adaptation to the human host. In 3. Hou X, Xiao M, Chen SCA, Kong F, Wang H et  al. Molecular epide- order to address this question, development and application miology and antifungal susceptibility of Candida glabrata in China of sensitive and accurate metagenomics platforms might be (August 2009 to July 2014): a multi-center study. Front Microbiol 2017;8:1–9. a useful solution. Once established, subjecting human and environmental-derived samples might elucidate the real 4. Healey KR, Zhao Y, Perez WB, Lockhart SR, Sobel JD et  al. Prevalent mutator genotype identified in fungal pathogen biological niches of these species. Candida glabrata promotes multi-drug resistance. Nat Commun 2016;7:11128. Conclusion 5. Lamoth F, Lockhart SR, Berkow EL, Calandra T. Changes in the epidemiological landscape of invasive candidiasis. J Antimicrob In summary, we report four clinical isolates of C. nivariensis Chemother 2018;73:i4–i13. among a set that was presumptively identified as C. glabrata and 6. Alcoba-Flórez J, Méndez-Alvarez S, Cano J, Guarro J, Pérez- that were all recovered from blood samples. Taking advantage Roth E et al. Phenotypic and molecular characterization of Candida of accurate means of identification, such as molecular assays nivariensis sp. nov., a possible new opportunistic fungus. J Clin Microbiol 2005;43:4107–4111. and MALDI-TOF MS will aid in unravelling the epidemiology 7. Correia A, Sampaio P, James S, Pais C. Candida bracarensis sp. of these cryptic yeast species. Utilization of these techniques nov., a novel anamorphic yeast species phenotypically similar to will even have a significant impact on our general knowledge Candida glabrata. Int J Syst Evol Microbiol 2006;56:313–317. of these species. For instance, although it was thought that 8. Gabaldón T, Martin T, Marcet-Houben M, Durrens P, Bolotin-Fuku- C. nivariensis was isolated in 2005 in Spain for the first time [6], hara M et al. Comparative genomics of emerging pathogens in the with the aid of accurate means of identification, the first docu- Candida glabrata clade. BMC Genomics 2013;14:623. mented isolates of this species dated back to 1996 in Argentina 9. Arastehfar A, Fang W, Pan W, Liao W, Yan L et al. Identification of nine cryptic species of Candida albicans, C. glabrata, and C. parap- [31]. Once isolated, we will have the possibility to take a global silosis complexes using one-step multiplex PCR. BMC Infect Dis initiative to study the lacking pieces of the puzzles of genotyping, 2018;18:1–9. antifungal susceptibility patterns, and virulence of members of 10. Hou X, Xiao M, Chen SCA, Wang H, Yu SY et  al. Identification and this cryptic complex species. antifungal susceptibility profiles of Candida nivariensis and Candida bracarensis in a multi-center Chinese collection of yeasts. Front Microbiol 2017;8:1–8. Funding information 11. Marchetta A, Gerrits van den Ende B, Al-Hatmi AMS, Hagen F, This project has received funding from the European Union’s Horizon Zalar P et al. Global Molecular Diversity of the Halotolerant Fungus 2020 research and innovation programme under the Marie Sklo- Hortaea werneckii. Life 2018;8:31. dowska-Curie grant agreement no. 642095, National Health Depart- 12. Arastehfar A, Fang W, Pan W, Lackner M, Liao W et  al. Yeast ment of China [2018ZX10101003], National Natural Science Foundation panel multiplex PCR for identification of clinically important yeast of China [31770161], Second Military Medical University [2017JZ47] species: stepwise Diagnostic strategy, useful for developing coun- and Shanghai Science and Technology Committee [14DZ2272900 and tries. Diagn Microbiol Infect Dis 2019;93:112–119. 14495800500]. 13. Cassagne C, Cella AL, Suchon P, Normand AC, Ranque S et  al. Author contributions Evaluation of four pretreatment procedures for MALDI-TOF MS Authors declared that they have actively participated in this study and yeast identification in the routine clinical laboratory. Med Mycol they are fully aware of the contents expressed in this manuscript. A. A. 2013;51:371–377. and A. F. have participated in study design, performing experiments, 14. Stielow JB, Lévesque CA, Seifert KA, Meyer W, Iriny L et  al. One data analysis, and drafting and revising the manuscript. M. R. S., H. Z., fungus, which genes? development and assessment of universal S. K., M. R., M. J. N., K. Z. and A. C. participated in perforing experiments, primers for potential secondary fungal DNA barcodes. Persoonia collection of isolates and clinical data, and revised the manuscript. 2015;35:242–263. P. J. H. and C. B. helped with the antifungal susceptibility testing, data analysis, and revision of the manuscript. F. H., P. W. and T. B. have partici- 15. Alexander BD, Johnson MD, Pfeiffer CD, Jiménez- pated in study design, data analysis, drafting and revision of the manu- Ortigosa C, Catania J et  al. Increasing echinocandin resistance in script, and funded the project. Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations. Clin Conflicts of interest Infect Dis 2013;56:1724–1732. The authors declare that there are no conflicts of interest. 16. Shields RK, Nguyen MH, Press EG, Updike CL, Clancy CJ. Anidu- Ethical statement lafungin and micafungin MIC breakpoints are superior to that of This study was approved by the ethical committees of Tehran, Mashhad, caspofungin for identifying FKS mutant Candida glabrata strains Isfahan, and Shiraz Universities of Medical Sciences (IR MUMS fm and echinocandin resistance. Antimicrob Agents Chemother REC.1397.268, and IR. TUMS. .SPH.REC.1396.4195). Consent forms 2013;57:6361–6365. 776 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 17. McCarty TP, Pappas PG. Invasive candidiasis. Infect Dis Clin North 25. Tay ST, Lotfalikhani A, Sabet NS, Ponnampalavanar S, Sulaiman S Am 2016;30:103–124. et al. Occurrence and characterization of Candida nivariensis from a culture collection of Candida glabrata clinical isolates in Malaysia. 18. Bouza E, Muñoz P. Epidemiology of candidemia in intensive care Mycopathologia 2014;178:307–314. units. Int J Antimicrob Agents 2008;32:S87–S91. 26. Li J, Shan Y, Fan S, Liu X. Prevalence of Candida nivariensis and 19. Swoboda-Kopeć E, Sikora M, Golas M, Piskorska K, Gozdowski D Candida bracarensis in vulvovaginal candidiasis. Mycopathologia et al. Candida nivariensis in comparison to different phenotypes of 2014;178:279–283. Candida glabrata. Mycoses 2014;57:747–753. 27. López-Soria LM, Bereciartua E, Santamaría M, Soria LM, 20. Bishop JA, Chase N, Magill SS, Kurtzman CP, Fiandaca MJ et  al. Hernández-Almaraz JL et al. Primer caso de fungemia asociada a Candida bracarensis detected among isolates of Candida glabrata catéter por Candida nivariensis en la Península Ibérica. Rev Iberoam by peptide nucleic acid fluorescence in situ hybridization: suscep- Micol 2013;30:69–71. tibility data and documentation of presumed infection. J Clin Micro- biol 2008;46:443–446. 28. Shields RK, Kline EG, Healey KR, Kordalewska M, Perlin DS et al. Spontaneous mutational frequency and FKS mutation rates vary 21. Byun SA, Won EJ, Kim M et al. Multilocus Sequence Typing (MLST) by echinocandin agent against Candida glabrata. Antimicrob Agents genotypes of Candida glabrata bloodstream isolates in Korea: asso- Chemother 2019;63:1692–18. ciation with antifungal resistance, mutations in mismatch repair gene (MSH2), and clinical outcomes. Antimicrob Agents Chemother 29. Shields RK, Nguyen MH, Press EG, Kwa AL, Cheng S et  al. The 2018;13:1–10. presence of an FKS mutation rather than MIC is an independent risk factor for failure of echinocandin therapy among patients with 22. Fujita S-i, Senda Y, Okusi T, Ota Y, Takada H et al. Catheter-related invasive candidiasis due to Candida glabrata. Antimicrob Agents fungemia due to fluconazole-resistant Candida nivariensis. J Clin Chemother 2012;56:4862–4869. Microbiol 2007;45:3459–3461. 30. Limtong S, Kaewwichian R, Yongmanitchai W, Kawasaki H. Diver- 23. Sharma C, Wankhede S, Muralidhar S, Prakash A, Singh PK et al. sity of culturable yeasts in phylloplane of sugarcane in Thailand Candida nivariensis as an etiologic agent of vulvovaginal candidi- and their capability to produce indole-3-acetic acid. World J Micro- asis in a tertiary care hospital of New Delhi, India. Diagn Microbiol biol Biotechnol 2014;30:1785–1796. Infect Dis 2013;76:46–50. 31. Morales-López SE, Taverna CG, Bosco-Borgeat ME, 24. Singh A, Healey KR, Yadav P, Upadhyaya G, Sachdeva N et  al. Absence of azole or echinocandin resistance in Candida glabrata Maldonado I, Vivot W et al. Candida glabrata species complex prev- isolates in India despite background prevalence of strains with alence and antifungal susceptibility testing in a culture collection: defects in the DNA mismatch repair pathway. Antimicrob Agents first description of Candida nivariensis in Argentina. Mycopathologia Chemother 2018;62:195–18. 2016;181:871–878. 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Molecular characterization and antifungal susceptibility testing of Candida nivariensis from blood samples – an Iranian multicentre study and a review of the literature

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0022-2615
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10.1099/jmm.0.000963
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Abstract

Purpose. Identification of the emerging yeast species Candida nivariensis among presumptively identified Iranian Candida glabrata isolates. Methodology. Clinical C. glabrata species complex isolates from blood (n=100; 46.9%), vaginal swabs (n=20; 9.4%), bronchoal- veolar lavage (n=10; 4.7%) and sputum (n=12; 5.6%) from 68 patients from Iran were investigated. Isolates were characterized by CHROMagar, multiplex PCRs, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), amplified fragment length polymorphism (AFLP) fingerprinting, internal transcribed spacer (ITS)/large subunit (LSU) rDNA and FKS1/FKS2 sequencing, and the European Committee on Antimicrobial Susceptibility Testing broth microdilution method. A comprehensive literature review was conducted and all the relevant clinical and microbiological data were collected. Results. Four C. nivariensis isolates were recovered from blood samples of three subjects and were all consistently identified by nine-plex PCR, Bruker MALDI-TOF MS, and LSU and ITS rDNA sequencing. AFLP genotyping clustered the isolates into two groups. Sequencing of the FKS1 and FKS2 hotspots showed no accountable amino acid substitutions. All isolates were suscep- tible to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin and micafungin. Conclusion. In total, 4 out of 213 clinical C. glabrata species complex candidemia isolates were C. nivariensis. Improvement of the BioMerieux Vitek MS database is required to accurately identify C. nivariensis and it is advised to alternatively use CHROMagar and/or PCR-based techniques. As other species within the Nakaseomyces clade may cause infection and showed high MIC values for antifungals, inclusion of their spectra into the MALDI-TOF MS database seems relevant. Due to developing resistance to fluconazole and insufficient efficacy of caspofungin, the combination of catheter removal plus treatment with caspofungin, or voriconazole, or micafungin might be effective for patients. such as CHROMagar, can easily distinguish these species InTR oduCTIon from C. glabrata, molecular assays increased the accuracy Candida glabrata is the second most common cause of and shortened the identification turn-around time [9]. candidemia in the USA [1] and its prevalence is increasing Moreover, not only is little known about their virulence, worldwide [2, 3]. Acquisition of resistance to azoles, antifungal susceptibility and prevalence, but also rarity of echinocandins, amphotericin B and multidrug resistant spectra of these species has hampered the accuracy of Vitek (MDR) traits are alarming features of C. glabrata [3–5]. MS to correctly identify clinical isolates of C. bracarensis For a decade C. glabrata has been recognized as a cryptic and C. nivariensis [10]. Accordingly, identification and species complex containing the emerging opportunistic sharing data on nationwide and worldwide scales could yeast species C. nivariensis [6] and Candida bracarensis enrich our knowledge about various aspects of these species [7]. These species belong to the Nakeseomyces clade of and will contribute to diagnostic and even therapeutic the Saccharomycotina [8]. Comparative genomic studies improvement. showed that C. bracarensis and C. nivariensis are more related to the non-pathogenic yeast species Nakaseomyces Here, we retrospectively studied a large collection of clinical delphensis than to C. glabrata [8]. C. glabrata isolates recovered from Iranian patients. Subse- quently, amplified fragment length polymorphism (AFLP) Although some studies showed that time-consuming, fingerprinting [11], antifungal susceptibility patterning, using but relative simple phenotypic identification techniques, 000963 © 2019 The Authors This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 770 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 European Committee on Antimicrobial Susceptibility Testing diagnosis, some masses were visible around the mitral valve (EUCAST) broth microdilution, and sequencing of the FKS1 on his Trans esophageal echocardiography (TEE). With being and FKS2 hotspot 1 (HS1) regions were performed. Lastly, a suspicious of mitral valve endocarditis, a blood sample was comprehensive literature review was performed to collect taken and incubated in Bactec device (Bactec 9420, Becton relevant clinical and microbiological data of C. nivariensis cases. Dickinson, Franklin Lakes, NJ, USA). Broad spectrum anti- biotics, including cefepime 2 g/IV/TDS, gentamicin 80 mg/ IV/TDS, rifampin 300 mg/PO/BD and vancomycin 1 g/IV/ METHodS BD were prescribed aer b ft lood culture sampling. Aer 36 h, ft Retrieving data of published cases the blood culture was positive for Enterobacter spp. Vanco- mycin was discontinued and other antibiotics continued All published studies containing C. nivariensis cases from and his fever completely resolved aer 7 d ft ays. On 18 April 2005 (year of description of the species C. nivariensis) to fever started again, hence, the blood sample was taken and 1 October 2018 were retrieved. The keyword ‘Candida aer 48 h i ft t was positive for Candida spp. Caspofungin nivariensis’ without any limitations in languages and dates (70 mg/IV/stat and 50 mg/IV/daily) was added to the previous was searched in PubMed and Google search engines. antibiotic regimen and this treatment was continued for Clinical aspects, including year of publication and location, 10 days. His follow-up blood culture was negative aer 3 d ft ays. age, sex, sample type, risk factors and underlying conditions, Despite being partially recovered, due to his family member's antifungals used and duration of therapy, outcome and request, he was discharged while he was under treatment with microbiological data, including number of isolates, pheno- uco fl nazole (400 mg daily/PO) 22 days post admission. type on CHROMagar, antifungal susceptibility testing (AFST) protocol, and the MIC values for the antifungals fluconazole Case 2 (isolate 5n) (FLC), voriconazole (VRC), itraconazole (ITC), posaconazole e s Th ame patient (case 1) aer 30 d ft ays with fever complaint (PSC), caspofungin (CSP), micafungin (MCF), anidulafungin was referred to Ghaem hospital and admitted to the infection (ANF), amphotericin B (AMB), and 5-fluorocytosine (5FC) ward on 26 May 2015. He described his previous frequency were recorded. In order to assure the accuracy of collected of dysuria 5 days before admission and he was evaluated for clinical and microbiological data, they were checked and urosepsis. Urine analysis revealed the presence of many white recorded by two individuals to reach consensus. 5 −1 blood cells (leukocyturia) and yeast cells (>10 c.f.u. ml ) and accordingly AMB deoxycholate (75 mg/IV/ daily) was Patient information prescribed. A blood sample was taken and aer 6 d ft ays incu- Case 1 (isolate 2n) bation yielded yeast cells. Although, not fully recovered and A 67-year-old man, who underwent mitral and aortic valves' his general health status was not satisfactory for the medical repair, two months aer c ft ardiac surgery and with clinical team, the patient left the hospital and no information was neurological deficits compatible with cerebrovascular acci - available on his follow-up. dent (CVA) was admitted in Ghaem hospital, Mashad, Iran, on 5 April 2015. Brain MRI (T1 with contrast view) revealed Case 3 (isolate 35n) two ring enhancement masses on right frontal and temporal A 14-year-old girl with severe burnings was hospitalized lobes that suggested brain abscesses. Aer t ft his primary in the burn intensive care unit (ICU), Ghaem Hospital, Received 15 December 2018; Accepted 19 February 2019; Published 29 March 2019 1 2 Author affiliations: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; Department of Infectious Diseases and Tropical Medicine, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, 4 5 Iran; Department of Medical Mycology and Parasitology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Medical Mycology and Parasitology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; Department of Medical Mycology and Parasitology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Basic Sciences in Infectious Diseases Research Center, and Department of Medical Mycology and Parasitology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Medical Parasitology and Mycology, School of Medicine, Isfahan 10 11 University of Medical Sciences, Isfahan, Iran; BioAware Life Sciences Data Management Software, Belgium; CBMR Scientific Inc, Edmonton, Canada; Department of Dermatology, Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Medical Mycology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, PR China; Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands. *Correspondence: Weihua Pan, panweihua@ smmu. edu. cn; Ferry Hagen, f. hagen@ westerdijkinstitute. nl Keywords: Candida glabrata complex; multicenter study; Candida nivariensis; multi-plex PCR; MALDI-TOF MS; ribosomal DNA sequencing. Abbreviations: AFLP, amplified fragmentlength polymorphism; AMB, amphotericin B; AND, anidulafungin; BAL, Bronchoalveolar lavage; EUCAST, European Committee of Antifungal Susceptibility Testing; 5-FC, 5-fluorocytosine; FLC, fluconazole; ITC, itraconazole; ITS, internal transcribed spacer; LSU, large subunit; MALDI-TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry; MCF, micafungn; PSC, posaconazole; VRC, voriconazole. Four isolates of C. nivariensis were deposited in the culture collection of Westerdijk Fungal Biodiversity Institute (accession numbers CBS 15642– 15645). Sequences of the large subunit and internal transcribed sequences of rDNA and hotspot 1 of FKS1 and FKS2 were deposited in GenBank under the accession numbers, MK503934-MK503937, MK503955-MK503958, and MK509355-MK509362. †These authors contributed equally to this work Two supplementary tables are available with the online version of this article. 771 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 Mashhad, Iran, on 26 October 2015. The whole upper body Isfahan and Mashhad, were retrospectively collected. Isolates part, neck and face were involved with second to third degree were obtained from blood (n=71; 33.3%), urine (n=100; burns. In the beginning of her admission, she was alert and in 46.9%), vaginal swabs (n=20; 9.4%), bronchoalveolar lavage order to relieve the pain, fentanyl (50 µg/IV/infusion/hour) (BAL) fluid (n=10; 4.7%) and sputum (n=12; 5.6%) samples. Isolates were streaked on Saboraud dextrose agar at 37 C was prescribed for a few days and daily washing and dressing of wounds were performed. The first episode of fever was for 24–48 h. Colonies that showed white and pink colonies on CHROMagar were selected and subjected to a compre- noticed 7 days post admission. Physical examinations showed that there was a significant infection of the burn wounds. hensive multiplex 21-plex PCR [12]. Further analysis by a nine-plex PCR [9], Bruker MALDI-TOF MS (Microflex LT, Hence, wound drainage and blood were subjected to culture, and imipenem (500 mg/IV/QID) and vancomycin (1 g/IV/ Bruker Daltonics, Bremen, Germany), large subunit (LSU) and internal transcribed spacer (ITS) rDNA sequencing, BID) treatments were started. The blood and wound drainage culture yielded both Acinetobacter baumannii, vanco- antifungal susceptibility testing (EUCAST v9.0) and AFLP was performed. Identification by MALDI-TOF MS was mycin was replaced by colistin (9 mu/IV/loading and then 4.5 mu/IV/BID) and she was treated with both colistin and performed using a full extraction method [13]. In order to evaluate the accuracy of MALDI-TOF MS and the nine-plex imipenem. Aer ft 2 days, fever signs were eliminated and first debridement surgery was performed. On the fourteenth PCR, sequencing of LSU of ribosomal DNA (rDNA) using primers of LROR (5′-ACCCGCTGAACTTAAGC-3′) and admission day, the ae ff cted site was repaired with allogra ft skin surgery. en 3 Th days aer ft the surgery (seventeenth day LR5 (5′-TCCTGAGGGAAACTTCG-3′), and ITS rDNA using primers of ITS1 (5′-TCC GTA GGT GAA CCT GCG of admission), the patient became febrile and discharges were G-3′) and ITS4 (5′-TCC GTA GGT GAA CCT GCG G-3′) observed around the skin graft. A blood sample yielded an was performed [14]. imipenem and amikacin resistant strain of Pseudomonas aeruginosa and Candida spp., while the wound culture C. glabrata increasingly shows resistance to echinocandins remained negative. Imipenem treatment was stopped and that is mainly associated with acquisition of mutations in the meropenem (2 g/TDS/IV) and fluconazole (200 mg/daily/ hotspot 1 (HS1) of FKS1 and FKS2 genes [15]. C. nivariensis PO) were added to colistin. On the twenty-first day of admis- is phylogenetically closely related to C. glabrata, therefore, sion, blood cultures remained positive with Pseudomonas we sequenced HS1 of these two genes for the isolates of aeruginosa and Candida spp. On 26 November 2015, in spite C. bracarensis and C. nivariensis. Primers targeting HS1 of of the extensive debridement of the graft and the replace - FKS1 (389 bp) and FKS2 (718 bp) of C. glabrata were used ment of fluconazole with amphotericin B deoxycholate for this purpose (unpublished data). (50 mg/daily/IV) the patient succumbed. Genotyping of isolates of C. nivariensis using AFLP Case 4 (isolate 81) In order to investigate the genetic relationship and genotypic A 62-year-old female with left limb sarcoma was admitted with diversity of our isolates, AFLP was followed as described necrotizing fasciitis and severe sepsis in the emergency ward in before [11]. Besides our clinical isolates of C. nivariensis Imam Khomeini complex hospital, Tehran, Iran, on 23 July 2018. (n=4), CBS reference and type strains of C. nivariensis (CBS Treatment with meropenem (1 gr/TDS/IV) and vancomycin 9984, CBS 9985 and CBS 10161), C. bracarensis (CBS 10154) (1 g/IV/BID) was started and amputation of the left limb was and a clinical isolate of C. bracarensis (H111) generously immediately performed. During 24–26 July, despite antibiotic provided by Professor W. Liao (Shanghai, China), C. glabrata therapy and amputation, the patient was suffering from fever and (CBS 138), C. uthaithanina (CBS 10932), C. kungkrabaensis sepsis. Hence, being suspicious for an infection caused by other (CBS 10927), N. delphensis (CBS 2170) and N. bacillisporus micro-organisms, blood samples were taken and incubated in (CBS 7720) were included in the AFLP experiment. Bactec 9420 device (Becton Dickinson). On 26 July, following the isolation of Pseudomonas aeruginosa from a wound culture Antifungal susceptibility testing of the amputated leg discharges, colistin (9 mu/IV/Loading and To define the MIC of our isolates, antifungal susceptibility then 4.5 mu/IV/BID) and gentamycin (240 mg/IV/TDS) were testing was performed using broth microdilution procedure administered and treatment with vancomycin was stopped. of EUCAST version 9.0 ( www. eucast. org/ fileadmin/ src/ On 27 July, blood cultures yielded yeast cells and subsequent m e di a/ P D Fs/ EUCA ST_ fi les/ AFST/ C linic a l_ bre a kp oints/ PCR reported them as C. glabrata and antifungal therapy with Antifungal_ breakpoints_ v_ 9. 0_ 180212. pdf ). Amphotericin 300 mg of liposomal amphotericin B was started the following B (AMB), 5-flucytosine (5FC; Sigma Chemical Corporation, day. On 29 July, liposomal AMB was replaced with 50 mg of St. Louis, MO, USA), fluconazole (FLC), voriconazole (VCZ) caspofungin. On 8 August 2018, the patient died due to septic and anidulafungin (AND; Pfizer, New York, NY, USA), itra- shock and cardiac arrest. conazole (ITC; Santa Cruz Biotech, Dallas, TX, USA), posa- conazole (PSC; MSD, Kenilworth, NJ, USA), and micafungin Isolates, identification, and part sequencing of FKS1 (MCF; Astellas Pharma, Tokio, Japan) were included. As and FKS2 genes caspofungin is associated with interlaboratory variations Preserved clinical isolates of C. glabrata (2015–2018) from and obtained MIC values of this drug cannot differentiate four metropolitan cities of Iran, including, Tehran, Shiraz, wild-type and mutated strains of C. glabrata [16], we did not 772 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 include this drug in our antifungal susceptibility assessment. (n=2), sputum (n=2), CSF (n=2), pleural uid fl , urorstomy It is known that MIC values obtained for anidulafungin uid fl , lung biopsy, pelvic collection, nasal secretion, exit site and micafungin better predict the presence of mutations in swab, nail with each one sample (for three samples no source FKS1 and FKS2, and thus both echinocandins were included was mentioned). Candidemia (24%), vaginitis (22.66%) and [16]. AMB, AND, FLC and MCF were interpreted based on candiduria (8%) were among the most prevalent clinical mani- clinical breakpoints, while ITC, PSC, VRC and 5FC were festations caused by C. nivariensis. Broad-spectrum antibiotic interpreted based on epidemiological cut-off values. For anti - therapy and catheter insertion were the most encountered risk fungal susceptibility testing, apart from our clinical isolates factors for development of candidiasis due to C. nivariensis. of C. nivariensis (n=4), four reference and type strains of Among 18 patients with candidemia due to C. nivariensis, C. nivariensis (CBS 9983, CBS 9984, CBS 9985 and CBS treatment option and clinical outcome (death/survival) were 10161) were included. reported for only three and four patients, respectively. These three patients were primarily treated with fluconazole and during the course of infection, fluconazole was changed to RESuLTS voriconazole and micafungin followed by micafungin alone data obtained from published cases of (n=1), or catheter removal and CSP (n=1), or CSP treatment C. nivariensis (n=1). From these three patients two were treated with VCZ All clinically and microbiologically relevant data of published plus MCF or CSP plus catheter removal survived, while the literature and our cases are presented in Tables S1 and S2 one treated with CSP died. With respect to cases with vagi- (available in the online version of this article). In total, 75 nitis, FLC was the most frequently used antifungal (n=10/16), C. nivariensis isolates were found in 13 countries. Isolates of followed by miconazole (n=3/16), boric acid in combination C. nivariensis were recovered from four continents and with ITC and chlorhexidine (n=2/16), and FLC in combina- Europe with 39 isolates had the highest number of isolates, tion with nystatin (n=1/16). From these 16 vaginitis cases only followed by Asia with 30 isolates, America with 4 isolates and 12 mentioned clinical oucome, among them four out of eight Australia with only 1 isolate. Country-wise, China contained individuals administered with FLC, two out of three patients the highest number of isolates of C. nivariensis (n=20), treated with miconazole, and one individual treated with the followed by the UK (n=16), Poland (n=14), Spain (n=8), India combination of FLC and nystatin showed recurrent vaginitis. (n=6), Argentina (n=3), Malaysia (n=2), and Australia, Brazil, France, Indonesia, Italy and Japan reported only one isolate. Comparison of identification systems and e m Th ajority of infections caused by C. nivariensis were sequencing of HS1 FKS1 and FKS2 found in adults aged >20 years (89.65%) with a median age of Using 21-plex PCR, 213 clinical isolates were identified as 62 years. Two-thirds of cases of infections (n=29) (with C. glabrata. In contrast, the nine-plex PCR identified four known data of age and sex) were acquired by women and isolates of C. nivariensis and no C. bracarensis in agreement the rest by men (n=10). C. nivariensis isolates were recov- ered from a wide ranges of clinical samples, including blood with MALDI-TOF MS (green scores>2) and sequencing of LSU rDNA (Fig. 1). In total, 4 out of the 213 Iranian (n=18), vagina (n=17), urine (n=6), BAL and tracheal aspirate (n=4), peritoneal uid fl (n=6), oral samples (n=3), abscess C. glabrata species complex isolates were C. nivariensis and the (n=2), urine and renal catheter (n=2), vascular tip catheter rest were C. glabrata. Interestingly, all isolates of C. nivariensis Fig. 1. Successful identification of C. nivariensis isolates using nine-plex PCR. 773 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 Fig. 2. AFLP fingerprinting profiles generated for C. nivariensis isolates. were from blood samples and were obtained from Mashhad in 9983–9985 (Fig. 2). The isolates 2 N and 5 N that were isolated 2015 (n=3) and Tehran in 2018 (n=1) that comprised 4.41% – with an interval of >30 days – from the same patient were of the Iranian candidemia cases caused by C. glabrata. found to share the same AFLP genotype. Despite the fact that primers targeting HS1 of FKS1 and FKS2 Antifungal susceptibility testing were based on the corresponding sequences of C. glabrata, FKS2 was successfully amplified for all four isolates of C. nivariensis, Based on clinical breakpoints, all of our isolates showed a −1 while FKS1 showed only faint bands. However, subjecting these susceptible phenotype for FLC (0.0625–1 µg ml ), AND and −1 −1 amplicons to PCR sequencing yielded sequences with decent MCF (≤0.016 µg ml ) and AMB (0.5–1 µg ml ). Based on qualities. No mutations were observed in the HS1 for FKS2 and epidemiological cut-off value, our isolates were susceptible −1 −1 in FKS1 two of the isolates contained silent mutations. The first to ITC (≤0.016–0.0625 µg ml ), VRC (≤0.016–0.03 µg ml ), −1 −1 isolate carried a homozygote and a heterozygote mutations in PSC (≤0.016–0.125 µg ml ) and 5-FC (0.0625–0.125 µg ml ) the residues of 628 (A1884T) and 630 (C1888Y), and the second (Table 1). isolate harboured a mutation in the residue of 630 (C1888T). All these mutations were silent and did not ae ff ct the amino acid sequence of the HS1 of FKS1. dISCuSSIon Despite an increasing number of reported C. nivariensis cases Genotyping by AFLP from different countries, limited data is presented with regards AFLP analysis clustered our isolates with the CBS reference to antifungals' susceptibility pattern, virulence factors, genuine and type strains of C. nivariensis and they were grouped in distribution and epidemiology, and biological niches of this two discrete clusters. Three of our isolates (2 N, 5 N and 100B) species. Here, we have systematically screened clinical isolates of from Mashhad and Tehran clustered with CBS 10161, while C. glabrata collected from 2015 to 2018 to assess the nation- the other isolate from Mashhad (35 N) clustered with CBS wide epidemiology of C. nivariensis in Iran. Table 1. MIC of our isolates of C. nivariensis and CBS reference and type strains of C. bracarensis and C. nivariensis determined by EUCAST methodology −1 Strains MIC values (µg ml ) Fluconazole Itraconazole Voriconazole Posaconazole 5FC AMB Anidulafungin Micafungin CBS9983 1 0.06 0.6 0.125 0.06 0.5 ≤0.016 ≤0.016 CBS9984 1 0.125 0.6 0.125 0.06 1 ≤0.016 ≤0.016 CBS9985 1 0.06 0.03 0.125 0.06 0.5 ≤0.016 ≤0.016 CBS10161 0.125 ≤0.016 ≤0.016 ≤0.016 0.125 1 ≤0.016 ≤0.016 2N 0.06 ≤0.016 ≤0.016 ≤0.016 0.125 1 ≤0.016 ≤0.016 5N 0.125 ≤0.016 ≤0.016 ≤0.016 0.125 1 ≤0.016 ≤0.016 35 N 0.125 ≤0.016 ≤0.016 ≤0.016 0.125 0.5 ≤0.016 ≤0.016 81–7 1 0.06 0.03 0.125 0.06 1 ≤0.016 ≤0.016 774 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 the importance of using highly resolutive genotyping tech- C. nivariensis was exclusively found in blood niques, such as AFLP, in microbiological investigations. Using samples AFLP, our isolates clustered into two distinct genotypes of Isolates of C. nivariensis were only obtained from blood C. nivariensis, where three of our isolates (one from Tehran samples, and we did not find this species from other clinical and two from Mashhad from the same patient) clustered with samples, such as urine, vaginal swabs, BAL, sputum and stool. CBS 10161 and the fourth isolate (from Mashhad) clustered es Th e four isolates were recovered from three patients (two with CBS 9983, CBS 9984 and CBS 9985. females and one male) and for one of them within 30 days two isolates were recovered, which represented two episodes. Variability in antifungal susceptibility patterns and es Th e four cases were reported from two different hospitals. clinical outcomes e m Th edian age of infected patients was 62 years and it seems that C. glabrata and C. nivariensis tend to cause infection in Our C. nivariensis isolates were susceptible to all tested the elderlies [17]. All of our patients had received broad spec- drugs, including AMB, AND, FLC, ITC, MCF, PSC, VRC trum antibiotics, which is one of the highlighted risk factors and 5FC (Table  1). On the other hand, some clinical for development of candidemia [18]. isolates of C. nivariensis recovered from blood [23] and vaginal swabs [24] showed high MIC values for FLC and infected patients were not responsive to this drug. Data Appropriate identification tool is a prerequisite derived from the literature review revealed that there is a As a preliminary screening tool, all of the presumptive isolates strain and geographical variability for antifungal suscep- of C. glabrata were cultured on CHROMagar and isolates of tibility patterns of isolates of C. nivariensis and it seems C. glabrata and C. nivariensis showed pink and white colora- that this phenomenon is common for clinical isolates of tion, respectively, while a 21-plex PCR [12] identified them all C. glabrata, as well [15, 25]. Although, one of our patients as C. glabrata species complex. MALDI-TOF MS and nine-plex that was treated with CSP plus FLC and AMB deoxy- PCR, unequivocally identified four isolates of C. nivariensis in cholate survived, unfortunately no data was available on agreement with rDNA sequencing. In contrast, Vitek MS does his follow-up to ensure that he is alive. Moreover, two of not have the ability to identify isolates of C. nivariensis and our patients died despite treatment with FLC plus AMB C. bracarensis [19]. Hence, it is recommended that laborato- deoxycholate and AMB plus CSP. Considering that there ries equipped with this device reidentify isolates of C. glabrata is limited information with regards to treatment options with inexpensive alternative means of identification such as and outcomes of patients infected with C. nivariensis, it CHROMagar and/or PCR-based approaches. In our study, appears that the sole reliance on FLZ and CSP does not 4.41% (3/68) of candidemia cases caused by members of the seem enough to resolve candidemia and vaginitis caused cryptic complex species of C. glabrata were attributable to by this species [24, 26, 27], while a combination of VRC C. nivariensis. Although, most of studies have shown a low clinical plus MCF followed by micafungin alone [23] or CSP plus prevalence of C. bracarensis and C. nivariensis [10], the preva- catheter removal [27] for candidemia cases and oral VCZ lence we reported here is in agreement with the study conducted [24] for recurrent vaginitis cases showed successful treat- in Poland, where they did not find C. bracarensis, while they ment outcomes. A higher efficacy of MCF for clearance of showed that C. nivariensis constituted 6% of clinical isolates of infections caused by C. glabrata might be behind the fact C. glabrata [20]. The lower clinical prevalence of C. nivariensis that this drug causes mutations in HS1 of FKS1 and FKS2 and C. bracarensis is partially explained by genomic studies, approximately 11 times less than CSP and five times AND where it was shown that these two species are more closely [28]. Possible misidentification cases of C. nivariensis and related to the non-pathogenic members of the Nakaeseomyces C. bracarensis as C. glabrata and high MIC values for azole clades than to C. glabrata [8]. However, accelerated evolution of drugs might prompt clinicians to use echinocandins for their genome enable them to become more virulent [8] and with treatment of candidemia cases caused by C. nivariensis. On an increase in the population of immunocompromised patients the other hand, mutations in HS1 of FKS1 and FKS2 are a might lead to a higher prevalence of infections pertained to these predictor of therapeutic failure of echinocandins against two species. Moreover, all pathogenic attributes of C. glabrata C. glabrata [29], hence, screening of mutations in HS1 of are present even in environmental (non-pathogenic) species of FKS1 and FKS2 could be an imperative initiative to detect Nakaseomyces clade, for example N. delphensis [8]. Furthermore, resistance isolates of C. nivariensis and C. bracarensis to N. delphensis compared to clinical isolates of C. bracarensis was echninocandins. Lack of amino acid substitution in the HS1 found to be pan-azole-resistant (FLC=128, ITC>16, PSC>8, of FKS1 and FKS2 was in agreement with low MIC values −1 VRC>4 µg ml ) and its MIC values for echinocandins and AMB obtained for aniulafungin and micafungin. were higher or equal to that of C. bracarensis [21]. As a result, expanding appropriate and accurate means of identifications Where is the original biological niche of will result in a more comprehensive view on the distribution C. nivariensis? and epidemiology, antifungal susceptibility patterns, and even Comparative genomic studies have disclosed that C. glabrata pathogenicity of species within the Nakaseomyces clade. is well-adapted to the human gut as its main biological niche, Various genotypes of C. glabrata proved to differ in their while C. nivariensis might have adapted to other environmental antifungal susceptibility and mortality rates [22], highlighting niches [8]. This is in line with the findings of environmental 775 Arastehfar et al., Journal of Medical Microbiology 2019;68:770–777 were signed by patients and in order to prevent exposing their informa- sampling studies from Thailand that isolated C. nivariensis tion, clinical data and isolates derived from them were designated with from leaves of sugarcane [30], barks and soil [10], while specific codes. C. glabrata was not cultured. So far, all C. bracarensis isolates are from clinical sources, and the majority of C. nivariensis References 1. Lockhart SR, Iqbal N, Cleveland AA, Farley MM, Harrison LH isolates has a clinical source. This might raise the question if et  al. Species identification and antifungal susceptibility testing these two species are part of human mycobiota and they will of Candida bloodstream isolates from population-based surveil- invade the bloodstream, once there is an opportunity. This lance studies in two U.S. cities from 2008 to 2011. 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