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Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile G. Sadeghi a, M. Ebrahimi-Rad b, S.F. Mousavi c, M. Shams-Ghahfarokhi d, M. Razzaghi-Abyaneh a,* a
Department of Medical Mycology, Pasteur Institute of Iran, 1316943551 Tehran, Iran Department of Biochemistry, Pasteur Institute of Iran, 1316943551 Tehran, Iran c Department of Microbiology, Pasteur Institute of Iran, 1316943551 Tehran, Iran d Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, 14115-331 Tehran, Iran b
Received 17 May 2017; received in revised form 5 December 2017; accepted 15 December 2017
KEYWORDS Candidiasis; Non-Candida albicans species; ITS sequencing; Antifungal susceptibility; Broth microdilution; Fluconazole
Summary Objective. — Non-Candida albicans (NCA) species now account for a significant part of clinical candidiasis worldwide. In the present study, epidemiology and antifungal susceptibility profile of NCA isolated from various forms of candidiasis were studied with special focus on their phylogenetic relationship by ITS sequencing. Patients and methods. — Seventy-nine NCA isolates were isolated from skin and nail scrapings (67.0%), vaginal discharges (8.8%), blood (8.8%), sputa (5.0%), urine (5.0%), oral swabs (2.6%), biopsy and eye tumor, each (1.4%). These isolates were identified by morphological, biochemical and molecular (ITS sequencing) techniques. In vitro antifungal susceptibility of the isolates to fluconazole (FCZ) was tested according to the CLSI method (M27-S4). Results. — Among a total number of 79 cases of proven NCA infections, C. parapsilosis (36.8%) was the most prevalent species followed by C. glabrata (32.9%), C. orthopsilosis (11.4%), C. tropicalis (8.9%), C. krusei (5.0%) and C. guilliermondii (5.0%). The susceptibility to FCZ was assessed for C. parapsilosis (96.5%), C. orthopsilosis (88.9%), C. tropicalis (85.7%) and C. guilliermondii (50.0%). C. glabrata and C. krusei isolates were not susceptible to FCZ. NCA species were distributed in various phylogenetic clades including C. glabrata (1), C. tropicalis (3), C. parapsilosis (6) and C. orthopsilosis, C. krusei and C. guilliermondii (each 2).
* Corresponding author. E-mail addresses: [email protected], [email protected] (M. Razzaghi-Abyaneh). https://doi.org/10.1016/j.mycmed.2017.12.008 1156-5233/# 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Sadeghi G, et al. Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2017.12.008
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G. Sadeghi et al. Conclusion. — C. parapsilosis and C. glabrata were the most predominant NCA species involve in the etiology of candidiasis. C. orthopsilosis was reported from superficial candidiasis. Taken together, our results further substantiate the increasing importance of the involvement of NCA species in the etiology of candidiasis. # 2017 Elsevier Masson SAS. All rights reserved.
Introduction Candida species are widely distributed in nature and act as common saprophytic constituents of the normal human microflora. Some of these fungi can become opportunistic pathogens following a transition from a commensal to a pathogenic phase induced by alteration in the host environment. A number of factors have been implicated in this increased occurrence of candidiasis, but it is generally accepted that the increased and widespread use of certain medical practices, such as immunosuppressive therapies, invasive surgical procedures and use of broad-spectrum antibiotics are significant. Candidiasis can be divided into three groups: cutaneous (skin and its appendages), mucosal (oropharyngeal, esophageal, and vulvovaginal) and systemic (bloodstream infections, i.e., candidemia and other forms of invasive candidiasis). It currently represents the fourth leading cause of nosocomial infections, at 10%, and mortality due to systemic candidiasis remains high, ranging from 15 to 35% depending on the infecting Candida species [1]. Global surveillance programs (e.g. SENTRY and ARTEMIS) provide a tremendous amount of data regarding global trends in various aspects of NCA candidiasis including geographical variation in the frequency of species, distribution by type and age, as well as changes in the antifungal susceptibility of collected NCA isolates. Whereas, NCA species accounted for 10—40% of all systemic candidiasis from 1970 to 1990, this proportion reached 35—65% in the last two decades. A recent ten-year analysis of the worldwide distribution of these species indicated that C. glabrata remains the most common species and C. parapsilosis, C. tropicalis, C. krusei are frequently isolated. Significant geographic variation in the frequency of NCA species occurs. It has been shown that C. glabrata is more prominent in North America than Latin America; C. tropicalis was frequently isolated in Asia-Pacific, whilst C. parapsilosis remained more commonly recovered in North America than in Europe [1,2]. Candida albicans is the main cause of candidiasis. However, surveys from different institutions, cities, countries, and broad geographic regions have documented the emergence of the various NCA species as well as their potential to develop antifungal resistance [3— 5]. The apparent increased involvement of NCA species in human candidiasis may partly is related to improvements in diagnostic methods, such as morphological characteristics (germ tube test, chlamydospore development, chromogenic media with the ability to differentiate Candida species), carbohydrate assimilation as well as the introduction of molecular techniques in the routine diagnosis of fungaemia. Besides the conventional identification techniques, molecular-based technologies such as targeting yeast rDNA gene, operon, encoding the 18S, 5.8S, and 28S rDNA gene subunits, namely Internal Transcribed Spacer1 (ITS1), ITS2 and ITS4 is a
very effective method for confirmation of identity and phylogenetic analysis of Candida species [6]. In vitro antifungal susceptibility testing now plays an increasingly important role in guiding therapeutic decision making as an aid in drug development studies, and as a means of tracking the change of antifungal resistance in epidemiological studies. Microdilution methods are the gold standard or reference techniques. Azole antifungals such as FCZ are often preferred treatment for many Candida infections as they are inexpensive, exhibit limited toxicity and are available for oral administration. Over the past 10 years, there is extensive documentation of intrinsic and developed resistance to azole antifungals, especially FCZ among several Candida species mainly C. glabrata and C. krusei isolates [7]. The susceptibility pattern of Candida species to FCZ may be changed following by the increasing use of this antifungal, so in vitro susceptibility testing is necessary to help clinicians in the selection of appropriate therapy [8]. The present study represents the distribution and molecular epidemiology of non-Candida albicans species isolated from various clinical samples and evaluates the in vitro antifungal susceptibility of isolated Candida species to fluconazole.
Patients and methods Patients During the year 2014, 140 cases suspected to different types of candidiasis who referred to our department were studied. Clinical samples were prepared and examined according to the standard protocols.
Isolation and conventional identification of Candida species A portion of the specimens was inoculated on Sabouraud dextrose agar medium (SDA; E. Merck, Germany) supplemented by chloramphenicol (0.05 gL 1) and incubated at 37 8C for 24—48 h to generate the Candida species. All isolates were identified by a combination of morphological and biochemical criteria by germ tube test, chlamidospore formation, carbohydrate assimilation test by the commercial system ID32 C (biomérieux Marcy l’Etoile, France) and CHROMagar Candida (CHROMagar Candida, Paris, France). Reference strain of C. albicans ATCC 10231 was used as control in all later experiments.
Molecular identification of Candida species For DNA extraction, fresh colonies were collected upon culturing the isolates on SDA for 48 h at 37 8C. Genomic DNA was extracted using the Molecular Biology kit (Bio Basic
Please cite this article in press as: Sadeghi G, et al. Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2017.12.008
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Inc, Canada) according to the manufacturer’s instructions and stored at 20 8C until used. Amplification of the Internal Transcribed Spacer regions ITS1 and ITS4 was accomplished by adding 2 mL of the sample DNA, 0.4 mL (20 pmol/mL) of each 5’-TCCGTAGGTGAACCTGCGG-3’ (forward) and 5’-TCCTCCGCTTATTGATATGC-3’ (reverse), 9.7 mL deionized water, and 12.5 mL (250 U) of the PCR Master Kit (SinaClon Co.). The amplification was performed using the following protocol: 95 8C for 12 min, 30 cycles of 95 8C for 30 s, 54 8C for 30 s, and 72 8C for 100 s, and a final step at 72 8C for 10 min [9]. The amplicons were purified and the subsequent sequencing reaction was performed using Big Dye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI PRISM 377 Genetic Analyzer (Applied Biosystems). Nucleotide sequences were defined by alignment of forward and reverse sequences using MEGA version 7.0. Phylogenic analyses were conducted by unweighted pair group method with arithmetic averages (UPGMA) and neighbor-joining algorithms based on p-distance. The value of the cluster nodes was determined by bootstrapping with 1000 randomizations [10].
Antifungal susceptibility testing ALL the NCA isolates were tested for in vitro susceptibility to FCZ in sterile, disposable 96-well U shaped microplates as described by Clinical Laboratory Standard Institute (CLSI) document M27-S4 and epidemiological cutoff values (ECV) [11,12]. Reference powder (Sigma Aldrich, USA) of FCZ was used. Stock antifungal drug solution was prepared in dimethyl sulfoxide. It was diluted with RPMI 1640 with L-glutamine, without bicarbonate (PAA, Austria) supplemented with 2% dextrose and buffered to pH 7.0 with 0.165 M N-morpholinopropansulfonic acid (MOPS) (Sigma-Aldrich, USA) to obtain 2fold of the final concentration. The final concentration of the antifungal drugs in microplates was 0.031—64 mg/mL for FCZ. The drug containing microplate was inoculated with a suspension of yeast cells adjusted to the turbidity of a 0.5 McFarland standard and incubated for 24 h at 35 8C. MIC end points were read at the end of incubation period. Drug-free and yeast-free controls were included in the tests. Following incubation, the MIC was defined as the lowest concentration that produced a prominent decrease in turbidity compared with that of the drug-free controls. According to the M27-S4 document, the endpoint for FCZ was considered for the classification of the all isolates according to the MICs (mg/mL) as resistant (R): 8, susceptible dose dependent (SDD): 4 and susceptible(S): 2, except for C. glabrata that interpretive breakpoints was SDD: 32 and R: 64. As these parameters were not provided for C. krusei and C. guilliermondii in M27-S4 protocol [11], we used the epidemiological cutoff values for interpreting of the susceptibility of these species distributed in wild type or S: 2 and non-wild type or R > 2 [12].
Figure 1 ITS neighbour-joining tree showing the phylogenetic relationship among NCA isolates. Bootstrap percentages from 1000 replicates are shown in each node. Scale bar indicates the number of differences.
Results Patients The demographic information of NCA isolated obtained in our study is shown in Table 1. The mean age of patients with NCA
candidiasis was 46.5 years ranged from 5 to 88 years. Male to female gender ratio was 21/58. Cutaneous candidiasis comprised of nail specimens distributed in fingernail (40 cases, 75.5%) and toenail (11 cases, 20.7%) and the skin (2 cases, 3.8%).
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G. Sadeghi et al. Table 1 The detail of demographic characteristics of yeast strains and antifungal susceptibility pattern to fluconazole (FCZ) according to (CLSI) M27-S4 breakpoints. Candida Isolate No. 93-207 93-709 93-915 89-1179 93-811 177-671 176-749 202-645 184-743 215-651 206-213 171-662 178-495 207-494 168-597 173-658 165-659 174-750 204-496 203-605 90-1847 93-1270 93-2513 90-1482 89-3033 89-3031 93-156 93-187 93-335 93-640 93-771 93-1253 93-1377 93-1426 93-1443 93-1529 93-1560 93-1605 93-1844 93-2178 93-2327 93-2343 93-2480 93-2588 93-1434 93-173 93-358 93-466 93-1425 93-1610 93-1906 93-2097 93-2160 93-2405 89-772
Patient Sex-age F-33 M-73 F-36 F-30 M-42 F-40 F-39 M-68 M-63 F-50 F-52 F- 48 M-87 M-80 F-60 M-87 F-78 F-86 M-88 F-86 F-32 F-75 F-34 M-60 F-27 F-67 F-50 F-58 F-58 M-69 F-26 F-35 F-59 F-75 M-32 F-32 F-62 F-50 M-70 F-46 F-51 F-38 F-58 F-5 M-29 F-42 M-6 F-67 F-40 F-55 F-35 F-57 F-65 F-53 F-23
Species
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis
Specimens
Fingernail Fingernail Vaginal discharge Fingernail Biopsy Urine Blood Blood Blood Urine Vaginal discharge Urine Blood Blood Vaginal discharge Urine Vaginal discharge Blood Blood Vaginal discharge Vaginal discharge Sputum Sputum Groin Vaginal discharge Pharynx Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Leg palm Toenail Toenail Toenail Toenail Toenail Toenail Toenail Toenail Toenail
Type of candidiasis
Cutaneous Cutaneous Vulvovaginal Cutaneous Invasive Candiduria Candidemia Candidemia Candidemia Candiduria Vulvovaginal Candiduria Candidemia Candidemia Vulvovaginal Candiduria Vulvovaginal Candidemia Candidemia Vulvovaginal Vulvovaginal Oropharyngeal Oropharyngeal Cutaneous Vulvovaginal Oropharyngeal Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous
FCZ MIC (mg/mL)
Susceptibility pattern
0.25 2.0 0.25 64.0 0.5 1.0 2.0 1.0 2.0 0.25 2.0 4.0 2.0 2.0 1.0 2.0 4.0 2.0 1.0 0.25 8.0 2.0 16.0 32.0 16.0 8.0 1.0 0.25 0.5 2.0 1.0 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5 0.25 0.5 0.25 0.25 0.125 0.25 0.25 0.5 1.0 0.5 1.0 0.5 2.0 0.5 1.0 8.0
SDD SDD SDD R SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD S S S S S S S S S S S S S S S S S S S S S S S S S S S S R
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Table 1 (Continued )
Candida Isolate No. 93-1098 93-2713 93-701 93-936 93-1581 93-1729 93-2183 93-2212 93-2624 93-1357 93-1410 93-1857 93-2030 93-2040 93-2131 93-2191 93-1373 89-3001 90-1767 89-256 93-79 93-2742 93-2610 93-2699
Patient Sex-age F-55 M-57 F-33 F-24 F-52 F-71 F-21 F-37 F-33 F-43 F-57 F-67 F-42 F-40 F-41 F-32 M-68 M-64 M-72 M-62 M-66 F-54 F-68 M-32
Species
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis tropicalis tropicalis tropicalis tropicalis tropicalis tropicalis tropicalis krusei krusei krusei krusei guilliermondii guilliermondii guilliermondii guilliermondii
Specimens
Type of candidiasis
Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Mouth Eye tumor Sputum Sputum Fingernail Fingernail Toenail Toenail
Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Oropharyngeal Ocular Oropharyngeal Oropharyngeal Cutaneous Cutaneous Cutaneous Cutaneous
FCZ MIC (mg/mL)
Susceptibility pattern
1.0 1.0 0.25 0.5 1.0 4.0 1.0 1.0 1.0 0.25 0.25 0.5 0.5 1.0 0.5 16.0 0.5 64 64 64 64 16 0.25 0.0625
S S S S S SDD S S S S S S S S S R
S: susceptible; SDD: susceptible dose dependent; R: resistant, ( ) breakpoints not provided by CLSI documents M27-S4.
Isolation and conventional identification of Candida species As shown in Table 2, a total number of 79 NCA isolates identified in this study were distributed in skin and nail scrapings (67.0%), vaginal discharge (8.8%), blood (8.8%), sputum (5.0%), urine (5.0%), oral swab (2.6%), tissue biopsy (1.4%) and eye tumor samples (1.4%). The predominant NCA species on the basis of biochemical and molecular methods were C. parapsilosis (36.8%, 29/79), C. glabrata (32.9%, 26/79), C. orthopsilosis (11.4%, 9/79), C. tropicalis (8.9%, 7/79), C. krusei (5.0%, 4/79) and C. guilliermondii (5.0%, 4/79). Table 2
Molecular identification of Candida species All the C. orthopsilosis isolates were identified by ITS sequencing method, individually. The most common species in systemic infections was C. glabrata and in superficial candidiasis, C. parapsilosis. No C. metapsilosis strains were found. Besides that, identification of species by conventional methods was confirmed by typing of ITS genes in most cases, all of the C. orthopsilosis isolates were detected by this method, exclusively (Fig. 1). ITS sequences obtained in this study were aligned and a neighbor-joining tree was generated to monitor the clusters. In the phylogenic tree, there is a consistent separation among 79 isolates belonging to 6 dif-
Distribution of Candida species among clinical specimens.
Specimen Type Skin and nail scraping Vaginal discharge Sputum Urine Blood Biopsy Eye tumor Oral swabs Total
No.
53 7 4 4 7 1 1 2 79
Candida species C. parapsilosis
C. tropicalis
C. orthopsilosis
C. glabrata
C. krusei
C. guilliermondii
29 0 0 0 0 0 0 0 29
7 0 0 0 0 0 0 0 7
9 0 0 0 0 0 0 0 9
4 7 2 4 7 1 0 1 26
0 0 2 0 0 0 1 1 4
4 0 0 0 0 0 0 0 4
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ferent Candida species. Furthermore, we were able to observe low intra-specific variability (Fig. 1). There was no obvious relationship between phylogenetic distribution of Candida isolates and FCZ susceptibility profile. As shown in Fig. 1, the number of clades for each species included C. glabrata; 1, C. tropicalis; 3, C. orthopsilosis; 2, C. krusei; 2, C. parapsilosis; 6 and C. guilliermondii; 2. Totally, the NCA species aligned together in two main clusters apart from each other: one cluster containing C. guilliermondii strains and another that included the rest.
Antifungal susceptibility testing According to the results of antifungal susceptibility tests against FCZ shown in Table 3, the rank order of the six isolated NCA species according to FCZ susceptibility was C. parapsilosis (96.5%) > C. orthopsilosis (88.9%) > C. tropicalis (85.7%) > C. guilliermondii (75.0%) > C. glabrata and C. krusei (0). The most of C. glabrata isolates were susceptible dose dependent (SDD) (96.2%). All the C. krusei strains were resistant to FCZ.
Discussion In this study, 79 strains belonging to six NCA species were identified as the etiologic agents of candidiasis in suspected individuals referred to Department of Medical Mycology, Pasteur Institute of Iran. Overview of the literature from the last four decades highlights that the proportion of NCA species among Candida isolates recovered from patients is increasing in many countries, especially among patients with immunosuppressive disorder [2,4,13—18]. Our results showed that C. parapsilosis was the main cause among NCA species, especially in nail and skin specimens while C. glabrata was the most prevalent NCA species in systemic infections. These results are in accordance with the published reports from China, Turkey, Brazil and India but opposite to the results from Zambia which C. lusitaniae was the most common [19—23]. Resistance to FCZ has been shown to be more common in NCA species compared to C. albicans isolates in a population based laboratory studies such as C. krusei to FCZ and the greater tendency of species such as C. glabrata to develop antifungal resistance [24,25]. Our results showed that the rate of resistance to FCZ was lower than that of reported in Turkey, India, Zambia and China [19—23]. Table 3
In vitro susceptibility testing of 79 Candida isolates to fluconazole.
Candida species (No.)
Fluconazole MIC range (mg/mL)
Candida Candida Candida Candida Candida Candida
In the present study, accordance with previous reports from Iran, C. glabrata was the frequent NCA species in vaginal discharge specimens [25—28]. The most predominant NCA species in onychomycosis was C. parapsilosis whereas in a previous report from Iran by Khosravi et al., C. glabrata identified as the main etiologic agent [29]. These results were in accordance with the published reports from Iran by Mohammadi et al. [30] and Zaini et al. [31], and in contrast to Jafari-Nodoushan et al. [8] where C. tropicalis was reported as the most common NCA species from deferent forms of clinical specimens. In reports by Feng et al. [32] and Mohammadi et al. [30], C. orthopsilosis was only isolated from cutaneous samples, suggesting a possible pathogenic role of the species in superficial infections. No C. metapsilosis strains were found in the present study. Based on recent data, C. metapsilosis is an environmental and less virulent organism, whereas C. parapsilosis and C. orthopsilosis have adapted to mammalian niches [30]. However, contrary to our finding, some studies found high prevalence of C. metapsilosis, accompanied with low frequency of C. orthopsilosis [33]. Superficial infections of the nail and skin due to C. tropicalis are rarely seen [34], but in our study all of C. tropicalis strains were isolated from cases of onychomycosis. Candida guilliermondii is an infrequent species worldwide. However, as reported also by Kiraz et al. [35] and Mohammadi et al. [30], this species had the lowest rate of isolation (5.0%) in the present study. In the present study, all Candida isolates detected as C. parapsilosis by ID32C test were confirmed as C. orthopsilosis by ITS sequencing. Other Candida species were identical in both methods. A neighbor-joining tree generated from ITSsequencing showed the distribution of 79 NCA species in distinct identifiable branches. Low intra-specific variability was identified. The genetic relatedness between C. glabrata, C. tropicalis, C. orthopsilosis and C. krusei isolates were more evidenced than that of other species including C. parapsilosis and C. guilliermondii. In the present study, antifungal susceptibility of isolated NCA species to FCZ as a choice drug to cure a wide range of clinical candidiasis from superficial to mucosal and systemic was evaluated. In the Asia-Pacific region, FCZ resistance in C. tropicalis ranges from 0 to as high as 83%. The worldwide incidence of FCZ resistance in C. parapsilosis isolated from disseminated infections ranged between 2 and 5%. As C. krusei exhibits intrinsic resistance to FCZ, there is some
parapsilosis (29) orthopsilosis (9) krusei (4) glabrata (26) tropicalis (7) guilliermondii (4)
0.125—8 0.25—4 64 0.25—64 0.25—16 0.0625—16
Geometric mean
0.512095 0.925875 64 2.166528 0.742997 0.707106
Susceptibility (%) Resistant
Susceptible Dose Dependent
Susceptible
3.5 11.1
0 0
96.5 88.9
3.8 14.3
96.2 0
0 85.7
MIC: minimum inhibitory concentration, ( ) breakpoints not provided by CLSI documents M27-S4.
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Emergence of non-albicans Candida species controversy whether its increased infection rate is related to FCZ prophylaxis or previous treatment [31,36,37]. Susceptibility pattern of our Candida species to FCZ revealed that the most sensitive agent was C. parapsilosis (96.5%). All of C. krusei isolates were resistant. 96.2% of C. glabrata strains were susceptible-dose-dependent. Also, these patterns have been reported by Matehkolaei et al. [38]. In previous records from Iran, all of NCA isolates except to C. glabrata were susceptible to FCZ, completely [29]. Among the NCA species, C. tropicalis was usually less susceptible to FCZ than C. parapsilosis. C. glabrata is intrinsically more resistant to antifungal drugs especially to FCZ. Comparable pattern has been reported by Pereira et al. [24] and Mohammadi et al. [39]. Although FCZ was reported as the good agent in our report affecting the growth of the majority of isolates, however, resistance to this drug was common among isolates of C. krusei. There was no significant correlation between drug resistance and isolate location in neighbor-joining tree. Taken together, our results clearly show that NCA species gain increasing importance as the etiologic agents of different clinical forms of candidiasis from superficial to systemic as reported earlier by us and by other researchers [4,24,40,41]. We showed that ITS sequencing is a very useful method for confirmation of identity of Candida isolates initially identified by conventional methods at species level. All the C. orthopsilosis isolates were exclusively identified by this method. The present study is a new report implying the importance of C. orthopsilosis and C. tropicalis in the etiology of superficial candidiasis in Iran. While FCZ has provided a long and effective treatment for Candida-related infections, our results indicate that FCZ resistance in some Candida species can be considered as a problem of critical importance in the clinical setting. The regular investigation of antifungal susceptibility in medical centers should be performed in order to promote the effective management of candidiasis. Moreover, as causative agents in candidiasis vary according to geographical area, treatment should be based on studies carried out regularly in the same regions through well-planned studies.
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[5]
[6]
[7]
[8]
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[10]
[11]
[12]
[13]
[14]
Disclosure of interest
[15]
The authors declare that they have no competing interest.
[16]
Acknowledgements This work was financially supported by the Pasteur Institute of Iran. The authors wish to gratefully thank all the staffs of Medical Mycology Department of Pasteur Institute of Iran for their invaluable helps in patient sampling.
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[32] Feng X, Ling B, Yang G, Yu X, Reu D, Yao Z. Prevalence and distribution profiles of Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis responsible for superficial candidiasis in a Chinese university hospital. Mycopathologia 2012;173:229—34. [33] Ge YP, Bockhout T, Zhan P. Characterization of the Candida parapsilosis complex in East china; species distribution differs among cities. Med Mycol 2012;50:56—66. [34] Chai LY, Denning DW, Warn P. Candida tropicalis in human disease. Crit Rev Microbiol 2010;36:282—98. [35] Kiraz N, Oz Y. Species distribution and in vitro antifungal susceptibility of clinical Candida isolates from a university hospital in Turkey over a 5-year period. Med Mycol 2011;49: 126—31. [36] Gong X, Luan T, Wu X, Li G, Qiu H, Kang Y, et al. Invasive candidiasis in intensive care units in China; risk factors and prognoses of Candida albicans and non- albicans Candida infections. Am J Infect Control 2016;44:e59—63. [37] Pfaller MA, Rhomberg PR, Messer SA, Jones RN, Castanbeira M. Isavuconazole, micafungin, and 8 comparator antifungal agents,susceptibility profiles for common and uncommon opportunistic fungi collected in 2013; temporal analysis of antifungal drug resistance using CLSI species-specific clinical breakpoints and proposed epidemiological cutoff values. Diagn Microbial Infect Dis 2015;82:303—13. [38] Matehkolaei AR, Shafiei S, Zarei-Mahmoudabadi A. Isolation, molecular identification, and antifungal susceptibility profiles of vaginal isolates of Candida species. Iran J Microbiol 2016;8:410—7. [39] Mohammadi R, Ataei B. Candidiasis in pediatrics; identification and in vitro antifungal susceptibility of the clinical isolates. Iranian J Pediat Hematol Oncol 2015;6:43—51. [40] Sadeghi G, Abouei M, Alirezaee M, Tolouei R, Shams-Ghahfarokhi M, Razzaghi-Abyaneh M. A 4-year survey of dermatomycoses in Tehran from 2006—2009. J Mycol Med 2011;21: 260—5. [41] Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD. Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front Microbiol 2017;7:1—12.
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MYCMED-756; No. of Pages 8 Journal de Mycologie Médicale (2017) xxx, xxx—xxx
Available online at
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile G. Sadeghi a, M. Ebrahimi-Rad b, S.F. Mousavi c, M. Shams-Ghahfarokhi d, M. Razzaghi-Abyaneh a,* a
Department of Medical Mycology, Pasteur Institute of Iran, 1316943551 Tehran, Iran Department of Biochemistry, Pasteur Institute of Iran, 1316943551 Tehran, Iran c Department of Microbiology, Pasteur Institute of Iran, 1316943551 Tehran, Iran d Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, 14115-331 Tehran, Iran b
Received 17 May 2017; received in revised form 5 December 2017; accepted 15 December 2017
KEYWORDS Candidiasis; Non-Candida albicans species; ITS sequencing; Antifungal susceptibility; Broth microdilution; Fluconazole
Summary Objective. — Non-Candida albicans (NCA) species now account for a significant part of clinical candidiasis worldwide. In the present study, epidemiology and antifungal susceptibility profile of NCA isolated from various forms of candidiasis were studied with special focus on their phylogenetic relationship by ITS sequencing. Patients and methods. — Seventy-nine NCA isolates were isolated from skin and nail scrapings (67.0%), vaginal discharges (8.8%), blood (8.8%), sputa (5.0%), urine (5.0%), oral swabs (2.6%), biopsy and eye tumor, each (1.4%). These isolates were identified by morphological, biochemical and molecular (ITS sequencing) techniques. In vitro antifungal susceptibility of the isolates to fluconazole (FCZ) was tested according to the CLSI method (M27-S4). Results. — Among a total number of 79 cases of proven NCA infections, C. parapsilosis (36.8%) was the most prevalent species followed by C. glabrata (32.9%), C. orthopsilosis (11.4%), C. tropicalis (8.9%), C. krusei (5.0%) and C. guilliermondii (5.0%). The susceptibility to FCZ was assessed for C. parapsilosis (96.5%), C. orthopsilosis (88.9%), C. tropicalis (85.7%) and C. guilliermondii (50.0%). C. glabrata and C. krusei isolates were not susceptible to FCZ. NCA species were distributed in various phylogenetic clades including C. glabrata (1), C. tropicalis (3), C. parapsilosis (6) and C. orthopsilosis, C. krusei and C. guilliermondii (each 2).
* Corresponding author. E-mail addresses: [email protected], [email protected] (M. Razzaghi-Abyaneh). https://doi.org/10.1016/j.mycmed.2017.12.008 1156-5233/# 2017 Elsevier Masson SAS. All rights reserved.
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G. Sadeghi et al. Conclusion. — C. parapsilosis and C. glabrata were the most predominant NCA species involve in the etiology of candidiasis. C. orthopsilosis was reported from superficial candidiasis. Taken together, our results further substantiate the increasing importance of the involvement of NCA species in the etiology of candidiasis. # 2017 Elsevier Masson SAS. All rights reserved.
Introduction Candida species are widely distributed in nature and act as common saprophytic constituents of the normal human microflora. Some of these fungi can become opportunistic pathogens following a transition from a commensal to a pathogenic phase induced by alteration in the host environment. A number of factors have been implicated in this increased occurrence of candidiasis, but it is generally accepted that the increased and widespread use of certain medical practices, such as immunosuppressive therapies, invasive surgical procedures and use of broad-spectrum antibiotics are significant. Candidiasis can be divided into three groups: cutaneous (skin and its appendages), mucosal (oropharyngeal, esophageal, and vulvovaginal) and systemic (bloodstream infections, i.e., candidemia and other forms of invasive candidiasis). It currently represents the fourth leading cause of nosocomial infections, at 10%, and mortality due to systemic candidiasis remains high, ranging from 15 to 35% depending on the infecting Candida species [1]. Global surveillance programs (e.g. SENTRY and ARTEMIS) provide a tremendous amount of data regarding global trends in various aspects of NCA candidiasis including geographical variation in the frequency of species, distribution by type and age, as well as changes in the antifungal susceptibility of collected NCA isolates. Whereas, NCA species accounted for 10—40% of all systemic candidiasis from 1970 to 1990, this proportion reached 35—65% in the last two decades. A recent ten-year analysis of the worldwide distribution of these species indicated that C. glabrata remains the most common species and C. parapsilosis, C. tropicalis, C. krusei are frequently isolated. Significant geographic variation in the frequency of NCA species occurs. It has been shown that C. glabrata is more prominent in North America than Latin America; C. tropicalis was frequently isolated in Asia-Pacific, whilst C. parapsilosis remained more commonly recovered in North America than in Europe [1,2]. Candida albicans is the main cause of candidiasis. However, surveys from different institutions, cities, countries, and broad geographic regions have documented the emergence of the various NCA species as well as their potential to develop antifungal resistance [3— 5]. The apparent increased involvement of NCA species in human candidiasis may partly is related to improvements in diagnostic methods, such as morphological characteristics (germ tube test, chlamydospore development, chromogenic media with the ability to differentiate Candida species), carbohydrate assimilation as well as the introduction of molecular techniques in the routine diagnosis of fungaemia. Besides the conventional identification techniques, molecular-based technologies such as targeting yeast rDNA gene, operon, encoding the 18S, 5.8S, and 28S rDNA gene subunits, namely Internal Transcribed Spacer1 (ITS1), ITS2 and ITS4 is a
very effective method for confirmation of identity and phylogenetic analysis of Candida species [6]. In vitro antifungal susceptibility testing now plays an increasingly important role in guiding therapeutic decision making as an aid in drug development studies, and as a means of tracking the change of antifungal resistance in epidemiological studies. Microdilution methods are the gold standard or reference techniques. Azole antifungals such as FCZ are often preferred treatment for many Candida infections as they are inexpensive, exhibit limited toxicity and are available for oral administration. Over the past 10 years, there is extensive documentation of intrinsic and developed resistance to azole antifungals, especially FCZ among several Candida species mainly C. glabrata and C. krusei isolates [7]. The susceptibility pattern of Candida species to FCZ may be changed following by the increasing use of this antifungal, so in vitro susceptibility testing is necessary to help clinicians in the selection of appropriate therapy [8]. The present study represents the distribution and molecular epidemiology of non-Candida albicans species isolated from various clinical samples and evaluates the in vitro antifungal susceptibility of isolated Candida species to fluconazole.
Patients and methods Patients During the year 2014, 140 cases suspected to different types of candidiasis who referred to our department were studied. Clinical samples were prepared and examined according to the standard protocols.
Isolation and conventional identification of Candida species A portion of the specimens was inoculated on Sabouraud dextrose agar medium (SDA; E. Merck, Germany) supplemented by chloramphenicol (0.05 gL 1) and incubated at 37 8C for 24—48 h to generate the Candida species. All isolates were identified by a combination of morphological and biochemical criteria by germ tube test, chlamidospore formation, carbohydrate assimilation test by the commercial system ID32 C (biomérieux Marcy l’Etoile, France) and CHROMagar Candida (CHROMagar Candida, Paris, France). Reference strain of C. albicans ATCC 10231 was used as control in all later experiments.
Molecular identification of Candida species For DNA extraction, fresh colonies were collected upon culturing the isolates on SDA for 48 h at 37 8C. Genomic DNA was extracted using the Molecular Biology kit (Bio Basic
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Inc, Canada) according to the manufacturer’s instructions and stored at 20 8C until used. Amplification of the Internal Transcribed Spacer regions ITS1 and ITS4 was accomplished by adding 2 mL of the sample DNA, 0.4 mL (20 pmol/mL) of each 5’-TCCGTAGGTGAACCTGCGG-3’ (forward) and 5’-TCCTCCGCTTATTGATATGC-3’ (reverse), 9.7 mL deionized water, and 12.5 mL (250 U) of the PCR Master Kit (SinaClon Co.). The amplification was performed using the following protocol: 95 8C for 12 min, 30 cycles of 95 8C for 30 s, 54 8C for 30 s, and 72 8C for 100 s, and a final step at 72 8C for 10 min [9]. The amplicons were purified and the subsequent sequencing reaction was performed using Big Dye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI PRISM 377 Genetic Analyzer (Applied Biosystems). Nucleotide sequences were defined by alignment of forward and reverse sequences using MEGA version 7.0. Phylogenic analyses were conducted by unweighted pair group method with arithmetic averages (UPGMA) and neighbor-joining algorithms based on p-distance. The value of the cluster nodes was determined by bootstrapping with 1000 randomizations [10].
Antifungal susceptibility testing ALL the NCA isolates were tested for in vitro susceptibility to FCZ in sterile, disposable 96-well U shaped microplates as described by Clinical Laboratory Standard Institute (CLSI) document M27-S4 and epidemiological cutoff values (ECV) [11,12]. Reference powder (Sigma Aldrich, USA) of FCZ was used. Stock antifungal drug solution was prepared in dimethyl sulfoxide. It was diluted with RPMI 1640 with L-glutamine, without bicarbonate (PAA, Austria) supplemented with 2% dextrose and buffered to pH 7.0 with 0.165 M N-morpholinopropansulfonic acid (MOPS) (Sigma-Aldrich, USA) to obtain 2fold of the final concentration. The final concentration of the antifungal drugs in microplates was 0.031—64 mg/mL for FCZ. The drug containing microplate was inoculated with a suspension of yeast cells adjusted to the turbidity of a 0.5 McFarland standard and incubated for 24 h at 35 8C. MIC end points were read at the end of incubation period. Drug-free and yeast-free controls were included in the tests. Following incubation, the MIC was defined as the lowest concentration that produced a prominent decrease in turbidity compared with that of the drug-free controls. According to the M27-S4 document, the endpoint for FCZ was considered for the classification of the all isolates according to the MICs (mg/mL) as resistant (R): 8, susceptible dose dependent (SDD): 4 and susceptible(S): 2, except for C. glabrata that interpretive breakpoints was SDD: 32 and R: 64. As these parameters were not provided for C. krusei and C. guilliermondii in M27-S4 protocol [11], we used the epidemiological cutoff values for interpreting of the susceptibility of these species distributed in wild type or S: 2 and non-wild type or R > 2 [12].
Figure 1 ITS neighbour-joining tree showing the phylogenetic relationship among NCA isolates. Bootstrap percentages from 1000 replicates are shown in each node. Scale bar indicates the number of differences.
Results Patients The demographic information of NCA isolated obtained in our study is shown in Table 1. The mean age of patients with NCA
candidiasis was 46.5 years ranged from 5 to 88 years. Male to female gender ratio was 21/58. Cutaneous candidiasis comprised of nail specimens distributed in fingernail (40 cases, 75.5%) and toenail (11 cases, 20.7%) and the skin (2 cases, 3.8%).
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G. Sadeghi et al. Table 1 The detail of demographic characteristics of yeast strains and antifungal susceptibility pattern to fluconazole (FCZ) according to (CLSI) M27-S4 breakpoints. Candida Isolate No. 93-207 93-709 93-915 89-1179 93-811 177-671 176-749 202-645 184-743 215-651 206-213 171-662 178-495 207-494 168-597 173-658 165-659 174-750 204-496 203-605 90-1847 93-1270 93-2513 90-1482 89-3033 89-3031 93-156 93-187 93-335 93-640 93-771 93-1253 93-1377 93-1426 93-1443 93-1529 93-1560 93-1605 93-1844 93-2178 93-2327 93-2343 93-2480 93-2588 93-1434 93-173 93-358 93-466 93-1425 93-1610 93-1906 93-2097 93-2160 93-2405 89-772
Patient Sex-age F-33 M-73 F-36 F-30 M-42 F-40 F-39 M-68 M-63 F-50 F-52 F- 48 M-87 M-80 F-60 M-87 F-78 F-86 M-88 F-86 F-32 F-75 F-34 M-60 F-27 F-67 F-50 F-58 F-58 M-69 F-26 F-35 F-59 F-75 M-32 F-32 F-62 F-50 M-70 F-46 F-51 F-38 F-58 F-5 M-29 F-42 M-6 F-67 F-40 F-55 F-35 F-57 F-65 F-53 F-23
Species
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata glabrata parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis parapsilosis
Specimens
Fingernail Fingernail Vaginal discharge Fingernail Biopsy Urine Blood Blood Blood Urine Vaginal discharge Urine Blood Blood Vaginal discharge Urine Vaginal discharge Blood Blood Vaginal discharge Vaginal discharge Sputum Sputum Groin Vaginal discharge Pharynx Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Leg palm Toenail Toenail Toenail Toenail Toenail Toenail Toenail Toenail Toenail
Type of candidiasis
Cutaneous Cutaneous Vulvovaginal Cutaneous Invasive Candiduria Candidemia Candidemia Candidemia Candiduria Vulvovaginal Candiduria Candidemia Candidemia Vulvovaginal Candiduria Vulvovaginal Candidemia Candidemia Vulvovaginal Vulvovaginal Oropharyngeal Oropharyngeal Cutaneous Vulvovaginal Oropharyngeal Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous
FCZ MIC (mg/mL)
Susceptibility pattern
0.25 2.0 0.25 64.0 0.5 1.0 2.0 1.0 2.0 0.25 2.0 4.0 2.0 2.0 1.0 2.0 4.0 2.0 1.0 0.25 8.0 2.0 16.0 32.0 16.0 8.0 1.0 0.25 0.5 2.0 1.0 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5 0.25 0.5 0.25 0.25 0.125 0.25 0.25 0.5 1.0 0.5 1.0 0.5 2.0 0.5 1.0 8.0
SDD SDD SDD R SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD SDD S S S S S S S S S S S S S S S S S S S S S S S S S S S S R
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Table 1 (Continued )
Candida Isolate No. 93-1098 93-2713 93-701 93-936 93-1581 93-1729 93-2183 93-2212 93-2624 93-1357 93-1410 93-1857 93-2030 93-2040 93-2131 93-2191 93-1373 89-3001 90-1767 89-256 93-79 93-2742 93-2610 93-2699
Patient Sex-age F-55 M-57 F-33 F-24 F-52 F-71 F-21 F-37 F-33 F-43 F-57 F-67 F-42 F-40 F-41 F-32 M-68 M-64 M-72 M-62 M-66 F-54 F-68 M-32
Species
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis orthopsilosis tropicalis tropicalis tropicalis tropicalis tropicalis tropicalis tropicalis krusei krusei krusei krusei guilliermondii guilliermondii guilliermondii guilliermondii
Specimens
Type of candidiasis
Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Fingernail Mouth Eye tumor Sputum Sputum Fingernail Fingernail Toenail Toenail
Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous Oropharyngeal Ocular Oropharyngeal Oropharyngeal Cutaneous Cutaneous Cutaneous Cutaneous
FCZ MIC (mg/mL)
Susceptibility pattern
1.0 1.0 0.25 0.5 1.0 4.0 1.0 1.0 1.0 0.25 0.25 0.5 0.5 1.0 0.5 16.0 0.5 64 64 64 64 16 0.25 0.0625
S S S S S SDD S S S S S S S S S R
S: susceptible; SDD: susceptible dose dependent; R: resistant, ( ) breakpoints not provided by CLSI documents M27-S4.
Isolation and conventional identification of Candida species As shown in Table 2, a total number of 79 NCA isolates identified in this study were distributed in skin and nail scrapings (67.0%), vaginal discharge (8.8%), blood (8.8%), sputum (5.0%), urine (5.0%), oral swab (2.6%), tissue biopsy (1.4%) and eye tumor samples (1.4%). The predominant NCA species on the basis of biochemical and molecular methods were C. parapsilosis (36.8%, 29/79), C. glabrata (32.9%, 26/79), C. orthopsilosis (11.4%, 9/79), C. tropicalis (8.9%, 7/79), C. krusei (5.0%, 4/79) and C. guilliermondii (5.0%, 4/79). Table 2
Molecular identification of Candida species All the C. orthopsilosis isolates were identified by ITS sequencing method, individually. The most common species in systemic infections was C. glabrata and in superficial candidiasis, C. parapsilosis. No C. metapsilosis strains were found. Besides that, identification of species by conventional methods was confirmed by typing of ITS genes in most cases, all of the C. orthopsilosis isolates were detected by this method, exclusively (Fig. 1). ITS sequences obtained in this study were aligned and a neighbor-joining tree was generated to monitor the clusters. In the phylogenic tree, there is a consistent separation among 79 isolates belonging to 6 dif-
Distribution of Candida species among clinical specimens.
Specimen Type Skin and nail scraping Vaginal discharge Sputum Urine Blood Biopsy Eye tumor Oral swabs Total
No.
53 7 4 4 7 1 1 2 79
Candida species C. parapsilosis
C. tropicalis
C. orthopsilosis
C. glabrata
C. krusei
C. guilliermondii
29 0 0 0 0 0 0 0 29
7 0 0 0 0 0 0 0 7
9 0 0 0 0 0 0 0 9
4 7 2 4 7 1 0 1 26
0 0 2 0 0 0 1 1 4
4 0 0 0 0 0 0 0 4
Please cite this article in press as: Sadeghi G, et al. Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2017.12.008
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ferent Candida species. Furthermore, we were able to observe low intra-specific variability (Fig. 1). There was no obvious relationship between phylogenetic distribution of Candida isolates and FCZ susceptibility profile. As shown in Fig. 1, the number of clades for each species included C. glabrata; 1, C. tropicalis; 3, C. orthopsilosis; 2, C. krusei; 2, C. parapsilosis; 6 and C. guilliermondii; 2. Totally, the NCA species aligned together in two main clusters apart from each other: one cluster containing C. guilliermondii strains and another that included the rest.
Antifungal susceptibility testing According to the results of antifungal susceptibility tests against FCZ shown in Table 3, the rank order of the six isolated NCA species according to FCZ susceptibility was C. parapsilosis (96.5%) > C. orthopsilosis (88.9%) > C. tropicalis (85.7%) > C. guilliermondii (75.0%) > C. glabrata and C. krusei (0). The most of C. glabrata isolates were susceptible dose dependent (SDD) (96.2%). All the C. krusei strains were resistant to FCZ.
Discussion In this study, 79 strains belonging to six NCA species were identified as the etiologic agents of candidiasis in suspected individuals referred to Department of Medical Mycology, Pasteur Institute of Iran. Overview of the literature from the last four decades highlights that the proportion of NCA species among Candida isolates recovered from patients is increasing in many countries, especially among patients with immunosuppressive disorder [2,4,13—18]. Our results showed that C. parapsilosis was the main cause among NCA species, especially in nail and skin specimens while C. glabrata was the most prevalent NCA species in systemic infections. These results are in accordance with the published reports from China, Turkey, Brazil and India but opposite to the results from Zambia which C. lusitaniae was the most common [19—23]. Resistance to FCZ has been shown to be more common in NCA species compared to C. albicans isolates in a population based laboratory studies such as C. krusei to FCZ and the greater tendency of species such as C. glabrata to develop antifungal resistance [24,25]. Our results showed that the rate of resistance to FCZ was lower than that of reported in Turkey, India, Zambia and China [19—23]. Table 3
In vitro susceptibility testing of 79 Candida isolates to fluconazole.
Candida species (No.)
Fluconazole MIC range (mg/mL)
Candida Candida Candida Candida Candida Candida
In the present study, accordance with previous reports from Iran, C. glabrata was the frequent NCA species in vaginal discharge specimens [25—28]. The most predominant NCA species in onychomycosis was C. parapsilosis whereas in a previous report from Iran by Khosravi et al., C. glabrata identified as the main etiologic agent [29]. These results were in accordance with the published reports from Iran by Mohammadi et al. [30] and Zaini et al. [31], and in contrast to Jafari-Nodoushan et al. [8] where C. tropicalis was reported as the most common NCA species from deferent forms of clinical specimens. In reports by Feng et al. [32] and Mohammadi et al. [30], C. orthopsilosis was only isolated from cutaneous samples, suggesting a possible pathogenic role of the species in superficial infections. No C. metapsilosis strains were found in the present study. Based on recent data, C. metapsilosis is an environmental and less virulent organism, whereas C. parapsilosis and C. orthopsilosis have adapted to mammalian niches [30]. However, contrary to our finding, some studies found high prevalence of C. metapsilosis, accompanied with low frequency of C. orthopsilosis [33]. Superficial infections of the nail and skin due to C. tropicalis are rarely seen [34], but in our study all of C. tropicalis strains were isolated from cases of onychomycosis. Candida guilliermondii is an infrequent species worldwide. However, as reported also by Kiraz et al. [35] and Mohammadi et al. [30], this species had the lowest rate of isolation (5.0%) in the present study. In the present study, all Candida isolates detected as C. parapsilosis by ID32C test were confirmed as C. orthopsilosis by ITS sequencing. Other Candida species were identical in both methods. A neighbor-joining tree generated from ITSsequencing showed the distribution of 79 NCA species in distinct identifiable branches. Low intra-specific variability was identified. The genetic relatedness between C. glabrata, C. tropicalis, C. orthopsilosis and C. krusei isolates were more evidenced than that of other species including C. parapsilosis and C. guilliermondii. In the present study, antifungal susceptibility of isolated NCA species to FCZ as a choice drug to cure a wide range of clinical candidiasis from superficial to mucosal and systemic was evaluated. In the Asia-Pacific region, FCZ resistance in C. tropicalis ranges from 0 to as high as 83%. The worldwide incidence of FCZ resistance in C. parapsilosis isolated from disseminated infections ranged between 2 and 5%. As C. krusei exhibits intrinsic resistance to FCZ, there is some
parapsilosis (29) orthopsilosis (9) krusei (4) glabrata (26) tropicalis (7) guilliermondii (4)
0.125—8 0.25—4 64 0.25—64 0.25—16 0.0625—16
Geometric mean
0.512095 0.925875 64 2.166528 0.742997 0.707106
Susceptibility (%) Resistant
Susceptible Dose Dependent
Susceptible
3.5 11.1
0 0
96.5 88.9
3.8 14.3
96.2 0
0 85.7
MIC: minimum inhibitory concentration, ( ) breakpoints not provided by CLSI documents M27-S4.
Please cite this article in press as: Sadeghi G, et al. Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2017.12.008
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Emergence of non-albicans Candida species controversy whether its increased infection rate is related to FCZ prophylaxis or previous treatment [31,36,37]. Susceptibility pattern of our Candida species to FCZ revealed that the most sensitive agent was C. parapsilosis (96.5%). All of C. krusei isolates were resistant. 96.2% of C. glabrata strains were susceptible-dose-dependent. Also, these patterns have been reported by Matehkolaei et al. [38]. In previous records from Iran, all of NCA isolates except to C. glabrata were susceptible to FCZ, completely [29]. Among the NCA species, C. tropicalis was usually less susceptible to FCZ than C. parapsilosis. C. glabrata is intrinsically more resistant to antifungal drugs especially to FCZ. Comparable pattern has been reported by Pereira et al. [24] and Mohammadi et al. [39]. Although FCZ was reported as the good agent in our report affecting the growth of the majority of isolates, however, resistance to this drug was common among isolates of C. krusei. There was no significant correlation between drug resistance and isolate location in neighbor-joining tree. Taken together, our results clearly show that NCA species gain increasing importance as the etiologic agents of different clinical forms of candidiasis from superficial to systemic as reported earlier by us and by other researchers [4,24,40,41]. We showed that ITS sequencing is a very useful method for confirmation of identity of Candida isolates initially identified by conventional methods at species level. All the C. orthopsilosis isolates were exclusively identified by this method. The present study is a new report implying the importance of C. orthopsilosis and C. tropicalis in the etiology of superficial candidiasis in Iran. While FCZ has provided a long and effective treatment for Candida-related infections, our results indicate that FCZ resistance in some Candida species can be considered as a problem of critical importance in the clinical setting. The regular investigation of antifungal susceptibility in medical centers should be performed in order to promote the effective management of candidiasis. Moreover, as causative agents in candidiasis vary according to geographical area, treatment should be based on studies carried out regularly in the same regions through well-planned studies.
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Disclosure of interest
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The authors declare that they have no competing interest.
[16]
Acknowledgements This work was financially supported by the Pasteur Institute of Iran. The authors wish to gratefully thank all the staffs of Medical Mycology Department of Pasteur Institute of Iran for their invaluable helps in patient sampling.
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Please cite this article in press as: Sadeghi G, et al. Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2017.12.008