Molecular epidemiology, risk factors, species distribution, antifungal susceptibility and outcome of candidemia in the capital of Iran: a prospective study

Background Candidemia is a major cause of morbidity and mortality among patients receiving immunosuppressive therapy and those hospitalized with serious underlying diseases despite the commencement of antifungal therapy. We investigated the molecular epidemiology, clinical characteristics, comorbidity risk factors, species distribution, antifungal susceptibility prole, and outcome of candidemia to provide appropriate perspectives in Tehran, Iran. Methods A prospective observational study of all patients diagnosed with candidemia was performed at two referral teaching hospitals in Tehran. Demographic characteristics, underlying diseases, risk factors, clinical symptoms, and laboratory analyses were mined. One-hundred and fty-two Candida isolates from 89 patients with candidemia were recovered. The overall crude mortality was 47.2%. The most common underlying disease was sepsis (48.3%) followed by malignancy (46.1%), renal failure/ dialysis (43.8%), and Hypertension (40.0%). C. albicans (43.8%) was the most frequent causative agent followed by C. glabrata (21.3%), C. parapsilosis complex (15.7%), C. tropicalis (11.2%), and C. lusitaniae (3.4%). Result of antifungal susceptibility test show that activity of all the four azole antifungal agents was low against non-albicans Candida species, especially C. tropicalis. candidemia. in 2018. All methods were performed in accordance with the relevant guidelines and regulations. The studied population included all culture-positive Candida BSI patients irrespective of their age or gender. In this study, we dened nosocomial candidaemia as the occurrence of one or more Candida species culture-positive blood drawn at least 48 h after admission. We excluded nosocomial candidemia episodes that represented relapses but included fresh episodes that occurred during separate admissions as new cases. The patient’s baseline characteristics, clinical, laboratory, and microbiological data were collected upon conrmation of candidemia. Data were extracted from the patients’ hospital records using a standardized case report form and included the baseline characteristics comorbidities malignancy, diseases, human immunodeciency virus infection, viral hepatitis, sepsis, and neutropenia neutrophil count < 500 cells/mm 3 ]); invasive procedures (including the insertion of a central venous catheter [CVC], nasogastric tube, urinary catheterization, immunosuppressive therapy, and intubation) and risk factors as total parenteral nutrition (TPN) 72 onset of candidemia, use of broad-spectrum antibiotics etc; antifungal therapy, and outcome parameters - hospital mortality death within 30 days candidemia episode). risk factor B, uconazole, itraconazole, voriconazole, posaconazole, and echinocandins in vitro, especially in patients without a history of exposure to antifungal agents [34]. A recent study looked at the impact of the new CLSI breakpoints and demonstrated that applying revised uconazole breakpoints increased the rate of uconazole resistance in C. albicans, C. tropicalis, and C. parapsilosis [35]. The occurrence of uconazole resistance in C. tropicalis has been previously reported as 5.0–7.2% from two reports from the ARTEMIS study over 12 years [36, 37]. The high rate of azole non-susceptible C. tropicalis in this study was similar to other studies from Asia [38, 39]. Also among the uconazole-resistant C. tropicalis isolates in our study, six were resistant to voriconazole. Many candidemia studies revealed a signicant increase in azole-resistant C. tropicalis blood isolates and some reported pan-azole and amphotericin B resistant isolates [40]. An extensive candidemia study in India showed that C. tropicalis and C. auris isolates also carry MDR trait [41]. The susceptible dose-dependent (SDD) C. glabrata isolate is dened as uconazole MIC ≤ 32 mg/l, since 2012. Overall, 96.7% of C. glabrata isolates were categorized as SDD (MIC 50 = 16 mg/l, MIC 90 = 32 mg/l) that is consistent with studies from the Asian-Pacic region [42]. As demonstrated in the current study, no resistance to any of the antifungal agents was observed in C. guiliermondii, C. lusitaniae, C. kefyr, and C. krusei (except for the intrinsic uconazole resistance in C. krusei). Furthermore, resistance to echinocandins was very low, except for C. parapsilosis, which exhibited higher MICs than those of other Candida species. The increased frequency of NAC may also be attributable to the improved diagnostic technique, NAC species characterized with methods


Introduction
Despite advancement in clinical patient care, Candida species remain the most commonly encountered pathogens isolated from bloodstream infections (BSIs) globally and are associated with signi cant morbidity and mortality particularly, among hospitalized patients receiving immunosuppressive therapy or diagnosed with a serious underlying health condition [1,2]. Depending on the yeast species, the mortality rate may vary from 30 to 85% [3,4]. Although in general, C. albicans is still the leading cause of candidemia, a shift towards non-albicans Candida (NAC) species has been reported in recent years [5]. The changing face of candidemia is alarming because NAC species might be associated with increased mortality and antifungal drug resistance [6,7]. Although empirical therapy partly depends on the epidemiological data, risk assessment for candidemia is critical for clinicians to commence appropriate empirical antifungal therapy [8]. There are several risk factors associated with candidemia such as exposure to broad-spectrum antibiotics, surgical procedures, prolonged use of central venous catheters, dialysis, use of corticosteroids, and cytotoxic chemotherapy, among others [9]. Again, the diagnosis of candidemia remains a challenging task, despite the development attained in the diagnosis of fungal BSIs during recent years [10][11][12]. Disparities in the epidemiology of candidemia exist between countries and this seriously in uences the need for continuous surveillance to monitor the trend of the disease, the species distribution, and the emergence of antifungal drug resistance [13]. Although some studies have reported determinants of mortality in candidemia, their results were based on retrospective data and from a restricted viewpoint [14][15][16][17]. Accordingly, the reasons for the current poor outcome of candidemia are based on inadequate data. With this idea in mind, we investigated the molecular epidemiology, clinical (age, sex); microbiological parameters (Candida species); comorbidities (diabetes mellitus, pulmonary disease, chronic renal failure/hemodialysis, malignancy, cardiovascular diseases, human immunode ciency virus [HIV] infection, viral hepatitis, sepsis, and neutropenia [absolute neutrophil count < 500 cells/mm 3 ]); invasive procedures (including the insertion of a central venous catheter [CVC], nasogastric tube, urinary catheterization, immunosuppressive therapy, and intubation) and other risk factors such as total parenteral nutrition (TPN) within 72 h prior to the onset of candidemia, clinical manifestations, use of broad-spectrum antibiotics etc; antifungal therapy, and outcome parameters -hospital mortality (i.e., death within 30 days of the rst documented candidemia episode). In cases where a patient had more than one episode of candidemia, the rst episode was used in the risk factor analysis.

Clinical specimens and identi cation of Candida species
Blood samples were aseptically obtained from patients with suspected bloodstream infections. The samples were inoculated in aerobic blood culture medium bottles (BacT/ALERT® Culture Media/bioMerieux) and incubated within the automated Bactec system (BACT/ALERT® 3D). Through observation, we identi ed and then subcultured the initial positive blood cultures onto Sabouraud dextrose agar (SDA) supplemented with 0.5% chloramphenicol and incubated at 37ºC for 24 to 48 hours. Yeast-like colonies were sub-cultured on CHROMagar Candida medium (CHROMagar Company, Paris, France) to ensure purity, and then identi ed using the automated Vitek 2 YST ID Card system (bioMérieux, Marcy-L'Etoile, France), according to the manufacturer's instructions [18]. Molecular identi cation was conducted for all recovered isolates. Brie y, we extracted the genomic DNA from cultures grown on SDA using the Genomic Extraction Kit (GeneAll, Korea), according to the manufacturer's instructions, and stored at − 20 °C till next use. The internal transcribed spacer rDNA region (ITS1-5.8S-ITS2) was ampli ed and sequenced using ITS1 and ITS4 primers, as previously described by Leaw et al. [19]. Thereafter, we performed a bidirectional chain terminated Sanger sequencing with the same primers used for the ampli cation. We processed the sequence data using the Lasergene SeqMan software (version 9.0.4, DNASTAR) and aligned the results with the data in the GenBank database (https://blast.ncbi. nlm.nih.gov) and the Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands) research database (http://www.westerdijkinstitute.nl/). Identi cation was de ned by > 99.5% sequence similarity, with ≥ 95% query coverage.

Antifungal susceptibility testing
Antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute broth microdilution guidelines (CLSI-M27-A3 and M60) [20,21]. All tests were performed in duplicate, on two different days. C. krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were used as quality control strains, as recommended by the CLSI. Data interpretation was based on clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs) [21,22].

Statistical analyses
We used the SPSS statistical software version 22.0 (SPSS Inc., Chicago, IL, USA) for statistical analysis in this study. Whereas the median, mean, standard deviation (SD), maximum, and minimum values were used to describe quantitative data frequencies were used to describe categorical data. The Chi-square test or Fisher's exact test was used to evaluate categorical variables and the Student's t-test to evaluate continuous variables. Logistic regression analyses were performed to identify independent variables associated with candidemia due to C. albicans and non-C. albicans spp. and the nal outcome.

Results:
3.     Table 2. This analysis revealed that malignancy was an independent risk factor for candidemia (P = 0.013). In the multivariate analyses of risk factors for BSIs mortality, intubation (P = 0.001) and urinary catheterization (P = 0.03) were independent risk factors for mortality (Table 3).

Discussion
We focused on candidemia patients and found that C. albicans was the most prevalent of the candidemia episodes (43.8%). Among the NAC species, C. glabrata was the predominant species, followed by C. parapsilosis, C. tropicalis, and C. lusitaniae. In most previous studies around the world, C. albicans was the most common species isolated from candidemia, which is consistent with our study [13]. Despite this, in a systematic review, C. parapsilosis (30.8%) was the leading agent of candidemia in Iran [23]. Similar to previous studies, C. glabrata is the most common NAC species in this study [24][25][26]. However, in other studies, C. parapsilosis has been the most prevalent NAC species [27][28][29][30]. Moreover, the increasing mortality rate associated with the increased frequency of NAC might be linked to the failure in clinical cure rate resulting from either acquired or intrinsic resistance to the few antifungal drugs available to treat candidemia. For example, echinocandins are regarded as the rst-line drug for the treatment of C. glabrata BSI. However, the increasing reports on BSI caused by C. glabrata isolates resistant to both uconazole and the echinocandins are alarming. It has been shown that C. lusitaniae can rapidly acquire multidrug resistance traits (MDR) during the course of antifungal treatment with uconazole, amphotericin B, and caspofungin [31]. C. kefyr can cause serious infection in patients with hematologic malignancies and recently, resistance to amphotericin B have been reported in C. kefyr isolates [32]. C. norvegensis, is also shown to be azole resistant [33]. While 12.5% of the NAC species in this study were uconazoleresistant, only 4.2% of the C. albicans species were resistant to uconazole. Our study con rmed that primary uconazole resistance is uncommon in C. albicans. The majority of C. albicans are sensitive to amphotericin B, uconazole, itraconazole, voriconazole, posaconazole, and echinocandins in vitro, especially in patients without a history of exposure to antifungal agents [34]. A recent study looked at the impact of the new CLSI breakpoints and demonstrated that applying revised uconazole breakpoints increased the rate of uconazole resistance in C. albicans, C. tropicalis, and C. parapsilosis [35]. The occurrence of uconazole resistance in C. tropicalis has been previously reported as 5.0-7.2% from two reports from the ARTEMIS study over 12 years [36,37]. The high rate of azole non-susceptible C. tropicalis in this study was similar to other studies from Asia [38,39]. Also among the uconazole-resistant C. tropicalis isolates in our study, six were resistant to voriconazole. Many candidemia studies revealed a signi cant increase in azole-resistant C. tropicalis blood isolates and some reported pan-azole and amphotericin B resistant isolates [40]. An extensive candidemia study in India showed that C. tropicalis and C. auris isolates also carry MDR trait [41]. The susceptible dose-dependent (SDD) C. glabrata isolate is de ned as uconazole MIC ≤ 32 mg/l, since 2012. Overall, 96.7% of C. glabrata isolates were categorized as SDD (MIC 50 = 16 mg/l, MIC 90 = 32 mg/l) that is consistent with studies from the Asian-Paci c region [42]. As demonstrated in the current study, no resistance to any of the antifungal agents was observed in C. guiliermondii, C. lusitaniae, C. kefyr, and C. krusei (except for the intrinsic uconazole resistance in C. krusei). Furthermore, resistance to echinocandins was very low, except for C. parapsilosis, which exhibited higher MICs than those of other Candida species.
The increased frequency of NAC may also be attributable to the improved diagnostic technique, allowing NAC species to be characterized with methods that are more sensitive. In this study, all Candida isolates were identi ed using the DNA sequencing method to assess the exact epidemiological pattern of species distribution. The mean age of the patients in this study was 49.6 years. Most of these patients were over forty years old (68.5%). The Mean age in other studies varies from 40 to 65 years. Candidemia patients (usually with underlying conditions, such as diabetes, cancer, pulmonary and heart complications) mostly are admitted to ICUs. The overall crude mortality of 47.2% in our study is similar to that reported by other investigators from Iran [43][44][45] but considerably higher than the 26% quoted by Chen et al. [46]. Sixty patients took at least one antifungal drug and the mortality rate for these patients was 38.3% (23/60; 38.3%). However, the mortality rate among patients who did not receive antifungal drugs was 65.5% (19/29; 65.5%). This suggests that early diagnosis and timely antifungal administration can dramatically reduce the rate of mortality. We found that mortality was highest among ICU patients (65.7%) which is unsurprising, given the severity of underlying illness in this population. Evidence supports the fact that patients admitted in the ICU have higher mortality rates than those in other wards [47]. The major underlying diseases were malignancy, sepsis, renal failure/dialysis, and HTN. Diabetes mellitus, cardiovascular diseases, and pulmonary disorders were other underlying diseases. Consistently, similar underlying condition were documented in studies conducted in Turkey [48], China [9], and Australia [49]. Multivariate analyses of risk factors for BSIs caused by C. albicans and NAC species showed that malignancy was an independent risk factor for candidemia (P = 0.013). In the multivariate analyses of risk factors for BSIs mortality, intubation (P = 0.001) and urinary catheterization (P = 0.03) were independent risk factors for mortality. In a prospective study performed in the ICU of a tertiary care hospital in Athens, the authors noted that the administration of glucocorticoids, presence of CVCs, and candiduria were independent risk factors for candidemia caused by NAC species [50].
The current study had some limitations. First, in this study, two main medical centers in the capital of Iran had been selected; however, information from other centers has not been included in this study to obtain insights into the epidemiological status of candidemia in Tehran. Second, the results of the multivariate analyses might be in uenced by the sample size and the number of variables included in the models.
In conclusion, Candidemia, with a shift in species distribution towards non-albicans Candida species, remains a lethal disease. The results of this study provide important information regarding the distribution of Candida spp in patients with candidemia in Tehran, the capital of Iran for which there is a paucity of data regarding the epidemiology, risk factors, and antifungal susceptibility patterns of these species. Accurate knowledge of predisposing factors and epidemiological patterns can be an effective step in disease management. In this study, C. albicans is reported to be the most common species causing candidemia; however, an increasing frequency of NAC species could pose a serious challenge to treatment due to different antifungal susceptibility patterns. This report shows that candidemia is a signi cant source of morbidity in Tehran. Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Con ict of interest: