Impact of antifungal stewardship interventions on the susceptibility of Candida species in pediatric patients with malignancy

Background There is a worldwide concern with respect to the antimicrobial resistance and the inappropriate use of antifungal agents, which had led to an ever-increasing antifungal resistance. This study aimed to identify the antifungal susceptibility of Candida species isolated from the pediatric patients with cancer and also to evaluate the clinical impact of antifungal stewardship (AFS) interventions on antifungal susceptibility of Candida species. In this study, Candida species colonization were evaluated among hospitalized children with cancer in a tertiary teaching hospital, Shiraz 2017-18. The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) for polyenes (amphotericin B), echinocandins (caspofungin), and azoles (voriconazole, uconazole, posaconazole, and itraconazole). Antifungal susceptibility of Candida species was compared with our previous study results to determine the clinical impact of AFS interventions on the antifungal susceptibility. The prevalence of Candida albicans in the present study was signicantly higher than other Candida species. Candida albicans species were completely susceptible to the azoles. The sensitivity rate of C. albicans to amphotericin B and caspofungin was 93.1% and 97.1%, respectively. The results conrm the positive effect of optimized antifungal usage and bedside intervention on the susceptibility of Candida species after the implementation of the AFS program. C. albicans and C. glabrata exhibited signicant increase in sensitivity after the execution of AFS program. Improving the antifungal agent usage can improve antifungal susceptibility and reduce resistance. The AFS is recommended to be addressed, applied, and regularly assessed in centers. categorical variables and Fisher’s exact values statistically signicant. Pearson azoles remain the most effective choice when they are used wisely. Improving Candida species antifungal susceptibility after the implementation of AFS is promising. Knowledge of etiologic agents, along with the regular identication of antifungal susceptibility patterns is necessary in oncology settings.


Introduction
The prevalence of Candida infections is on the rise as a result of excessive usage of broad-spectrum antibiotics, HIV infection, malignancies, transplants, invasive procedures, and prolonged hospitalization [1][2][3]. More than 17 different species of Candida are recognized that can infect humans, and amongst them, 90% of all invasive infections are related to C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei [4,5].
Antifungal resistance usually occurs following selective pressure induced by the use or misuse of antifungal agents in high-risk patients, especially those with malignancy [6][7][8]. Candida infections epidemiology changes by the duration of the antifungal therapy and its type during preventive strategies, such as prophylaxis [9].
C. albicans susceptibility breakpoint is continuously updated by the Clinical and Laboratory Standards Institute (CLSI), which can be used as an indicator to manage systemic candidiasis [10]. Although C. albicans is the most common cause of Candida infections, the prevalence of non-albicans species is increasing [6]. The emergence of non-albicans Candida infections have become a global concern; however, as we described previously, change in its epidemiology after adherence to the AFS program was observed [11]. Similar positive effects could be expected by improving the susceptibility pattern of Candida spices after AFS implementation. Therefore, the aim of this study was to identify the antifungal susceptibility of Candida species amongst pediatric patients with Candida colonization and also to investigate the ASP intervention effects on antifungal susceptibility in pediatric patients with hematological malignancy.
The incidence of IFDs ranged from 7.7 to 12.5/1000 admissions during 2015-18 in our center. Invasive candidiasis (IC) is the most common form of IFDs (47.2%), and its annual incidence ranges 22.5-55.3%.

Methods And Materials
Page 3/15

Settings
The university hospital Amir is a tertiary oncology center in Shiraz, Iran, with more than 1.5 million inhabitants. The department of hematology/medical oncology has seven wards (100 active beds), an autologous transplant ward, and annually over 5000 pediatric patients are given admission. Population All children aged <18-year-old between 2017-18 with hematologic malignancy or solid organ tumors were included in this study. Samples were collected from oral/nasal secretions and urine/stool specimens. In children with severe thrombocytopenia or bleeding tendency, only urine and stool samples were collected. Weekly sampling was performed for patients with more than one-week of hospitalization. This cross-sectional study was approved by the Ethics Committee of Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences. After explaining the study objectives, informed consent of children's parents or guardians was obtained.

Mycological study
Urine, nasal, oral, and stool samples were cultured on Sabouraud Dextrose Agar (Merck, Germany) medium. The cultured samples were transferred to the Mycology Laboratory of Professor Alborzi Clinical Microbiology Research Center for identi cation and susceptibility testing. The fungi isolates were cultured twice on Potato Dextrose Agar medium at 35°C for seven days to ensure the culture purity, and then the cultures were read at 530 nm by a spectrophotometer. C. parapsilosis ATCC-22019 and C. krusei-ATCC-6258 were used as standard quality control CLSI-recommended strains.

Antifungal drugs
The amphotericin B, caspofungin, voriconazole, uconazole, posaconazole, and itraconazole powders (Sigma-Aldrich, Germany) were used to make serial dilutions. The broth microdilution method was applied to determine the susceptibility. CLSI M51-A, M59, and M60 followed in specifying the susceptibility pattern of isolates [10,12,13]. Inoculums suspension was prepared, using the spectrophotometric method at 530 nm. The dilutions of 1:1000 was prepared in RPMI 1640 medium (Sigma-Aldrich, England), supplemented with 1.5% agar and 2% glucose at pH 7.0, using 0.165 M-morpholine propane sulfuric acid (MOPS). The nal concentration was adjusted to 0.5 McFarland, with a volume of 100 μl conidium dilution, were added to wells and incubated at 35 °C. Positive and negative wells (wells without drug and yeast, respectively) were considered for each species, and the minimum inhibitory concentration (MIC) was read visually after 24 and 48 hours. For amphotericin B, complete growth inhibition was considered as MIC value. Then, MIC 50 and MIC 90 values of the isolated species were calculated. The susceptibility pattern of each isolate was analyzed based on CLSI breakpoints [10].
Antifungal Stewardship program principles in Amir medical oncology center AFS is a "strategic planning" that can be summarized in learning, training, and continuous practice to improve evidence-based skills in the management of IFDs in high-risk patients. By the sustained adherence to the AFS, indiscriminate use of antifungal agents, drug resistance, side effects and costs will be reduces. The AFS has been executed in our center since June 2015. Characteristics of AFS interventions summarized in Table 1. It should be noted that the diagnosis and treatment of the IFDs was signi cantly improved after the implementation of the AFS. Changing from empiric therapy to pre-emptive antifungal treatment strategies was accomplished by the application of nonculture-based methods, such as galactomannan (GM) antigen test and polymerase chain reaction (PCR). Therapeutic drug monitoring and antifungal susceptibility testing has become the standard of care for monitor serum voriconazole concentrations and targeted therapy since the early 2016. We used the original data from our recent investigation, which was conducted before the implementation of AFS during 2011-2012 in pediatric patients with malignancy (period-1; p1) [14]. This was done to compare the clinical impact of AFS interventions on antifungal susceptibility of different Candida spices with the present study (period-2; p2).

Results
From May 2017 to November 2018, 482 oral/nasal samples, and urine and stool specimens were collected from 136 pediatric patients with hematological malignancies or solid organ tumors. Most patients were male (53.3%), and the mean age was 7.57 years (Median: 6.5, Std. Deviation: ± 4.85, range from 4.8 months to 18 years). Eighty-two cases were detected having at least one Candida spp. colony, and amongst them 59 were C. albicans colonies.
Among the 102 C. albicans isolates collected during 2017-18 surveillance program, all were susceptible to the azole agents. The sensitivity rate of C. albicans to amphotericin B and caspofungin was 93.1% (95) and 97.1% (99), respectively.
Among the seven C. krusei isolates, 28.6% were resistant to caspofungin. All the C. krusei isolates were sensitive to amphotericin B, voriconazole. For itraconazole, 85.7% were sensitive, and 14.3% were susceptible dose-dependent.
All the ve C. parapsilosis isolates were sensitive to amphotericin B, caspofungin. For itraconazole, 80% were sensitive, and 20% were susceptible dose-dependent. In total, 80% of C. parapsilosis were found to be susceptible to uconazole.
Among all the tested strains of C. glabrata and C. tropicalis, none of them were resistant to any of the antifungal agents.
CLSI breakpoints are not available for C. Kefyr and C. famata. Susceptibility of different antifungals to C. kefyr and C. famata is provided in Table 3.   Fig. 1).

Discussion
Amongst the 136 studied cases, 59.9% (82) were colonized with at least one Candida species. Most of them were colonized with C. albicans 59 (72%). C. krusei, C. kefyr, C. glaberata, C. parapsilosis, C. tropicalis, and C. famata were the least common Candida species. Detail information regarding the colonization pattern of the studied cases can be found in our recently published paper [11].
According to the results, all C. krusei, C. glaberata, C. parapsilosis, and C. tropicalis isolates were susceptible to amphotericin B (the most active agent for the treatment of non-albicans Candida species). Caspofungin exhibited high resistance against C. krusei and C. glaberata isolates (28.6% and 25%, respectively). Accordingly, amphotericin B can be considered as a more active agent than caspofungin for the treatment of non-albicans Candida species, especially, C. krusei and C. glaberata.
Accordingly, in this study the colonized isolates of C. Kefyr and C. famata were susceptible to the tested antifungal agents.
Therapeutic options for fungal infections are limited even before the global rise of antifungal resistance [23,24]; hence, judicious prescription of available choices, especially non-azole antifungals, should be considered in high-risk settings, such as oncology centers. Our results con rmed that caspofungin and amphotericin B are more active antifungal agents compare to azoles in some isolates, such as C. parapsilosis.
The emergence of azole-resistant C. glabrata is also of signi cant concern in the setting that use uconazole prophylaxis [9].
Clinical impact of antifungal stewardship interventions on antifungal susceptibility of Candida species during the two study periods Epidemiological changes in the Candida colonization pattern was described in our previous report. Brie y, we found a signi cant reduction in non-albicans species colonization after the implementation of AFS. During period 1 (p1), 46.5% (88) of the studied cases (n = 188) were colonized, while in the 2nd period, the colonization rate reached 59.9% (P value = 0. 0.017) [11]. In total, 25.3% (23) of the cases were receiving antifungal prophylaxis during the 2nd period, mainly with the liposomal formulation of amphotericin B, while 54% were on antifungal prophylaxis during p1, mostly with uconazole or itraconazole [Difference 21.2%, 95% CI: 9.16-31.77%, P = 0.0007]. This success was achieved by controlling and restricting antifungal usage during p2.
Multidrug-resistant strains, including azole, caspofungin and amphotericin B resistant isolates were not found within the two study periods (Table 4 and Fig. 2).

Frequency of amphotericin B resistance C. albicans
Despite the signi cant reduction in uconazole and caspofungin-resistant, during p2, some increase in the incidence of amphotericin Bresistant C. albicans was detected during p2 (Table 5). A small but signi cant increase in amphotericin B-resistant C. albicans strains can be partially explained by changing antifungal preventive strategy to liposomal amphotericin B, since 2015. However, the frequency of amphotericin B-resistant C. albicans was not affected by liposomal amphotericin B prophylaxis between the two periods (p = 0.619). We also analyzed the rate of uconazole, voriconazole, itraconazole, caspofungin and amphotericin B resistance amongst the non-albicans colonized species between the two study periods. Signi cant decrease in uconazole, itraconazole and caspofungin resistance was found among the C. glaberata strains during the second study period compared with 2011-12. Also, a statistically signi cant reduction in amphotericin B resistance was found during p2 in C. krusei isolates (Table 6).

Clinical impact of Candida colonization in hospitalized patients with hematological malignancies
Most of Candida bloodstream infections, including central line-associated candidemia originate from endogenous host ora [26][27][28]. The clinical impact of Candida colonization on the short-term mortality rate of patients with hematological malignancies was reported in a previous study [29]. On the other hand, short-term survival was affected in patients with non-albicans species, including C. glabrata, C. kefyr, and C. krusei, compare with C. albicans [29,30]. Azole prophylaxis has a critical role in the development of either unsusceptible strains or even in the selection of yeast with intrinsic azole-resistant, such as C. krusei [6,9,31,32].
As discussed earlier, during p1, more than 35% of cases were colonized with non-albicans species, mostly C. glabrata and C. krusei. However, after the implementation of the AFS, non-albicans colonization decreased to less than 20%, mostly C. krusei and C. Parapsilosis, with a signi cant decrease in C. glabrata colonization [11]. C. glabrata is considered as the second most common gastrointestinal yeast ora, after C. albicans [33]. In our opinion, the shift toward more C. albicans colonization is the result of sustained adherence to the AFS and restricted azole prescription ( Table 1).
Comparison of the mean minimum inhibitory concentration (MIC) value of the six antifungal drugs between different Candida spp. during the two study periods In addition to the susceptibility results, we also compared the mean MIC value of each antifungal drug for C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. famata, and C. kefyr during two study periods (Fig. 4). As shown in Fig. 4, a signi cant reduction in mean FCZ-MIC was found for C. albicans, C. glabrata, and C. tropicalis in p2 compare to p1.
There are lots of data with respect to the positive effect of stewardship program on bacterial resistance [34][35][36][37]; however, due to its multifactorial development, antifungal resistance is more challenging to measure. Even in colonized patients, susceptibility patterns might change over time, especially in immunocompromised hosts [8]. Although the AFS has known short-term effects (e.g., reduction in antifungal consumption, costs and outcomes) on the management of IFDs and patient safety [38][39][40][41], its long-term effects has been described on resistance patterns [42]. Based on stewardship program metrics, change in resistance patterns, and pathogen-speci c resistance is the most di cult target to reach [34]. To the best of our knowledge, there is a shortage of reports on the improvement of antifungal susceptibility of Candida species overtimes after the implementation of AFS. Hence, the results of our study highlight the importance of strict adherence to the stewardship programs amongst the cancer patients.

Limitations
This study was limited by the small number of samples. Additionally, further studies using next generation sequencing are needed to detect AFS effects on antifungal resistance genes in Candida spices.

Conclusion
C. albicans is the most prevalent colony amongst the pediatric patients with malignancy, and azoles remain the most effective choice when they are used wisely. Improving Candida species antifungal susceptibility after the implementation of AFS is promising. Knowledge of etiologic agents, along with the regular identi cation of antifungal susceptibility patterns is necessary in oncology settings.

Declarations
Ethics and consent to participate The study was approved by medical ethics committee of Professor Alborzi clinical microbiology research center. All individuals (or their parents) in the study population were informed about the current study, with written consents obtained before enrolment in the present study.

Consent for publication Not applicable
Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors do not have any nancial or other relationships which could be regarded as a con ict of interest.

Funding
Not applicable Author contributions: Study concept and design: AA and HSS; Acquisition of data: AA, JH, HSS and GF; Mycological analysis: BP, JH and GF; Statistical Analysis: AA, HS, and NS, Analysis and interpretation of data: AA and HSS; Drafting of the manuscript: AA, Critical revision of the manuscript for important intellectual content: AA and BP; Study supervision: AA and BP. All individuals listed as (co)-authors have met the authorship criteria, and nobody who quali es for authorship has been omitted from the list. The nal manuscript was corrected and approved by all authors. Figure 1 24-hour and 48-hour MIC distribution with a histogram of the isolated C. albicans  Frequency of uconazole-resistant, caspofungin-resistant and uconazole/caspofungin-resistant strains of C. albicans during the two study periods