Evaluation of Candida colonization index, molecular identification, and antifungal susceptibility pattern of Candida species isolated from critically ill pediatric patients: A single-center study in Iran

Background and Purpose: Given the high mortality rate of invasive candidiasis in hospitalized pediatric patients, it is crucial to establish a predictive system to achieve early diagnosis and treatment of patients who are likely to benefit from early antifungal treatment. This study aimed to assess the Candida colonization index, species distribution, and antifungal susceptibility pattern of Candida strains isolated from pediatric patients with high Candida colonization index (CI). Materials and Methods: This study was carried out at the Children’s Medical Center in Tehran-Iran. In total, 661 samples were collected from 83 patients. The Candida CI was calculated according to the descriptions of previous studies. The isolates were identified using polymerase chain reaction-based techniques. The Clinical and Laboratory Standard Institute protocol M60 was used to conduct the antifungal susceptibility test. Results: A colonization index greater than 0.5 was confirmed in 29 cases (58% of positive samples) with two children developing candidemia. Candida albicans (n=53, 49.5%) was the most common Candida species in patients with CI > 0.5. Except for acute lymphoblastic leukemia, no risk factors were linked to a high index in colonized children (P > 0.05). Twelve isolates (7.01%) were multi-azole resistant with high MICs against both isavuconazole and ravuconazole and seven strains (4.09%) were echinocandins resistant. Conclusion: In pediatric intensive care units, patients are at risk of fungal infection, particularly candidemia. In this study, more than half of the children with positive yeast cultures had CI > 0.5, and 6.8% developed candidemia.


Introduction
andidemia is the second foremost cause of sepsis-related mortality in children [1][2][3].Several publications have reported that the candidemia epidemiology, control, and outcome of candidemia differ noticeably and cannot be generalized to children [4].Furthermore, given the high mortality rate of invasive candidiasis in intensive care unit (ICU) patients, it is crucial to establish an alarm system against this pathogenic yeast to achieve early diagnosis and treatment [5].
Understanding the link between the Candida colonization index (CI) and invasive candidiasis (IC) has been a priority for clinicians for decades; therefore, the colonization index has been proposed as the "missing link" and one of the most widely used scores is Candida CI [5].Pittet et al. demonstrated that the Candida CI expresses the intensity of colonization and is the ratio of the number of non-blood distinct body sites screening positive for Candida species to the total number of distinct body sites tested [2].
In fact, multiple-site colonization with Candida spp.colonization is widely established as a key risk factor (5-30% of colonized patients) [6,7] for invasive fungal infection in seriously ill patients.Hence, a prognostic value, which could be colonization density, is needed for the diagnosis of systemic candidiasis [8,9].Candida colonization has been reported in up to 60% of critically ill pediatric patients after 4-7 days in the ICU [10][11][12].Early empirical management of acute candidiasis improves the chance of survival but might lead to antifungal overuse, which increases financial costs and also resistance in Candida isolates [13].
Adult patients and specific groups of diseases have been the focus of studies on this score.As a result, this study aimed to demonstrate the applicability of this score in pediatric patients at great risk for invasive candidiasis in pediatric/infant ICU (PICU and IICU) as well as in bone-marrow transplantation (BMT) units.The antifungal susceptibility profile of strains isolated from pediatric patients with high Candida colonization index was also determined.

Patients and sampling
This research was performed at the Children's Medical Center in Tehran at the ICU and BMT unit from March 2021 to September 2021.In total, 661 samples were gathered from 83 patients.The age of patients ranged from newborn to 21 years old (https://www.aap.org/).The patients who were supposed to stay for > 3 days were included in this study, from which, individuals with less than 7 days of hospitalization were excluded (See chart 1).This study was approved by the Ethics Committee of Mazandaran University of Medical Sciences (Code IR.MAZUMS.REC.1397.2771).Written informed consent was obtained from parents.Samples from Candida colonization-prone body sites, such as the oral cavity, skin-enclosed catheters, ear, and throat, as well as nasal swab and urine culture were collected at the time of admittance and also every week during their stay.Swab samples were obtained by using swab sticks whereas urine samples were collected as mid-stream urine or via a urinary catheter.On the admission day and days 7, 14, and 28, the Candida colonization index was calculated for each patient.
All samples were cultured on Sabouraud dextrose agar (HIMEDIA, India) with chloramphenicol as well as chromogenic medium (CHROMagar Candida, CHROMagar Microbiology, France).Each bottle contained a blood sample from a patient suspected of having candidemia which then was incubated at 35 ⁰ C in the automated BACTEC™ 9120 blood culture system (Becton Dickinson, USA).
The amplifications were conducted based on the following: initial denaturation at 95 ⁰ C for 5 min followed by 35 cycles of denaturation at 95 ⁰ C for 15 s, annealing at 56 ⁰ C for 30 s, and elongation at 72 ⁰ C for 30 s.After the final cycle, the samples were incubated for 5 min at 72 ⁰ C [16].The PCR products were first run on 1.2% agarose gels, visualized with UV after ethidium bromide staining, and then were digested with 5 U of the restriction enzyme MspI (Fermentas, Vilnius, and Lithuania) followed by gel electrophoresis on 2% agarose gel.Candida albicans and Candida parapsilosis species complexes were distinguished by amplification of the Hyphal wall protein and intein-containing vacuolar ATPase precursor genes, respectively, using previously described protocols [17,18].

Candida colonization index evaluation
The Candida colonization index was analyzed as the proportion of the number of colonized body sites (other than blood culture) with Candida spp.upon the total number of cultured sites.Colonization was considered high when CI ≥ 0.5 [2].
A suspension of fresh yeast colonies was prepared in a sterile saline solution with a transmittance range of 75-77%.A working suspension was made by 1:100 dilution which resulted in 2.5-5×10 3 CFU/ml.Drugfree and yeast-free wells were included as positive and negative controls, respectively.The microdilution plates were incubated at 35 °C and examined visually after 24 h.Candida krusei (ATCC6258) and C. parapsilosis (ATCC 22019) were used as quality controls.

Statistical analysis
The data were recorded in Microsoft Excel 2007 (Microsoft Corp, Redmond, WA, USA) and SPSS software (version 16; SPSS Inc., Chicago, IL, USA) was used for the analysis.Categorical variables were compared using the Fisher exact test to determine differences in their dispersals among the subgroups.Student's t-test was applied to evaluate the geometric means yielded from antifungal susceptibility testing (AFST).The P values less than 0.05 were considered statistically significant.

Results
A total of 206 yeast isolates were collected from different body sites of patients.The majority of the yeast isolates (n=104) were obtained from the urine.Other isolates were extracted from blood (n=52), vaginal swabs (n=45), ear discharge (n=2), tracheal aspirate (n=2), tissue (n=2), skin (n=1), nail (n=1), sputum (n=1), and cerebrospinal fluid (n=1).Among 206 yeast isolates, 200 isolates were identified as single species, and the most dominant species was C. albicans, with a number of 120 (59%).Out of 206 yeast cultures, six isolates were identified as a mixture of different Candida species by the PCR-RFLP method.Three mixtures combined C.

Candida colonization index evaluation
This study was performed on 83 patients (children/infants) from ICUs as well as BMT units.Overall, 661 swab and urine cultures were obtained from 83 patients.It should be noted that 177 out of 661 samples were collected from 50 individuals (26.8%) and were considered positive cultures with the least grown yeasts from one sample site.Moreover, 25 and 58 samples were isolated from female and male patients, respectively.
The most reported sites with yeast growth were the oral cavity, ear, and nasal samples.In total, 50 (16 BMT+34 ICU) patients had yeast growth and the CI was found to be ≥ 0.5 in 29 (8 BMT+21 ICU) of them (58% and 35% of positive and total samples, respectively).It was found that colonization occurred most strongly two weeks after admission.Table 1 summarizes the demographic data of patients with CI > 0.5.Among pediatric patients with CI > 0.5, 12 patients (ICU: 7/21, 33.3%, BMT: 5/8, 62.5%, total: 12/29, 41.4%) suffered from acute lymphoblastic leukemia (ALL) as the predisposing factor, whereas Curr Med Mycol, 2022, 8(4): 15-21 this parameter was reported in three patients.Therefore, the chance of Candida colonization with CI > 0.5 in pediatric patients who suffered from ALL is significantly higher (Odd's ratio with 95% CI was 4.4, P<0.05).
The CI > 0.5 was reported most in male patients, compared to female patients (n=18, 62% vs. n=11, 38%).However, being male or female did not significantly increase the possibility of Candida colonization occurrence with CI > 0.5 (Odd's ratio 1.27, P>0.05 for males and 0.81, P>0.05 for females, respectively).In total, 11 patients (38%) were below one year old (considered infant); nevertheless, age was not observed as a significant effective parameter as the values of odd's ratio values were 0.66 (P>0.05) and 1.5 (P>0.05) for pediatric patients over and under one year old, respectively.
It is noteworthy that two (2/29, 6.8%) out of the total patients with CL>0.5 who suffered from ALL (see the demographic data of patient no.11 in Table 1) and thalassemia (see the demographic data of patient no.20 in Table 1) developed candidemia during weeks one and three, respectively.Regarding molecular species, identification, and AFST results, multi-azole-resistant Candida albicans and FLZ-resistant C. parapsilosis were isolated from blood culture bottles, respectively.

Antifungal Susceptibility Testing
Table 2 summarizes the minimum inhibitory concentration (MIC) range, MIC50, MIC90, and    2 provides additional information about the AFST performed on all isolated species.The GM of C. albicans isolates, which was reported to be the most prevalent isolated species, was found to be significantly different between the patients in the BMT unit and ICU (P=0.004).Candida albicans isolated from patients in ICU had higher GM against all tested antifungals, compared to C. albicans isolated from BMT units.According to MIC50 values of the species isolated from ICUs, CAS with a MIC value of 0.016 µg/ml was reported as the most effective agent.

Discussion
The overall rate of fungal colonization (CI>0.5)was 35%.Therefore, it can be said that roughly, the majority of patients are at risk of improving their invasive candidiasis.As a result, one ALL patient (1/29, 3.4%) developed candidemia during week three.In line with the findings of this study, those of the research performed by Altintop et al. (2018) indicated a 33.3% Candida colonization rate in patients in PICU [5].In their study, C. albicans was the predominant species followed by C. parapsilosis and C. glabrata Candida albicans (n=53, 29.77%), C. krusei (n=43, 24.16%), C. parapsilosis (n=34, 19.10%), and C. glabrata (n=15, 8.42%).
Furthermore, Hamzavi estimated the Candida colonization incidence rate at 59.9% (82/136) in patients with hematological malignancy and found that C. albicans (72.0%) was the most common Candida species in colonized patients.They also reported that Candida colonization did not have a relationship with age, gender, oncologic diseases, and degree of neutropenia [21].The species distribution differed from the findings of a previous study conducted at the Children's Medical Center in Tehran [15].
According to Charsizadeh et al., Candida albicans and C. parapsilosis were the most commonly isolated species from ICU patients with invasive candidiasis.Although C. albicans was the most common species overall in the current study, C. krusei was the most common species in patients in PICU.The high rate of C. krusei isolation from hospitalized pediatrics in PICU is cause for concern.It was also reported as the third most isolated species in patients with CI > 0.5.
The patients in the BMT unit must stay in that unit for several days or even months; therefore, the number of involved patients is lower than that of the ICU unit.The most important reasons for the low rate of positive samples were prophylactic antifungal use and daily bathing.
According to Solomkin et al., colonization at more than two sites has been previously proposed as a key to the earlier initiation of antifungal therapy in highrisk surgical populations.Previous studies also suggested that early systemic antifungal therapy may be beneficial in patients who have been colonized at more than two sites but do not have candidemia [22,23].Based on AFST results, more than 10% of the isolates were FLZ-resistant.The selection of resistant species during prophylaxis and antifungal therapy, as well as the increased number of infections caused by drug-resistant fungal species and lack of satisfactory outcomes, highlight the need for more effective prevention and treatment strategies based on new antifungal drugs and fungal species-specific diagnoses.
The main limitation of the current study was the relatively small number of enrolled pediatric patients and time constraints in terms of following up with the patients.More studies are needed to understand the impact of risk factors on CI values and the complicated journey of Candida spp.from colonization to infection in pediatric patients.

Conclusion
In pediatric intensive care units, patients are at risk of fungal infection, particularly candidemia.In this study, more than half of the children with positive yeast cultures had CI > 0.5, and 6.8% developed candidemia.

Chart 1 .
General flowchart of the present study.BMT: bone-marrow transplantation, ICU: intensive care unit, PICU: pediatric intensive care unit, NICU: neonatal intensive care unit albicans and C. glabrata (MY 1, MY 2, and MY 3).The fourth was the mixture of C. glabrata and C. tropicalis (MY 4), the fifth mixture combined C. parapsilosis and C. tropicalis (MY 5), and the sixth mixture was the combination of C. glabrata, C. albicans, and C. tropicalis (MY 6).

Table 1 .
Demographic data of patients and distribution of the yeast species with colonization index ≥ 0.5.

Table 2 .
Geometric mean, MIC50, and MIC90 values were obtained by testing the susceptibility of isolated Candida species.(MIC50 and MIC90 of the isolated species of less than 5 were not calculated) One, two, and four strains of C. albicans, C. krusei, and C. parapsilosis, respectively, were resistant to all echinocandins (total: 4.09%) (see Table2).According to the new ECV (CLSI M59 2 nd Ed, 2018), five strains of C. parapsilosis, four strains of C. krusei, and nine strains of C. albicans had high MICs against AmB (10.52%).Table