Emergence of non-albicans Candida species and antifungal resistance in intensive care unit patients

Ravinder Kaur, Megh Singh Dhakad, Ritu Goyal, Rakesh KumarDepartment of Microbiology，Lady Hardinge Medical College and Associated Hospitals，New Delhi 110001，IndiaDepartment of Microbiology，Maulana Azad Medical College and Associated Lok Nayak Hospitals，New Delhi 11000，IndiaDepartment of Obstetrics and Gynaecology，Maulana Azad Medical College and Associated Lok Nayak Hospitals，New Delhi 11000，IndiaDepartment of Anaesthesiology，Maulana Azad Medical College and Associated Lok Nayak Hospitals，New Delhi 11000，India



Emergence of non-albicans Candida species and antifungal resistance in intensive care unit patients

Ravinder Kaur1*,#, Megh Singh Dhakad2,#, Ritu Goyal3, Rakesh Kumar41Department of Microbiology，Lady Hardinge Medical College and Associated Hospitals，New Delhi 110001，India
2Department of Microbiology，Maulana Azad Medical College and Associated Lok Nayak Hospitals，New Delhi 110002，India3Department of Obstetrics and Gynaecology，Maulana Azad Medical College and Associated Lok Nayak Hospitals，New Delhi 110002，India
4Department of Anaesthesiology，Maulana Azad Medical College and Associated Lok Nayak Hospitals，New Delhi 110002，India

ARTICLE INFO

Article history：

Received 17 Sep 2015

Receivedinrevisedform18 Dec,2nd

revised form 21 Dec 2015

Accepted 28 Dec 2015

Available online 22 Mar 2016

Keywords：

Antifungal resistance

Candida species

Colonization

Intensive care unit patients

ABSTRACT

Objective：To evaluate the epidemiology of candidiasis and the antifungal susceptibility profile of Candida species isolated from the intensive care unit（ICU）patients.

Methods：The study used a qualitative descriptive design. Relevant samples depending on organ system involvement from 100 ICU patients were collected and processed. Identification and speciation of the isolates was conducted by the biochemical tests. Antifungal susceptibility testing was carried out as per CLSI-M27-A3 document.

Results：Ninety Candida isolates were isolated from the different clinical samples: urine （43.3%）, tracheal aspirate（31.1%）, urinary catheter（12.2%）, endotracheal tube（7.8%）, abdominal drains（3.3%）, sputum（2.2%）. The incidence of candidiasis caused by nonalbicans Candida（NAC）species（63.3%）was higher than Candida albicans（36.7%）. The various NAC species were isolated as: Candida tropicalis（41.1%）, Candida glabrata（10%）, Candida parapsilosis（6.7%）, Candida krusei（3.3%）and Candida kefyr （2.2%）. The overall isolation rate of Candida species from samples was 53.3%. Antifungal susceptibility indicated that 37.8%and 7.8%of the Candida isolates were resistant to fluconazole and amphotericin B, respectively.

Conclusions：Predominance of NAC species in ICU patients along with the increasing resistance being recorded to fluconazole which has a major bearing on the morbidity and management of these patients and needs to be further worked upon.

Original article http://dx.doi.org/10.1016/j.apjtb.2015.12.019

Tel: +91 011 2340 8185, +91 9811809868, +91 9871272728

E-mail: drkaur@hotmail.com

The study protocol was performed according to the Helsinki declaration and approved by the Institutional Ethics Committee of Maulana Azad Medical College & Associated Hospitals, New Delhi, India. Informed written consent was obtained from the patients/guardians.

Foundation Project: Supported by the University Grants Commission（UGC）, Government of India, New Delhi-110002, India［Grant: Rs. 908100/- and Grant number: F.31-260/2005（SR）］.

Peer review under responsibility of Hainan Medical University. The journal implements double-blind peer review practiced by specially invited international editorial board members.

#These authors contributed equally to this work.

1. Introduction

Nosocomial infections constitute a serious public health problem and are among the major causes of morbidity and mortality leading to increased hospitalization time and consequently, generating high costs for patient treatment［1］. In the recent years, the incidence of nosocomial candidiasis has increased throughout the world, starting from tertiary care centers and spreading to community hospitals［2］. The epidemiological surveillance program in the United States has shown that 5%–10%of patients who admitted in the hospitals acquire nosocomial infection. Of these, about 80%of fungal infections were caused by Candida species. According to studies by Centers for Disease Control, Candida albicans （C. albicans）is the sixth most common cause of nosocomial infections［1］.

The frequent use of broad-spectrum antibiotics, central venous catheters, urinary catheters, prosthetic devices and abdominal surgery in intensive care unit（ICU）patients requiring emergency care［3］, put patients at a high risk of infection with Candida species［4］. Furthermore, ICU admission itself hasbecome an independent risk factor for the development of Candida species infection［5］. The most common species implicated was C. albicans recently, when the incidence of non-albicans Candida species（NAC）has risen dramatically ［6］. In recent decades, several countries around the world have witnessed a change in the epidemiology of Candida infections, characterized by a progressive shift from a predominance of C. albicans to NAC species［7］.

A worrisome trend is the increasing number of reports of fluconazole resistance among species that are typically fluconazole-susceptible, such as C. albicans. There are several reports regarding this phenomenon, but larger epidemiologic studies have failed to show a definite geographic or temporal trend toward fluconazole resistance, despite of heavy azoles usage. This epidemiologic shift has greatly impacted the therapeutic choices for initial and definite therapy for this disease［8］. Since there is scanty data on candidiasis in North India, the main objective of present study was to perform a study on the epidemiology of candidiasis and evaluate the antifungal susceptibility profile of the Candida species isolated from the ICU patients at our tertiary care hospital.

2. Materials and methods

2.1. Design and setting

A prospective study was focused on epidemiology of candidiasis in ICU patients and to evaluate the antifungal susceptibility profile of Candida species isolated from the ICU patients. Relevant samples depending on organ system involvement were collected from the ICU patients of a 1 500 bedded tertiary care hospital and processed in the Department of Microbiology in an Indian medical college.

2.2. Ethics approval

Ethics committee approval was granted by the Institutional Ethics Committee of the College & Associated Hospitals, India. Written consent was taken from the patients and they were informed that their participation was voluntary and that they withdrawfromthestudyatanystagewithoutincurringanypenalty.

2.3. Participants

All hospitalized medical/post-operative patients admitted to ICU for＞48 h undergoing therapy for one or more acute organ system failure or requiring intensive post-operative monitoring was studied. Detailed clinical history and investigations of each patient was recorded prospectively and analyzed.

2.4. Collection of samples

Relevant clinical samples depending on organ system involvement were collected as: urine, sputum, tracheal aspirate, urinary catheter, endotracheal tube, abdominal drains. All samples were inoculated on Sabouraud dextrose agar slants containing gentamicin（0.02 mg/mL）and cycloheximide（0.5%）. One set of inoculated slants was incubated at 25°C and the other at 37°C, and they were examined every other day for growth up to 4–6 weeks.

2.5. Microscopy，culture and identification

Identification and speciation of the isolates were done by colony morphology, Gram's staining, germ tube formation, corn meal agar with Tween 80, HiCrome Candida agar and enzymatic triphenyltetrazolium chloride reduction test. For further characterization each isolate was subjected to carbohydrate assimilation and fermentation tests as per standard recommended procedures［9］.

2.6. Antifungal susceptibility test

The in vitro minimal inhibitory concentrations（MICs）of the fluconazole and amphotericin B was done by the broth microdilution method as per Clinical and Laboratory Standards Institute（CLSI）M27-A3 document using Roswell Park Memorial Institute medium and 3-（N-morpholino）propane sulfonic acid buffer. The concentration ranges tested were 0.125–128.000 μg/ mL for fluconazole and 0.016–16.000 μg/mL for amphotericin B. Candida parapsilosis（ATCC 22019）（C. parapsilosis）and Candida krusei（ATCC 6258）（C. krusei）from the American Type Culture Collection（ATCC）used as quality control with each batch of clinical isolates［10］. The MIC breakpoints recommended by CLSI guidelines were followed［10］. For fluconazole, MIC breakpoints were as follows: sensitive（MIC 8 μg/mL）；susceptible-dose dependent（MIC 16–32 μg/mL）；resistant（MIC≥64 μg/mL）. For amphotericin B, isolates with MICs of 1 μg/mL were categorized as resistant［10］.

3. Results

The age（mean±SD）of the patients was 37.90±17.20 years （range 8–81 years）. Candida colonization was seen in 57.3% patients, with 90 Candida isolates from different samples （Figure 1）.

Figure 1. Distribution of Candida spp. in different samples（n = 90）.

The majority of patients（77%）had one or more risk factors at the time of the diagnosis of candidiasis（Table 1）. The mean age of patients with candidiasis（case group）was higher than the patients admitted to the same ICU with no candidiasis （control group）. Case group was divided into two sub-groups: first（CG1）–patients with C. albicans colonization and second（CG2）–patients with NAC colonization. The time of hospital stay（mean）in CG2（19.5 days）＞CG1（9.8 days）＞controls（6.3 days）with no statistically significant difference. In control, CG1 and CG2 group, the percentage of patients with an indwelling device and prolonged antibiotic therapy was significantly higher than control and CG1 groupwhile prolonged antibiotic therapy and surgery in CG2 group （Table 1）.

Table 1 Risk factors for candidiasis among the ICU patients.

Among the isolates, C. albicans accounted for 36.7%and NAC species for 63.3%of all the Candida colonization cases. Among the 63.3%NAC species isolates, the most common was Candida tropicalis（C. tropicalis）（41.1%）followed by Candida glabrata（C. glabrata）（10%）, C. parapsilosis（6.7%）, C. krusei （3.3%）and Candida kefyr（C. kefyr）（2.2%）.

Predominance of C. albicans was seen in urine（16.7%）followed by tracheal aspirate（14.4%）, catheter（3.3%）, endotracheal and abdominal drains（1.1%in each）. NAC was also predominant in urine（26.7%）followed by tracheal aspirate （16.7%）, catheter（8.9%）, endotracheal（6.7%）and sputum （2.2%）. Among NAC spp.: C. tropicalis（15.6%）, C. glabrata （5.6%）and C. krusei（2.2%）were dominant in urine samples while C. parapsilosis（2.2%）and C. kefyr（1.1%）were predominant in urine and tracheal aspirate samples.

Candida colonization rate was highest in 41–50 years （75%）age group, followed by 0–10 years（66.67%）,＞60 years（55.6%）, 31–40 years（54.5%）, 21–30 years（46.2%）, 51–60 years（46.2%）and 11–20 years（36.8%）age group （Table 2）.

Table 2 Age wise distribution of patients and Candida species isolated from the patients.

Table 3 Susceptibility to fluconazole and amphotericin B of colonized isolates of Candida species. n（%）.

The antifungal susceptibility testing showed that 34 Candida isolates（37.8%）were resistant to fluconazole. Out of the 34 resistant Candida isolates, 12（35.3%）were C. albicans and 22 （64.7%）were NAC species as: C. tropicalis 15（68.2%）, C. krusei and C. glabrata 3（13.6%）each and C. parapsilosis 1 （4.5%）. However, 7（7.8%）Candida isolates showed resistance to amphotericin B；2（28.6%）were C. albicans and 5（71.4%）were NAC species as: C. tropicalis 3（60%）, C. krusei 2（40%）as shown in Table 3.

4. Discussion

In recent years, there had been a remarkable increase in the use of indwelling devices in hospitals. Candida species are important nosocomial pathogens. Modern medical procedures including implantation of various kinds of devices contribute to the risk factors for developing candidiasis［11］.

In present study, the majority of patients（77%）had one or more risk factors at the time of the diagnosis of candidiasis. The most common risk factors in these patients were indwelling devices accounting for 98%, followed by prolonged antibiotic therapy（64%）, surgery（58%）, duration of stay＞1 week（42%）, immunosuppressants use（36%）, diabetes mellitus（32%）, multiple organ system involvement（30%）, peritonitis and trauma （18%）each, pneumonia（16%）, tuberculosis（14%）, smoking （12%）, previous surgery, neurological involvement and malignancy（10%）each, renal failure（6%）, heart disease（4%）of patients with Candida colonization.

However, a study at USA reported that the presence of prior intravenous antibiotics（95.8%）, prior surgical procedure （73.9%）, diabetes mellitus（37.3%）, cancer（21.1%）, alcoholism （16.9%）, neutropenia（0.7%）, renal failure（6.3%）, corticosteroid therapy（5.6%）, parenteral nutrition（9.2%）were the common risk factors in adult intensive care patients［12］. According to Arslankoylu et al. on symptomatic and asymptomatic candidiasis in a pediatric ICU, reported the use of broad spectrum antibiotics and parenteral nutrition were the most common risk factors followed by central venous catheter （96.8%）, blood transfusion（96.8%）, surgery（45.2%）and use of corticosteroids（35.5%）［13］.

In India, a study by Singh et al. reported that the most common risk factor was presence of urinary catheter（63.2%）, mechanical ventilation（63.2%）, peritoneal dialysis（63.2%）, followed by central line insertion（47.4%）, diabetes mellitus （26.3%）and use of corticosteroids（21.1%）of patients with fungal colonization of ICU patients［14］. In another Indian study by Jain et al., prolonged antibiotic therapy（100%）, diabetes mellitus（38.6%）and previous surgery（52.9%）were the universal risk factors reported, where 100%of catheterized ICUs patients had previous history of antibiotics. Antibiotics increase the risk of colonization of Candida spp. by suppressing endogenous flora［15］. Likewise, Sahni et al. reported that the antibiotics use, mechanical ventilation and central catheters significantly related to candidaemia［16］. High rate of colonization has been reported in critically ill surgical patients［13］. Urinary catheterization and broad-spectrum antibiotics use were the most common risk factors. On the other hand, Harvey and Myers observed that the most common risk factors in adult patients were central catheterization and blood transfusion［17］.

In this study, CG1（patients with C. albicans colonization）had risk factors of duration of stay＞1 week in ICU（36.8%）and immunosuppressant（31.6%）, and CG2（patients with NAC colonization）had risk factors of multiple organ system involvement（35.5%）, indwelling devices（96.8%）, prolonged antibiotic therapy（77.4%）, duration of stay＞1 week（45.2%）, use of immunosuppressant（38.7%）and diabetes mellitus （35.5%）which were significantly higher and statistically significant（P＜0.05）than the control group. The number of patients with central line insertion（81.3%）and urinary catheter （85.7%）were significantly more in fungal infection group, whereas those with peritoneal dialysis（63.2%）were significantly more in fungal colonization group. However, there was no significant difference in the mean duration of presence of these risk factors in both the groups［14］.

We observed a predominance of NAC species（63.3%）however C. albicans was the most frequently isolated species （36.7%）. This is also consistent with the emergence of predominance of NAC species worldwide［14］. The preponderance of C. tropicalis in our study, is consistent with a prospective study by Chakrabarti et al., where C. tropicalis was the most common（42.1%）yeast isolated［18］.

Wereportalowerrateof C.glabrata（10%）and C.parapsilosis （6.7%）almost similar to a study among adult diabetes mellitus patientsby Al-Attasand Amro,inwhichtheyalsoreportedalower rate of C. glabrata（11.1%）and C. parapsilosis（6.7%）［19］. Traditionally, C. tropicalis has been the second and C. glabrata the third or fourth most common Candida species isolated［20］. Our study clearly shows that C. krusei（3.3%）and C. kefyr （2.2%）are relatively rare as seen in rest of India［18,21,22］. In last few decades, there have been numerous reports documenting the rare isolation of C. kefyr and C. krusei［21–24］.

In this study, Candida colonization rate was 50%, which is similar to the colonization rate of 51%found in a previous international multicentric study from 75 countries［25］. Candida colonization rate due to NAC isolates（63.3%）was higher than C. albicans isolates（36.7%）. In our study, urinary tract Candida colonization was the most common.

After considering the corresponding sign of η during each period, the quadratic and cubic polynomials were factor-ized into the following forms, so defining the observed window functions as quadratic and cubic binomials:

In our study, highest rate of Candida colonization was seen in 41–50 years（75.00%）followed by 0–10 years（66.67%）,＞60 years（55.60%）, could be due to lowered host defenses at extremes of age. This finding is also supported by many other researchers'. Prolonged antibiotic use increases the risk of colonization of Candida species by suppressing the endogenous flora［15］. Higher rate of Candida colonization in 0–9 years could be understood as they are more susceptible to infections due to various reasons including weak immune systems and in the＞60 years age group this could correlate with prevalence of debilitating conditions, decreased immune status and aging［26］.

The most common site of Candida isolation was urine （43.3%）, followed by tracheal aspirate（31.1%）, urinary catheter （12.2%）, endotracheal tube（7.8%）, abdominal drains（3.3%）and sputum（2.2%）. Singh et al., reported that in medical and surgical critical care section patients, the most common site or specimen of fungus isolation in patients was also urine（74.7%）, followed by blood（20.8%）［14］. In a study by Jain et al., C. tropicalis was predominant in catheterized ICU patients［15］similar to our study where C. tropicalis was most common isolate from catheterized and endotracheal patients.

In this study, C. albicans was most commonly isolated from urine and tracheal samples. Most observational studies of candiduria have reported C. albicans to be the predominant species isolated［27］. However, Paul et al., reported C. tropicalis as the most prevalent NAC species causing candiduria［28］.

In our study, colonization of ICU patients with Candida species was common, occurring at least once in 73%of patients, a figure not dissimilar to that found in prior studies［12］. As the length of stay in ICUs and the frequency of invasive procedures increases, the incidence of Candida colonization and Candida infection increases［13］. Most patients suffer no ill effects due to Candida colonization because of its low-level virulence；however, in some patients with suppressed defenses, the organisms invade and cause illness［13］. Stamos and Rowley［29］reported that Candida colonization was observed after 7 days of hospitalization, which is similar to our results.

In our study 37.8%Candida isolates demonstrated resistance to fluconazole, concordant to a study by Deorukhkar et al.［27］. In our study, the resistance rate for fluconazole（37.8%）was more compared to amphotericin B（7.8%）. In the present study, C. tropicalis isolates were found to be more resistant to fluconazole（38.5%）. The increase in the rate of fluconazole resistance in C. tropicalis is of concern because this species is one of the most commonly isolated NAC species and fluconazole is the most common antifungal agent used for the treatment of various types of candidiasis［27］. In this study, C. albicans（36.4%）isolates was also found to be resistant to fluconazole, compared to the different resistance rates reported in other studies（10.5–21%）［30］.

A hundred percent resistance was reported for C. krusei, with its greatest potential to acquire resistance to fluconazole because of its intrinsic resistance toward azoles and poor susceptibility to all other antifungals, including amphotericin B［31,32］. Also, the resistance of C. glabrata to fluconazole was consistently higher （16.7%）with increased resistance（19.2%）reported by Bassetti et al.［20］. A reduced antifungal susceptibility in NAC species and a correlation with routine fluconazole prophylactic use has been suggested［20］. Extensive fluconazole use is one of the possible causes, for the increased resistance to the drug as well as for the progressive substitution of albicans species with non-albicans drug resistant strains as principal etiologic agent of infection［33］. The sensitivity pattern of Candida species as revealed in this study shows that amphotericin B to be suitable drug for empirical therapy unlike fluconazole with a high resistance in Candida species［30］.

The outcomes of our study, highlight the predominance of NAC species colonization in ICU patients similar to the trends in the western countries, a cause of concern in our country as reported by other workers too［14］. The major risk factors were use of indwelling devices, prolonged antibiotic therapy and surgery. The increase in resistance being recorded to fluconazole has a major bearing on the morbidity and management of these patients and needs to be further worked upon. Identification and antifungal susceptibility of Candida isolates from ICU patients will help in building a data center of the prevalent Candida species along with their antifungal resistance profile and will go a long way in the management of these serious patients.

We declare that we have no conflict of interest.

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*Corresponding author:Ravinder Kaur, Director Professor & HOD, Department of Microbiology, Lady Hardinge Medical College and Associated Hospitals, New Delhi 110001, India.