Candidemia in intensive care unit patients: A one year study from a tertiary care center in South IndiaS Giri1, AJ Kindo2, J Kalyani3
1 Department of Gastrointestinal Sciences, Wellcome Trust Research Laboratory, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India
3 Department of Microbiology, Sri Muthukumaran Medical College, Chennai, Tamil Nadu, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.118036
Source of Support: None, Conflict of Interest: None
Background: The occurrence of candidemia is on a rise worldwide. Non-albicans Candida species have emerged as major causes of candidemia in many countries. Added to it is the problem of antifungal resistance in Candida isolates. Objectives: To find out the prevalence of candidemia in our intensive care unit (ICU) setup along with the antifungal susceptibility pattern of Candida isolates and various risk factors associated with candidemia. Materials and Methods: All Candida isolates from blood stream infections of ICU patients were included in the 1 year study period (November 2008-October 2009). The isolates were speciated using various phenotypic tests. Antifungal susceptibility testing was done by disk diffusion methods according to Clinical and Laboratory Standards Institute guidelines and also using CANDIFAST. Various risk factors associated with the development of candidemia were looked into. Results: A total of 39 Candida isolates were isolated during the study period of 1 year (prevalence of 0.65%). Candida tropicalis (74.35%) was the most common isolate followed by Candida albicans, Candida parapsilosis, Candida krusei and Candida glabrata. All the 39 Candida isolates (100%) were sensitive to amphotericin B while 12 isolates (30.8%) were resistant to fluconazole. The risk factors commonly associated with candidemia patients were long term antibiotic therapy (64.1%), use of central venous catheters (56.4%), urinary catheters (53.9%), steroid therapy (35.9%) and diabetes mellitus (33.3%). Conclusion: Candidemia is emerging as a significant problem in hospitalized patients, especially in ICU setups. Non-albicans Candida species are the major cause of candidemia as found in our study and few other studies in India. Multicentric studies involving many hospitals are required to know the true prevalence of candidemia and the status of antifungal drug resistance among Candida isolates in our country.
Keywords: Antifungal susceptibility, candidemia, immunosuppression
The incidence and prevalence of candidemia is on a rise in many countries worldwide. Candida species are the fourth leading cause of nosocomial blood stream infection (BSI) in the United States.  In India, the picture is not very clear due to lack of multicentric studies although there are a few studies indicating the increasing trend of candidemia in some tertiary care hospitals.  The mortality rate associated with candidemia worldwide is also high ranging from 10% to 49%. 
More than 17 different species of Candida have been reported to be etiologic agents of invasive candidiasis in humans. Although more than 90% of invasive diseases by Candida are caused by 5 species - Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei- the number of reported species continues to grow.  Although C. albicans still remains the most common cause of candidemia worldwide, there has been an increase in the isolation of non-albicans Candida species.  In the few available studies from India, C. tropicalis has been the most common species of Candida isolated from blood. ,
Many studies have established independent risk factors for candidemia on the basis of multivariate analyses.  The important independent risk factors include use of broad-spectrum antimicrobials, cancer chemotherapy, mucosal colonization by Candida species, indwelling vascular catheters like central venous catheters (CVCs) and others.  Other predisposing factors include immunocompromised states such as diabetes mellitus and malignancy. 
With the emergence of non-albicans species of Candida worldwide, especially C. glabrata and C. krusei, antifungal drug resistance has become a major cause of concern in the management of candidemia. Resistance to fluconazole and other triazoles is very high among these species of Candida.  Other non-albicans Candida species like C. tropicalis and C. parapsilosis have been found to have variable susceptibility pattern to the azole group of drugs. There have been a few reports of Candida species being resistant to amphotericin B and echinocandins also. 
The present study was undertaken to look into the various aspects of candidemia occurring in intensive care unit (ICU) patients. This included the prevalence of candidemia in our ICU set up, the causative species of Candida along with the antifungal susceptibility pattern of the Candida isolates and the various predisposing factors associated with candidemia.
This study was conducted at Sri Ramachandra Medical College and Research Institute, Chennai during the period November 2008 to October 2009. Ethical clearance for the study was obtained from the Institutional Ethics Committee, Sri Ramachandra University. All blood isolates received from ICU patients during this period were screened for candidemia. Children and adults admitted in ICUs were included in the study if Candida organisms were isolated from a single blood culture.
Blood samples were collected from patients after all aseptic precautions. 1, 2 or sometimes even 3 samples were collected at the same time from the patients. Blood culture was performed using the automated blood culture system (VersaTREK). Once a blood culture bottle showed positive growth, initially a gram stain was done from the broth in the bottle to look for yeast cells. Then, it was subcultured for isolation and speciation.
Each positive blood sample was inoculated onto two plates of Sabouraud dextrose agar (SDA) with gentamicin and incubated at 25°C and 37°C. The colonies appeared within 24 hours in all the cases. Then, several phenotypic tests were performed for speciation of the Candida isolates. These included germ tube test, sugar fermentation and assimilation tests, tetrazolium reduction medium and CHROMagar Candida (CHROMagar, Paris, France). Antifungal susceptibility testing was also performed by disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines.  The antifungal disks tested were amphotericin B (100 units) and fluconazole (25 mcg). Quality control tests using reference strain C. albicans ATCC 90028 were done on each day that susceptibility tests were performed. The CANDIFAST kit (International Microbio, France) was also used to identify the various pathogenic Candida species as well as the testing of their resistance to various antifungal agents.
Relevant clinical details were collected for the patients who had candidemia. These included age, sex, clinical diagnosis, history of previous antibiotic therapy and presence of other predisposing factors such as CVC, mechanical ventilation, steroid therapy, total parenteral nutrition (TPN) and any immunocompromised conditions like diabetes, malignancy, neutropenia and others.
The demographic and clinical characteristics of patients with candidemia who expired and who survived were compared using Fisher's exact test for univariate analysis. P<0.05 (2 tailed) were considered statistically significant. Odds ratio (OR) was calculated to evaluate the strength of association. To determine the independent effects of these variables on the outcome in candidemia patients, multiple regression analysis was performed for variables which were found to have a statistically significant association (P<0.05) with the outcome after univariate analysis. GraphPad Instat version 3.06 (GraphPad Inc, San Diego, CA, USA) was used for all statistical analysis. Statistical significance was accepted if the P values were less than 0.05. All reported P values were based on two-tailed tests.
Blood samples from a total of 5976 ICU patients were received in the central laboratory of Sri Ramachandra Medical College for culture during the study period of 1 year (November 2008-October 2009). 1, 2 or even multiple samples were received simultaneously from each patient which were considered as a single sample for the study. Out of the 5976 ICU cases whose blood samples were received during the study period, 677 (11.32%) samples were culture positive. Among the positive samples, 637 (10.65%) were bacteremia and 40 (0.67%) were cases of fungemia. Out of the 40 cases of fungemia, 39 (0.65%) were cases of candidemia while a single case of Aspergillus flavus in blood was diagnosed during the period. Hence the overall prevalence of Candida species in blood samples during the study period was 0.65%.
The most common isolate from blood samples during the study period was Staphylococcus aureus, followed by Escherichia More Details coli, Klebsiella pneumoniae, Acinetobacter species and Pseudomonas species respectively. Candida species was the sixth most common isolate from blood samples. Out of the 39 blood isolates of various Candida species, the most common was C. tropicalis (74.35%). It was followed by C. albicans, C. parapsilosis, C. krusei and C. glabrata [Figure 1]. Majority of the infected patients were males (61.54%). Age of the patients having candidemia ranged from 3 days to 79 years with a mean of 35.14 years. The length of ICU stay for candidemia cases ranged from 9 days to 86 days with a mean of 26.4 days.
A number of predisposing factors were found among the candidemia cases. The majority of patients had multiple predisposing factors that have been associated with candidemia. For 22 cases (56.4%), ≥3 risk factors were identified. The risk factors which were more commonly found to be associated with candidemia in our study were long term antibiotic therapy (64.1%), use of CVCs (56.4%), urinary catheters (53.9%), steroid therapy (35.9%) and diabetes mellitus (33.3%). Other risk factors which were found to be associated with candidemia were mechanical ventilation (28.2%), prior surgery (25.6%), TPN (23.1%), preterm babies with low birth weight (LBW) (17.9%) and malignancy (7.7%). Neutropenia was found only in 2.5% of cases.
The time period of antibiotic usage prior to the development of candidemia varied from 4 days to 31 days with a mean of 19.45 days. Multiple antibiotics (≥2) were used in 30 cases (76.9%). The most common antibiotics used were the beta lactams (in 94.87% of cases). Other antibiotics commonly used were metronidazole (33.33%), aminoglycosides (30.77%), vancomycin (20.51%), linezolid (17.95%) and fluoroquinolones (12.82%).
Antifungal susceptibility testing of all 39 Candida isolates was performed by disk diffusion method according to CLSI guidelines (CLSI document M44-A2).  The resistant isolates were tested using CANDIFAST (International Microbio, France) also. All the 39 Candida isolates (100%) were sensitive to amphotericin B. 12 Candida isolates (30.8%) were resistant to fluconazole.
Out of the 39 candidemia cases, 9 (23.8%) expired while 30 cases recovered with anti-fungal therapy. A total of 10 potential risk factors for mortality in candidemia patients were analyzed by univariate analysis [Table 1]. Among them, resistance to fluconazole (P=0.0141) and the presence of mechanical ventilation (P=0.0006) were the only risk factors whose association with mortality was found to be statistically significant. Since only two of the risk factors (fluconazole resistance and mechanical ventilation) were found to have statistically significant association with mortality, a bivariate regression analysis was performed, which revealed that both these variables were independently associated with mortality in patients with candidemia [Table 1].
The prevalence of Candida species in BSI has increased worldwide in the last three decades. In our study, the overall prevalence of Candida species in BSI of ICU patients was found to be 0.65% in the 1 year study period. Candida species was found to be the 6 th most common cause of BSI in ICU patients in our study. There are many reports on the prevalence of Candida in BSI from different parts of the world. In the United States, a 7 year long study has reported Candida species to be the 4 th most common cause of BSI in hospitals.  A 10 year study on the epidemiology of candidemia in Switzerland found Candida species to be the 7 th most common cause of BSI in tertiary care hospitals.  An increased prevalence of candidemia has also been noted in Brazil and some European nations like Norway, Iceland, Denmark  and Finland. 
However, the scenario is not very clear in India and most other Asian countries due to lack of multicentric studies on candidemia. In a 13 year long study on candidemia in a tertiary care hospital in Thailand, the prevalence of Candida species was found to be 6.14% among isolates for blood culture.  There are very few studies in India indicating the occurrence of Candida species as a causative agent of BSI. A study by Verma et al. from SGPGI, Lucknow ranked Candida species as 8 th among all isolates causing BSI.  In a 5 year study from All India Institute of Medical Sciences (AIIMS), New Delhi, Xess et al., found a prevalence of 6% for Candida species in BSI.  Sahni et al., from Maulana Azad Medical College, New Delhi found the incidence rate of candidemia to be 6.9%.  Another New Delhi based study found that the percentage of Candida species among positive blood culture isolates was 18%.  A study in South India reported an incidence rate of 5.7% for candidemia among children with hematological malignancies.  Another study from Rohtak, North India reported an isolation rate of 8.1% for Candida species from cases of neonatal septicemia. 
The number of species of Candida isolated from BSI has been increasing during the last few years and many rare species have been reported to cause candidemia in different parts of the world. More than 17 species of Candida have been implicated in human infections till date and the list of reported species continues to grow.  It not only reflects the changing patterns of BSI worldwide, but also the rapid developments made in the field of diagnostic mycology, which has made it easier to detect and diagnose even rare fungal pathogens. It also denotes a substantial increase in the group of immunocompromised patients in the last few years who are more susceptible to opportunistic fungal infections.
In our study, C. tropicalis accounted for the maximum number of cases (74.35%). It was followed by C. albicans (10.25%), C. parapsilosis (7.69%), C. krusei (5.13%) and C. glabrata (2.56%). Among the Candida species, C. albicans has been the most common isolate from BSI worldwide. The percentage of C. albicans isolated from blood has varied from 50% (in the SENTRY surveillance program 1997-2000) to 71% (Fungal Disease Registry, Canada 1992-1994).  In a study from Spain, a nationwide sentinel surveillance of candidemia in 40 tertiary care hospitals reported the isolation of C. albicans in 49.08% of all cases of candidemia.  However during the last few years, the isolation of non-albicans Candida species from candidemia cases has been steadily increasing in different parts of the world. A number of international surveillance programs like the ARTEMIS antifungal surveillance program have noted a decreasing trend in the isolation of C. albicans although it still remains the most commonly isolated species overall.  The ARTEMIS surveillance study showed an increase in Candida species like C. tropicalis (4.6% in 1997-7.5% in 2003) and C. parapsilosis (4.2% in 1997-7.3% in 2003). 
In the Indian scenario, C. tropicalis has emerged as the most common cause of nosocomial candidemia in the past few decades and epidemiological studies have implicated C. tropicalis in as many as 67-90% of cases of candidemia. , The increased use of fluconazole has been determined to be the major cause of predominance of non-albicans Candida, especially C. tropicalis over C. albicans.  In 2 studies from South India by Shivaprakasha et al.,  and Adhikary et al.,  C. tropicalis was found to be the most common cause of candidemia (35.6% and 39.7% respectively). Xess et al., from AIIMS, New Delhi also found C. tropicalis to be the most common species of Candida in blood isolates. 
The risk factors associated with invasive candidiasis and candidemia in particular have not changed substantially in the past two decades. Prior to 1990, much importance was given to factors like neutropenia and malignancy in the development of candidemia, but in recent years, much emphasis has been laid on other risk factors like long period of hospitalization in ICUs (especially surgical ICUs). 
In our study, the most common risk factor associated with candidemia was long term antibiotic therapy (64.10%). The most common antibiotic used were the beta lactams (in 94.87% of cases) followed by other antibiotics like metronidazole, aminoglycosides and vancomycin. The mean time period of antibiotic usage prior to the development of candidemia was 19.45 days and multiple antibiotics (≥2) were used in 30 cases (76.9%). Long term antibiotic therapy especially with multiple antibiotics has been found to be an independent risk factor in the development of candidemia in many studies worldwide.  This is due to the fact that long term use of antibiotics like betalactams and vancomycin lead to depletion of the normal bacterial flora, which then leads to overgrowth of opportunistic fungal pathogens. Few Indian studies have also implicated long term antibiotic therapy as a common risk factor in the development of candidemia. Xess et al., reported prior use of antibiotics to be the most common predisposing factor in candidemia patients (71.2%).  Another 5 year long study by Chowta et al., found that prolonged use of antibiotics was the second most common risk factor for the development of candidemia (34.5%). 
The second most common predisposing factor in candidemia cases in our study was the use of CVCs (56.4%). Indwelling vascular catheters (especially CVCs) have been implicated as an important risk factor in candidemia patients worldwide. Candida species strongly adhere to materials used in intravascular devices and provide a potential nidus of infection. , C. parapsilosis has been particularly implicated to cause intravascular catheter related infections in neonates and pediatric age group. 
Urinary catheters were present in 53.90% of patients in our study. Xess et al., from AIIMS also found the use of urinary catheters to be an important predisposing factor in candidemia patients (55.6%).  Candida infections of the urinary tract are strongly associated with the presence of a urinary catheter. Candiduria has been found to be a risk factor for candidemia and can sometimes be an indicator of impending sepsis with Candida species in patients admitted to hospitals, especially those in ICUs. A few studies have suggested that as many as 10% of all candiduria cases may be associated with candidemia. 
Steroid therapy in hospitalized patients have been implicated as a risk factor for the development of candidemia as they cause immunosuppression.  In our study, 35.9% of patients had been administered steroids for various clinical conditions before developing candidemia. Diabetes has long been known as an important predisposing factor for opportunistic infections, especially fungal infections as it causes long term immunosuppression. In our study, 33.3% of the patients were diabetic. Diabetic patients are at an increased risk of colonization by Candida species, which can then lead to disseminated disease affecting various organ systems.  Other less commonly associated risk factors in our study included mechanical ventilation, prior surgery, TPN, preterm babies with LBW, malignancy and neutropenia.
The antifungal susceptibility and resistance pattern of all Candida isolates from blood was evaluated. All the 39 Candida isolates were found to be susceptible to amphotericin B while fluconazole resistance was seen in 12 (30.8%) Candida isolates. Resistance pattern to azole group of antifungal drugs (e.g., fluconazole) among Candida isolates from blood have been extensively studied in various countries of the world. Many potential mechanisms of azole resistance have been proposed. Alteration of drug efflux, reduced intracellular accumulation of fluconazole due to changes in CDR (Candida drug resistance) genes and increased expression of ATP-binding cassette transporter gene are some of the mechanisms responsible for development of azole resistance in Candida isolates. , Although fluconazole still remains a safe and effective choice for the treatment of candidemia, an increasing trend of fluconazole resistance in Candida isolates from blood has been reported in many studies.  This is mainly due the changing spectrum of Candida species causing candidemia from C. albicans to non-albicans Candida species, especially C. glabrata and C. krusei.
In India, the scenario of antifungal drug resistance to Candida isolates from blood is not very clear due to lack of multicentric studies over a period of many years. Xess et al., from AIIMS found fluconazole resistance to be present in 11.7% of Candida isolates.  In another study from New Delhi by Kothari et al., fluconazole resistance was reported in 36% of candidemia isolates.  An earlier study from a tertiary care center in North India by Gupta et al., showed fluconazole resistance in isolates of C. glabrata only (37.5%).  A study from Chennai by Kumar et al., found that 17.2% of all strains causing invasive candidiasis in children with onco-hematological diseases were resistant to fluconazole.  However, few studies from India have also reported very low percentages of resistance to different azoles. Capoor et al., in a study from New Delhi have reported a resistance rate of 4.9% for fluconazole and 3.9% for itraconazole among isolates of Candida.  Another study from North India by Goel et al., showed 4.5% of all candidemia isolates to be resistant to fluconazole.  Further multicentric based studies over a long period are necessary to evaluate the true picture of azole resistance in India.
Resistance to amphotericin B among Candida isolates was not found in our study. There are very few reports of amphotericin B resistance in Candida isolates from cases of candidemia in India.  Although amphotericin B has a rapid cidal action against most strains of Candida species (especially C. albicans), it is not the first choice for treatment of cases of candidemia because of the nephrotoxicity associated with it. The lipid formulations of amphotericin B have a better side-effect profile. However in a developing country like India, fluconazole is more widely used than other antifungal drugs for treatment of candidemia because it is significantly less expensive. Another advantage of fluconazole is that it is available in both intravenously administered and oral formulations with high bioavailability. Echinocandins are highly expensive and cannot be afforded by a majority of the Indian population.
Univariate analysis for 10 potential risk factors for mortality in candidemia patients in our study found fluconazole resistance (P=0.0141) and mechanical ventilation (P=0.0006) to be the only risk factors which were significantly associated with mortality. Subsequently, a bivariate regression analysis for the two risk factors was performed, which showed that both these risk factors were independently associated with mortality in cases of candidemia [Table 1]. There are few studies which have found these two factors (mechanical ventilation and fluconazole resistance) to be significantly associated with mortality in cases of candidemia. , A recent study by Guimarães et al., found mechanical ventilation to be significantly associated with mortality in elderly patients with candidemia.  In another study by Baddley et al., the association between fluconazole resistance and mortality in patients with candidemia was found to be significant. 
In conclusion, candidemia in hospitalized patients (especially ICU patients) is emerging as a significant problem worldwide. Various risk factors have contributed to this increase in candidemia in the hospital settings. The increase in resistance to antifungal agents among Candida isolates has resulted in increased mortality and morbidity. Further studies are necessary to understand the changing epidemiology of candidemia in India and other countries.