J Med Microbiol 56 (2007), 255-259; DOI: 10.1099/jmm.0.46817-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Species distribution and antifungal susceptibility of Candida bloodstream isolates in Kuwait: a 10-year study
Eiman M. Mokaddas,
Noura A. Al-Sweih and
Zia U. Khan
Department of Microbiology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat, Kuwait 13110
Correspondence
Zia U. Khan
Ziauddin{at}hsc.edu.kw
Received 4 July 2006
Accepted 13 October 2006
Bloodstream infections due to Candida species are important complications in severely ill hospitalized patients. This study presents data on species distribution and antifungal susceptibility profiles of Candida bloodstream isolates obtained from Kuwait during a 10-year period. All the bloodstream isolates were identified to species level by the germ tube test and carbohydrate assimilation profile using the VITEK 2 yeast identification system. Using E-test strips for amphotericin B, fluconazole, 5-flucytosine and voriconazole, MICs were determined on RPMI agar supplemented with 2 % glucose. The MIC breakpoints for resistance were based on Clinical and Laboratory Standards Institute criteria or those published by reference laboratories, and were as follows: amphotericin B, >1 µg ml–1; fluconazole, 
64 µg ml–1; 5-flucytosine, 32 µg ml–1; and voriconazole, 4 µg ml–1. In all, 607 bloodstream yeast isolates were obtained over the past 10 years in Kuwait. Candida albicans was the predominant species (39.5 %), followed by Candida parapsilosis (30.6 %), Candida tropicalis (12.4 %), Candida glabrata (5.6 %) and Candida krusei (1.6 %). All C. albicans, C. tropicalis and C. glabrata isolates were susceptible to amphotericin B. Of 186 isolates of C. parapsilosis tested, only four (2 %) exhibited an MIC for amphotericin B of >1 µg ml–1. Resistance to fluconazole was observed in nine (3.8 %) C. albicans isolates, two (5.8 %) C. glabrata isolates and four (40 %) C. krusei isolates. Resistance to 5-flucytosine was observed in two (0.8 %) C. albicans isolates, seven (9.3 %) C. tropicalis isolates, three (1.6 %) C. parapsilosis isolates and all ten (100 %) C. krusei isolates. All the isolates of C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei were susceptible to voriconazole, including those resistant to fluconazole. Although amphotericin B and fluconazole are widely used in clinical practice in Kuwait, resistance to these drugs remained low.
Abbreviations: CLSI, Clinical and Laboratory Standards Institute.
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INTRODUCTION
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Candida species are one of the major causes of nosocomial bloodstream infections worldwide (Jarvis, 1995; Pfaller et al., 2001). Despite the availability of an expanded antifungal armamentarium, the mortality associated with invasive Candida infections remains high, ranging between 19 and 49 % (Blot et al., 2002; Alonso-Valle et al., 2003; Gudlaugsson et al., 2003; Morgan, 2005). The incidence and associated mortality due to candidaemia can be influenced by several factors including characteristics of the population at risk, standard of the healthcare facilities available, distribution of Candida species and prevalence of resistance (Hobson, 2003; Eggimann et al., 2003). Hence, epidemiological information available for one centre or geographical region may not be applicable to others (Hobson, 2003). The increased isolation rates of non-albicans Candida species and a gradual shift in the antifungal susceptibility profile, especially against azole antifungal agents, have underlined the need to monitor laboratory data for possible emergence of resistance and to select the most appropriate antifungal agent for therapy (Sanglard & Odds, 2002; Eggimann et al., 2003). Candida species have been reported as important nosocomial pathogens in several studies reported from the Middle East (Nampoory et al., 1996; Khan & Chugh, 2000; Al-Essa et al., 2000; Mokaddas et al., 2000; Rennert et al., 2000; Bukharie, 2002; Ahmad et al., 2002, 2003; Ellis et al., 2003; Al-Jasser & Elkhizzi, 2004). However, comprehensive studies on antifungal susceptibility of Candida species from this region are lacking. In this communication, we present data on five major Candida species isolated from candidaemic patients during 1996–2005 and their antifungal susceptibility profiles.
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METHODS
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Bloodstream yeast isolates.
The bloodstream yeast isolates referred to the Mycology Reference Laboratory for identification and antifungal susceptibility testing from January 1996 to December 2005 were included in the study. These isolates originated from different categories of patients admitted to tertiary care hospitals in Kuwait. The isolates were initially tested with the germ tube test. All the germ tube positive isolates were provisionally identified as Candida albicans or Candida dubliniensis. The germ tube negative isolates were identified by carbohydrate assimilation tests using the VITEK 2 yeast identification system (bioMérieux). None of the isolates was identified as C. dubliniensis by the VITEK 2 system or morphological characteristics on sunflower seed agar (Khan et al., 2004).
Antifungal susceptibility by the E-test.
The in vitro activity of the antifungal agents was determined by the E-test (AB Biodisk), in accordance with the manufacturer's instructions. The E-test was performed by inoculating a 150 mm Petri dish containing 60 ml RPMI agar supplemented with 2 % glucose and buffered to pH 7.0 with MOPS. The inoculum was applied with cotton swabs using growth suspension prepared in 0.85 % NaCl with turbidity adjusted to 0.5 McFarland standard. Plates were incubated for 24 h at 35 °C and read after 24 h. For Candida glabrata, the incubation time was 48 h. Reference strains C. albicans ATCC 90028 and Candida parapsilosis ATCC 22019 were used for quality control. Interpretive susceptibility criteria for fluconazole and 5-flucytosine were those recommended by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, 2002). For fluconazole, isolates showing MICs 
8.0 µg ml–1 were regarded as susceptible, 16–32 µg ml–1 as dose-dependent susceptible and 64 µg ml–1 as resistant. For 5-flucytosine, isolates showing MICs 4 µg ml–1 were considered as susceptible, 8–16 µg ml–1 as intermediate and 32 µg ml–1 as resistant. For voriconazole, recently approved CLSI breakpoints were followed: 1.0 µg ml–1 regarded as susceptible, 2.0 µg ml–1 as dose-dependent and 4 µg ml–1 as resistant (Pfaller et al., 2003, 2005). Due to the lack of defined breakpoints for amphotericin B, isolates showing an MIC of 1.0 µg ml–1 were taken as susceptible and those with MIC >1 µg ml–1 were considered as resistant (Rex et al., 1997; Ostrosky-Zeichner et al., 2003a).
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RESULTS AND DISCUSSION
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During the 10-year study period, a total of 607 bloodstream yeast isolates were received in the Mycology Reference Laboratory for identification. The species distribution of Candida species was as follows: C. albicans, 39.5 % (n=240); C. parapsilosis, 30.6 % (n=186); Candida tropicalis, 12.4 % (n=75); C. glabrata, 5.6 % (n=34); Candida krusei, 1.6 % (n=10); and other yeast species, 10.2 % (n=62) (Table 1
). Of these, 198 (32.6 %) isolates were received for identification during 1996–2000 as compared to 409 (67.4 %) during 2001–2005, representing a more than twofold increase in the latter period. The most prominent increase was observed in the isolation rate of C. tropicalis (3.4-fold), followed by C. albicans (3.1-fold), C. glabrata (1.8-fold) and C. parapsilosis (1.4-fold). The ratio of C. albicans to non-albicans isolates in the two study periods was 59 : 139 and 181 : 228, respectively. The greater than twofold increase in candidaemia cases detected between the two study periods may be a reflection of its rising incidence. Recent epidemiological studies from the USA and Europe have suggested that the annual incidence of candidaemia in some centres might have stabilized or decreased (Trick et al., 2002; Hobson, 2003; Eggimann et al., 2003; Morgan, 2005). However, this does not appear to be the case in Kuwait. Here it may be mentioned that our data are based on the number of bloodstream isolates received in the Reference Laboratory for identification and susceptibility testing from different tertiary care hospitals and not on isolation rates/1000 admissions.
Consistent with the published reports from different parts of the world (Eggimann et al., 2003; Hobson, 2003), C. albicans was the predominant species, followed by C. parapsilosis. A relatively greater proportion of C. parapsilosis isolates in our study may be related to the large number of isolates received from paediatric patients where small outbreaks of candidaemia due to this species had occurred. Of 138 candidaemia cases recorded during 1998–2003 in a neonatal intensive care unit of the maternity hospital, Kuwait, 48.2 % were caused by C. parapsilosis (N. A. Al-Sweih, unpublished results). Considerable differences in the distribution of Candida species were also reported in different published studies from the region (Table 2). These differences may be attributed to several factors including the type of patient population studied, sample size, duration of study and presence of intravascular catheters. In a solitary report from Saudi Arabia based on 32 candidaemic patients, C. parapsilosis was the predominant species; notably 66 % of these patients had a central venous catheter in place (Bukharie, 2002).
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Table 2. Distribution of Candida species isolated from candidaemic patients in different studies reported from the Middle East
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The data on E-test susceptibility for amphotericin B, fluconazole, 5-flucytosine and voriconazole against five Candida species are presented in Table 3. Except for four (2.2 %) isolates of C. parapsilosis which showed an MIC of 1 µg ml–1, all Candida isolates were susceptible to amphotericin B. Resistance against 5-flucytosine was observed in 0.8 % of C. albicans isolates, 9.3 % of C. tropicalis isolates, 1.6 % of C. parapsilosis isolates and all the isolates of C. krusei. Nine (3.8 %) C. albicans isolates and two (5.8 %) C. glabrata isolates exhibited resistance to fluconazole. None of the isolates was found to be resistant to voriconazole, including those that showed resistance to fluconazole (Table 3).
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Table 3. In vitro susceptibility of Candida bloodstream isolates to amphotericin B, 5-flucytosine, fluconazole and voriconazole
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A comparison of the MIC90 values for amphotericin B for different Candida species suggested that C. albicans tended to have higher susceptibility (0.25 µg ml–1) than C. tropicalis (0.38 µg ml–1), C. krusei (0.38 µg ml–1), C. parapsilosis (0.5 µg ml–1) and C. glabrata (0.38 µg ml–1). These observations were consistent with several studies comparing MICs for bloodstream isolates of C. albicans with those for non-albicans Candida species (Simor et al., 1997; Ostrosky-Zeichner et al., 2003a; Lu et al., 2004). While resistance to 5-flucytosine for C. albicans, C. parapsilosis and C. glabrata was generally low (<2 %), 9.3 % of C. tropicalis isolates and all the C. krusei isolates were found to be resistant to this drug. In early studies, up to 6.5 % of C. albicans isolates in Europe and 33 % in the USA were found to be intrinsically resistant (Scholer, 1980), which apparently precluded use of this drug as a single therapeutic agent. However, more recent surveys based on CLSI methodology demonstrated much lower levels of 5-flucytosine resistance (Pfaller et al., 1998, 2002; Barchiesi et al., 2000). Pfaller et al. (2002) determined the in vitro activity of 5-flucytosine against 8803 clinical isolates, representing 18 Candida species, which were obtained from 200 medical centres worldwide. Regardless of the Candida species tested, 90 % of isolates showed MICs of 1 µg ml–1. While 97 % of C. albicans isolates and 92 % of C. tropicalis isolates were susceptible to 5-flucytosine, 28 % of 184 C. krusei isolates were resistant and 67 % showed intermediate susceptibility. In another study of 2000 Candida bloodstream isolates from the USA, Ostrosky-Zeichner et al. (2003a) found that 6 % of C. tropicalis and 12 % of C. krusei isolates were resistant to this drug, whereas resistance among other Candida species was less than 5 %. It is apparent that Candida species have variable susceptibilities to 5-flucytosine, and C. krusei is relatively less susceptible with resistance rates ranging between 7 and 44 % in different studies (Medoff & Kobayashi, 1980; Pfaller et al., 2002; Quindos et al., 2004). In this context, demonstration of 100 % resistance to 5-flucytosine in our C. krusei isolates is noteworthy.
Susceptibility to fluconazole was similar to that seen in other major surveillance studies reported from Europe and the USA (Sanglard & Odds, 2002; Ostrosky-Zeichner et al., 2003a). In our isolates, with the exception of C. krusei where resistance to fluconazole was 40 % (4/10), the other Candida species were found to be quite susceptible (Table 3
). Although C. krusei had a low prevalence (1.6 %) among bloodstream isolates, its resistance to fluconazole may have therapeutic implications. Since some of the isolates of C. krusei could be intrinsically resistant to fluconazole, it is not clear whether these isolates developed resistance during chemoprophylaxis. Ostrosky-Zeichner et al. (2003a) found that 34 % of 50 C. krusei bloodstream isolates were resistant to fluconazole, which is similar to what we observed in Kuwait. Interestingly, Candida species isolates exhibiting resistance to fluconazole were not resistant to voriconazole. Hence voriconazole, due to its wider species coverage, could be used in the treatment of candidaemia cases caused by fluconazole-resistant strains of C. krusei (Ostrosky-Zeichner et al., 2003b).
In summary, we have presented data on species distribution and antifungal susceptibility profiles of Candida bloodstream isolates received in the Mycology Reference Laboratory over a 10-year period. The percentage prevalence of different Candida species was largely similar to what has been reported in most published studies (Eggimann et al., 2003). Non-C. albicans yeast species constituted 60.5 % of the isolates. Although amphotericin B and fluconazole are widely used in clinical practice in Kuwait, there was no evidence of enhanced resistance.
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ACKNOWLEDGEMENTS
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The authors are thankful to all the microbiologists for referring bloodstream yeast isolates to the Mycology Reference Laboratory. Excellent technical support received from R. Chandy and Daad Farhat is acknowledged.
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INTRODUCTION
METHODS
