Prevalence of Candida Species, Including Candida dubliniensis, in Singapore

Abstract

We present data on the prevalence of Candida species isolated from inpatients in three Singapore hospitals and from vaginal samples collected at community clinics. Strikingly, ∼20% of the isolates from blood and vaginal samples were fluconazole-resistant species. By analyzing species-specific gene sequence signatures, we identified Candida dubliniensis from both groups of patients.

Frequent use of immunosuppressive treatments either alone or in combination with broad-spectrum antibiotics predisposes patients to subsequent opportunistic fungal infections. The majority of these infections are caused by Candida albicans and other Candida spp. (2, 9) and include numerous fungemic episodes. Tremendous health care implications in terms of cost (21), morbidity, and mortality (9) are associated with fungemia. In the United States, Candida spp. are ranked the fourth most common group of microorganisms causing nosocomial bloodstream infections (BSI), accounting for 8% of all septicemias (19).

Numerous studies on the prevalence of different Candida spp. have led to the general consensus that C. albicans is the most commonly isolated species. However, there has been a growing trend of recovery of non-albicans species. One possible explanation is the increased use of fluconazole and other azoles, which has positively selected for some less sensitive Candida species, such as C. glabrata and C. krusei (1-4, 9, 10, 15-18, 22, 23, 28). In contrast, others have reported that there were no significant changes in the prevalence of Candida species with fluconazole use (11) and that C. albicans is present at a stable level and is still the predominant species in systemic infections (5, 19).

Candida dubliniensis, a recently defined species (26), is an emerging pathogen garnering attention for its ability to develop in vitro resistance to fluconazole (13). Jabra-Rizk et al. have recently reported its prevalence in a cohort consisting exclusively of human immunodeficiency virus-infected individuals (8). However, this species has not been found in the general hospital population despite extensive studies (5, 10, 14).

In this study we investigated the prevalence of different Candida spp. isolated from inpatients of three hospitals and from vaginal swabs collected at community clinics in Singapore. In combination with phenotypic properties, we analyzed species-specific gene sequence signatures to identify C. dubliniensis.

Study design.

We prospectively collected yeasts isolated from inpatients of three large general hospitals over a 6-month period (1 July to 30 December 2001), excluding isolates from urine specimens and high vaginal swabs (HVS). We also collected yeast isolates from HVS from community clinics all over Singapore. All these organisms were reexamined for the identity of Candida species in one laboratory.

Organism identification.

We used germ tube production to screen for C. albicans and C. dubliniensis. Yeasts that failed to form germ tubes were identified to the species level by API Candida and API 20C AUX (bioMérieux, Marcy l'Etoile, France) complemented with morphology on cornmeal Tween 80 agar (BBL, Cockeysville, Md.). All putative isolates of C. albicans and C. dubliniensis were differentiated by a multiplex-PCR method. Three pairs of oligonucleotide primers were used simultaneously in each PCR. The first pair, DUBF (5′-GTATTTGTCGTTCCCCTTTC-3′) and DUBR (5′-GTGTTGTGTGCACTAACGTC-3′), is specific for C. dubliniensis and amplifies a 288-bp DNA fragment from ACT1 gene (6); the second pair, CAL5 (5′-TGTTGCTCTCTCGGGGGCGGCCG-3′) and NL4CAL (5′-AAGATCATTATGCCAACATCCTAGGTA/TAA-3′), is specific for C. albicans 25S rRNA gene, amplifying a 175-bp DNA fragment (12); and the third pair, RNAF (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and RNAR (5′-GGTCCGTGTTTCAAGACG-3′), amplifies a 610-bp fragment from the 25S rRNA gene of all fungal species (7), which serves as a positive control. A standard PCR mixture of 25 μl contained 20 pmol of each of the primers, 2.5 mM MgCl₂, 10 mM Tris-HCl (pH 9.0), 10 mM KCl, 0.1% (vol/vol) Triton X-100, 2.5 U of Taq DNA polymerase, and 10 ng of DNA. The PCR program started with 6 min of heating at 95°C and continued with 30 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C. At the end of the cycling the reactions were incubated further at 72°C for 10 min. Figure 1 presents an example of the PCR products from different isolates. The isolates that yielded the C. dubliniensis-specific 288-bp PCR product were further confirmed by determining the nucleotide sequence signatures of the V3 variable region in the 25S rRNA gene. A pair of primers, V3-11 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and V3-12 (5′-GGTCCGTGTTTCAAGACG-3′), was used to amplify a 600-bp fragment containing the V3 region for subsequent DNA sequence analysis. All the sequences obtained were in agreement with the C. dubliniensis-specific sequence (26; sequences not shown).

FIG. 1.

Multiplex-PCR identification of C. dubliniensis. PCR products were analyzed by 1.5% agarose gel electrophoresis. A 100-bp DNA ladder was used as DNA size marker (M). Lanes 1 to 5 show type strains acquired from the American Type Culture Collection used here as a control. Lane 1, C. dubliniensis (ATCC MY 646); lane 2, C. tropicalis (ATCC 750); lane 3, C. parapsilosis (ATCC 22019); lane 4, C. krusei (ATCC 6258); lane 5, C. albicans (ATCC 24433); lanes 6 to 10, five isolates (ZW677-81) that yielded the C. dubliniensis-specific 288-bp band.

A total of 775 isolates were obtained from 737 specimens during the 6-month period. More than one yeast species were recovered from ∼16% of specimens, including 2 of 72 blood cultures. Approximately 60% of the mixed cultures contained C. albicans and a non-albicans species. The most predominant species isolated together with C. albicans was C. glabrata (72%). The significance of this finding is that C. glabrata may replace C. albicans under selective pressure of fluconazole, resulting in infections refractory to the current fluconazole-based treatment.

C. dubliniensis has caused great concern with its inducible fluconazole resistance. C. dubliniensis has been reported in Singapore (27), but our present study documents, for the first time, its prevalence in both hospitalized inpatients and outpatients visiting community clinics (Table 1). We did not identify any C. dubliniensis from blood specimens, although two cases were previously reported from Singapore (27). Since we used species-specific oligonucleotide primers in PCR to differentiate C. albicans from C. dubliniensis, it is unlikely that we misidentified C. dubliniensis as C. albicans. It is more likely that C. dubliniensis fungemia is rare. Studies focusing exclusively on candidemia with bigger cohorts and longer study periods did not find C. dubliniensis isolates either (5, 10, 14), although molecular methods were not used in these studies. In our study, the API 20C AUX misidentified 12 of 20 C. dubliniensis isolates as C. albicans. Similar misidentification by using API 20C AUX has been noted before (20). As previously reported (25), most isolates of C. dubliniensis were recovered from respiratory specimens.

TABLE 1.

Distribution of Candida spp. by specimen type

Species	No. (%) of isolates in specimens from:				Total n (%)
	Inpatients			Outpatients
	Blood	Respiratory	Othersa	HVS
C. albicans	24 (33.3)	118 (54.6)	28 (37.8)	311 (75.3)	481 (62.1)
C. glabrata	11 (15.3)	37 (17.1)	10 (13.4)	81 (19.9)	139 (17.9)
C. tropicalis	16 (22.2)	32 (14.8)	11 (14.9)	5 (1.2)	64 (8.3)
C. parapsilosis	14 (19.4)	8 (3.7)	18 (24.2)	5 (1.2)	45 (5.8)
C. dubliniensis	0 (0)	12 (5.5)	3 (4.1)	5 (1.2)	20 (2.7)
C. krusei	2 (2.8)	3 (1.4)	0 (0)	3 (0.6)	8 (1.0)
C. famata	3 (4.2)	3 (1.4)	2 (2.8)	0 (0)	8 (1.0)
C. guilliermondii	2 (2.8)	1 (0.5)	0 (0)	2 (0.4)	5 (0.6)
Otherb	0 (0)	2 (1)	2 (2.8)	1 (0.2)	5 (0.6)
Total	72	216	74	413	775

^aIncludes wound swabs, central venous pressure catheter tips, surgically excised tissues, bodily fluids, and aspirations.

^bIncludes one C. norvegenesis, one C. pelliculosa, one C. lusitaniae, and two C. rugosa isolates.

The distribution of Candida spp. in BSI in our study differs from that reported in other studies (1, 5, 10, 16, 19). This emphasizes the value of local epidemiologic studies (18). In particular, the prevalence of C. albicans was comparatively low (33.3% versus 52 to 56%), although it was still the predominant species. The reasons for this are unclear. It may be related to differences in utilization of antifungal and other agents that exert selective pressures. The most prevalent species after C. albicans were C. tropicalis (22.2%), C. parapsilosis (19.4%), and C. glabrata (15.3%). C. krusei, which is intrinsically resistant to fluconazole, was uncommon (2.7%). This concurs with previous findings (19). C. glabrata and C. krusei together made up 18% of all BSI cases. We determined the sensitivities of all isolates to 8, 16, 32, and 64 μg of fluconazole/ml and found that 100% (6 of 6) of C. krusei and 29% (41 of 139) of C. glabrata isolates were resistant to 64 μg of fluconazole/ml. In marked contrast, only 1% (5 of 481) of C. albicans isolates were resistant to 64 μg of fluconazole/ml, while 97% (465 of 481) were sensitive to 8 μg of fluconazole/ml (complete data on resistance of all isolates to antifungal drugs will be published elsewhere). This result is consistent with previous reports on the higher resistance of C. krusei and C. glabrata than C. albicans to fluconazole (1-4, 9, 10, 15-18, 22, 23). Our result suggests that amphotericin B or itraconazole should be the drug of choice in the empirical treatment of candidemia, rather than fluconazole.

In our community isolates from HVS, C. albicans accounted for 75.3% and C. glabrata accounted for 19.9% of the isolates. This is in contrast to BSI data showing that other species accounted for 52% of all isolates. C. dubliniensis, C. tropicalis, and C. parapsilosis were found in equal numbers (1.2%) in HVS. We did not gather data on the use of antifungals in the population studied. The prevalence of C. glabrata may represent unadulterated flora or be due to selective pressure from previous azole therapy. Our data are similar to those reported by Schuman et al. (24). The clinical relevance is the same as that for BSI in that clinicians might expect failures with use of the popular one-dose oral fluconazole when treating vaginal candidiasis because of the high prevalence of C. glabrata.

The distribution of species in different mucosal sites was interesting and raises questions about the anatomical origin of species found in BSI and their relative pathogenicity. C. albicans accounted for 75% of isolates from HVS and 55% from respiratory samples but disproportionately fewer from BSI (33%). C. glabrata accounted for similar proportions in both mucosal sites and BSI (respiratory, 17%; HVS, 19%; blood, 15%). C. tropicalis, which accounted for 22% of BSI isolates, was found in respiratory samples in proportions (15%) similar to those of C. glabrata (17%) but was almost absent from HVS (1.2%). This may be because the HVS were from community patients while the respiratory samples were from inpatients. However, there is no immediately obvious reason why hospitalization should increase the proportion of the normally fluconazole-susceptible C. tropicalis at the expense of other Candida species. C. parapsilosis accounted for a large proportion of BSI isolates (19%) but only a small proportion of respiratory (3.7%) and HVS (1.2%) isolates. This is a rather unexpected result. The portal of entry of Candida spp. into the bloodstream may be damaged mucosal surfaces or central intravascular catheters. These catheters are commonly inserted close to the neck, where contamination with oropharyngeal secretions is common. Our data suggest that C. parapsilosis and C. tropicalis may be more successful than C. albicans and C. glabrata in invading mucosal surfaces or in colonizing intravascular catheters.

In summary, we have documented the prevalence of C. dubliniensis and other Candida spp. in both the general hospital and community populations in Singapore. In particular, our data show a high prevalence of fluconazole-resistant species, C. glabrata and C. krusei in BSI and C. glabrata in HVS, which should influence the choice of empirical therapy of candidiasis for both community patients and hospital inpatients.