Introduction

In recent years, non-albicans Candida (NAC) species have emerged as important pathogens, causing 35–65% of all cases of nosocomial candidaemia [1]. The most common NAC species isolated from blood are C. tropicalis, C. glabrata, C. krusei and C. parapsilosis. These taken together account for one-half of all the Candida spp. isolated from blood cultures [1]. Although isolated rarely, C. rugosa has been cited as a distinct emerging cause of candidaemia [2]. C. rugosa, best known for its industrial uses [3], is also an established pathogen causing bovine mastitis [4]. Minces et al. [2] recently reviewed the available literature on C. rugosa candidaemia and highlighted its unique aspects.

The rise in frequency of candidaemia due to NAC has been documented previously at our hospital [5]. In a 5-year retrospective study done by us, C. tropicalis was the most frequently isolated species from cases of candidaemia overall, with C. parapsilosis being the predominant species in the fifth year [5]. C. rugosa has not been reported earlier from our centre. However, recently, we encountered a cluster of cases of C. rugosa candidaemia in trauma patients admitted to the trauma care intensive care unit (ICU), which occurred over a short period. We conducted a retrospective evaluation of these cases. This study describes the clinical and epidemiologic features of these cases of C. rugosa candidaemia. In addition, we also determined the antifungal susceptibility profile of the C. rugosa isolates and subjected a majority of them to a molecular typing method (random amplified polymorphic DNA; RAPD) to ascertain the strain variability.

Materials and methods

This study reports an outbreak of C. rugosa bloodstream infection (BSI) in an ICU at the 190-bed level I trauma centre of the All India Institute of Medical Sciences, which itself is a 2,500-bed, tertiary care, teaching hospital of North India. During the period from July 2008 to September 2009, a total of 28 blood cultures from 19 patients were found to be positive for C. rugosa. We also retrospectively evaluated blood samples received during this period in our laboratory from cases of septicaemia to assess the prevalence of candidaemia.

As a policy of our centre, the current recommendation for the diagnosis of fungaemia in adult trauma patients is to draw at least 10 ml of blood per blood culture sample (to be inoculated into a single blood culture bottle). Two blood cultures (drawn from two or three different sites) are sent within a 24-h period per episode. For the diagnosis of candidaemia/bacteraemia, 5–10 ml of blood was collected in BacT/ALERT FA aerobic blood culture bottles (bioMérieux Inc., Marcy l’Etoile, France). These bottles were incubated and monitored regularly by the BacT/ALERT system (bioMérieux Inc.) [6]. All positive samples were processed for microbial identification by standard methods. Blood culture bottles positive for yeast cells were subcultured on Sabouraud’s dextrose agar (SDA) for further identification. Speciation of these isolates was done by both conventional and automated methods. Conventional methods included the germ tube test, morphology on cornmeal agar (CMA) (HiMedia, Mumbai, India), reduction of triphenyl tetrazolium chloride (TTC) dye (HiMedia), assimilation of various sugars and growth in the presence of actidione [7]. Growth on chromogenic medium CHROMagar Candida [8] and the Vitek-2 system ID-YST cards were also used for identification [9]. Antifungal susceptibility to amphotericin B and fluconazole was performed using the broth microdilution method (M27-A2) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. Quality control was ensured by testing the CLSI recommended quality control strains C. parapsilosis ATCC 22019 (fluconazole minimum inhibitory concentration [MIC] range 2–8 μg/ml) and C. krusei ATCC 6258 (fluconazole MIC range 16–64 μg/ml). Antifungal susceptibility to amphotericin B, fluconazole, flucytosine and voriconazole was also performed by the Vitek-2 system using AST-YST cards [11]. The MIC breakpoints recommended by the CLSI guidelines were followed. For fluconazole and voriconazole, the MIC breakpoints are as follows: sensitive, MIC of ≤8 μg/ml (fluconazole) and ≤1 μg/ml (voriconazole); susceptible dose-dependant (SDD), MIC of 16–32 μg/ml (fluconazole) and 2 μg/ml (voriconazole); resistant, MIC of ≥64 μg/ml (fluconazole) and ≥4 μg/ml (voriconazole) [10, 12]. For amphotericin B, isolates with MICs of ≥1 μg/ml are categorised as resistant [10]. For flucytosine, each isolate is assigned to a susceptibility category according to the following MIC breakpoints recommended by the CLSI: susceptible ≤4 μg/ml, SDD 8–16 μg/ml; and resistant ≥32 μg/ml.

RAPD was done on C. rugosa isolates using by using M13 primer [13]. Polymerase chain reaction (PCR) was performed in a reaction volume of 50 μl which consisted of 30 ng of DNA, 2.5 mM (each) dNTPs (Biotools), 5 U of Taq polymerase (Biotools), 5 μl of 10× reaction buffer (75 mM Tris HCl pH 9.0; 2 mM MgCl2; 50 mM KCl; 20 mM (NH4)2SO4), 3 mM magnesium acetate and 30 pmol of M13 primer (5′-GAG GGT GGC GGT TCT-3′). The initial denaturation was for 3 min at 94°C, followed by 40 cycles of PCR with denaturation for 30 s at 94°C, annealing for 1 min at 52°C and extension for 30 s at 72°C, followed by a final extension step at 72°C for 6 min. PCR products were run in 1.2% agarose in 1× TBE and run for 6 h at 60 V. Amplified products were visualised under UV light and bands were analysed manually.

Results

During the period from July 2008 to September 2009, a total of 28 blood samples from 19 patients were found to be positive for C. rugosa. The isolates were identified as C. rugosa by the following method. On SDA white to cream coloured, smooth, glabrous colonies were formed initially, which became wrinkled upon prolonged incubation. These were germ tube-negative. CMA showed the presence of ellipsoidal to elongated budding blastoconidia measuring 6–10 μm by 2–3.5 μm in size. Short pseudohyphae were noticed occasionally. On TTC, dark red coloured colonies were obtained. CHROMagar showed light blue green, rough colonies with pale borders. The carbohydrate assimilation profile (modified auxanographic method) of the isolates revealed the following (+ assimilated; − not assimilated): dextrose +, maltose −, sucrose −, lactose −, galactose −, trehalose −, raffinose −, cellobiose −, melibiose +, inositol −, xylose +. This result matched the assimilation profile of C. rugosa. All isolates were identified as C. rugosa by the Vitek-2 system (99% probability, excellent identification).

The demographic and clinical data of the 19 cases of C. rugosa candidaemia are summarised in Table 1. All of the patients underwent multiple invasive medical procedures and were put on broad-spectrum antibiotics therapy during the course of their stay. On average, almost all of the patients had at least 2 weeks of ICU stay before developing C. rugosa fungaemia (14 ± 6.65 days, mean ± standard deviation [SD]) pointing to the nosocomial nature of these infections (Table 1). The total duration of stay ranged from 8 to 76 days (28.4 ± 16.75, mean ± SD). C. rugosa was isolated from both blood and urine in 6 (31.5%) patients. In one patient, C. rugosa was isolated from bronchoalveolar lavage (BAL) fluid and central venous pressure (CVP) catheter tip also. None of the patients were on antifungal prophylaxis prior to the onset of fungaemia.

Table 1 Demographic and clinical data of the patient cohort
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We performed RAPD on 17 isolates from 17 patients using the M13 primer to determine the strain variability. Visual analyses of the bands of these 17 isolates of C. rugosa showed identical patterns (Fig. 1). In an attempt to analyse strain diversity, we further compared the RAPD patterns of the C. rugosa strains belonging to this cluster with C. rugosa isolates unrelated to this cluster of cases. RAPD was performed again with the M13 primer on 13 isolates belonging to this cluster, along with two blood isolates of C. rugosa obtained from two different patients admitted to our trauma centre in the months of April and May 2010 and a strain from a different Candida species (C. albicans) (Fig. 2). A similar result was obtained with the 13 strains belonging to the same cluster sharing the same high-intensity band patterns. These band patterns differed considerably from those of the two unrelated C. rugosa isolates.

Fig. 1
figure 1

Electrophoretic random amplified polymorphic DNA (RAPD) pattern of 17 Candida rugosa isolates using the M13 core sequence (5′-GAG GGT GGC GGT TCT-3′). Lane 1 is the 100-bp DNA ladder and lanes 2–18 are 17 different isolates of C. rugosa

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Fig. 2
figure 2

Electrophoretic RAPD pattern of 13 C. rugosa (numbers 1–13) isolates using the M13 core sequence (5′-GAG GGT GGC GGT TCT-3′). A and B are two isolates of C. rugosa unrelated to this cluster. Ca is the C. albicans strain. Lane 1 is the 100-bp DNA ladder and lanes 2–18 are 17 different isolates of C. rugosa

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Antifungal susceptibility testing by both microbroth dilution and Vitek-2 AST revealed that isolates from all patients were susceptible to 5-flucytosine, voriconazole and amphotericin B. Four isolates (4/19, 21%) were resistant to fluconazole. Fluconazole was started in 11 patients within 24 h of laboratory confirmation of fungaemia, and therapy was later modified to amphotericin B in two and caspofungin in one patient on the basis of the antifungal susceptibility report (Table 1). The central venous catheter was removed and antifungal therapy was continued for a period of 14 days. In total, seven (7/19, 36.8%) patients died (six died before the initiation of antifungal therapy and one patient died later during the course of hospital stay). Two patients cleared the infection after removal of the intravenous catheter and were not given any antifungal therapy due to the absence of clinical signs and symptoms of sepsis. As mentioned above, one of these patients succumbed later during the course of hospital stay. Response was considered to be favourable when clinical symptoms and signs of sepsis subsided and repeat blood culture(s) was sterile. Antifungal therapy was modified in three patients on the basis of their isolates’ antifungal susceptibility testing reports that showed fluconazole resistance or susceptibility in the SDD range (one patient with isolate resistant to fluconazole died before antifungal therapy could be modified).

An analysis of all of the samples of blood received for culture received in the microbiology laboratory of the trauma centre during the same period was also done to assess the true magnitude of candidaemia in general and C. rugosa BSI in particular. A total of 2,219 blood culture samples were received during this period. Of these, 509 (22.9%) gave a positive signal. Candida spp. were isolated in pure culture from 75 of these 509 (15%) positive blood cultures. Of these, in seven (1.3%) positive blood samples, Candida spp. were isolated along with Gram-positive cocci and Gram-negative bacilli, signifying polymicrobial infection. Out of the 75 Candida isolates, C. rugosa accounted for 28 (37%), followed by C. tropicalis and C. parapsilosis accounting for 16 (21%) each, whereas C. albicans, C. glabrata and C. krusei accounted for 11 (15%), 3 (4%) and 1 (1.3%), respectively.

Discussion

The findings of our study confirm several previous observations made regarding C. rugosa. First, as suggested recently by Minces et al. [2], the frequency of C. rugosa as a cause of candidaemia is increasing. These infections are especially common among patients in ICUs. They are associated with typical risk factors such as indwelling central vascular catheters, broad-spectrum antibiotics therapy and prior surgery. In our experience, all C. rugosa candidaemic episodes were documented in ICUs. Prior to this cluster, C. rugosa had never been identified as a cause of infection in our institution.

The gradual emergence of resistance to azoles in C. rugosa has also been noted. In the ARTEMIS DISK Antifungal Surveillance Program, 40.5 and 61.4% of the C. rugosa isolates were susceptible to fluconazole and voriconazole, respectively [14]. In another study, Colombo et al. have reported an outbreak of C. rugosa candidaemia refractory to amphotericin B therapy, despite the apparent in vitro susceptibility of the isolates to amphotericin B. In this study, none of the patients with C. rugosa fungaemia treated with this agent survived [15]. Isolates of C. rugosa resistant to amphotericin B have also been reported previously [16]. In our study, 21% of C. rugosa isolates were resistant to fluconazole, whereas 100% in vitro susceptibility to amphotericin B was noted. Thirteen out of the 19 patients (68.4%) with C. rugosa candidaemia died. Of these, six had received antifungal therapy after the confirmation of fungaemia. Other factors such as late onset of antifungal therapy and co-morbid conditions could have accounted for the high mortality seen in these patients, despite their isolates showing in vitro sensitivity to polyenes and other antifungal agents (azoles). Thus, considering the fact that most of these patients were seriously ill, one can at best conjecture that fungaemia was one of the attributed causes of death among a host of many causes. Another interesting finding in our study was the identical band patterns of our isolates obtained after performing RAPD using the M13 primer, which differed considerably from those of C. rugosa isolates that were unrelated to this cluster. However, a limitation of this study was the inability to obtain a larger number of such unrelated C. rugosa isolates, which would have made it possible to evaluate the strain diversity of this species. Moreover, being a retrospective study, it was not possible to trace the possible source of C. rugosa infection. Therefore, it was not possible to confirm the outbreak nature of this cluster of cases of candidaemia. Nevertheless, to the best of our knowledge, this is the largest cluster of cases of C. rugosa candidaemia reported from a single institution in the English literature and further highlights the ability of this rarely isolated species to cause serious infections in humans.

In conclusion, C. rugosa is an emerging nosocomial bloodstream pathogen that appears in sporadic clusters usually associated with invasive medical procedures. Fungaemia due to this species may be associated with high mortality. The emergence of azoles resistance in this pathogen is also a matter of concern. The best empiric antifungal therapy for treating C. rugosa candidaemia is unclear, as breakthrough infections and treatment failures are well recognised during therapy with polyene agents and data for echinocandins are limited [17, 18]. Additional epidemiologic and clinical information is needed to provide optimal preventive and therapeutic measures for this serious Candida infection.