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REVIEW ARTICLE

Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance
Sonia Silva1, Melyssa Negri1, Mariana Henriques1, Rosario Oliveira1, David W. Williams2 & 1 Joana Azeredo
Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal; and 2Tissue Engineering & Reparative Dentistry, School of Dentistry, Heath Park, Cardiff, UK
1

Correspondence: Mariana Henriques, Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar 4710-057, Braga, Portugal. Tel.: 1351 253 604 408; fax: 1351 253 604 429; e-mail: mcrh@deb.uminho.pt Received 29 December 2010; revised 31 March 2011; accepted 6 May 2011. Final version published online 6 June 2011. DOI:10.1111/j.1574-6976.2011.00278.x Editor: Martin Kupiec Keywords Candida species; candidosis; epidemiology; virulence factors; antifungal resistance.

Abstract The incidence of infections caused by Candida species (candidosis) has increased considerably over the past three decades, mainly due to the rise of the AIDS epidemic, an increasingly aged population, higher numbers of immunocompromised patients and the more widespread use of indwelling medical devices. Candida albicans is the main cause of candidosis; however, non-C. albicans Candida (NCAC) species such as Candida glabrata, Candida tropicalis and Candida parapsilosis are now frequently identied as human pathogens. The apparent increased emergence of these species as human pathogens can be attributed to improved identication methods and also associated with the degree of diseases of the patients, the interventions that they were subjected and the drugs used. Candida pathogenicity is facilitated by a number of virulence factors, most importantly adherence to host surfaces including medical devices, biolm formation and secretion of hydrolytic enzymes (e.g. proteases, phospholipases and haemolysins). Furthermore, despite extensive research to identify pathogenic factors in fungi, particularly in C. albicans, relatively little is known about NCAC species. This review provides information on the current state of knowledge on the biology, identication, epidemiology, pathogenicity and antifungal resistance of C. glabrata, C. parapsilosis and C. tropicalis.

MICROBIOLOGY REVIEWS

Introduction
In last 30 years there has been a signicant increase in the incidence of fungal infections in humans (Lass-Fl orl, 2009). Such infections may either be supercial, affecting the skin, hair, nails and mucosal membranes, or systemic, involving major body organs (Ruping et al., 2008). A number of factors have been implicated in this increased occurrence of fungal disease, but it is generally accepted that the increased and widespread use of certain medical practices, such as immunosuppressive therapies, invasive surgical procedures and use of broad-spectrum antibiotics are signicant (Samaranayake et al., 2002; Hagerty et al., 2003; Kojic & Darouiche, 2004). Of the fungi regarded as human pathogens, the members of the genus Candida are the most frequently recovered from human fungal infection. The Candida genus contains over 150 heterogeneous species (Calderone, 2002), but only a
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minority have been implicated in human candidosis. Additionally, it is known that approximately 65% of Candida species are unable to grow at a temperature of 37 1C, which precludes these species from being successful pathogens or indeed commensals of humans (Calderone, 2002). Of the Candida species isolated from humans, Candida albicans is the most prevalent under both healthy and disease (Calderone, 2002; Samaranayake et al., 2002) conditions. However, while mycological studies have shown that C. albicans represents over 80% of isolates from all forms of human candidosis (Calderone, 2002) in the last two decades, the number of infections due to non-C. albicans Candida (NCAC) species has increased signicantly (Kauffman et al., 2000; Manzano-Gayosso et al., 2008; Ruan & Hsueh, 2009). The apparent increased involvement of NCAC species in human candidosis may partly be related to improvements in diagnostic methods, such as the use of chromogenic media with the ability to differentiate Candida species, as well as
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the introduction of molecular techniques in the routine diagnosis of fungemia (Liguori et al., 2009). However, the high prevalence of NCAC species in disease could also be a reection of their inherently higher level of resistance to certain antifungal drugs (Gonzalez et al., 2008) compared with C. albicans, as this would promote their persistence, possibly to the detriment of C. albicans, in mixed species infections treated with traditional antifungal agents. Unfortunately, compared with C. albicans there are relatively few studies examining the virulence factors of NCAC species. This review therefore provides information on the current state of knowledge on the biology, identication, epidemiology, pathogenicity and antifungal resistance of Candida glabrata, Candida parapsilosis and Candida tropicalis, three of the most frequent causes of candidosis after C. albicans.

Biology of NCAC species


Candida comprises an extremely heterogeneous group of fungal organisms that can all grow as yeast morphology. Macroscopically, colonies of Candida, on the routinely used Sabouraud dextrose agar (SDA), are cream to yellow in colour. Depending on the species, colony texture may be smooth, glistening or dry, or wrinkled and dull. Under standard conditions with optimal nutrients, yeast grow in log phase as budding cells (blastoconidia), which are spherical to oval in shape and are approximately 25 37 mm in size (Fig. 1) (Larone, 2002). Moreover, certain species, such as C. albicans and Candida dubliniensis, can produce a

lamentous type of growth, such as true hyphae (Fig. 1) or more frequently, pseudohyphae (Fig. 1). The distinction between hyphae and pseudohyphae is related to the way in which they are formed. Pseudohyphae are formed from yeast cells or hyphae by budding (Fig. 1), but the new growth remains attached to the parent cell and elongates, resulting in laments with constrictions at the cellcell junctions. There are no internal cross walls (septa) associated with pseudohyphae (Fig. 1). In comparison, true hyphae are formed from yeast cells or even as branches of existing hyphae. The development of true hyphae is initiated by a germ tube projection (Fig. 1), which elongates and then branches with dened septa that divide the hyphae into separate fungal units (Fig. 1). Candida albicans and C. dubliniensis are truly polymorphic, due to their ability to form hyphae and/or pseudohyphae, and these species are also referred to germ tube positive, a diagnostic feature (Table 1) (Calderone, 2002). In contrast, C. glabrata is not polymorphic, growing only as blastoconidia (yeast) (Table 1; Fig. 2). Historically, this species was originally classied in the genus Torulopsis due to its lack of pseudohyphal formation. However, in 1978, it was determined that the ability to form pseudohyphae was not a reliable distinguishing factor for members of the genus Candida and it was proposed that Torulopsis glabrata could be classied in the genus Candida, due to its association with human infection (Fidel et al., 1999). With regard to C. parapsilosis, this species does not produce true hyphae, but can generate pseudohyphae that are characteristically large and curved, and often referred to as giant cells

Fig. 1. Epiuorescence photocomposition of the different morphological growth forms of Candida albicans stained with calcouor white: (A) blastoconidia; (B1) reproduction by budding; (B2) germ tube formation; (C1) pseudohyphae formation; (C2) yeast form; (C3) hyphae formation. Internal cross walls (septa).

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Table 1. Morphological characteristics of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata species Species C. albicans C. tropicalis C. parapsilosis C. glabrata Germ tube production 1 Hyphae/pseudohyphae 1/1 /1 /1 / Yeasts size (mm) 46 610 48 511 2.54 2.59 14 CHROM-agar colony colour Blue-green Dark blue White White, Pink-purple

Fig. 2. Candida species macroscopic colonies on cornmeal Tween 80 and microscopy structure on SDA. Microscopic structures: (a) Candida glabrata; (b) Candida parapsilosis; (c) Candida tropicalis; macroscopic colonies: (d) C. glabrata; (e) C. parapsilosis; (f) C. tropicalis.

(Fig. 2) (Larone, 2002; Trofa et al., 2008). In contrast, on corn meal Tween 80 agar and at 25 1C after 72 h, C. tropicalis produces oval blastospores, pseudohyphae depending on some reports, true hyphae (Fig. 2) (Calderone, 2002; Larone, 2002; Yoshio & Kouji, 2006). It should also be highlighted that C. glabrata cells (14 mm) are noticeably smaller than the blastoconidia of C. albicans (46 mm), C. tropicalis (48 mm) and C. parapsilosis (2.54 mm) (Larone, 2002) (Table 1). On SDA (Fig. 2) C. glabrata forms glistening, smooth, and cream-coloured colonies, which are largely indistinguishable from those of other Candida species except for their relative size, which can be quite small (Fig. 2). Furthermore, C. parapsilosis, when grown on SDA, yields white, creamy, shiny and smooth/wrinkled colonies (Fig. 2). On the same medium, C. tropicalis forms colonies that are cream-coloured with a slightly mycelial border (Fig. 2) (Calderone, 2002). Concerning the biochemistry of Candida species, C. glabrata ferments and assimilates only glucose and trehalose, which contrasts with C. albicans, which ferments and/or assimilates a number of sugars with the notable exception of sucrose (Odds, 1988). Additionally, C. tropicalis has the ability to ferment and assimilate sucrose and maltose
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(Martin, 1979). Interestingly, C. parapsilosis was rstly classied as a species of Monilia, due to its inability to ferment maltose (Odds, 1988; Trofa et al., 2008). A main distinguishing genetic characteristic of C. glabrata is that it has a haploid genome, in contrast to the diploid genome of C. albicans and several other NCAC species (Fidel et al., 1999). Genetically, C. tropicalis has the highest similarity to C. albicans, and C. glabrata the least (Butler et al., 2009). It is through the advent of molecular genetics that new identication methods for Candida have been developed, leading to the identication of new species together with their increased recognition in human infection. Before 2005, C. parapsilosis was separated into three groups (IIII), but further studies revealing genomic differences that have led to the separation of these groups into closely related, but distinct species, namely, C. parapsilosis, Candida orthopsilosis and Candida metapsilosis (Tavanti cser et al., 2007a, b). et al., 2005; Ga

Laboratory identification of NCAC species


The laboratory diagnosis of candidoses continues to be problematic. Microbiological conrmation can be difcult
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as blood cultures can be negative in up to 50% of autopsyproven cases of deep-seated candidoses, or may only become positive late in the infection (Ellepola & Morrison, 2005). Positive cultures from urine or mucosal surfaces do not necessarily indicate invasive disease although may occur during systemic infection (Ellepola & Morrison, 2005). Furthermore, differences in virulence between Candida species as well as in their susceptibility to antifungal drugs make identication important for clinical management. Laboratory diagnosis has improved with the advent of new methods for Candida isolation and identication. Technologies such as species-specic FISH (Alexander et al., 2006), antibody and antigen detection (Pfaller, 1992; Ellepola & Morrison, 2005) and molecular approaches for typing and detection of fungal pathogens (Ellepola & Morrison, 2005) have all been used successfully. However, many of these approaches have not yet been standardized or validated in large clinical trials and therefore are not widely used in clinical laboratory settings (Ellepola & Morrison, 2005). Laboratory surveillance of at-risk patients could result in earlier initiation of antifungal therapy if sensitive and specic diagnostic tests, which are also cost effective, become widely available. The clinical symptoms of fungemia are not indicative of particular Candida species and may be induced by other microorganisms. The laboratory identication of Candida is therefore essential for establishing a denitive diagnosis. A standard approach to the laboratory diagnosis generally involves nonmolecular methods, although PCR is increasingly being used.

Non-PCR based methods of Candida identification


CHROMagars Candida (CHROMagars, Paris, France), is a relatively new differential agar medium for Candida species identication and has been particularly useful in the presumptive identication of C. albicans, C. tropicalis and Candida krusei upon primary culture of clinical specimens. On CHROMagars Candida, C. glabrata colonies appear white, pink or purple in contrast to C. albicans colonies, which are blue-green, while C. parapsilosis colonies are white and C. tropicalis dark blue (Table 1). Moreover, it is possible to detect coinfection with different Candida species on primary culture plates and this can have importance in infection management strategies (Ellepola & Morrison, 2005; Furlaneto-Maia et al., 2007). After Candida isolation, species can also be identied by carbohydrate assimilation and fermentation tests as well as morphological characteristics such as germ tube and chlamydospore development (Fig. 2). In addition, more rapid and less laborious phenotypic identication methods have become available. Perhaps the most widely used methods for
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Candida species identication are those based on the format of carbohydrate assimilation and/or enzyme detection within plastic wells of commercially available kits. Examples of such biochemical tests include the API 20C AUX (API Candida) Auxacolor (Bio-Rad) and the Uni-Yeast-Tek kit (Ellepola & Morrison, 2005). These tests generate reliable identication for the most common species of Candida, while identication of other Candida species may not be so accurate. For example, the differentiation of C. dubliniensis from C. albicans often requires the use of supplemental biochemical or morphological tests for denitive identication (Verweij et al., 1999; Ellepola & Morrison, 2005). Additional methods for Candida species identication include tests that allow the detection of an isolate in 1 day, such as the RapID Yeast Plus System (Innovative Diagnostic Systems, Norcross), the Fongiscreen test (Sano Diagnostics Pasteur, France) and the automated Rapid Yeast Identication Panel (Dade Microscan). However, as mentioned above, most of these tests tend to be most accurate for the identication of the more frequently encountered yeast pathogens (Ellepola & Morrison, 2005). The diagnosis of invasive candidosis should include a collection of adequate volumes of blood and an agar-based blood culture method for optimal detection of candidemia (Pfaller, 1992). Several advances in blood culturing techniques have been developed, which appear to have improved the sensitivity and/or reduced the time required to obtain a positive blood culture. Two automated methods for monitoring of blood culture bottles, based on colour (BacT/ ALERT 3D, Organon Teknika Corp., Durham, NC) and uorescence (BACTEC 9240, Becton Dickinson), have been developed recently (Ellepola & Morrison, 2005). The identication of typical blastospores and pseudohyphae of Candida species on microscopic examination of tissue remains the unequivocal standard for the diagnosis of invasive or disseminated candidoses. Unfortunately, the usefulness of this approach is frequently limited by sampling problems (isolation source and sample size) (Pfaller, 1992). The use of uorescent antibody, acridine orange or calcouor-white staining (Pfaller, 1992; Ellepola & Morrison, 2005) may enhance the sensitivity of microscopic examination. However, the production of uorescent antibodies specic for the identication of individual Candida species has proved to be extremely difcult. A relatively recent laboratory method based on PNA FISH targeting the 26S rRNA gene allows the reliable detection of C. albicans from NCAC species, within 2.5 h of yeast growth detection in blood culture, with a sensitivity of 99% and specicity of 100% (Rigby et al., 2002). According to recent studies PNA FISH also results in substantial cost savings for hospitals, making the method both an effective and affordable one for the laboratory diagnosis of candidoses (Rigby et al., 2002; Ellepola & Morrison, 2005; Alexander et al., 2006).
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PCR-based methods of Candida identification


The molecular-based technology that has undoubtedly had the greatest impact in the clinical diagnosis of Candida infections is PCR. This technique can detect highly limited quantities of microbial nucleic acid from blood, tissue specimens as well as cultured microorganisms. Over the last decade, numerous studies have been performed to investigate the effectiveness of PCR in diagnosis of systemic infection caused by Candida (Williams et al., 1995, 2001; Chen et al., 2000; Carvalho et al., 2007; Orazio et al., 2009). In PCR, a pair of synthetic oligonucleotides homologous to specic sequences serves to prime the amplication of target DNA. The most important feature of any PCR primers used directly on clinical samples is that they are specic and do not amplify host DNA or that of other microorganisms. To improve the sensitivity of PCR, many investigators have designed primers that amplify regions of DNA that are repeated in the fungal genome. The most commonly used target for yeast diagnostic PCR primers is the rRNA gene operon, encoding the 18S, 5.8S, and 28S rRNA gene subunits, namely internal transcribed spacer 1 (ITS1), ITS2 and ITS4 (Fell et al., 1992; Sullivan et al., 1995; Williams et al., 1995, 2001; Haynes & Westerneng, 1996; Chen et al., 2000). More recently, multiplex targets, coupled to real-time PCR, have been used successfully (Sampaio et al., 2005; Carvalho et al., 2007; Orazio et al., 2009) for Candida species identication. Despite the increased development of new molecular approaches, the great majority of clinical diagnosis of candidosis are based on nonmolecular methodologies due the reduced amount of PCR equipment in hospital laboratories, the problems with sample preparation and environmental contamination and the lack of standardized protocols for PCR methodologies.

Epidemiology and risk factors in NCAC species infection


The mortality rates associated with different microorganisms have declined with the early administration of empirical antibiotics and antifungal agents. However, despite this, systemic fungal infections are increasingly recognized as important causes of morbidity and mortality. Candida species are among the most frequently recovered fungi from blood cultures of hospitalized patients (Pfaller et al., 1998, 2010). In fact, an increasing incidence of fungal infections with Candida species has been noted in immunocompromised patients, including those in intensive care, postsurgical units and suffering from cancer (Kiehn et al., 1980; Samaranayake et al., 2002; Hagerty et al., 2003). Candida species are most frequently isolated from the oral cavity, and vulvovaginal and urinary tracts and are detected in approxi 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

mately 3155% of healthy individuals. Historically, C. albicans has accounted for 7080% of clinical isolates, with other NCAC species only rarely encountered (Odds, 1988; Calderone, 2002; Samaranayake et al., 2002). Nevertheless, over the last 1030 years NCAC species have emerged as important opportunistic pathogens of humans and the reasons for this might be related to improved diagnostic methods or altered medical practices, as mentioned above. Regardless of the basis of this change, recent epidemiological data reveal a mycological shift, and while C. albicans remains the most common causative agent, its relative incidence in infection is declining with the increasing prevalence of other species such as C. glabrata, C. tropicalis and C. parapsilosis (Chandra et al., 2001; Colombo et al., 2003; Bassetti et al., 2006). In a study on the epidemiology of invasive candidosis, Pfaller & Diekema (2007) observed that C. albicans, C. glabrata, C. tropicalis and C. parapsilosis collectively accounted for about 95% of identiable Candida infections. Moreover, in the 1980s, according to Kiehn et al. (1980), C. albicans constituted 68% of Candida isolates from sites other than blood in cancer patients, while C. tropicalis, C. parapsilosis and C. glabrata accounted only for 12%, 10% and 3.0% of the isolates, respectively. Table 2 presents epidemiologic studies published between 2000 and 2010, concerning oral candidosis, candiduria and candidemia. In more recent studies, most cases of fungemia have been signicantly associated with NCAC species (Bassetti et al., 2006; Colombo et al., 2007; Chakrabarti et al., 2009; Pfaller et al., 2010). However, it is important to emphasize that there are signicant variations in Candida species isolation depending on the geographical region and patient group, with some NCAC species being more prevalent, even compared with C. albicans, in certain countries (Colombo et al., 2007). The incidence of C. glabrata is higher in adults than in children, and lower in neonates (Krcmery & Barnes, 2002). In contrast, C. parapsilosis appears to be a signicant problem in neonates, transplant recipients and patients receiving parenteral nutrition (Trofa et al., 2008). Furthermore, C. tropicalis is commonly associated with patients with neutropenia and malignancy (Colombo et al., 2007). For many years C. glabrata was considered a relatively nonpathogenic saprophyte of the normal ora of healthy individuals and certainly not readily associated with serious infection in humans. However, following the widespread and the increased use of immunosuppressive therapies together with broad-spectrum antibiotic treatment, the frequency of mucosal and systemic infections caused by C. glabrata has increased signicantly (Hajjeh et al., 2004). Although the mortality rate associated with Candida infections varies with the type of patient and with the causative agent, the incidence rates of candidosis infections attributed
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Table 2. Selected epidemiological studies published from 2000 to 2010, concerning the distribution of Candida species isolates among various types of candidosis Candidosis Oral candidosis References Gonzalez Gravina et al. (2007) Martins et al. (2010a, b) Luque et al. (2009) Kauffman et al. (2000) Kobayashi et al. (2004) Passos et al. (2005) Binelli et al. (2006) Chen et al. (2008) lvarez-Lerma et al. A (2003) Dorko et al. (2002) Hazen et al. (1986) Chakrabarti et al. (2009) Colombo et al. (2007) Costa-de-Oliveira et al. (2008) Bassetti et al. (2006) Miranda et al. (2009) Tortorano et al. (2006) Trick et al. (2002) Pfaller et al. (2010) Period of observation FebruaryMay 2003 May 20052006 19992001 JuneAugust 2006 19981999 During 2004 19992003 20042005 19971999 During 1999 20082009 Region/ country Venezuela Portugal Argentine USA Brazil Brazil Brazil Australia Spain Slovakia USA India Brazil Portugal Italy Brazil Europe USA Europe/ Asia/ American Number of strains 43 53 530 45 43 23 65 389 94 126 282 182 473 1239 C. albicans (%) 42.3 79 60.7 51.8 35.5 70 52 85.2 68.4 61.7 21 26.3 38 35 40 42 53 59 50 C. tropicalis (%) 12.8 4.8 4.5 7.9 22.3 4.6 43.5 36 6.3 38 48 9 33 7 10 9.8 C. parapsilosis (%) 14.9 6.5 4.1 11.1 4.6 4.4 0.5 24.5 12 23 26.5 23 16 14 11 17.4 C. glabrata (%) 2.1 4.8 5.6 15.6 8.8 7 17.3 27.8 8.2 3 10.5 9 15 2 14 12 17.4

Candiduria

Candidemia

-, Not mentioned.

to NCAC species were 14% for C. glabrata and C. parapsilosis and 7% for C. tropicalis according to a European Confederation of Medical Mycology survey (Tortorano et al., 2006). Recently, Chen et al. (2008) reported that C. glabrata was a causative agent of candiduria in Australia. This is extremely important, because, compared with other NCAC species infection, the mortality rate associated with C. glabrata is the highest (Abi-Said et al., 1997; Krcmery, 1999b). Until recently, few studies had evaluated independent risk factors associated with nosocomial C. glabrata acquisition and subsequent disease. Although C. glabrata is known to be present in patients ora, relatively little is known about the hospital reservoirs of C. glabrata, with likely sources of infection involving a complex interaction of both environmental and human reservoirs. Two studies (Isenberg et al., 1989; Vazquez et al., 1993) have indicated hand carriage on hospital personnel as possible sources of infection. Thus, similar to other nosocomial pathogens, C. glabrata may also be acquired, directly or indirectly, from contaminated environmental surfaces. However, the role of carriage by personnel in dissemination of C. glabrata remains to be claried. Lately, the most frequent combination of mixed species infection by Candida species is C. glabrata and C. albicans, which has been found in approximately 70% of the patients with oral candidosis (Redding et al., 2002).
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Candida parapsilosis, despite being initially considered a nonpathogenic species, was initially identied as the causative agent of a fatal case of endocarditis in an intravenous drug user in 1940 (Joachim & Polayes, 1940). Furthermore, over the past decade, the incidence of C. parapsilosis in infections has increased drastically. In fact, reports indicate that C. parapsilosis is often the second most frequently isolated Candida species from blood cultures (Almirante et al., 2006; Colombo et al., 2007; Costa-de-Oliveira et al., 2008). Furthermore, C. parapsilosis is one of the fungi most frequently isolated from human hands (Bonassoli et al., 2005) and the second most commonly isolated Candida species from normally sterile body sites of hospitalized patients. This species accounts for 15.5% of Candida isolates in North America, 16.3% in Europe, and 23.4% in Latin America, outranked only by C. albicans (51.5%, 47.8% and 36.5%, respectively) and C. glabrata (21.3%) in North America (Messer et al., 2006). However, C. parapsilosis fungemia has a lower mortality rate (4%) compared with that caused by C. albicans and C. glabrata (Kossoff et al., 1998). The increased incidence of C. parapsilosis infections has been attributed to a variety of risk factors, similar to other Candida species, including the organisms selective growth capabilities in hyperalimentation solutions and its high
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ability to colonize intravascular devices and prosthetic materials. Additionally, patients requiring prolonged used of a central venous catheter or indwelling devices, such as cancer patients, are at increased risk of infection with C. parapsilosis. A recent Spanish study of 72 patients with invasive C. parapsilosis identied vascular catheterization (97%), prior antibiotic therapy (91%), parenteral nutrition (54%), prior surgery (46%), prior immunosuppressive therapy (38%), malignancy (27%), transplant receipt (16%), neutropenia (12%) and prior colonization (11%), as risk factors for infection (Almirante et al., 2006). In a report of 64 episodes (between 2002 and 2003) of C. parapsilosis candidemia in Brazilian hospitals, the primary risk factors were neutropenia, the use of central venous catheters and cancer chemotherapy (Brito et al., 2006). The population at greatest risk for nosocomial infection with C. parapsilosis is that of extremely low-birth-weight neonates (Solomon et al., 1984; Voss et al., 1994). In fact, colonization of the skin or gastrointestinal tract is frequently the rst step in the pathogenesis of invasive candidosis, and neonates are especially prone to such infections given their compromised skin integrity, susceptibility to gastrointestinal tract infection, long-term need for central venous or umbilical catheters and prolonged endotracheal intubation (Benjamin et al., 2000). Furthermore, C. parapsilosis has been isolated from approximately one-third of neonates with gastrointestinal colonization by Candida species (Saiman et al., 2001) and from oropharynges of 23% of healthy neonates (Contreras et al., 1994). Furthermore, in contrast to other NCAC species, the rates of mortality in low-birth-weight neonates caused by C. parapsilosis are drastically higher and sometimes equivalent to those associated with C. albicans (Trofa et al., 2008). Candida tropicalis is one of the three most commonly lvarez-Lerma et al., 2003; Binelli isolated NCAC species (A et al., 2006; Colombo et al., 2007; Hasan et al., 2009). Usually, C. tropicalis is considered the third most frequently isolated NCAC species from blood and urine cultures (Table lvarez-Lerma et al., 2003). 2) (Kauffman et al., 2000; A Moreover, in a recent epidemiology study conducted in 12 Brazilian medical centres, C. tropicalis was the second most frequently recovered Candida species, accounting for 3348% of all candidemia cases (Colombo et al., 2007; Miranda et al., 2009). Additionally, C. tropicalis is often found in patients admitted to intensive care units, especially in patients requiring prolonged catheterization, receiving broad-spectrum antibiotics or with cancer (Kauffman et al., 2000; Rho et al., 2004; Colombo et al., 2007; Nucci & Colombo, 2007). Furthermore, C. tropicalis appears to display a higher potential for dissemination in neutropenic individuals compared with C. albicans and other NCAC species (Colombo et al., 2007). According to Kontoyiannis et al. (2001), there are distinct differences in the presentation and risk factors of C. tropica 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

lis and C. albicans fungemia, with the former more persistent and leading to longer intensive care unit stays during the course of infection. This may imply a higher virulence and greater resistance to commonly used antifungals by C. tropicalis when compared with C. albicans. In fact, some epidemiologic studies (Krcmery, 1999a; Kontoyiannis et al., 2001; Eggimann et al., 2003; Colombo et al., 2007) documented that C. tropicalis was associated with higher mortality than other NCAC species and C. albicans. This propensity of C. tropicalis for dissemination and the associated high mortality may be related to the virulence factors exhibited by this species such as biolm formation, proteinases secretion and dimorphism (Krcmery, 1999b; Negri et al., 2010a).

Pathogenicity and virulence factors of NCAC species


There remains a debate over what actually constitutes a virulence factor. It can be argued that all the traits required for establishing disease are virulence factors; however, strictly speaking, virulence factors are those that interact directly with host cells causing damage (Haynes, 2001). The pathogenicity of Candida species is mediated by a number of virulence factors, including adherence and biolm formation on host tissue as well as medical devices, the ability to evade host defences and the production of tissue-damaging hydrolytic enzymes (e.g. proteases, phospholipases and haemolysins). Infection models of candidosis in animals suggest that C. albicans is the most pathogenic species (Samaranayake & Samaranayake, 2001), and in vitro investigations indicate that it also expresses higher levels of putative virulence factors compared with other species (Jayatilake et al., 2006). Furthermore, it is important to emphasize that these yeasts are not usual pathogens of these animals and therefore such studies do not necessarily reect the reality of pathogenicity of Candida species. Moreover, Candida species can colonize and cause disease at several anatomically distinct sites including the skin, oral cavity, gastrointestinal tract, vagina and vascular system. In order to establish infection, opportunistic pathogens have to evade the immune system, survive, reproduce in the host environment, and in the case of systemic infection, disseminate to new tissues and organs.

Adhesion and biofilm formation


The primary event in Candida infection is adherence to host surfaces, which is required for initial colonization. Adherence contributes to persistence of the organism within the host, and is considered essential in the establishment of disease. Furthermore, Candida species can also adhere to the surfaces of medical devices and form biolms. Several factors have been implicated in inuencing adhesion,
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including the prole of cell wall proteins (Chafn, 2008) and cell surface physicochemical properties (Anil et al., 2001; Henriques et al., 2002). Candida cell surface proteins that are involved in specic adherence are described as adhesins. In C. glabrata, a major group of adhesins is encoded for by the EPA (epithelial adhesin) gene family (De las Penas et al., 2003). The overall structure of Epa proteins is similar to that of the ALS (agglutinin-like sequence) proteins of C. albicans. Although there are few studies concerning C. glabrata Epa proteins, it is known that EPA1p is a calcium-dependent lectin that binds to N-acetyl lactosamine-containing glycol conjugates (Cormack et al., 1999). Furthermore, despite the large number of EPA genes, it has been shown that deletion of merely Epa1p reduces adherence in vitro (De las Penas et al., 2003). In addition, although EPA6 is not expressed in vitro, its expression increases during in vivo urinary tract infection, suggesting that C. glabrata is capable of adapting to different environmental conditions (Domergue et al., 2005). Furthermore, a bioinformatic search of pathogen-specic gene families of Candida species revealed a number of genes for putative cell wall adhesins-like-proteins in C. parapsilosis. This study included genes for ve Als proteins and six for Pga 30 (predicted glycosylphosphatidylinositol-anchored protein 30) (Butler et al., 2009). Unfortunately, there has been no further work in studying the role that these proteins play in C. parapsilosis adhesion. Concerning, proteins from the C. tropicalis cell wall, at least three Alsp have been identied through Western blot analysis with anti-Als antibodies (Hoyer et al., 2001); however, to the authors knowledge, no further work has been undertaken in this area. The fungal cell surface is the site of physicalchemical interactions with host tissues or medical devices leading to its adherence (Cannon & Chafn, 1999). Previous studies of the cell wall of Candida have suggested a relationship between cell surface hydrophobicity and adherence (Panagoda et al., 2001). In a study with a limited number of C. glabrata isolates, this species was found to exhibit a degree of hydrophobicity similar to C. albicans (Hazen et al., 1986). Interestingly, while the hydrophobicity of C. albicans was extremely sensitive to specic growth conditions, numerous isolates of C. glabrata were relatively insensitive to the same growth conditions (Kikutani & Makino, 1992). In addition, Camacho et al. (2007) did not nd a correlation between the hydrophobicity and adherence for Candida cells on siliconized latex catheters, demonstrating that cell hydrophobicity alone was not a predictor for adhesion levels. As reported for C. glabrata, Panagoda et al. (2001) showed that the initial adhesion of C. parapsilosis and C. tropicalis cells was associated with surface hydrophobicity. Initial attachment of Candida to host or/and medical devices is followed by cell division, proliferation and subFEMS Microbiol Rev 36 (2012) 288305

sequent biolm development (Ramage et al., 2006). Biolms are described as surface-associated communities of microorganisms embedded within an extracellular matrix. It is now considered that biolms represent the most prevalent growth form of microorganisms (Al-Fattani & Douglas, 2006; Silva et al., 2009a). Biolm formation is an important virulence factor for a number of Candida species, as it confers signicant resistance to antifungal therapy by limiting the penetration of substances through the matrix and protecting cells from host immune responses (Donlan & Costerton, 2002; Mukherjee & Chandra, 2004). Moreover, biolms formed by C. albicans, C. parapsilosis, C. tropicalis and C. glabrata isolates have been associated with higher morbidity and mortality rates compared with isolates unable to form biolms (Kumamoto, 2002). It is assumed that the formation of mature biolms and subsequent production of extracellular matrix is strongly dependent on species, strain and environmental conditions (pH, medium composition, oxygen) (Ramage et al., 2006; Jain et al., 2007). Recently, Silva et al. (2010b) showed that C. glabrata produced a higher biolm biomass on silicone surfaces in the presence of urine, compared with C. parapsilosis and C. tropicalis. The opposite was found for biolms formed in Sabouraud dextrose broth (Silva et al., 2009a). These results are in accordance with Shin et al. (2002) who reported that biolm formation by C. glabrata was lower compared with other NCAC species, when grown in rich culture media. Candida tropicalis clinical isolates have been classied as being extensive biolm formers on silicone and latex catheter (Fig. 3) (Redding et al., 2002; Silva et al., 2009a; Negri et al., 2010b). Biolms are readily formed by C. parapsilosis cells grown in media containing high glucose and lipid concentrations, and can be associated with the increased prevalence of this organism in bloodstream infections of patients receiving parenteral nutrition (Nosek et al., 2009). The selective preference of this species for plastic medical devices is of particular interest, as biolm formation enhances the capacity of the organism to colonize catheters and intravascular lines (Weems, 1992; Trofa et al., 2008). In contrast to C. albicans, C. parapsilosis biolms are thinner, less structured and consist exclusively of aggregated blastospores (Kuhn et al., 2002). These biolm features are in accordance with those recently reported by Silva et al. (2009a). Lattif et al. (2009) demonstrated that, like C. parapsilosis, the two newly identied Candida species (C. orthopsilosis and C. metapsilosis) were also able to form biolms. Little is known about the matrix composition of NCAC species biolms, but according to Baillie & Douglas (2000), C. albicans biolm matrix is mainly composed of carbohydrates, proteins, phosphorus and hexosamines. Silva et al. (2009a) reported that the extracellular matrices of C. parapsilosis biolms contained large amounts of carbohydrates
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(a)

(b)

Fig. 3. Scanning electron microscopy images of Candida tropicalis biolms formed on (a) silicone and (b) latex catheter in articial urine at 24 h. Scale bar = 20 mm.

with correspondingly low levels of proteins. In the same study, C. glabrata biolm matrices were found to have high levels of both proteins and carbohydrates, while C. tropicalis biolm matrices had low levels of carbohydrates and proteins compared with the other NCAC species. Interestingly, the biolm matrix composition is highly strain dependent, a phenomenon that has not been observed in related yeasts (Silva et al., 2009a). Furthermore, Al-Fattani & Douglas (2006) showed that matrix material extracted from biolms of C. tropicalis and C. albicans contained carbohydrates, proteins, hexosamine, phosphorus and uronic acid. However, the major component in C. tropicalis biolm matrices was hexosamine (27%). The same authors also reported that these biolms partially detached after treatment with lipase type VII and chitinase, which is in contrast to biolms of C. albicans that detached only after treatment with proteinase K, chitinase, DNase I or b-N-aceytyglucosamidase. DNA has been described as a component of the extracellular matrix in bacteria biolms (Allesen-Holm et al., 2006; Vilain et al., 2009). In Candida species, there is scarce knowledge concerning the contribution of extracellular DNA to biolm matrix and overall structure. Recently, Martins et al. (2010a, b) highlighted the importance of DNA in C. albicans biolm formation, integrity and structure. However, there is a lack of knowledge concerning NCAC species and the role of extracellular DNA on biolm composition. While extensive work has been performed on the C. albicans genes involved in adhesion/colonization and biolm formation, little is known about equivalent controlling genes in C. glabrata, C. parapsilosis and C. tropicalis. However, two recent studies involving the study of C. parapsilosis lipase knockout mutants found that these had a decreased ability to form biolms. The C. parapsilosis mutants produced signicantly less biolm than the wild cser et al., 2007a). Moreover, the BCR type strain (Ga (biolm and cell wall regulator) gene was also deemed cser et al., necessary for proper biolm formation (Ga 2007b). Notably, the biolm-decient C. parapsilosis lipase mutants were less virulent in tissue culture infection models and in mice (G acser et al., 2007b). As documented above, the cell wall seems to play a crucial role in colonization and infection, and therefore, elucidation of its structure and
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composition may lead to a better understanding of NCAC pathogenicity, and also aid in identifying therapeutic targets.

Hydrolytic enzymes
Destruction of host tissues by Candida species may be facilitated by the release of hydrolytic enzymes into the local environment. Secreted aspartyl proteinases (Saps), phospholipases, lipases (LIPs) and haemolysins are the enzymes most frequently implicated in Candida species pathogenicity. Saps facilitate invasion and colonization of host tissues by disruption of the host mucosal membranes (R uchel, 1999) and by degrading important immunological and structural et al., 2001). In the case of defence proteins (Pichova C. glabrata, only one study has shown that this species is capable of proteinase production, but the type of proteinase was not specied (Chakrabarti et al., 1991). For C. parapsilosis, three SAP genes have been identied (SAPP1-3), two of which remain largely uncharacterized (Merkerova et al., 2006). The Sapp1p isoenzyme has, however, been biochemi et al., 2001; cally characterized (Fusek et al., 1994; Pichova Dostal et al., 2005), and SAPP2 encodes a functional proteinase that constitutes only 20% of the Saps isolated from a culture supernatant (Fusek et al., 1994). It has been reported (Silva et al., 2009b) that SAPP1-3 genes expression varies with different clinical isolates of C. parapsilosis when grown in contact with an oral epithelium and even in planktonic growth forms. However, there is a trend relating Sap production and site of strain isolation, because both vaginal and skin isolates of C. parapsilosis exhibit higher in vitro Sap activity than blood isolates (Cassone et al., 1995; Dagdeviren et al., 2005). Candida parapsilosis has been shown to be poorly invasive of an oral epithelium, but can nevertheless induce signicant damage, which was related to specic SAP gene expression (Silva et al., 2009b). As with C. albicans, in vitro studies reveal that C. tropicalis secretes high levels of Saps in a medium containing bovine serum albumin as the sole source of nitrogen (Zaugg et al., 2001; Negri et al., 2010b). Furthermore, C. tropicalis possesses at least four genes encoding Saps, designated as SAPT1 to SAPT4 (Togni et al., 1991; Zaugg et al., 2001). To date, Sapt1p is the only enzyme that has been puried from culture supernatant, biochemically characterized and
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crystallized (Togni et al., 1991; Symersky et al., 1997). The presence of Saps secreted by C. tropicalis has also been reported on the surface of fungal elements penetrating tissues during disseminated infection and evading macrophages after phagocytosis of yeast cells (Borg & Ruchel, 1990; R uchel et al., 1992). Recently, Silva et al. (2010b) demonstrated that, like C. albicans (Lermann & Morschhauser, 2008; Naglik et al., 2008), Sap expression during C. tropicalis colonization of an oral epithelium was not associated with invasion and tissue damage. In addition to Saps, enzymes categorized as phospholipases are often considered to be involved in Candida pathogenicity. Phospholipases are enzymes that hydrolyze phospholipids into fatty acids. The production of all classes of phospholipases have been described for Candida species and are suggested to contribute to host cell membrane damage, which could also expose receptors to facilitate lu & Y adherence (Ghannoum, 2000; Kantarcio ucel, 2002). The most widely used diagnostic method for phospholipase determination is based on yeast growth in an egg yolk agar media. Several studies indicate that NCAC species produce extracellular phospholipases (Furlaneto-Maia et al., 2007; Cafarchia et al., 2008; Galan-Ladero et al., 2010), but at signicantly lower levels compared with C. albicans (Ghannoum, 2000). There have been contradictory ndings, with some investigators reporting phospholipase activity in 51% of the strains assayed (Ghannoum, 2000) and others nding no phospholipase activity in the examined strains (Kantar lu & Y cio ucel, 2002). According to recent studies, while C. tropicalis appears to have a reduced ability to produce extracellular phospholipases, production is highly strain dependent (Furlaneto-Maia et al., 2007; Cafarchia et al., 2008; Galan-Ladero et al., 2010; Negri et al., 2010b). Furthermore, contrary to the few studies on C. tropicalis and C. parapsilosis (Kumar et al., 2009), no studies have been reported concerning C. glabrata phospholipase production. Lipases are involved in the hydrolysis of triacylglycerols. In C. albicans, 10 genes encoding for lipases have been identied and it has been shown that C. albicans CaLIP8 mutants were signicantly less virulent in a murine intrave cser et al., 2007b). For C. paranous infection model (Ga psilosis, CpLIP1 and CpLIP2 have been reported, with the latter known to encode for an active protein (Neugnot et al., 2002). Recently, G acser et al. (2007a) demonstrated that a lipase inhibitor signicantly reduced tissue damage during C. parapsilosis infection of a reconstituted human tissue, and that CpLIP1/CpLIP2 mutants formed thinner and less complex biolms. Sequences similar to C. albicans (LIP1-10) were also detected in C. tropicalis, but not in C. glabrata. However, no studies have been performed to investigate the role of these genes in the virulence of C. tropicalis (Fu et al., 1997). Pathogenic microorganisms can grow in the host using haemin or haemoglobin as a source of iron. Haemolysins are
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used by Candida species to degrade haemoglobin and facilitate recovery of the elemental iron from host cells. Thus, haemolysins are considered key virulence factors enabling pathogen survival and persistence in the host (Manns et al., 1994; Watanabe et al., 1999; Luo et al., 2004). Furthermore, it is known that C. albicans has the ability to utilize iron to produce a factor that can release haemoglobin by lysing erythrocytes (Manns et al., 1994; Watanabe et al., 1999). Production of this haemolytic factor may be regulated by the presence of glucose in the growth medium. Candida glabrata, C. parapsilosis and C. tropicalis are all able to produce haemolysins in vitro, inducing partial or total erythrocyte lyses, although the extent of this is both strain and species dependent (Luo et al., 2004). Other authors (Furlaneto-Maia et al., 2007; Kumar et al., 2009; Negri et al., 2010b) only observed production of haemolysins by C. albicans. Although haemolysins are known to be putative virulence factors contributing to pathogenicity in Candida species, the genetic expression of haemolytic activity of Candida is poorly understood at present, but a study conducted by Luo et al. (2004) showed that a haemolysinlike protein (HLP) gene was associated with the haemolytic activity of C. glabrata.

Filamentous growth
Hyphae are believed to play an important role in tissue invasion, and in vitro research has shown that C. albicans lacking hyphal formation exhibited lower ability to invade tissue compared with wild-type C. albicans strains (Jayatilake et al., 2006). Furthermore, lamentous forms (hyphae and/or pseudohyphae) of Candida species also demonstrate increased resistance to phagocytosis compared with yeast (Gow et al., 2002). The morphological forms exhibited by C. tropicalis are similar to those shown by C. albicans, but despite this few studies have explored the importance of C. tropicalis morphology on virulence. However, Silva et al. (2010a) demonstrated recently that only lamentous forms of C. tropicalis were able to invade an oral epithelium. In the case of C. parapsilosis, it has been found that hyphal transition occurs in a strain-dependent manner (Enger et al., 2001), and contrary to C. albicans and C. tropicalis, the ability of C. parapsilosis to invade an oral epithelium did not correlate with pseudohyphal production (Silva et al., 2009b).

Antifungal therapies and mechanisms of resistance of NCAC species


Compared with antibiotics, the development of antifungal agents has been relatively limited. This can be attributed to several factors including inherent problems in the identication of an effective agent that acts on eukaryotic fungal cell type without being toxic to host cells. Resistance to
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Table 3. Common antifungal agents used in the management of candidosis, mode of action and susceptibilities of Candida species Susceptibility of NCAC species Antifungal Polyenes Amphotericin B Azoles Fluconazole Itraconazole Voriconazole Posoconazole Ravuconazole 5-Flucytosine Echinocandins Caspofungin C. albicans C. parapsilosis C. tropicalis C. glabrata Disruption of fungal cell membrane S S S Inhibition of ergosterol synthesis S S S S S S S S S S S S S S S Inhibition of DNA and protein synthesis S S S Inhibition of b1,3-D-glucan synthesis S S S S to I SDD to R SDD to R S S S S S

Adapted from references Rex et al., 1995, 1997, 2000; Diekema et al., 2002; Roling et al., 2002; Eggimann et al., 2003; S-DD, susceptible-dose dependent; I, intermediate; R, resistant.

antifungal drugs is an increasingly recognized phenomenon and can be dened clinically as the persistence of signs and symptoms of the infection despite the presence of a tolerable level of the drug. Depending on the drug and the Candida species, the mechanism of antifungal resistance can either be inherent (present without previous exposure to the antifungal) or acquired, where resistance develops in a previously susceptible organism after a period of exposure to the agent. The classication of these drugs is currently based on their target of activity (Table 3). Polyene antifungals, such as amphotericin B, are fungicidal due to their ability to interact with the ergosterol component within the cell membrane to generate pores, causing cell membrane leakage leading to loss of cytoplasmic content. Azoles are another class of antifungal agents that inhibit the biosynthesis of ergosterol by interfering with the fungal enzyme, lanosterol demethylase. A key function of this enzyme is to convert lanosterol to ergosterol and the inhibition of this leads to depletion of the sterol in the fungal cell membrane. Azole antifungals have a fungistatic or fungicidal activity against Candida species and the most frequently used azole antifungals are uconazole and itraconazole. 5-Flucytosine is an antifungal that enters the fungal cell through a cytosine permease and is then converted by the fungus into 5-uorouracil. This nucleoside analogue gets incorporated into RNA molecules and subsequently interferes with the synthesis of proteins within the fungal cell. Several antifungals have been developed that target cell wall components, for example b-1,3-D-glucan, a key component of the fungal cell wall is not present within mammalian cells. Interference with the enzyme b-1,3-D-glucan synthetase can inhibit the synthesis of b-1,3-D-glucan. A
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group of antifungals demonstrating this mode of action are the echinocandins, and three drugs (caspofungin, micofungin and anidulafungin) have shown antifungal activity against Candida species. Amphotericin B is generally regarded to have the broadest spectrum of antifungal activity and is used in cases of serious and invasive Candida infections, such as in the treatment of systemic infection in hospitalized patients. Resistance to amphotericin B remains uncommon during treatment, but reports of isolates exhibiting elevated minimum inhibitory concentration (MIC) have become more frequent (Pfaller & Diekema, 2007). Resistance to polyenes is believed to result from the alteration of sterol content or composition in the cell membrane (Ghannoum & Rice, 1999). Lupetti et al. (2002) described that among Candida species, polyene resistance was usually due to defective ergosterol biosynthesis, and most likely resulted from mutation in the ERG3 gene that produces altered d5,6-sterol desaturase activity. In addition to ERG3 gene, mutation in ERG11 (the gene that produces lanosterol 14a-demethylase) and in ERG6 (a gene that is required for normal membrane function, but is not essential for sterol biosynthesis) may generate polyene resistance. Importantly, C. glabrata isolates have been identied with mutations in the ERG6 gene (Vandeputte et al., 2007). The development of the azole antifungals enhanced the treatment options for fungal infections and their reduced host toxicity has led to their widespread use. Consequently, with this extensive use, it is perhaps not surprising that resistance to these agents, particularly uconazole, has been encountered (Rex et al., 1995; Pfaller & Diekema, 2007). Resistance to the azoles can result from quantitative or qualitative modications of target enzymes, reduced access of the drug to the target or a combination of these mechanisms. Qualitative modications in target enzymes result from point mutations in ERG11, the gene responsible for production of 14a-demethylase, which is the principal target of the azoles. The other primary mechanism by which Candida species resist the effects of azole antifungals involves the active efux of the drug out of the cell via the activation of two kinds of efux transport proteins encoded by either MDR or CDR genes (Lupetti et al., 2002; Ghannoum & Rice, 1999; Sanglard & Odds, 2002). Candida glabrata may be intermediately resistant to all azoles and about 20% of strains develop resistance during therapy and prophylaxis with uconazole (Pfaller & Diekema, 2007). Susceptibility testing has shown that uconazole is active against several Candida species, including C. albicans, C. parapsilosis and C. tropicalis (Pfaller & Diekema, 2007). Itraconazole is moderately active against most medically important uconazole-susceptible and -resistant Candida species, with the exception of C. glabrata (Pfaller et al., 2005). Voriconazole exerts fungicidal activity against most
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yeasts and certain opportunistic fungi, specically against some NCAC species (Groll et al., 2001). This agent is generally active against Candida species including uconazole-resistant C. albicans and C. glabrata (Pfaller & Diekema, 2007). With the exception of C. tropicalis, voriconazole is more active than uconazole against medically important Candida species (Pfaller & Diekema, 2007). Posaconazole exerts fungistatic activity against some NCAC species, including C. glabrata, C. tropicalis and C. parapsilosis (Scozo et al., 2007). Flucytosine has a narrow spectrum of activity, and several mechanisms of resistance are possible due to the multiple intracellular enzymatic steps required for its action. These include alterations in the target enzymes UMP pyrophosphorylase, cytosine permease and cytosine deaminase, or increased production of pyrimidines (Atkinson & Israel, 1973). The antifungal spectrum of ucytosine is extremely narrow: Candida species, Cryptococcus species and Aspergillus species (Polak et al., 1982; Vermes et al., 2000). Furthermore, due to the multiple steps in its mode of action, including transport into the cell and deamination of the active compound, ucytosine is normally used only in combination with other agents, including amphotericin B and uconazole (Vermes et al., 2000). As a class, the echinocandins are the most recent addition to the antifungal arsenal, and to date their use has been very limited to assess whether signicant resistance will develop to these agents. Microorganisms that demonstrate inherent resistance to echinocandins either generate insufcient target enzyme b-1,3-D-glucan synthase or produce an alternate form of the enzyme with reduced echinocandin binding. All echinocandins exert fungicidal activity against Candida species. The echinocandins are highly active against C. albicans, C. glabrata and C. tropicalis both in vitro and in vivo (Pfaller et al., 2003, 2005; Bayegan et al., 2010; Kuchar nov a et al., 2010). It is important is to emphasize that the MIC values for echinocandins tend to be higher for C. parapsilosis than for most other common Candida species, particularly C. albicans (Walsh, 2002). From the clinical perspective, the most important feature of Candida biolms is their role in increasing tolerance to conventional antifungal therapy. The reduced susceptibility of C. albicans biolms to antifungal agents was rst reported in 1995 (Hawser et al., 1995). Furthermore, several groups have demonstrated that biolm cells drastically increase their tolerance to the most commonly used antifungal agents (uconazole and amphotericin B) (Ramage et al., 2001). Biolms of NCAC species, such as C. tropicalis, C. parapsilosis and C. glabrata, have also been shown to exhibit reduced antifungal susceptibility (Hawser & Douglas, 1994, 1995). Although the mechanisms of biolm drug resistance are not fully understood, the current consensus is that biolm tolerance is a complex multifactorial
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phenomenon involving different molecular mechanisms, restricted penetration of the drug through the matrix and the presence of so-called persister cells within the biolm, which survive exposure to the agent (Lewis, 2001; Donlan & Costerton, 2002; Douglas, 2003).

Concluding remarks
Changes in the host are generally required for opportunistic yeast to alter from harmless commensal microorganisms to potentially life-threatening human pathogens. Management of candidosis involves the identication and control of host factors that may predispose one to infection. Furthermore, Candida species can exhibit several virulence factors such as adherence, biolm formation and secretion of hydrolytic enzymes that both increase their persistence within the host as well as cause host cell damage. Therefore, the increase in the incidence and antifungal resistance of NCAC species, specically C. glabrata, C. parapsilosis and C. tropicalis, and the unacceptably high morbidity and mortality associated with these species, make it essential to further enhance our knowledge on the virulence and resistance mechanisms associated with these species. Studies in this area will contribute towards the identication of new targets for novel therapeutics against these recently emerged pathogens.

Acknowledgements
The authors acknowledge FCT, Portugal, for supporting S.S.s work through grant SFRH/BD/28341/2006 and CAPES, Brazil, for supporting M.N.s work through grant BEX-4642/06-6. We would like to thank Designer Fabio Grassi for helping in the improvement of the images.

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