3 Especies de Candida Generalidades 2011

R E V I E W A R T I C L E

Candidaglabrata,Candida parapsilosis andCandida tropicalis:biology, epidemiology,pathogenicityandantifungal resistanceSonia Silva1, Melyssa Negri1, Mariana Henriques1, Rosario Oliveira1, David W. Williams2 &Joana Azeredo1

1Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal; and 2Tissue Engineering & Reparative

Dentistry, School of Dentistry, Heath Park, Cardiff, UK

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: [email protected]

Received 29 December 2010; revised 31 March

2011; accepted 6 May 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 immunocompro-

mised 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 identified as human pathogens. The

apparent increased emergence of these species as human pathogens can be

attributed to improved identification 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, biofilm forma-

tion 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, identification, epidemiology, pathogenicity and antifungal resistance of

C. glabrata, C. parapsilosis and C. tropicalis.

Introduction

In last 30 years there has been a significant increase in the

incidence of fungal infections in humans (Lass-Florl, 2009).

Such infections may either be superficial, 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 significant (Sa-

maranayake 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

minority have been implicated in human candidosis. Addi-

tionally, 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) condi-

tions. 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 significantly (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

c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

FEMS Microbiol Rev 36 (2012) 288–305

Final version published online 6 June 2011.

MIC

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REV

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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

reflection 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, identifica-

tion, 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 sphe-

rical to oval in shape and are approximately 2–5� 3–7mm in

size (Fig. 1) (Larone, 2002). Moreover, certain species, such

as C. albicans and Candida dubliniensis, can produce a

filamentous 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 filaments with constrictions at the

cell–cell 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 defined septa that divide the hyphae into

separate fungal units (Fig. 1).

Candida albicans and C. dubliniensis are truly poly-

morphic, 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 classified in the genus Torulopsis

due to its lack of pseudohyphal formation. However, in

1978, it was determined that the ability to form pseudohy-

phae was not a reliable distinguishing factor for members of

the genus Candida and it was proposed that Torulopsis

glabrata could be classified 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 character-

istically large and curved, and often referred to as ‘giant cells’

Fig. 1. Epifluorescence photocomposition of the different morphological growth forms of Candida albicans stained with calcofluor white: (A)

blastoconidia; (B1) reproduction by budding; (B2) germ tube formation; (C1) pseudohyphae formation; (C2) yeast form; (C3) hyphae formation.�Internal cross walls (septa).

FEMS Microbiol Rev 36 (2012) 288–305 ª 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Non-Candida albicans Candida species pathogenicity 289

(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

(1–4 mm) are noticeably smaller than the blastoconidia of

C. albicans (4–6 mm), C. tropicalis (4–8mm) and C. para-

psilosis (2.5–4mm) (Larone, 2002) (Table 1). On SDA (Fig.

2) C. glabrata forms glistening, smooth, and cream-co-

loured 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. para-

psilosis, 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

(Martin, 1979). Interestingly, C. parapsilosis was firstly

classified 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 identification methods for Candida have been

developed, leading to the identification of new species

together with their increased recognition in human infec-

tion. Before 2005, C. parapsilosis was separated into three

groups (I–III), but further studies revealing genomic differ-

ences that have led to the separation of these groups into

closely related, but distinct species, namely, C. parapsilosis,

Candida orthopsilosis and Candida metapsilosis (Tavanti

et al., 2005; Gacser et al., 2007a, b).

Laboratory identification of NCAC species

The laboratory diagnosis of candidoses continues to be

problematic. Microbiological confirmation can be difficult

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.

Table 1. Morphological characteristics of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata species

Species Germ tube production Hyphae/pseudohyphae Yeasts size (mm) CHROM-agar colony colour

C. albicans 1 1/1 4–6�6–10 Blue-green

C. tropicalis � � /1 4–8�5–11 Dark blue

C. parapsilosis � � /1 2.5–4�2.5–9 White

C. glabrata � � /� 1–4 White, Pink-purple

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 36 (2012) 288–305Published by Blackwell Publishing Ltd. All rights reserved

290 S. Silva et al.

as blood cultures can be negative in up to 50% of autopsy-

proven 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 identification important for clinical management.

Laboratory diagnosis has improved with the advent of

new methods for Candida isolation and identification.

Technologies such as species-specific 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 specific 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 identification of Candida is

therefore essential for establishing a definitive diagnosis. A

standard approach to the laboratory diagnosis generally

involves nonmolecular methods, although PCR is increas-

ingly being used.

Non-PCR based methods of Candidaidentification

CHROMagars Candida (CHROMagars, Paris, France), is a

relatively new differential agar medium for Candida species

identification and has been particularly useful in the pre-

sumptive identification 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 identified by

carbohydrate assimilation and fermentation tests as well as

morphological characteristics such as germ tube and chla-

mydospore development (Fig. 2). In addition, more rapid

and less laborious phenotypic identification methods have

become available. Perhaps the most widely used methods for

Candida species identification are those based on the format

of carbohydrate assimilation and/or enzyme detection with-

in 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

identification for the most common species of Candida,

while identification 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 definitive identifica-

tion (Verweij et al., 1999; Ellepola & Morrison, 2005).

Additional methods for Candida species identification

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 (Sanofi Diagnostics

Pasteur, France) and the automated Rapid Yeast Identifica-

tion Panel (Dade Microscan). However, as mentioned

above, most of these tests tend to be most accurate for the

identification 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 techni-

ques 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 mon-

itoring of blood culture bottles, based on colour (BacT/

ALERT 3D, Organon Teknika Corp., Durham, NC) and

fluorescence (BACTEC 9240, Becton Dickinson), have been

developed recently (Ellepola & Morrison, 2005).

The identification of typical blastospores and pseudohy-

phae 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 fluorescent antibody, acridine orange or calco-

fluor-white staining (Pfaller, 1992; Ellepola & Morrison,

2005) may enhance the sensitivity of microscopic examina-

tion. However, the production of fluorescent antibodies

specific for the identification of individual Candida species

has proved to be extremely difficult.

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

specificity 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).



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 investi-

gate 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 specific sequences serves to prime the amplification of

target DNA. The most important feature of any PCR

primers used directly on clinical samples is that they are

specific 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

identification.

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 labora-

tories, the problems with sample preparation and environ-

mental contamination and the lack of standardized

protocols for PCR methodologies.

Epidemiology and risk factors in NCACspecies infection

The mortality rates associated with different microorgan-

isms have declined with the early administration of empiri-

cal 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 immunocompro-

mised patients, including those in intensive care, postsurgi-

cal 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-

mately 31–55% of healthy individuals. Historically, C.

albicans has accounted for 70–80% of clinical isolates, with

other NCAC species only rarely encountered (Odds, 1988;

Calderone, 2002; Samaranayake et al., 2002). Nevertheless,

over the last 10–30 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 ac-

counted for about 95% of identifiable 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

significantly 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 significant 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 significant

problem in neonates, transplant recipients and patients

receiving parenteral nutrition (Trofa et al., 2008). Further-

more, 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 flora 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 significantly (Hajjeh et al., 2004).

Although the mortality rate associated with Candida infec-

tions varies with the type of patient and with the causative

agent, the incidence rates of candidosis infections attributed


292 S. Silva et al.

to NCAC species were 14% for C. glabrata and C. parapsilosis

and 7% for C. tropicalis according to a European Confedera-

tion 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 patient’s flora, relatively little is known about the

hospital reservoirs of C. glabrata, with likely sources of

infection involving a complex interaction of both environ-

mental 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

clarified. 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).

Candida parapsilosis, despite being initially considered a

nonpathogenic species, was initially identified as the causa-

tive 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 organism’s selective growth

capabilities in hyperalimentation solutions and its high

Table 2. Selected epidemiological studies published from 2000 to 2010, concerning the distribution of Candida species isolates among various types of

candidosis

Candidosis References

Period of

observation

Region/

country

Number

of strains

C. albicans

(%)

C. tropicalis

(%)

C. parapsilosis

(%)

C. glabrata

(%)

Oral candidosis Gonzalez Gravina et al.

(2007)

February–May

2003

Venezuela 43 42.3 12.8 14.9 2.1

Martins et al. (2010a, b) May 2005–2006 Portugal 53 79 4.8 6.5 4.8

Luque et al. (2009) - Argentine - 60.7 4.5 - 5.6

Candiduria Kauffman et al. (2000) - USA 530 51.8 7.9 4.1 15.6

Kobayashi et al. (2004) - Brazil 45 35.5 22.3 11.1 8.8

Passos et al. (2005) - Brazil 43 70 4.6 4.6 7

Binelli et al. (2006) 1999–2001 Brazil 23 52 43.5 - 17.3

Chen et al. (2008) June–August 2006 Australia 65 85.2 - 4.4 27.8

Alvarez-Lerma et al.

(2003)

1998–1999 Spain 389 68.4 36 0.5 8.2

Dorko et al. (2002) - Slovakia 94 61.7 6.3 24.5 -

Candidemia Hazen et al. (1986) - USA 126 21 38 12 3

Chakrabarti et al. (2009) - India - 26.3 - 10.5

Colombo et al. (2007) - Brazil 282 38 48 23 9

Costa-de-Oliveira et al.

(2008)

During 2004 Portugal - 35 - 26.5 -

Bassetti et al. (2006) 1999–2003 Italy 182 40 9 23 15

Miranda et al. (2009) 2004–2005 Brazil - 42 33 16 2

Tortorano et al. (2006) 1997–1999 Europe 473 53 7 14 14

Trick et al. (2002) During 1999 USA - 59 10 11 12

Pfaller et al. (2010) 2008–2009 Europe/

Asia/

American

1239 50 9.8 17.4 17.4

-, Not mentioned.



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 identified 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 pri-

mary 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 first 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 approxi-

mately one-third of neonates with gastrointestinal coloniza-

tion 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

isolated NCAC species (Alvarez-Lerma et al., 2003; Binelli

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

2) (Kauffman et al., 2000; Alvarez-Lerma et al., 2003).

Moreover, in a recent epidemiology study conducted in 12

Brazilian medical centres, C. tropicalis was the second most

frequently recovered Candida species, accounting for

33–48% 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 dis-

play 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-

lis and C. albicans fungemia, with the former more persis-

tent 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 biofilm formation,

proteinases secretion and dimorphism (Krcmery, 1999b;

Negri et al., 2010a).

Pathogenicity and virulence factors ofNCAC 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 biofilm forma-

tion 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 reflect the reality of patho-

genicity 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. Adher-

ence 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 biofilms. Several

factors have been implicated in influencing adhesion,


294 S. Silva et al.

including the profile of cell wall proteins (Chaffin, 2008) and

cell surface physicochemical properties (Anil et al., 2001;

Henriques et al., 2002).

Candida cell surface proteins that are involved in specific

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 infec-

tion, suggesting that C. glabrata is capable of adapting to

different environmental conditions (Domergue et al., 2005).

Furthermore, a bioinformatic search of pathogen-specific

gene families of Candida species revealed a number of genes

for putative cell wall adhesins-like-proteins in C. parapsilo-

sis. This study included genes for five 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 identified 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 physical–chemical

interactions with host tissues or medical devices leading to

its adherence (Cannon & Chaffin, 1999). Previous studies of

the cell wall of Candida have suggested a relationship

between cell surface hydrophobicity and adherence (Pana-

goda 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 specific 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 find a correlation between the

hydrophobicity and adherence for Candida cells on silico-

nized latex catheters, demonstrating that cell hydrophobi-

city 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 sub-

sequent biofilm development (Ramage et al., 2006). Biofilms

are described as surface-associated communities of micro-

organisms embedded within an extracellular matrix. It is

now considered that biofilms represent the most prevalent

growth form of microorganisms (Al-Fattani & Douglas,

2006; Silva et al., 2009a). Biofilm formation is an important

virulence factor for a number of Candida species, as it

confers significant resistance to antifungal therapy by limit-

ing the penetration of substances through the matrix and

protecting cells from host immune responses (Donlan &

Costerton, 2002; Mukherjee & Chandra, 2004). Moreover,

biofilms formed by C. albicans, C. parapsilosis, C. tropicalis

and C. glabrata isolates have been associated with higher

morbidity and mortality rates compared with isolates un-

able to form biofilms (Kumamoto, 2002). It is assumed that

the formation of mature biofilms and subsequent produc-

tion of extracellular matrix is strongly dependent on species,

strain and environmental conditions (pH, medium compo-

sition, oxygen) (Ramage et al., 2006; Jain et al., 2007).

Recently, Silva et al. (2010b) showed that C. glabrata

produced a higher biofilm biomass on silicone surfaces in

the presence of urine, compared with C. parapsilosis and C.

tropicalis. The opposite was found for biofilms formed in

Sabouraud dextrose broth (Silva et al., 2009a). These results

are in accordance with Shin et al. (2002) who reported that

biofilm formation by C. glabrata was lower compared with

other NCAC species, when grown in rich culture media.

Candida tropicalis clinical isolates have been classified as

being extensive biofilm formers on silicone and latex

catheter (Fig. 3) (Redding et al., 2002; Silva et al., 2009a;

Negri et al., 2010b). Biofilms 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 infec-

tions of patients receiving parenteral nutrition (Nosek et al.,

2009). The selective preference of this species for plastic

medical devices is of particular interest, as biofilm 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 biofilms are thinner,

less structured and consist exclusively of aggregated blastos-

pores (Kuhn et al., 2002). These biofilm 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 identified Candida species (C.

orthopsilosis and C. metapsilosis) were also able to form

biofilms.

Little is known about the matrix composition of NCAC

species biofilms, but according to Baillie & Douglas (2000),

C. albicans biofilm matrix is mainly composed of carbohy-

drates, proteins, phosphorus and hexosamines. Silva et al.

(2009a) reported that the extracellular matrices of C. para-

psilosis biofilms contained large amounts of carbohydrates



with correspondingly low levels of proteins. In the same

study, C. glabrata biofilm matrices were found to have high

levels of both proteins and carbohydrates, while C. tropicalis

biofilm matrices had low levels of carbohydrates and pro-

teins compared with the other NCAC species. Interestingly,

the biofilm 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 biofilms

of C. tropicalis and C. albicans contained carbohydrates,

proteins, hexosamine, phosphorus and uronic acid. How-

ever, the major component in C. tropicalis biofilm matrices

was hexosamine (27%). The same authors also reported that

these biofilms partially detached after treatment with lipase

type VII and chitinase, which is in contrast to biofilms

of C. albicans that detached only after treatment with

proteinase K, chitinase, DNase I or b-N-aceytyglucosami-

dase. DNA has been described as a component of the

extracellular matrix in bacteria biofilms (Allesen-Holm

et al., 2006; Vilain et al., 2009). In Candida species, there is

scarce knowledge concerning the contribution of extracel-

lular DNA to biofilm matrix and overall structure. Recently,

Martins et al. (2010a, b) highlighted the importance of DNA

in C. albicans biofilm formation, integrity and structure.

However, there is a lack of knowledge concerning NCAC

species and the role of extracellular DNA on biofilm

composition.

While extensive work has been performed on the

C. albicans genes involved in adhesion/colonization and

biofilm formation, little is known about equivalent control-

ling 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 biofilms. The C. parapsilosis

mutants produced significantly less biofilm than the wild-

type strain (Gacser et al., 2007a). Moreover, the BCR

(biofilm and cell wall regulator) gene was also deemed

necessary for proper biofilm formation (Gacser et al.,

2007b). Notably, the biofilm-deficient C. parapsilosis lipase

mutants were less virulent in tissue culture infection models

and in mice (Gacser 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

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), phospho-

lipases, 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 (Ruchel, 1999)

and by degrading important immunological and structural

defence proteins (Pichova et al., 2001). In the case of

C. glabrata, only one study has shown that this species is

capable of proteinase production, but the type of proteinase

was not specified (Chakrabarti et al., 1991). For C. para-

psilosis, three SAP genes have been identified (SAPP1-3), two

of which remain largely uncharacterized (Merkerova et al.,

2006). The Sapp1p isoenzyme has, however, been biochemi-

cally characterized (Fusek et al., 1994; Pichova et al., 2001;

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 significant damage, which was related to

specific 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 purified from

culture supernatant, biochemically characterized and

(a) (b)

Fig. 3. Scanning electron microscopy images of

Candida tropicalis biofilms formed on (a) silicone

and (b) latex catheter in artificial urine at 24 h.

Scale bar = 20 mm.


296 S. Silva et al.

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 macro-

phages after phagocytosis of yeast cells (Borg & Ruchel,

1990; Ruchel et al., 1992). Recently, Silva et al. (2010b)

demonstrated that, like C. albicans (Lermann & Morsch-

hauser, 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 phospholi-

pases 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

adherence (Ghannoum, 2000; Kantarciolu & Yucel, 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

significantly lower levels compared with C. albicans (Ghan-

noum, 2000). There have been contradictory findings, with

some investigators reporting phospholipase activity in 51%

of the strains assayed (Ghannoum, 2000) and others finding

no phospholipase activity in the examined strains (Kantar-

ciolu & Yucel, 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). Further-

more, 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

identified and it has been shown that C. albicans CaLIP8

mutants were significantly less virulent in a murine intrave-

nous infection model (Gacser et al., 2007b). For C. para-

psilosis, CpLIP1 and CpLIP2 have been reported, with the

latter known to encode for an active protein (Neugnot et al.,

2002). Recently, Gacser et al. (2007a) demonstrated that a

lipase inhibitor significantly reduced tissue damage during C.

parapsilosis infection of a reconstituted human tissue, and

that CpLIP1/CpLIP2 mutants formed thinner and less com-

plex biofilms. 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

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 regu-

lated 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 haemolysin-

like 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 (Jayati-

lake et al., 2006). Furthermore, filamentous 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 filamentous 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 ofresistance 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 identifi-

cation of an effective agent that acts on eukaryotic fungal

cell type without being toxic to host cells. Resistance to



antifungal drugs is an increasingly recognized phenomenon

and can be defined 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 anti-

fungal) or acquired, where resistance develops in a pre-

viously susceptible organism after a period of exposure to

the agent.

The classification 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 demethy-

lase. 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 fluconazole

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-fluorouracil. This nucleoside analogue gets

incorporated into RNA molecules and subsequently inter-

feres 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 com-

ponent of the fungal cell wall is not present within mamma-

lian cells. Interference with the enzyme b-1,3-D-glucan

synthetase can inhibit the synthesis of b-1,3-D-glucan. A

group of antifungals demonstrating this mode of action are

the echinocandins, and three drugs (caspofungin, micofun-

gin 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 biosynth-

esis, 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 iden-

tified 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 fluconazole, has been

encountered (Rex et al., 1995; Pfaller & Diekema, 2007).

Resistance to the azoles can result from quantitative or

qualitative modifications of target enzymes, reduced access

of the drug to the target or a combination of these mechan-

isms. Qualitative modifications 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 in-

volves the active efflux of the drug out of the cell via the

activation of two kinds of efflux transport proteins encoded

by either MDR or CDR genes (Lupetti et al., 2002; Ghan-

noum & 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 fluconazole (Pfaller & Diekema, 2007).

Susceptibility testing has shown that fluconazole 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 fluconazole-susceptible and -resistant Candida

species, with the exception of C. glabrata (Pfaller et al.,

2005). Voriconazole exerts fungicidal activity against most

Table 3. Common antifungal agents used in the management of

candidosis, mode of action and susceptibilities of Candida species

Antifungal

Susceptibility of NCAC species

C. albicans C. parapsilosis C. tropicalis C. glabrata

Polyenes Disruption of fungal cell membrane

Amphotericin B S S S S to I

Azoles Inhibition of ergosterol synthesis

Fluconazole S S S SDD to R

Itraconazole S S S SDD to R

Voriconazole S S S S

Posoconazole S S S S

Ravuconazole S S S S

5-Flucytosine Inhibition of DNA and protein synthesis

S S S S

Echinocandins Inhibition of b1,3-D-glucan synthesis

Caspofungin 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.


298 S. Silva et al.

yeasts and certain opportunistic fungi, specifically against

some NCAC species (Groll et al., 2001). This agent is

generally active against Candida species including flucona-

zole-resistant C. albicans and C. glabrata (Pfaller & Dieke-

ma, 2007). With the exception of C. tropicalis, voriconazole

is more active than fluconazole 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 pyropho-

sphorylase, cytosine permease and cytosine deaminase, or

increased production of pyrimidines (Atkinson & Israel,

1973). The antifungal spectrum of flucytosine is extremely

narrow: Candida species, Cryptococcus species and Aspergil-

lus species (Polak et al., 1982; Vermes et al., 2000). Further-

more, due to the multiple steps in its mode of action,

including transport into the cell and deamination of the

active compound, flucytosine is normally used only in

combination with other agents, including amphotericin B

and fluconazole (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 significant resistance will develop

to these agents. Microorganisms that demonstrate inherent

resistance to echinocandins either generate insufficient

target enzyme b-1,3-D-glucan synthase or produce an alter-

nate form of the enzyme with reduced echinocandin bind-

ing. 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;

Kucharınova 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 biofilms is their role in increasing tolerance to

conventional antifungal therapy. The reduced susceptibility

of C. albicans biofilms to antifungal agents was first reported

in 1995 (Hawser et al., 1995). Furthermore, several groups

have demonstrated that biofilm cells drastically increase

their tolerance to the most commonly used antifungal

agents (fluconazole and amphotericin B) (Ramage et al.,

2001). Biofilms 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 biofilm

drug resistance are not fully understood, the current con-

sensus is that biofilm tolerance is a complex multifactorial

phenomenon involving different molecular mechanisms,

restricted penetration of the drug through the matrix and

the presence of so-called ‘persister’ cells within the biofilm,

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 identification and control of host

factors that may predispose one to infection. Furthermore,

Candida species can exhibit several virulence factors such as

adherence, biofilm 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,

specifically 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 con-

tribute towards the identification 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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3 Especies de Candida Generalidades 2011