ISRN Preventive MedicineVolume 2013 (2013), Article ID 763628, 13 pageshttp://dx.doi.org/10.5402/2013/763628

Review Article

Candida Infections and Their Prevention

M. Anaul Kabir¹ and Zulfiqar Ahmad²

¹Molecular Genetics Laboratory, School of Biotechnology, National Institute of Technology Calicut, Calicut 673601, India²Department of Biological and Environmental Sciences, Alabama A&M University, Normal, AL 35762, USA

Received 13 September 2012; Accepted 4 October 2012

Academic Editors: R. G. Masterton, A. S. Mustafa, and M.-Y. Zhang

Copyright © 2013 M. Anaul Kabir and Zulfiqar Ahmad. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Infections caused by Candida species have been increased dramatically worldwide due to the increase in immunocompromised patients. For the prevention and cure of candidiasis, several strategies have been adopted at clinical level. Candida infected patients are commonly treated with a variety of antifungal drugs such as fluconazole, amphotericin B, nystatin, and flucytosine. Moreover, early detection and speciation of the fungal agents will play a crucial role for administering appropriate drugs for antifungal therapy. Many modern technologies like MALDI-TOF-MS, real-time PCR, and DNA microarray are being applied for accurate and fast detection of the strains. However, during prolonged use of these drugs, many fungal pathogens become resistant and antifungal therapy suffers. In this regard, combination of two or more antifungal drugs is thought to be an alternative to counter the rising drug resistance. Also, many inhibitors of efflux pumps have been designed and tested in different models to effectively treat candidiasis. However, most of the synthetic drugs have side effects and biomedicines like antibodies and polysaccharide-peptide conjugates could be better alternatives and safe options to prevent and cure the diseases. Furthermore, availability of genome sequences of Candida  albicans and other non-albicans strains has made it feasible to analyze the genes for their roles in adherence, penetration, and establishment of diseases. Understanding the biology of Candida species by applying different modern and advanced technology will definitely help us in preventing and curing the diseases caused by fungal pathogens.

1. Introduction

Candida species are associated with human beings for quite long time as harmless commensals. They are commonly found on the mucosal surfaces of gastrointestinal and genitourinary tracts and skin of humans. However, they become opportunistic pathogens in immunologically weak and immunocompromised patients. As opportunistic pathogens, they can cause local mucosal infections and sometimes, systemic infections in which Candida species can spread to all major organs and colonize in these organs [1, 2]. The systemic infections can be life threatening among the individuals having severely paralyzed immune system such as AIDS patients, people undergoing chemotherapy and radiotherapy treatment for cancers, and patients undergoing organ transplants. As the number of immunocompromised patients is increasing worldwide due to change in life style and improvement in medical facilities, infections caused by Candida species and mainly by Candida albicans have been increased dramatically in the last two decades. This has posed a serious and daunting challenge to the effective management of candidiasis and cost has been increased manifold. It is estimated that in the United States itself the excess cost due to candidemia is between $1 and $2 billion per year [3, 4]. Here we briefly review different aspects of Candida infections, antifungals for treatment of candidiasis, drug resistance, and certain preventive measures.

2. Candida Infections

Candida species can cause superficial and local mucosal infections and the best known of these is commonly called thrush. Such infections generally affect gastrointestinal, vaginal, esophageal, and oralpharygeal mucosae. Besides, most of the women suffer from vulvovaginal Candidiasis (VVC) at least once in their life time [5]. Some women experience repeated recurrences of this infection and it is known as recurrent vulvovaginal candidiasis (RVVC). The oral-pharyngeal candidiasis (OPC) is common among the HIV-infected patients and it is considered as an important marker for the onset of AIDS as well. OPC also affects oral cancer patients and debilitated patients who produce less amount of saliva [6]. However, it can cause a severe, life-threatening bloodstream infection that leads to colonization of Candida in internal organs (disseminated candidemia) which poses serious health problem in these individuals. Mortality rate for these patients is observed between 30% and 50% [7, 8]. Candida infections in the United States are the fourth most common hospital acquired infections and the second most common cause due to such infections [7]. Among the Candida species, C. albicans causes most of the candidemia, followed by non-albicans strains such as Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei [9]. C. glabrata is responsible for about 16% of all bloodstream infections whereas C. krusei accounts for 2% of all the clinical Candida isolates [10, 11].

3. Antifungal Drugs and Mechanism of Action

For the effective treatment of superficial mucosal infections and systemic life-threatening fungal diseases, a considerably large number of antifungal drugs have been developed and used for clinical purposes (Table 1). Though fungal infections were known for centuries, antifungal drugs were not available till 1930s. The first antifungal drug griseofulvin was isolated as a metabolic product from the mold Penicillium griseofulvum in 1939. However, it took several years to prove its efficacy in curing fungal infections and it was not used for clinical purposes till 1958 [12]. Subsequently, antifungal drug in the category of polyene, amphotericin B, was introduced for clinical purpose in 1960 which was much more effective and even today it is considered as one of the best antifungals [13]. However, to counter the growing challenges of fungal infections and increasing demands of appropriate drugs, many potential antifungal drugs have been developed since 1960s onward and are being used to treat fungal infections. Here we will give brief descriptions of some of these drugs.

Table 1: Commonly used antifungal drugs and their targets/mode of action.

Antifungals
Targets/mode of action
References

Azoles
Ergosterol biosynthesis (inhibition of ERG11 gene product, lanosterol 14α-demethylase)
[14–20]

    Fluconazole (FLC)

    Itraconazole (ITC)

    Voriconazole (VCZ)

    Posaconazole (POS) 

    Ravuconazole

    Isavuconazole

    Pramiconazole

    Albaconazole

    Miconazole

    Ketoconazole

Polyenes
Cell membrane ergosterol (increased permeability and oxidative damage)
[21, 22]

    Amphotericin B

    Nystatin

Echinocandins
Cell wall biosynthesis, inhibition of GSC1 gene product, β(1,3)-glucan synthase
[23–26]

    Caspofungin

    Micafungin

    Anidulafungin

Allylamines
Ergosterol biosynthesis (inhibition of ERG1 gene product squalene epoxidase)
[27]

    Terbinafine

    Naftifine

Fluorinated pyrimidine
DNA and RNA synthesis (misincorporation of 5-fluorouracil)
[28, 29]

analogs

    5-fluorocytosine

3.1. Azole Antifungal Drugs

Azole drugs are one of the most common classes of drugs used for treatment of Candida infections worldwide for both mucosal and systemic infections. The azole derivatives were introduced as antifungals in 1960s and it is most rapidly expanding (Table 1) [30–32]. Azole drugs are categorized as imidazole or triazoles depending upon the presence of two or three nitrogens in the five-membered azole ring. Most of the azole derivatives are fungistatic having broad spectrum against yeast and filamentous fungi. These antifungals target ergosterol biosynthetic pathway and thereby inhibit the growth of fungi [14–17]. Ergosterol is the major component of fungal cell wall and acts as a bioregulator for maintaining fluidity and asymmetry of cell membrane and overall integrity of the cell wall [33–35]. Azole drugs such as fluconazole, itraconazole, voriconazole, and posaconazole inhibit the lanosterol 14α-demethylase encoded by the gene ERG11 and decrease the level of ergosterol required for cell membrane [18–20]. On the other hand, the precursors of ergosterol, such as lanosterol, 4,14-dimethly zymosterol, and 24-methylene dihydrolanosterol, are accumulated inside the cell and integrated into plasma membrane resulting in the altered structure and function of the membrane. Subsequently, it increases water penetration and drug uptake into the cell [36, 37]. The azole-induced altered plasma membrane structure also leads to several other responses in the cell including inactivation of vacuolar ATPases (V-ATPase), inhibition of hyphal development, and change in the oxidative and nitrosative stresses [38–40].

3.2. Polyenes

The polyene antibiotics, produced by Streptomyces species, have broader spectrum than many other antifungal drugs and they are fungicidal in nature instead of fungistatic like azole drugs [41–43]. The most commonly used polyenes are amphotericin B, nystatin, and natamycin. These drugs act by binding specifically to ergosterol present in the plasma membrane and thereby affecting the integrity of cell membrane that results in cell death. Matsumori et al. have shown that amphotericin B has direct intermolecular interaction with ergosterol whereas it scarcely interacts with mammalian counterpart, cholesterol [44]. This intermolecular interaction has also been supported by other evidences such as higher affinity of amphotericin B to ergosterol-containing membranes than to sterol-free and cholesterol membranes [45, 46]. The complex formation between the polyenes and ergosterol causes disruption in the membrane by forming membrane-spanning ion channels that result in the increased permeability of the membrane, leakage of essential components, and death of the pathogens [21, 47]. Several studies have also suggested that polyenes can cause oxidative damages to the cell that contributes to their fungicidal activity [22, 41, 48]. Undoubtedly, amphotericin B has broad specificity against many fungal pathogens; however, it has considerably high toxic effect on human cells leading to renal failure in the patients undergoing this treatment. For reducing this toxicity but retaining the full activity of amphotericin B, new formulations, such as liposome, lipid complexes, and colloidal dispersions, have been made and obtained promising outcomes [49–55].

3.3. Echinocandins

These compounds are a class of lipoproteins, discovered in the 1970s, having fungicidal activity against Candida both in vivo and in vitro [56–58]. The commonly used echinocandins, for clinical purposes are caspofungin, micafungin, and anidulafungin [23–26]. These drugs are specific noncompetitive inhibitors of the enzyme β-(1,3)-glucan synthase, a membrane heterodimeric protein, responsible for the synthesis of β-glucan [59]. A recent study has shown that anidulafungin, a semisynthetic echinocandin has better efficacy compared to commonly used fluconazole for systemic candidiasis caused by C. albicans [60]. This echinocandin has more effective global response compared to fluconazole and cleans the bloodstream infections quite faster. Moreover, after treatment with this drug, a fewer persistent infections have been observed [60]. This interesting outcome might be attributed to the fungicidal activity of echinocandin (anidulafungin) which could have better response in the patients compared to fungistatic fluconzaole. However, this observation cannot be extrapolated to other systemic infections and the patient’s immunological status might contribute to efficacy of drugs used.

3.4. Allylamines

The most commonly used allylamines for clinical purposes include naftifine and terbinafine [27]. Allylamines are noncompetitive inhibitors of squalene epoxidase and are effective against many fungal agents including azole-resistant Candida strains [61]. The enzyme squalene epoxidase is encoded by the gene ERG1 located early in the ergosterol biosynthetic pathway [62]. Cells treated by allylamines accumulate squalene while becoming deficient in ergosterol (essential component of cell membranes) as subsequent steps in the ergosterol biosynthetic pathway are blocked. Furthermore, studies with isolated squalene epoxidase indicated that this enzyme is indeed the target of allylamines [61]. The fungal cell death by allylamines may not be due to the depletion of ergosterol in the cell as such, rather it could be because of accumulation of squalene that results in the formation of altered plasma membrane and disruption of membrane organization. This leads to increased permeability of membrane resulting in the cell death [27, 63]. It has also been demonstrated that naftifine has anti-inflammatory properties such as reduction in polymorphonuclear leukocyte chemotaxis and reduction in superoxide production. Though naftinfine has shown good efficacy for fungal treatments and relief of inflammatory signs and symptoms, it has some adverse effects like burning or stinging at the site of application [64].

3.5. Fluorinated Pyrimidine Analog

The 5-fluorocytosine (flucytosine or 5-FC) is a derivative of cytosine (essential component of nucleic acids) and was first synthesized in 1957 as anti-tumor drug [65]. However, its efficacy was not proven in cancer treatment. Later, it was tested for its antifungal activity, and subsequently, it was used for treatment of fungal infections in 1968 especially for candidiasis and cryptococcosis [66, 67]. Flucytosine does not have any antifungal activity as such, rather its metabolite 5-fluorouracil (5-FU) is considered to be toxic for the fungal cell. It may be asked why 5-FU is not administered to patients suffering from candidiasis when it has toxic activity rather than giving 5-FC. The reason is that 5-FU is toxic to mammalian cell, whereas 5-FC is quite safe. Here mode of action of 5-FC is discussed briefly. 5-FC is taken up by Candida species through cytosine permease and once inside the cell, it is rapidly converted into 5-FU [28, 29]. This 5-FU can exert its toxic effect by adopting two different pathways inside the cell. In one pathway, 5-FU is converted into 5-fluorodeoxyuridine monophosphate (FdUMP) which is proven to be potential inhibitor of thymidylate synthase, an essential enzyme for biosynthesis of thymidine [68, 69]. As a result, DNA synthesis gets blocked in fungal cells and it is unable to go for cell division. Another mechanism is through the conversion of 5-FU into 5-fluorouridine monophosphate and subsequently into 5-fluorouridine triphosphate which is incorporated into RNA in place of normal uridine triphosphate. In turn, this inhibits the protein synthesis in fungal cell (Figure 1) [68, 70, 71]. Therefore, both processes prove to be lethal for fungal pathogens and, subsequently, they are eliminated from the site of infection.

Figure 1: Schematic diagram of the effect of 5-Fluorocytosine (5-FC) on the fungal cell. Genes for three enzymes are given in italic capital letters in the bracket. Mutations in these genes make the cells resistant to 5-FC.

4. Drug Resistance

Though infections caused by Candida species are treated with different antifungal drugs available as mentioned above, the drug resistance is posing a serious problem to the health of individual patients and management of health care system becomes difficult. Studies have shown that several factors including pumping out of drugs from fungal cells, modification of the targets by incorporating point mutations in the genes, modification of key enzymes for biosynthetic pathways, and modulation of transcription factors play important roles for this phenomenon (Figure 2) [72, 73]. These mechanisms are discussed below briefly.

Figure 2: Probable mechanisms of drug resistance in Candida species. (1) Drugs are pumped out by efflux pump. (2) Drug targets such as enzymes are overproduced and drugs cannot inhibit the enzymatic reactions. (3) Due to mutations, the structures of enzymes or other proteins are altered and drugs cannot bind to it. (4) Crucial enzymatic function that is inhibited by drug can be bypassed. (5) Drugs may be degraded and are used as carbon source. (6) Drugs may be modified by enzymes and become nontoxic. (7) Drugs are degraded and become nonfunctional. (8) Extracelluar enzyme may degrade the drugs outside the fungal cell and make them inactive. (9) Altered membrane may inhibit the entry of drugs into cell and drugs cannot function.

4.1. Efflux Pumps

Efflux pumps remain the major reason for drug resistance in almost all the Candida species as they have broad specificity and thought to be a prominent factor for drug resistance in clinical isolates. There are two major classes of efflux pumps, ABC (ATP binding cassette) transporter and MFS (major facilitator superfamily) pump. These transmembrane proteins transport different substrates across membranes using two different energy sources. The ABC transporters use ATP as energy source whereas MFS pump utilizes proton-motive force across the membrane. Among the ABC transporters, Cdr1p and Cdr2p have been well studied and they play an active and critical role in drug resistance in C. albicans [74–77]. Also, the role of Mdr1p (member of MFS pump) has been demonstrated in drug resistance in Candida species. Several studies have shown that azole-resistance of clinical isolates of Candida species is always associated with the overexpression of Cdr1p and Cdr2p as well as Mdr1p. In addition to azole drugs, Cdr1p and Cdr2p are also implicated in the drug resistance to topical antifungals such as terbinafine and amorrolfine [78]. Now the question is how do these pumps efflux drugs from fungal cells? Structure-function analysis of Cdr1p and Cdr2p shows that these ABC transporters have two distinct domains, the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs). It is suggested that two TMDs in the homodimer generate inward-facing drug binding cavity in which drugs can bind either from lipid bilayer or from cytoplasm. Subsequently, binding of two ATP molecules to two NBDs induces conformational changes in TMDs resulting in the opening of drug binding cavities extracellularly and closing intracellularly. This allows the bound drugs to be effluxed from the cell. Again, the hydrolysis of bound ATP resets this pump in the drug-binding mode. Thus, it completes one cycle and this is repeated to efflux drugs from fungal cell making it resistant to that particular drug. In the absence of crystal structure of ABC transporter, the above mechanism has been deduced from ABC transporter Sav1866 of Staphylococcus aureus whose crystal structure is available along with AMP-PNP [73, 79–81].

4.2. Mutations in the Target Sites

Mutations have been observed in a number of genes in clinical strains of Candida species which are resistant to antifungal drugs. For becoming resistant to a particular drug, a specific mutation in a specific gene has to occur. For example, mutations in the gene, ERG11, encoding sterol 14α-demethylase can reduce the binding of azole drugs to this enzyme resulting in the increased resistance of the Candida strains to these drugs [82, 83]. Cross-resistance to different azole drugs has also been observed in the strains having mutations in ERG11 [84]. Mutations that affect the uptake of 5-FC or its conversion into 5-FU and incorporation into growing nucleic acid chains have been implicated in the drug resistance as well [85, 86]. For example, most of the 5-FC-resistant Candida strains have mutations in FUR1 gene that encodes uracil phosphoribosyl transferase and the mutant version of this enzyme prevents the conversion of 5-FU to FdUMP. Studies have shown that mutation in FUR1 occurs at 301 bp position of the gene resulting in amino acid change from arginine to cysteine at 101 position in Fur1p [87]. Also the mutations, glycine to aspartate at position 28 and serine to leucine at position 29 in the enzyme cytosine deaminase, encoded by the gene FCA1, have been implicated in the resistance to 5-FC for C. albicans [88]. Furthermore, in a recent study, mechanism of resistance to 5-FC with respect to mutations in FCA1 has been analyzed extensively in C. glabrata [89]. Mutations are also found in the gene FCA1 (also known as FCY1) of clinical isolates of C. dubliniensis and C. lusitaniae which are resistant to 5-FC [85, 90]. Similarly, mutations in the gene FKS1 that encodes a subunit of β-1,3-glucan synthase complex can cause resistance to echinocandin drugs as well [91].

5. Diagnosis and Prevention of Candidiasis

Prevention of candidiasis and its management broadly depends on two important and critical factors. One is the early detection and identification of Candida strains. Second is the use of appropriate antifungal drugs. For example, C. albicans is quite sensitive to azole drugs whereas non-albicans strains such as C. glabrata and C. krusei are resistant to this antifungal. Here we will give brief account of these two factors.

5.1. Identification of Candida Species

The early detection of the strains certainly facilitates the use of antifungal drugs in cost-effective manner and will have positive impact on overall health of the patients [92]. The techniques for identification of Candida species must be rapid and strain-specific that not only separate Candida from other microbial pathogens rather it should also be able to distinguish them from other important fungal pathogens such as Aspergillus fumigatus, Cryptococcus neoformans, and other yeasts. For the identification of Candida species, different methods have been developed and used since 1950s; however, most of them were based on culturing the strains and searching for different phenotypes which is considered to be time-consuming [93–96]. As a result, treatment of candidiasis faced major problems and mortality and morbidity rates were quite high. However, different advanced technologies have now been developed and are being used for rapid and accurate identification of Candida strains that help in early diagnosis, treatment and management of candidemia and other infections caused by Candida species. Here, three of the advanced techniques for Candida identification have been discussed briefly.

5.1.1. Polymerase Chain-Based Candida Detection

A large number of different kinds of protocols have been developed over the last five decades to identify different fungal strains present in clinical specimens. The detection and speciation techniques include germ tube test, chromogenic test, enzymatic test, immunological test (identification of antigen or antibody), and fermentation tests [97, 98]. However, in many cases, these tests were not sensitive enough to give the accurate result, and subsequently, it delayed the antifungal therapy. Furthermore, blood culture test is considered to be “gold standard” for identification of Candida species, but it takes 24–48 hours to give the positive signal. Moreover, this method may not be sensitive enough to identify the strains from different tissue specimens due to low number of cells present in different internal organs especially in case of invasive candidiasis. However, this problem has been addressed effectively by applying polymerase chain reaction (PCR) for identifying Candida species. Recent years have witnessed the development of very sensitive PCR machines that are able to detect and separate large number of fungal pathogens including A. fumigatus, C. neoformans, and C. albicans. Different Candida DNA markers such as 5.8S rRNA genes, 18S rRNA gene, small subunit rRNA gene, noncoding internal transcribed spacer (ITS) of rRNA genes, and lanosterol demethylase gene have been used in PCR amplification for detection of Candida species [99–105]. This technique has been improved further to separate C. albicans from other Candida by PCR amplification of 5.8S rRNA gene followed by DNA enzyme immunoassay with C. albicans-specific oligonucleotide probe [106]. Later on, real-time PCRs were developed for rapid and accurate identification of different Candida species which is more sensitive and less time-consuming [107–110]. Real-time PCRs have been improved further to identify different Candida species within reasonably short time [111–113]. For example, Metawally et al. adopted real-time PCR in which rRNA gene complex has been used as target sequence for amplification that differentiates between fluconazole-sensitive and resistant Candida strains. This technique is able to identify the most commonly encountered Candida species in blood cultures such as C. albicans, Candida parapsilosis, Candida tropicalis, C. dubliniensis, C. glabrata, and C. krusei in less than 3 hours [111]. On the other hand, Innings et al. have been able to identify eight Candida species such as C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. famata, C. dubliniensis, and C. guilliermondii, commonly found in blood culture, by amplifying RNaseP RNA gene RPR1 sequence [112]. Recently it has been shown that there is no significant difference between SeptiFast (a commercially available molecular diagnosis of sepsis based on PCR) and blood culture method in the identification of pathogens in sepsis patients. However, the combination of both methods might be quite helpful for patients with suspected sepsis and especially those who are undergoing antibiotic treatment in an internal medicine ward in hospital [114]. In another study, two commercially available universal rRNA gene PCR plus sequencing test, SepsiTest and universal microbe detection (UMD), were evaluated for suspected infectious endocarditis (IE). These tests proved to be of immense value for rapid diagnosis of IE, particularly for cases of culture-negative infections [115].

5.1.2. MALDI-TOF-MS for Candida Detection

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was introduced by Karas and Hillenkamp in the late 1980s for mass determination of proteins [116]. This technique proved to be extremely powerful for the analysis and identification of other large biomolecules such as nucleic acids, carbohydrates, and lipids [117–119]. This has been extensively used to profile, characterize, and identify proteins and other molecules from intact and disrupted cells. Subsequently, the power of this spectrometry has been exploited for the rapid identification of clinically important bacteria and yeasts [120–122]. In the recent years, this technology has been applied to Candida biology as well as for rapid, accurate, and cost-saving identification of different Candida species and also for their speciation [123–127]. For example, MALDI-TOF intact cell mass spectrometry (MALDI-TOF-ICMS) has been extremely useful for separating Candida species that are not easy to differentiate in the conventional phenotypic growth or biochemical reactions. This technology has been applied to separate Candida species such as C. parapsilosis, C. orthopsilosis, and C. metapsilosis as well as closely related species like C. dubliniensis, C. albicans and C. glabrata in a time-saving manner [125]. In another study, Sendid et al. have compared the suitability of MALDI-TOF-MS for the identification of Candida species with that of conventional identification (CI) methods such as morphological, biochemical, or immunological procedures. Concordance between MALDI-TOF-MS and CI was found to be 98–100% for medically important pathogens and was able to separate closely related Candida species such as C. albicans, C. dubliniensis, and other Candida strains [127]. Taken together, these studies have clearly shown the potential of MALDI-TOF-MS for rapid, accurate and cost-saving identification of Candida species that will lead to appropriate antifungal therapy in a timely manner.

5.1.3. DNA Microarray for Candida Detection

DNA microarray has revolutionized the understanding of molecular functioning of different genes in all the organisms including humans. Though it was invented with respect to the analysis of gene expression, it is now applied to understand different aspects of molecular biology including rapid detection and identification of different viruses, bacteria, and fungi of medical importance for proper diagnosis and therapy [128–130]. In brief, probes (oligonucleotides, short fragment of DNA or cDNA) can be spotted on solid matrix (glass slides, plastic, or biochip) and the targets are amplified from mRNA or genomic DNA and labeled with different fluorophores. Fluorescently labeled target sequences are hybridized with probes and the signals generated from interaction between targets and probes are analyzed. The strength of the signal from a particular spot of array will depend on the amount of target sequence present in that spot binding to the probe.

In the oligonucleotide microarray method, specific probes targeted to internal transcribed spacer 2 (ITS2) can be used for hybridization with fungal DNA amplified by PCR from different species. This technique is sensitive enough to discriminate among different fungal pathogens at species level and can detect as minimum as 15 pg of DNA/mL in the sample [131]. This method has been further improved by enhancing the hybridization signals with gold nanoparticles and silver deposition and detection using flatbed scanner [132]. This advanced method has been very sensitive and can detect C. albicns in the sample as low as 10 cells/mL. For the rapid identification of microbes in bloodstream infections (BSI), DNA-microarray-based Prove-it Sepsis assay has been evaluated and found to be 98-99% sensitive compared to conventional blood-culture tests. It takes less than 3 hours from DNA extraction to BSI diagnosis [130]. Undoubtedly, DNA microarray with its different variants will be quite helpful for rapid and accurate detection and identification of different fungal pathogens including Candida species. This will certainly complement other available methods for proper diagnosis of fungal infections.

5.2. Prevention and Treatment of Candidiasis with Biomedicine

Though the number of antifungal drugs is rapidly increasing and they are used to treat Candida infections for both mucosal and invasive, the outcome is not satisfactory so far. Moreover, most of the antifungal drugs have substantial amount of toxic effect on human cells. Therefore, it has been imperative to find an alternative to the conventional drugs to treat the infected patients. Besides, it will be better to prevent the onset of the diseases instead of curing it. This can be done by adopting certain immunization strategies as it is done for many other bacterial infections [133–136]. Though the concept of protection through antibody has been controversial for quite long time, a large amount of data is coming out in favor of its use to prevent and also to cure the diseases. This alternative method is gaining its importance in the context of growing number of immunocompromised patients who are sensitive to toxic effect of conventional drugs. For treating the Candida infections, antibodies have been generated against cell wall polysaccharides, heat shock protein, secreted proteins, and peptides [137–141]. The synthetic glycopeptide vaccine against disseminated candidiasis has been found to be quite effective in mice [140]. Furthermore, synthetic glycopeptide conjugates were made by combining fungal cell wall beta-mannan trisaccharide and six 14 mer peptides from six different proteins such as enolase, phosphoglycerate kinase, fructose-bis-phosphate aldolase, hyphal wall protein-1, methyl tetrahydropteroyltriglutamate, and glyceraldehydes-3-phosphate dehydrogenase [140]. Furthermore, it has been demonstrated that vaccine and monoclonal antibody E2-9 (IgM) against peptide, Fba (derived from fructose bis phosphate aldolase), can protect mice from candidiasis [142]. Also, antibodies raised against beta glucan (elicited by peptide conjugate) are able to protect mice that are challenged with C. albicans possibly by inhibiting the fungal growth and its adherence to mammalian cell [143, 144]. Among the antibodies that are used for prevention as well as for curing of Candida infections, Mycograb (human recombinant antibody generated against Hsp90) has been of utmost importance in the last one decade. This antibody has been used in combination with other antifungal drugs and produced quite encouraging result [145, 146]. Matthews et al. have reported that Mycograb is active against a range of Candida species such as C. albicans, C. krusei, and C. tropicalis and it has synergistic effect on amphotericin B [139]. In another study, Mycograb was used in combination with lipid-associated formulation of amphotericin B for the treatment of invasive candidiasis and shows promising result [145]. However, recently, it has been shown that potentiation of amphotericin B appears to be nonspecific protein effect rather than the effect of antibody [146]. Efungumab (monoclonal antibody against Hsp90) has been tested in combination with other antifungal drugs for treatment of Candida infections and also for prevention [147–150]. Furthermore, as the complete genome sequences of quite good number of Candida species including C. albicans, and C. glabrata, C. dubliniensis are available, it is possible to develop genetically engineered Candida strains which are avirulent and can be used for immunization as vaccines. Also, Candida-specific genes or their protein products can be used as biomedicine to prevent candidiasis. Taken together, it seems plausible to take an alternative method for vaccination and prevention of Candida infections.

6. Conclusion

It is well accepted that Candida infections are on the rise and it needs to be handled with due care to decrease the rate of morbidity and mortality for immunocompromised patients. For the proper management of the Candida infections, multiple strategies must be adopted in a cost-effective and time-saving manner. First strategy will be to prevent the onset of disease by immunization/vaccination of the susceptible individuals by applying knowledge gained from genomics, proteomics, and transcriptomics of Candia and related species. Second strategy is to treat the Candida infections seriously and promptly. Any delay for antifungal therapy may lead to disseminated candidemia and systemic candidiasis in which different internal organs will be highly colonized by Candida strains. Again for proper antifungal therapy, it is imperative to identify the Candida species at early stage of infections. The conventional methods such as phenotypic, morphological, biochemical, and immunological should be replaced with highly advanced technologies like MALDI-TOF-MS and real-time PCR and DNA microarray in clinical setup. Identification and speciation facilities should be developed in such a way so that whole process will be rapid, accurate, cost-effective, and time-saving.

Once the strains are identified, appropriate antifungal drugs can be administered to the patients and level of fungal strains can be monitored in clinical specimens. However, almost all the Candida strains isolated from infected individuals are becoming resistant to the commonly used antifungal drugs. In this regard, combination of two or more drugs has been suggested and tested for C. albicans and other Candida species and found to be synergistic for amphotericin B/ketoconazole, 5-FC/ketoconazole, and other combinations as well [151, 152]. The drug combination therapy was also tested in mice model and patients [153, 154]. In a recent study, Tavanti et al. have shown that clinical isolates of C. glabrata (low susceptibility to azole drugs) are susceptible to human cationic peptide hepcidin (Hep-20) (100–200 μg/mL). However, increased antifungal activity was observed when combined with amphotericin B and a synergistic effect was found for Hep20/caspofungin and He-20/fluconazole combinations [155].

Another measure to counter the rising drug resistance of the strains is to use inhibitors for efflux pumps in combination with commonly used drugs. The inhibitors for ABC pumps such as milbemycins, enniatin, FK506, FK520, and unnarmicins can be used along with azole drugs to reverse the drug resistance [73, 156–159]. Recently, Hayama et al. have assessed the therapeutic potential of D-octapeptide derivative RC21v3 (an inhibitor of Cdr1p) in a murine oral candidiasis infection model and have shown its potential in combination with fluconazole [160]. This suggests that this inhibitor has a potential in treating oral candidiasis. In another study, Holmes et al. have reported the identification of the monoamine oxidase A inhibitor, clorgyline, as inhibitor of ABC and MFS pumps in clinical isolates of C. albicans and C. glabrata [161].

However, for the prevention of onset of the disease and to treat the Candida infections effectively, the understanding of the complete life cycle of C. albicans and other Candida species is required. In this regard, the functions of all the ORFs and specially the Candida-specific genes/ORFs should be assigned. This will help in developing potential antifungal drugs in terms of antibody, proteins, DNA, or the whole-organism itself for the prevention of this disease.

Acknowledgment

The authors express their gratitude to Department of Science and Technology (Government of India) for the research Grant SR/SO/BB-038/2009 which was sanctioned to M. A. Kabir.

References

F. C. Odds, Candida and Candidosis, Bailliere Tindall, London, UK, 2nd edition, 1988.

M. H. Miceli, J. A. Díaz, and S. A. Lee, “Emerging opportunistic yeast infections,” The Lancet Infectious Diseases, vol. 11, no. 2, pp. 142–151, 2011.

M. A. Pfaller, P. G. Pappas, and J. R. Wingard, “Invasive fungal pathogens: current epidemiological trends,” Clinical Infectious Diseases, vol. 43, no. 1, pp. S3–S14, 2006.

L. S. Wilson, C. M. Reyes, M. Stolpman, J. Speckman, K. Allen, and J. Beney, “The direct cost and incidence of systemic fungal infections,” Value in Health, vol. 5, no. 1, pp. 26–34, 2002.

J. D. Sobel, “Vaginitis,” The New England Journal of Medicine, vol. 337, no. 26, pp. 1896–1903, 1997.

M. Ruhnke, “Skin and mucous infections,” in Candida and Candidiasis, R. Calderone, Ed., pp. 307–325, ASM Press, Washington, DC, USA, 2002.

M. A. Pfaller, R. N. Jones, S. A. Messer, M. B. Edmond, and R. P. Wenzel, “National surveillance of nosocomial blood stream infection due to Candida albicans: frequency of occurrence and antifungal susceptibility in the SCOPE program,” Diagnostic Microbiology and Infectious Disease, vol. 31, no. 1, pp. 327–332, 1998.

C. C. Kibbler, S. Seaton, R. A. Barnes et al., “Management and outcome of bloodstream infections due to Candida species in England and Wales,” Journal of Hospital Infection, vol. 54, no. 1, pp. 18–24, 2003.

M. A. Pfaller and D. J. Diekema, “Epidemiology of invasive candidiasis: a persistent public health problem,” Clinical Microbiology Reviews, vol. 20, no. 1, pp. 133–163, 2007.

M. A. Pfaller, D. J. Diekema, L. Steele-Moore et al., “Twelve years of fluconazole in clinical practice: global-trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida,” Clinical Microbiology and Infection, vol. 10, supplement 1, pp. 11–23, 2004.

M. A. Pfaller, D. J. Diekema, D. L. Gibbs et al., “Candida krusei, a multidrug-resistant opportunistic fungal pathogen: geographic and temporal trends from the ARTEMIS DISK Antifungal Surveillance Program, 2001 to 2005,” Journal of Clinical Microbiology, vol. 46, no. 2, pp. 515–521, 2008.

S. Budavari, The Merck Index, Merck & Co., Rahway, NJ, USA, 1989.

A. K. Gupta, D. N. Sauder, and N. H. Shear, “Antifungal agents: an overview. Part I,” Journal of the American Academy of Dermatology, vol. 30, no. 5, pp. 677–698, 1994.

M. Borgers, “Mechanism of action of antifungal drugs, with special reference to the imidazole derivatives,” Reviews of Infectious Diseases, vol. 2, no. 4, pp. 520–534, 1980.

H. Van Den Bossche, J. M. Ruysschaert, and F. Defrise-Quertain, “The interaction of miconazole and ketoconazole with lipids,” Biochemical Pharmacology, vol. 31, no. 16, pp. 2609–2617, 1982.

D. J. Sheehan, C. A. Hitchcock, and C. M. Sibley, “Current and emerging azole antifungal agents,” Clinical Microbiology Reviews, vol. 12, no. 1, pp. 40–79, 1999.

D. C. Lamb, D. E. Kelly, M. R. Waterman, M. Stromstedt, D. Rozman, and S. L. Kelly, “Characteristics of the heterologously expressed human lanosterol 14α-demethylase (other names: P45014DM, CYP51, P45051) and inhibition of the purified human and Candida albicans CYP51 with azole antifungal agents,” Yeast, vol. 15, no. 9, pp. 755–763, 1999.

C. A. Hitchcock, K. Dickinson, S. B. Brown, E. G. V. Evans, and D. J. Adams, “Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14α-sterol demethylase purified from Candida albicans,” Biochemical Journal, vol. 266, no. 2, pp. 475–480, 1990.

R. Courtney, S. Pai, M. Laughlin, J. Lim, and V. Batra, “Pharmacokinetics, safety, and tolerability of oral posaconazole administered in single and multiple doses in healthy adults,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 9, pp. 2788–2795, 2003.

A. J. Carrillo-Muñoz, G. Giusiano, P. A. Ezkurra, and G. Quindós, “Antifungal agents: mode of action in yeast cells,” Revista Espanola de Quimioterapia, vol. 19, no. 2, pp. 130–139, 2006.

J. Bolard, “How do the polyene macrolide antibiotics affect the cellular membrane properties?” Biochimica et Biophysica Acta, vol. 864, no. 3-4, pp. 257–304, 1986.

M. Sokol-Anderson, J. E. Sligh, S. Elberg, J. Brajtburg, G. S. Kobayashi, and G. Medoff, “Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B,” Antimicrobial Agents and Chemotherapy, vol. 32, no. 5, pp. 702–705, 1988.

D. S. Perlin, “Current perspectives on echinocandin class drugs,” Future Microbiology, vol. 6, no. 4, pp. 441–457, 2011.

N. A. Kartsonis, J. Nielsen, and C. M. Douglas, “Caspofungin: the first in a new class of antifungal agents,” Drug Resistance Updates, vol. 6, no. 4, pp. 197–218, 2003.

P. H. Chandrasekar and J. D. Sobel, “Micafungin: a new echinocandin,” Clinical Infectious Diseases, vol. 42, no. 8, pp. 1171–1178, 2006.

M. A. Pfaller, D. J. Diekema, L. Boyken et al., “Effectiveness of anidulafungin in eradicating Candida species in invasive candidiasis,” Antimicrobial Agents and Chemotherapy, vol. 49, no. 11, pp. 4795–4797, 2005.

N. S. Ryder, “Mechanism of action and biochemical selectivity of allylamine antimycotic agent,” Annals of the New York Academy of Sciences, vol. 544, pp. 208–220, 1988.

A. Polak and M. Grenson, “Evidence for a common transport system for cytosine, adenine and hypoxanthine in Saccharomyces cerevisiae and Candida albicans,” European Journal of Biochemistry, vol. 32, no. 2, pp. 276–282, 1973.

A. Polak and H. J. Scholer, “Mode of action of 5 fluorocytosine and mechanisms of resistance,” Chemotherapy, vol. 21, no. 3-4, pp. 113–130, 1975.

R. A. Fromtling, “Overview of medically important antifungal azole derivatives,” Clinical Microbiology Reviews, vol. 1, no. 2, pp. 187–217, 1988.

H. Vanden Bossche, “Biochemical targets for antifungal azole derivatives: hypothesis on the mode of action,” Current Topics in Medical Mycology, vol. 1, pp. 313–351, 1985.

J. Xu, Y. Cao, J. Zhang et al., “Design, synthesis and antifungal activities of novel 1,2,4-triazole derivatives,” European Journal of Medicinal Chemistry, vol. 46, no. 7, pp. 3142–3148, 2011.

M. A. Ghannoum and L. B. Rice, “Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance,” Clinical Microbiology Reviews, vol. 12, no. 4, pp. 501–517, 1999.

F. C. Odds, A. J. P. Brown, and N. A. R. Gow, “Antifungal agents: mechanisms of action,” Trends in Microbiology, vol. 11, no. 6, pp. 272–279, 2003.

R. A. Akins, “An update on antifungal targets and mechanisms of resistance in Candida albicans,” Medical Mycology, vol. 43, no. 4, pp. 285–318, 2005.

M. C. Cruz, A. L. Goldstein, J. R. Blankenship et al., “Calcineurin is essential for survival during membrane stress in Candida albicans,” The EMBO Journal, vol. 21, no. 4, pp. 546–559, 2002.

F. Abe, K. Usui, and T. Hiraki, “Fluconazole modulates membrane rigidity, heterogeneity, and water penetration into the plasma membrane in Saccharomyces cerevisiae,” Biochemistry, vol. 48, no. 36, pp. 8494–8504, 2009.

Y. Q. Zhang, S. Gamarra, G. Garcia-Effron, S. Park, D. S. Perlin, and R. Rao, “Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs,” PLoS Pathogens, vol. 6, no. 6, Article ID e1000939, 2010.

F. C. Odds, A. Cockayne, J. Hayward, and A. B. Abbott, “Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae,” Journal of General Microbiology, vol. 131, no. 10, pp. 2581–2589, 1985.

D. M. Arana, C. Nombela, and J. Pla, “Fluconazole at subinhibitory concentrations induces the oxidative- and nitrosative-responsive genes TRR1, GRE2 and YHB1, and enhances the resistance of Candida albicans to phagocytes,” The Journal of Antimicrobial Chemotherapy, vol. 65, no. 1, pp. 54–62, 2010.

J. Brajtburg, W. G. Powderly, G. S. Kobayashi, and G. Medoff, “Amphotericin B: current understanding of mechanisms of action,” Antimicrobial Agents and Chemotherapy, vol. 34, no. 2, pp. 183–188, 1990.

H. A. Gallis, R. H. Drew, and W. W. Pickard, “Amphotericin B: 30 years of clinical experience,” Reviews of Infectious Diseases, vol. 12, no. 2, pp. 308–329, 1990.

M. D. Moen, K. A. Lyseng-Williamson, and L. J. Scott, “Liposomal amphotericin B: a review of its use as empirical therapy in febrile neutropenia and in the treatment of invasive fungal infections,” Drugs, vol. 69, no. 3, pp. 361–392, 2009.

N. Matsumori, K. Tahara, H. Yamamoto et al., “Direct interaction between amphotericin B and ergosterol in lipid bilayers as revealed by ²H NMR spectroscopy,” Journal of the American Chemical Society, vol. 131, no. 33, pp. 11855–11860, 2009.

N. M. Witzke and R. Bittman, “Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles,” Biochemistry, vol. 23, no. 8, pp. 1668–1674, 1984.

R. Mouri, K. Konoki, N. Matsumori, T. Oishi, and M. Murata, “Complex formation of amphotericin B in sterol-containing membranes as evidenced by surface plasmon resonance,” Biochemistry, vol. 47, no. 30, pp. 7807–7815, 2008.

R. S. Al-Dhaheri and L. J. Douglas, “Apoptosis in Candida biofilms exposed to amphotericin B,” Journal of Medical Microbiology, vol. 59, no. 2, pp. 149–157, 2010.

M. L. Sokol-Anderson, J. Brajtburg, and G. Medoff, “Amphotericin B-induced oxidative damage and killing of Candida albicans,” Journal of Infectious Diseases, vol. 154, no. 1, pp. 76–83, 1986.

J. P. Adler-Moore and R. T. Proffitt, “Development, characterization, efficacy and mode of action of AmBisome, a unilamellar liposomal formulation of amphotericin B,” Journal of Liposome Research, vol. 3, no. 3, pp. 429–450, 1993.

F. Meunier, “New methods for delivery of antifungal agents,” Reviews of Infectious Diseases, vol. 11, pp. S1605–1612, 1989.

L. H. Hanson and D. A. Stevens, “Comparison of antifungal activity of amphotericin B deoxycholate suspension with that of amphotericin B cholesteryl sulfate colloidal dispersion,” Antimicrobial Agents and Chemotherapy, vol. 36, no. 2, pp. 486–488, 1992.

P. C. Gokhale, R. J. Barapatre, S. H. Advani, N. A. Kshirsagar, and S. K. Pandya, “Successful treatment of disseminated candidiasis reistant to amphotericin B by liposomal amphotericin B: a case report,” Journal of Cancer Research and Clinical Oncology, vol. 119, no. 10, pp. 569–571, 1993.

G. Lopez-Berestein, R. Mehta, and R. Hopfer, “Effects of sterols on the therapeutic efficacy of liposomal amphotericin B in murine candidiasis,” Cancer Drug Delivery, vol. 1, no. 1, pp. 37–42, 1983.

M. N. Oda, P. L. Hargreaves, J. A. Beckstead, K. A. Redmond, R. Van Antwerpen, and R. O. Ryan, “Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B,” Journal of Lipid Research, vol. 47, no. 2, pp. 260–267, 2006.

N. Linder, G. Klinger, I. Shalit et al., “Treatment of Candidaemia in premature infants: comparison of three amphotericin B preparations,” Journal of Antimicrobial Chemotherapy, vol. 52, no. 4, pp. 663–667, 2003.

J. S. Tkacz, “Glucan biosynthesis in fungi and its inhibition,” in Emerging Targets in Antibacterial and Antifungal Chemotherapy, J. Sutchliffe and N. H. Georgopapadakou, Eds., pp. 495–523, Chapman & Hall, New York, NY, USA, 1992.

A. Cassone, R. E. Mason, and D. Kerridge, “Lysis of growing yeast-form cells of Candida albicans by echinocandin: a cytological study,” Sabouraudia Journal of Medical and Veterinary Mycology, vol. 19, no. 2, pp. 97–110, 1981.

T. J. Walsh, J. W. Lee, P. Kelly et al., “Antifungal effects of the nonlinear pharmacokinetics of cilofungin, a 1,3-β-glucan synthetase inhibitor, during continuous and intermittent intravenous infusions in treatment of experimental disseminated candidiasis,” Antimicrobial Agents and Chemotherapy, vol. 35, no. 7, pp. 1321–1328, 1991.

R. F. Hector, “Compounds active against cell walls of medically important fungi,” Clinical Microbiology Reviews, vol. 6, no. 1, pp. 1–21, 1993.

A. C. Reboli, A. F. Shorr, C. Rotstein et al., “Anidulafungin compared with fluconazole for treatment of candidemia and other forms of invasive candidiasis caused by Candida albicans: a multivariate analysis of factors associated with improved outcome,” BMC Infectious Diseases, vol. 11, article 261, 2011.

N. S. Ryder and B. Favre, “Antifungal activity and mechanism of action of terbinafine,” Reviews in Contemporary Pharmacotherapy, vol. 8, no. 5, pp. 275–287, 1997.

B. Favre and N. S. Ryder, “Cloning and expression of squalene epoxidase from the pathogenic yeast Candida albicans,” Gene, vol. 189, no. 1, pp. 119–126, 1997.

N. H. Georgopapadakou and A. Bertasso, “Effects of squalene epoxidase inhibitors on Candida albicans,” Antimicrobial Agents and Chemotherapy, vol. 36, no. 8, pp. 1779–1781, 1992.

A. K. Gupta, J. E. Ryder, and E. A. Cooper, “Naftifine: a review,” Journal of Cutaneous Medicine and Surgery, vol. 12, no. 2, pp. 51–58, 2008.

C. Heidelberger, N. K. Chaudhuri, P. Danneberg et al., “Fluorinated pyrimidines, a new class of tumour-inhibitory compounds,” Nature, vol. 179, no. 4561, pp. 663–666, 1957.

E. Titsworth and E. Grunberg, “Chemotherapeutic activity of 5-fluorocytosine and amphotericin B against Candida albicans in mice,” Antimicrobial Agents and Chemotherapy, vol. 4, no. 3, pp. 306–308, 1973.

D. Tassel and M. A. Madoff, “Treatment of Candida sepsis and Cryptococcus meningitis with 5-fluorocytosine. A new antifungal agent,” JAMA, vol. 206, no. 4, pp. 830–832, 1968.

A. R. Waldorf and A. Polak, “Mechanisms of action of 5-fluorocytosine,” Antimicrobial Agents and Chemotherapy, vol. 23, no. 1, pp. 79–85, 1983.

R. B. Diasio, J. E. Bennett, and C. E. Myers, “Mode of action of 5-fluorocytosine,” Biochemical Pharmacology, vol. 27, no. 5, pp. 703–707, 1978.

H. J. Scholer, “FlucytosineIn,” in Antifungal Chemotherapy, D. C. E. Speller, Ed., pp. 35–106, Wiley, Chichester, UK, 1980.

A. Vermes, H. J. Guchelaar, and J. Dankert, “Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions,” Journal of Antimicrobial Chemotherapy, vol. 46, no. 2, pp. 171–179, 2000.

J. Morschhäuser, “Regulation of multidrug resistance in pathogenic fungi,” Fungal Genetics and Biology, vol. 47, no. 2, pp. 94–106, 2010.

R. D. Cannon, E. Lamping, A. R. Holmes et al., “Efflux-mediated antifungal drug resistance,” Clinical Microbiology Reviews, vol. 22, no. 2, pp. 291–321, 2009.

R. Prasad, P. De Wergifosse, A. Goffeau, and E. Balzi, “Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals,” Current Genetics, vol. 27, no. 4, pp. 320–329, 1995.

D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, “Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters,” Antimicrobial Agents and Chemotherapy, vol. 39, no. 11, pp. 2378–2386, 1995.

D. Sanglard, F. Ischer, M. Monod, and J. Bille, “Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene,” Microbiology, vol. 143, no. 2, pp. 405–416, 1997.

R. Prasad and A. Goffeau, “Yeast ATP-binding cassette transporters conferring multidrug resistance,” Annual Review of Microbiology, vol. 66, pp. 39–63, 2012.

M. Niimi, K. Niimi, Y. Takano et al., “Regulated overexpression of CDR1 in Candida albicans confers multidrug resistance,” Journal of Antimicrobial Chemotherapy, vol. 54, no. 6, pp. 999–1006, 2004.

R. J. P. Dawson and K. P. Locher, “Structure of a bacterial multidrug ABC transporter,” Nature, vol. 443, no. 7108, pp. 180–185, 2006.

R. J. P. Dawson and K. P. Locher, “Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP,” FEBS Letters, vol. 581, no. 5, pp. 935–938, 2007.

H. W. Pinkett, A. T. Lee, P. Lum, K. P. Locher, and D. C. Rees, “An inward-facing conformation of a putative metal-chelate-type ABC transporter,” Science, vol. 315, no. 5810, pp. 373–377, 2007.

S. L. Kelly, D. C. Lamb, and D. E. Kelly, “Y132H substitution in Candida albicans sterol 14α-demethylase confers fluconazole resistance by preventing binding to haem,” FEMS Microbiology Letters, vol. 180, no. 2, pp. 171–175, 1999.

D. C. Lamb, D. E. Kelly, T. C. White, and S. L. Kelly, “The R467K amino acid substitution in Candida albicans sterol 14α- demethylase causes drug resistance through reduced affinity,” Antimicrobial Agents and Chemotherapy, vol. 44, no. 1, pp. 63–67, 2000.

D. Sanglard, F. Ischer, L. Koymans, and J. Bille, “Amino acid substitutions in the cytochrome P-450 lanosterol 14α- demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents,” Antimicrobial Agents and Chemotherapy, vol. 42, no. 2, pp. 241–253, 1998.

M. Florent, T. Noël, G. Ruprich-Robert et al., “Nonsense and missense mutations in FCY2 and FCY1 genes are responsible for flucytosine resistance and flucytosine-fluconazole cross-resistance in clinical isolates of Candida lusitaniae,” Antimicrobial Agents and Chemotherapy, vol. 53, no. 7, pp. 2982–2990, 2009.

N. Papon, T. Noël, M. Florent et al., “Molecular mechanism of flucytosine resistance in Candida lusitaniae: contribution of the FCY2, FCY1, and FUR1 genes to 5-fluorouracil and fluconazole cross-resistance,” Antimicrobial Agents and Chemotherapy, vol. 51, no. 1, pp. 369–371, 2007.

A. R. Dodgson, K. J. Dodgson, C. Pujol, M. A. Pfaller, and D. R. Soll, “Clade-specific flucytosine resistance is due to a single nucleotide change in the FUR1 gene of Candida albicans,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 6, pp. 2223–2227, 2004.

W. W. Hope, L. Tabernero, D. W. Denning, and M. J. Anderson, “Molecular mechanisms of primary resistance to flucytosine in Candida albicans,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 11, pp. 4377–4386, 2004.

T. D. Edlind and S. K. Katiyar, “Mutational analysis of flucytosine resistance in Candida glabrata,” Antimicrobial Agents and Chemotherapy, vol. 54, no. 11, pp. 4733–4738, 2010.

B. A. McManus, G. P. Moran, J. A. Higgins, D. J. Sullivan, and D. C. Coleman, “A Ser29Leu substitution in the cytosine deaminase Fca1p is responsible for clade-specific flucytosine resistance in Candida dubliniensis,” Antimicrobial Agents and Chemotherapy, vol. 53, no. 11, pp. 4678–4685, 2009.

D. S. Perlin, “Resistance to echinocandin-class antifungal drugs,” Drug Resistance Updates, vol. 10, no. 3, pp. 121–130, 2007.

K. W. Garey, M. Rege, M. P. Pai et al., “Time to initiation of fluconazole therapy impacts mortality in patients with candidemia: a multi-institutional study,” Clinical Infectious Diseases, vol. 43, no. 1, pp. 25–31, 2006.

M. T. Prospero and A. C. Reyes, “The efficacy of corn meal agar, soil extract agar and purified polysaccharide medium in the morphological identification of Candida albicans,” Acta Medica Philippina, vol. 12, no. 2, pp. 69–74, 1955.

C. L. Taschdjian, J. J. Burchall, and P. J. Kozinn, “Rapid identification of Candida albicans by filamentation on serum and serum substitutes,” A.M.A. Journal of Diseases of Children, vol. 99, pp. 212–215, 1960.

M. J. Denny and B. M. Partridge, “Tetrazolium medium as an aid in the routine diagnosis in Candida,” Journal of Clinical Pathology, vol. 21, no. 3, pp. 383–386, 1968.

K. R. Joshi, D. A. Bremner, D. N. Parr, and J. B. Gavin, “The morphological identification of pathogenic yeasts using carbohydrate media,” Journal of Clinical Pathology, vol. 28, no. 1, pp. 18–24, 1975.

A. M. Freydiere, R. Guinet, and P. Boiron, “Yeast identification in the clinical microbiology laboratory: phenotypical methods,” Medical Mycology, vol. 39, no. 1, pp. 9–33, 2001.

A. Laín, N. Elguezabal, S. Brena et al., “Diagnosis of invasive candidiasis by enzyme-linked immunosorbent assay using the N-terminal fragment of Candida albicans hyphal wall protein 1,” BMC Microbiology, vol. 7, article 35, 2007.

A. R. Holmes, R. D. Cannon, M. G. Shepherd, and H. F. Jenkinson, “Detection of Candida albicans and other yeasts in blood by PCR,” Journal of Clinical Microbiology, vol. 32, no. 1, pp. 228–231, 1994.

T. Sakai, K. Ikegami, E. Yoshinaga, R. Uesugi-Hayakawa, and A. Wakizaka, “Rapid, sensitive and simple detection of Candida deep mycosis by amplification of 18s ribosomal RNA gene; Comparison with assay of serum β-D-glucan level in clinical samples,” Tohoku Journal of Experimental Medicine, vol. 190, no. 2, pp. 119–128, 2000.

H. G. M. Niesters, W. H. F. Goessens, J. F. M. G. Meis, and W. G. V. Quint, “Rapid, polymerase chain reaction-based identification assays for Candida species,” Journal of Clinical Microbiology, vol. 31, no. 4, pp. 904–910, 1993.

J. P. Burnie, N. Golbang, and R. C. Matthews, “Semiquantitative polymerase chain reaction enzyme immunoassay for diagnosis of disseminated candidiasis,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 16, no. 5, pp. 346–350, 1997.

S. Nho, M. J. Anderson, C. B. Moore, and D. W. Denning, “Species differentiation by internally transcribed spacer PCR and HhaI digestion of fluconazole-resistant Candida krusei, Candida inconspicua, and Candida norvegensis strains,” Journal of Clinical Microbiology, vol. 35, no. 4, pp. 1036–1039, 1997.

G. Morace, M. Sanguinetti, B. Posteraro, G. L. Cascio, and G. Fadda, “Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis,” Journal of Clinical Microbiology, vol. 35, no. 3, pp. 667–672, 1997.

P. Burgener-Kairuz, J. P. Zuber, P. Jaunin, T. G. Buchman, J. Bille, and M. Rossier, “Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-α-demethylase (L1A1) gene fragment,” Journal of Clinical Microbiology, vol. 32, no. 8, pp. 1902–1907, 1994.

R. Wahyuningsih, H. J. Freisleben, H. G. Sonntag, and P. Schnitzler, “Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis,” Journal of Clinical Microbiology, vol. 38, no. 8, pp. 3016–3021, 2000.

P. L. White, A. Shetty, and R. A. Barnes, “Detection of seven Candida species using the Light-Cycler system,” Journal of Medical Microbiology, vol. 52, no. 3, pp. 229–238, 2003.

M. C. Hsu, K. W. Chen, H. J. Lo et al., “Species identification of medically important fungi by use of real-time LightCycler PCR,” Journal of Medical Microbiology, vol. 52, no. 12, pp. 1071–1076, 2003.

T. M. Pryce, I. D. Kay, S. Palladino, and C. H. Heath, “Real-time automated polymerase chain reaction (PCR) to detect Candida albicans and Aspergillus fumigatus DNA in whole blood from high-risk patients,” Diagnostic Microbiology and Infectious Disease, vol. 47, no. 3, pp. 487–496, 2003.

A. Imhof, C. Schaer, G. Schoedon et al., “Rapid detection of pathogenic fungi from clinical specimens using LightCycler real-time fluorescence PCR,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 22, no. 9, pp. 558–560, 2003.

L. Metwally, G. Hogg, P. V. Coyle et al., “Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR,” Journal of Medical Microbiology, vol. 56, no. 7, pp. 964–970, 2007.

A. Innings, M. Ullberg, A. Johansson et al., “Multiplex real-time PCR targeting the RNase P RNA gene for detection and identification of Candida species in blood,” Journal of Clinical Microbiology, vol. 45, no. 3, pp. 874–880, 2007.

M. Kasai, A. Francesconi, R. Petraitiene et al., “Use of quantitative real-time PCR to study the kinetics of extracellular DNA released from Candida albicans, with implications for diagnosis of invasive candidiasis,” Journal of Clinical Microbiology, vol. 44, no. 1, pp. 143–150, 2006.

L. Pasqualini, A. Mencacci, C. Leli et al., “Diagnostic performance of a multiple real-time PCR assay in patients with suspected sepsis hospitalized in an internal medicine ward,” Journal of Clinical Microbiology, vol. 50, no. 4, pp. 1285–1288, 2012.

C. Kühn, C. Disqué, H. Mühl, P. Orszag, M. Stiesch, and A. Haverich, “Evaluation of commercial universal rRNA gene PCR plus sequencing tests for identification of bacteria and fungi associated with infectious endocarditis,” Journal of Clinical Microbiology, vol. 49, no. 8, pp. 2919–2923, 2011.

M. Karas and F. Hillenkamp, “Laser desorption ionization of proteins with molecular masses exceeding 10 000 daltons,” Analytical Chemistry, vol. 60, no. 20, pp. 2299–2301, 1988.

U. Pieles, W. Zurcher, M. Schar, and H. E. Moser, “Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides,” Nucleic Acids Research, vol. 21, no. 14, pp. 3191–3196, 1993.

W. Mo, T. Takao, H. Sakamoto, and Y. Shimonishi, “Structural analysis of oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino)ethyl ester by matrix-assisted laser desorption/ionization mass spectrometry,” Analytical Chemistry, vol. 70, no. 21, pp. 4520–4526, 1998.

B. Fuchs and J. Schiller, “MALDI-TOF MS analysis of lipids from cells, tissues and body fluids,” Sub-Cellular Biochemistry, vol. 49, pp. 541–565, 2008.

M. A. Claydon, S. N. Davey, V. Edwards-Jones, and D. B. Gordon, “The rapid identification of intact microorganisms using mass spectrometry,” Nature Biotechnology, vol. 14, no. 11, pp. 1584–1586, 1996.

A. M. Haag, S. N. Taylor, K. H. Johnston, and R. B. Cole, “Rapid identification and speciation of Haemophilus bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,” Journal of Mass Spectrometry, vol. 33, no. 8, pp. 750–756, 1998.

J. Qian, J. E. Cutler, R. B. Cole, and Y. Cai, “MALDI-TOF mass signatures for differentiation of yeast species, strain grouping and monitoring of morphogenesis markers,” Analytical and Bioanalytical Chemistry, vol. 392, no. 3, pp. 439–449, 2008.

C. Marinach-Patrice, A. Fekkar, R. Atanasova et al., “Rapid species diagnosis for invasive candidiasis using mass spectrometry,” PLoS ONE, vol. 5, no. 1, Article ID e8862, 2010.

Y. Yan, Y. He, T. Maier et al., “Improved identification of yeast species directly from positive blood culture media by combining sepsityper specimen processing and microflex analysis with the matrix-assisted laser desorption ionization biotyper system,” Journal of Clinical Microbiology, vol. 49, no. 7, pp. 2528–2532, 2011.

C. Santos, N. Lima, P. Sampaio, and C. Pais, “Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry to detect emerging pathogenic Candida species,” Diagnostic Microbiology and Infectious Disease, vol. 71, no. 3, pp. 304–308, 2011.

T. Spanu, B. Posteraro, B. Fiori et al., “Direct MALDI-TOF mass spectrometry assay of blood culture broths for rapid identification of Candida species causing bloodstream infections: an observational study in two large microbiology laboratories,” Journal of Clinical Microbiology, vol. 50, no. 1, pp. 176–179, 2012.

B. Sendid, P. Ducoroy, N. Francois, et al., “Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals,” Medical Mycology. In press

Z. B. Zheng, Y. D. Wu, X. L. Yu, and S. Q. Shang, “DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses,” Journal of Medical Virology, vol. 80, no. 6, pp. 1042–1050, 2008.

S. F. Al-Khaldi, M. M. Mossoba, M. M. Allard, E. K. Lienau, and E. D. Brown, “Bacterial identification and subtyping using DNA microarray and DNA sequencing,” Methods in Molecular Biology, vol. 881, pp. 73–95, 2012.

A. Aittakorpi, P. Kuusela, P. Koukila-Kähkölä, et al., “Accurate and rapid identification of Candida, frequently associated with fungemia, by PCR and microarray-based PROVE-ITTM Sepsis assay,” Journal of Clinical Microbiology, vol. 50, no. 11, pp. 3635–3640, 2012.

A. Huang, J. W. Li, Z. Q. Shen, X. W. Wang, and M. Jin, “High-throughput identification of clinical pathogenic fungi by hybridization to an oligonucleotide microarray,” Journal of Clinical Microbiology, vol. 44, no. 9, pp. 3299–3305, 2006.

W. Lu, D. Gu, X. Chen et al., “Application of an oligonucleotide microarray-based nano-amplification technique for the detection of fungal pathogens,” Clinical Chemistry and Laboratory Medicine, vol. 48, no. 10, pp. 1507–1514, 2010.

A. Cassone, F. De Bernardis, and A. Torososantucci, “An outline of the role of anti-Candida antibodies within the context of passive immunization and protection from candidiasis,” Current Molecular Medicine, vol. 5, no. 4, pp. 377–382, 2005.

A. Cassone, F. De Bernardis, and G. Santoni, “AntiCandidal immunity and vaginitis: novel opportunities for immune intervention,” Infection and Immunity, vol. 75, no. 10, pp. 4675–4686, 2007.

W. Magliani, S. Conti, A. Cassone, F. De Bernardis, and L. Polonelli, “New immunotherapeutic strategies to control vaginal candidiasis,” Trends in Molecular Medicine, vol. 8, no. 3, pp. 121–126, 2002.

H. E. Rowlands, K. Morris, and C. Graham, “Human recombinant antibody against Candida,” Pediatric Infectious Disease Journal, vol. 25, no. 10, pp. 959–960, 2006.

J. E. Cutler, “Defining criteria for anti-mannan antibodies to protect against candidiasis,” Current Molecular Medicine, vol. 5, no. 4, pp. 383–392, 2005.

F. De Bernardis, M. Boccanera, D. Adriani, E. Spreghini, G. Santoni, and A. Cassone, “Protective role of antimannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats,” Infection and Immunity, vol. 65, no. 8, pp. 3399–3405, 1997.

R. C. Matthews, G. Rigg, S. Hodgetts et al., “Preclinical assessment of the efficacy of mycograb, a human recombinant antibody against fungal HSP90,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 7, pp. 2208–2216, 2003.

H. Xin, S. Dziadek, D. R. Bundle, and J. E. Cutler, “Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 36, pp. 13526–13531, 2008.

Q. Yang, L. Wang, D. N. Lu et al., “Prophylactic vaccination with phage-displayed epitope of C. albicans elicits protective immune responses against systemic candidiasis in C57BL/6 mice,” Vaccine, vol. 23, no. 31, pp. 4088–4096, 2005.

H. Xin and J. E. Cutler, “Vaccine and monoclonal antibody that enhance mouse resistance to candidiasis,” Clinical and Vaccine Immunology, vol. 18, no. 10, pp. 1656–1667, 2011.

A. Torosantucci, P. Chiani, C. Bromuro et al., “Protection by anti-β-glucan antibodies is associated with restricted β-1,3 glucan binding specificity and inhibition of fungal growth and adherence,” PLoS ONE, vol. 4, no. 4, Article ID e5392, 2009.

N. Kondori, L. Edebo, and I. Mattsby-Baltzer, “A novel monoclonal antibody recognizing β(1-3) glucans in intact cells of Candida and Cryptococcus,” APMIS, vol. 116, no. 10, pp. 867–876, 2008.

R. Herbrecht, C. Fohrer, and Y. Nivoix, “Mycograb for the treatment of invasive candidiasis,” Clinical Infectious Diseases, vol. 43, no. 8, article 1083, 2006.

D. L. Richie, M. A. Ghannoum, N. Isham, K. V. Thompson, and N. S. Ryder, “Nonspecific effect of mycograb on amphotericin B MIC,” Antimicrobial Agents and Chemotherapy, vol. 56, no. 7, pp. 3963–3964, 2012.

B. Wirk, “Heat shock protein inhibitors for the treatment of fungal infections,” Recent Patents on Anti-Infective Drug Discovery, vol. 6, no. 1, pp. 38–44, 2011.

J. Cabezas, O. Albaina, D. Montaez, M. J. Sevilla, M. D. Moragues, and J. Pontn, “Potential of anti-Candida antibodies in immunoprophylaxis,” Immunotherapy, vol. 2, no. 2, pp. 171–183, 2010.

Z. Krenova, Z. Pavelka, P. Lokaj et al., “Successful treatment of life-threatening Candida peritonitis in a child with abdominal non-hodgkin lymphoma using efungumab and amphotericin B colloid dispersion,” Journal of Pediatric Hematology/Oncology, vol. 32, no. 2, pp. 128–130, 2010.

R. Karwa and K. A. Wargo, “Efungumab: a novel agent in the treatment of invasive candidiasis,” Annals of Pharmacotherapy, vol. 43, no. 11, pp. 1818–1823, 2009.

F. C. Odds, “Interactions among amphotericin B, 5-fluorocytosine, ketoconazole, and miconazole against pathogenic fungi in vitro,” Antimicrobial Agents and Chemotherapy, vol. 22, no. 5, pp. 763–770, 1982.

K. R. Smith, K. M. Lank, W. E. Dismukes, and C. G. Cobbs, “In vitro comparison of cilofungin alone and in combination with other antifungal agents against clinical isolates of Candida species,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 10, no. 7, pp. 588–592, 1991.

A. Polak, “Combination therapy of experimental candidiasis, cryptococcosis, aspergillosis and wangiellosis in mice,” Chemotherapy, vol. 33, no. 5, pp. 381–395, 1987.

M. Scheven, K. Junemann, H. Schramm, and W. Huhn, “Successful treatment of a Candida albicans sepsis with a combination of flucytosine and fluconazole,” Mycoses, vol. 35, no. 11-12, pp. 315–316, 1992.

A. Tavanti, G. Maisetta, G. Del Gaudio et al., “Fungicidal activity of the human peptide hepcidin 20 alone or in combination with other antifungals against Candida glabrata isolates,” Peptides, vol. 32, no. 12, pp. 2484–2487, 2011.

A. R. Holmes, Y. H. Lin, K. Niimi et al., “ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates,” Antimicrobial Agents and Chemotherapy, vol. 52, no. 11, pp. 3851–3862, 2008.

E. Lamping, B. C. Monk, K. Niimi et al., “Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae,” Eukaryotic Cell, vol. 6, no. 7, pp. 1150–1165, 2007.

C. Gauthier, S. Weber, A. M. Alarco et al., “Functional similarities and differences between Candida albicans Cdr1p and Cdr2p transporters,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 5, pp. 1543–1554, 2003.

K. Tanabe, E. Lamping, K. Adachi et al., “Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium,” Biochemical and Biophysical Research Communications, vol. 364, no. 4, pp. 990–995, 2007.

K. Hayama, H. Ishibashi, S. A. Ishijima, et al., “A d-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model,” FEMS Microbiology Letters, vol. 328, no. 2, pp. 130–137, 2012.

A. R. Holmes, M. V. Keniya, I. Ivnitski-Steele et al., “The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates,” Antimicrobial Agents and Chemotherapy, vol. 56, no. 3, pp. 1508–1515, 2012.