Candidiasis: A Fungal Infection- Current ... - Ingenta Connect

Send Orders for Reprints to [email protected] Infectious Disorders – Drug Targets, 2015, 15, 42-52

42

Candidiasis: A Fungal Infection- Current Challenges and Progress in Prevention and Treatment Umme Hani*, Hosakote G. Shivakumar, Rudra Vaghela, Riyaz Ali M. Osmani and Atul Shrivastava Department of Pharmaceutics, JSS College of Pharmacy, JSS University, Sri Shivarathreeshwara Nagar, Mysore-570 015, Karnataka, India Abstract: Despite therapeutic advances candidiasis remains a common fungal infection most frequently caused by C. albicans and may occur as vulvovaginal candidiasis or thrush, a mucocutaneous candidiasis. Candidiasis frequently occurs in newborns, in immune-deficient people like AIDS patients, and in people being treated with broad spectrum antibiotics. It is mainly due to C. albicans while other species such as C. tropicalis, C. glabrata, C. parapsilosis and C. krusei are increasingly isolated. OTC antifungal dosage forms such as creams and gels can be used for effective treatment of local candidiasis. Whereas, for preventing spread of the disease to deeper vital organs, candidiasis antifungal chemotherapy is preferred. Use of probiotics and development of novel vaccines is an advanced approach for the prevention of candidiasis. Present review summarizes the diagnosis, current status and challenges in the treatment and prevention of candidiasis with prime focus on host defense against candidiasis, advancements in diagnosis, probiotics role and recent progress in the development of vaccines against candidiasis.

Keywords: Antifungal agents, C. albicans, candidiasis, host defense, probiotics, vaccine. 1. INTRODUCTION The eukaryotic organism fungi have a distinct nucleus with the DNA (genetic material) and enclosed by a nuclear membrane. The cell wall of true fungi is composed of chitin as shown in (Fig. 1) [1]. Candida, yeast like fungus, is a common organism present in the reproductive and gastrointestinal mucosa and can be isolated from the oral cavity and hence upto 80% of the healthy population is found to be prone to the most common fungal infections such as candidiasis. Candida species produce infections that range from non-life threatening mucocutaneous illnesses to invasive conditions which may involve severe infection to destruction of vital organs [2, 3]. Historically, botanist Christine Marie Berkhout was the first to describe about the genus Candida and species C. albicans, which has evolved over the years. Mycotorula and Torulopsis are obsolete expressions for the genus Candida. Previously, these species had been referred to as Monilia albicans and Oidium albicans. Out of the possible 150 different species of the genus Candida, few have been known to cause human infections; with C. albicans being the most pathogenic amongst all. Few other species found to be pathogenic to humans includes C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, C. dubliniensis, C. kefyr, C. guilliermondii and C. stellatoidea [4]. C. albicans is a dimorphic fungus primarily existing and the propagating through its phenotype blastophore also called as blastoconidia. C. albicans has ability to transforms into *Address correspondence to this author at the Department of Pharmaceutics, JSS College of Pharmacy, JSS University, Sri Shivarathreeshwara Nagar, Mysore-570 015, Karnataka, India; Tel: 919738746926; Fax: 918212548394; E-mail: [email protected]

2212-3989/15 $58.00+.00

different morphologies such as yeast, hyphae and pseudohyphae upon perception of environmental signals which have importance in virulence [5, 6]. Elongated, ellipsoidal cells that are attached to one another are referred to as psuedohyphae, while cells that are considered to be true hyphae are characterized by a cylindrical cellular morphology and are separated by perpendicular septal walls [7]. Candidiasis or thrush is a fungal infection (presence of parasitic infection in or on any part of body i.e., mycosis) caused by any of the species from the genera Candida, amongst which C. albicans is the most common causative species. The infection is technically referred to as candidosis, moniliasis as well as oidiomycosis and also commonly called as yeast infection. These infections are broad spectrum, ranging from superficially oral thrush and vaginitis to deep cited systemic, which are mostly life threatening. Systemic infections are commonly referred to as candidemia and are one of the prominent co-infections in immuno-compromised patients such as those suffering from cancer, HIV etc. as well as patients in non-trauma emergency surgery, with extensive use of powerful antibiotics and immune-suppressive therapies during organ transplant or anti-leukemia therapies [8, 912]. Depending on the area infected, signs and symptoms of candidiasis vary, which are briefly depicted in (Fig. 2). A number of antifungal drugs are found to be efficient and are available for the treatment of candidiasis. Amphotericin B has served as a standard treatment for five decades but its usage is often limited due to toxic effects, fluconazole is as effective as amphotericin B with superior safety but certain non-albicans candida species are less susceptible to fluconazole. New agents from echinocandins class, such as caspofungin, target the fungal cell wall and retain the activity against isolates with resistance to azoles or polyenes [13]. © 2015 Bentham Science Publishers

Candidiasis: A Fungal Infection- Current Challenges and Progress

Infectious Disorders – Drug Targets, 2015, Vol. 15, No. 1

43

Fig. (1). Schematic diagram of C. albicans cell wall.

Fig. (2). Signs and symptoms of candidiasis.

Apart from the conventional antifungal therapy use of probiotics and development of new efficient vaccines is an advanced approach in the direction of prevention and treatment of candidiasis. Till date a number of protective and highly immunogenic vaccine formulations have been developed against candidiasis and relative further research is still going on. Present review summarizes the diagnosis, current status and challenges in the treatment and prevention of candidiasis with prime focus on host defense against candidiasis, probiotics role and recent progress in the development of vaccines against candidiasis.

gal treatment as well. When compared to mucocutaneous candidiasis, diagnosis of disseminated candidiasis is difficult due to non-specific clinical presentation. For effective therapeutic outcome, accurate identification of Candida species is very essential. Conventional methods for the diagnosis of candidiasis are less sensitive and time consuming, hence, immunodiagnostic and molecular techniques can be recommended for early and specific diagnosis. Sachin et al. have discussed in detail the laboratory approach for the diagnosis of candidiasis [14], some of the methods are listed below: 2.1. Direct Examination

2. DIAGNOSIS Diagnosis is very crucial not only for confirmation of infection but for species identification and to initiate antifun-

Direct microscopic examination is a cost effective, rapid method for the diagnosis of candidiasis. It involves scraping or swabbing of the affected area followed by placing the swab on the microscopic slide. Further, 10% potassium hy-

44 Infectious Disorders – Drug Targets, 2015, Vol. 15, No. 1

droxide (KOH) solution is added in order to dissolve the skin cells without affecting the integrity of the Candida cells. This allows complete visualization of pseudohyphae as well as the budding yeasts cells, which corresponds to various Candida species. In case of disseminated candidiasis, direct demonstration of elements of fungi is done by wet and fixed mounts. Addition of calcofluor white allows rapid recognition of fungal components by binding to chitin and cellulose in fungal cell wall and on excitation by long wave ultraviolet rays, it fluoresces. Histological specimens are stained with periodic acid-schiff or Gomori S methenamine silver stains [14]. 2.2. Culture Method Candida species readily grows on Sabouraud dextrose agar media, the most commonly used media for the isolation of Candida species, which permits the growth of Candida and suppresses the growth of some bacteria because of low pH. This method includes rubbing of the sterile swab onto the infected area followed by streaking it on a Sabouraud dextrose agar medium and incubating at 37°C for a couple of days in order to allow the development of colonies. On the basis of colony characterization, microscopic morphology, physiological or biochemical characteristics, speciation of Candida is done [10, 11]. A range of differential media available for Candida speciation includes Pagano-Levin agar, Nickerson s medium [15], phosphomolybdate agar and chromogenic medium [16]. Chromogenic media (Candida ID, CHROM agar, Candiselect 4 and Candida media) are also implied for speedy identification of Candida [17]. Chromogenic substrates present in these media react with yeast cells by enzymes secreted to give particular colony characteristic and pigmentations which are species specific and thus allow easy speciation [18]. 2.3. Diagnosis of Invasive Candidiasis (IC) Diagnosis of invasive candidiasis can be carried against recombinant fructose bisphosphate aldolase and C. albicans enolase using antibodies as evaluated by Li et al. It was found that it could be a powerful tool for diagnosing IC, when they are used in combination [12]. 2.4. Culture Recent developments in blood culturing techniques have improved the sensitivity as well as reduced the time required to obtain a positive blood culture. The developments include centrifugation tubes as well as automated monitoring of blood-cultured bottles. Specific detergents are being used in order to release the trapped fungi within the host phagocytic cells thereby enhancing the yield of Candida spp. by lysis centrifugation system [12]. The lytic mixture is responsible for the disruption as well as inactivation of host cells and some of the antimicrobial agents, which possess a threat to the viability of the fungi. 2.5. (1→3)-β-D-Glucan Detection Method The cell walls of Candida species contain (1→3)-β-Dglucan (BDG) as a structural component. As this polysaccha-

Hani et al.

ride is not found in bacteria, viruses, or mammals, its presence in the circulation of patients has been used as an indicator of invasive disease. An assay to detect BDG has been developed, which utilizes the activation of a clotting pathway found in amoebocyte lysates of the Japanese horseshoe crab, Tachypleus tridentalis [19]. 2.6. Serological Methods The serological methods commonly used include agar gel diffusion (AGD), counter immunoelectrophoresis (CEP), whole cell agglutination (AGGL), latex agglutination (LAT), indirect fluorescent antibody, and complement fixation. Of these, the AGGL test, AGD test and CEP test are the most frequently employed [20, 21]. One of the most common serological tests for systemic candidiasis is AGGL test developed by Hasenclever and Mitchell [22]. This test detects antibody specific primarily to the mannan component of the Candida cell wall [23]. As very low numbers of yeasts are observed using direct microscopic examination in peritoneal candidiasis, Corrales et al. explored clinical utility and potential of molecular methods and serum (1-3)-β-d-glucan (BDG) estimation as novel tools in its diagnosis. They have compared performance of a polymerase chain reaction (PCR) with that of automated culture method using peritoneal fluids from peritonitis patients for Candida spp. detection. The PCR assay and the culture method reflected good agreement with promising sensitivities as 93.5% and 74.19% respectively. It has been found that serum BDG levels were high in peritoneal fluids of patients with Candida spp. than those without the yeast [24]. In another approach, Levesque et al. demonstrated the importance of BDG as a biomarker in the diagnosis of invasive candidiasis particularly after liver transplantation, which asserts high morbidity and mortality around the globe [25]. Considering the steady increase of invasive Candida infection incidences in intensive care requiring patients and difficulties associated with their diagnosis, Kautzky et al. have proposed clinical scoring systems (like four risk factorbased Candida score and/or Candida colonization index) to be extremely valuable tools in the diagnosis and selection of higher risk patients for immediate systemic antifungal therapy. They reported that both the Candida colonization index and Candida score were incredibly practical tools; with cutoff standards ≥ 0.5 and ≥ 2.5 respectively [26]. Apart from the conventional diagnostic methods, Ardizzoni et al. have developed a ten protein array based immunoassay for assessing antibody (IgG) responses towards recognized immunogenic proteins of C. albicans with a trial in invasive candidiasis patients. Results of their studies suggested probable implication of microarray immunoassay along with conventional diagnostics, for a rapid and sensitive detection of invasive candidiasis patients [27]. 3. HOST DEFENSE AGAINST CANDIDA ALBICANS A postulated pathway of response to infections caused by C. albicans is sketched in (Fig. 3). Upon interaction of C. albicans with epithelial cells, it triggers the release of cytokines and chemokines, which in turn causes activation of



45

Fig. (3). Schematic illustration of known and postulated pathways of response towards infection with C. albicans.

inflammatory as well as immune cells such as phagocytes, antigen presenting cells (APCs) and T-cells. Fungus cells are engulfed and killed by phagocytic cells by respiratory burst as well as cytokine release. Patients suffering from chronic mucocutaneous candidiasis express defective genes in type-I interferon pathway. This pathway plays a significant role in anti-Candida host defense in humans as it is a signature of Candida-induced inflammation. Pattern-recognition receptors such as Toll-like receptors and C-type lectin receptors facilitate the recognition of C. albicans. These recognition receptors interact with the cell wall components of C. albicans, which are primarily expressed by Antigen Presenting Cells (APCs) [13]. Gauglitz et al. earlier discussed the various patternrecognition receptors that facilitate the recognition of the special structures of C. albicans, responsible for activation of intracellular signal, which ultimately leads to efficient host defense [14]. Macrophage migration towards C. albicans is dependent upon the glycosylation status of fungal cell wall rather than cell viability or morphogenic switching from yeast to hyphal forms. For determination of engulfment of C. albicans by macrophages, spatial orientation of the hypha and its attachment to macrophage are the important parameters that are taken into consideration. Host defence against Candida was improved using a rational novel therapeutic approach of adjuvant immunotherapy [28, 29]. A study was carried out on patients suffering from rare immunedeficiencies such as dectin-1 deficiency, CARD9 deficiency, chronic mucocutaneous candidiasis, hyper-IgE or Job's syndrome and chronic granulomatous disease; the results explained the role of dectin-1 and its signaling pathway, which involves interleukins 17 and 22, defensins and phagocytic cells for defense against Candida [6]. The susceptibility to recurrent Candida infections can be a monogenetic Men-

delian trait. The sensing of Candida cell wall components and the consecutive intracellular signaling in myeloid cells via CARD9, and also the role of Th17 cells and their cytokines take the center stage in the human host defense against Candida [30]. Neutrophils (PMNs) are responsible for the playing an important role host defense against acute invasive and disseminated candidiasis. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and granulocyte colony stimulating factor (G-CSF) are involved in staffing of the PMNs at the site of invasive infection. Combination therapy with recombinant G-CSF and fluconazole exhibits additive effect on fungal reduction in the organs. In case of sub-acute or chronic disseminated Candida infection, recombinant G-CSF was found to be less effective; which indicates that neutrophil staffing and activation play a crucial role in acute as well as life threatening candidiasis. For invasive Candida infection, other host defense mechanisms control the outcome [31]. 4. TREATMENT OF CANDIDIASIS Treatments for candidiasis for managing Candida infections are usually based upon the anatomic location of the infection, immune status of the patient, risk factors for patients with infection, species responsible and lastly, upon the susceptibility of the Candida species towards the anti-fungal drug. Classification of antifungal agents used for the treatment of candidiasis is given below: 4.1. Classification 4.1.1. Systemic Antifungal Agents A. Polyenes: Amphotericin B. B. Pyrimidine analogue: Flucytosine.


Hani et al.

C. Triazoles: Fluconazole, Itraconazole, Voriconazole, Ravuconazole, Posaconazole, Ketoconazle.

Anti-fungal agents disrupt the fungal cell wall by targeting the ergosterol component via binding with sterols, formation of pores in the membrane, creating a transmembrane ion channel and causing the membrane to become leaky, which results in loss of intracellular K+ ions (polyene antifungals) or by directly inhibiting ergosterol biosynthesis. Enzyme CYP P450 α- demethylase in the fungi is responsible for conversion of lanosterol to ergosterol. The basic nitrogen of azole ring involves the formation of a bond with the heme iron of the fungal P450; thereby preventing substrate and oxygen binding. C-14 α-demethylase inhibition consequently accumulates sterols comprising of C14 methyl group, leading to changes in permeability and malfunctioning of membrane embedded proteins. These azoles have minimum affinity towards mammalian P450’s. The overall effect is fungistatic, which at higher concentrations may be fungicidal.

D. Echinocandins: Caspofungin, Anidulafungin, Micafungin. 4.1.2. Topical Antifungal Agents A. Topical azoles: Terconazole, Butaconazole, Miconazole, Clotrimazole, Tioconazole, Sulconazole, Oxiconazole and Econazole. B. Topical allylammines: Terbinafine and Naftifine. One of the selection criteria for anti-fungal agents is Candida species susceptibility. Table 1 depicts in vitro susceptibility of Candida isolates from blood cultures to most often used anti-fungal agents based on resistance testing by CLSI-M27-A3 method [32]. Commercially available antifungal dosage forms for curing varied types of candidiasis are listed out in Table 2.

Few anti-fungal agents act by targeting the microtubule; which affects the formation of mitotic spindle, or by inhibition of DNA transcription ultimately affecting cell division [20]. For example flucocystine converts to 5-fluorouracil, which is an antimetabolite responsible for inhibition of thymidylate synthetase and DNA synthesis.

4.2. Mechanism of Action of Antifungal Agents General mechanism of action for anti-fungal drugs includes:

The treatment recommendations for mucocutaneous candidiasis by Infectious Diseases Society of America (IDSA) are summarized in Table 3 [33].

a) Cell wall formation inhibition. b) Disruption of cell membrane. c) Cell division inhibition.

5. RECENT ADVANCES IN PROBIOTICS FOR PREVENTION AND TREATMENT OF CANDIDIASIS

Inhibition of fungal cell biosynthesis has not been successful and effective. Attempts have been made to interfere with fungal cell wall synthesis, using some of the chemical agents, which have demonstrated excellent anti-fungal activity in vitro. However, failure to develop these agents to useful drugs has verified to be a difficult task and hence ultimately development of new agents to target β-glucan synthesis has been focused. Echinocandins are the recent class of antifungal agents that act by inhibiting β-(1-3)D-glucan at the fungal cell wall level, resulting in complete disruption of the cell wall and consequently cell death. (Fig. 4) depicts mechanisms of action of some most popular antifungal drugs. Table 1.

Lilley and Stillwell, 1965, were the first ones to describe about substances secreted by one microorganism, which in turn is responsible for stimulating the growth of another. Fuller (1989) redefined probiotics as ‘a live microbial feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance’ [34]. Probiotics are defined as living microorganisms, which when administered in adequate amounts, provide beneficial effects to the host [35]. Probiotics are emerging as a new approach to counteract vaginal candidiasis. They lower intravaginal pH thereby forming a barrier against different types of yeasts [21]. Probiotics inherit property of inhibiting pathogen

In-vitro susceptibility of diverse Candida species.

Candida species

AMB

5-FC

FCZ

ITZ

VCZ

PCZ

AFG

CFG

MFG

Candida albicans

S

S

S

S

S

S

S

S

S

Candida glabrata

S

S

I-R

S-I-R

S-I-R

S-I-R

S

S

S

Candida tropicalis

S

S

S-I

S

S

S

S

S

S

Candida parapsilosis

S

S

S

S

S

S

I

I

I

Candida krusei

S

R

R

I-R

S-I-R

S-I-R

S

S

S

Candida guilliermondii

S

S

S

S

S

S

R

R

R

Candida lusitaniae

S-I-R

S

S

S

S

S

S

S

S

Where, AMB: Amphotericin-B formulations; 5-FC: flucytosine; FCZ: fluconazole; ITZ: itraconazole; VCZ: voriconazole; PCZ: posaconazole; AFG: anidulafungin; CFG: caspofungin; MFG: micafungin; S: susceptible; I: intermediate; R: resistant.


Table 2.


47

Commercially available antifungal dosage forms for candidiasis.

Type of candidiasis

Class

Generic name

Brand name

Dosage form

Oral

Azole antifungals

Miconazole

Oravig

Buccal Tablets ®

Buccal Tablets

®

Oral gels

Loramyc Daktarin

Vaginal

Azole antifungals

Clotrimazole

Mycelex Troche

Troches

Fluconazole

Diflucan

Oral suspension

Miconazole nitrate

Gyne-Lotrimin

Cream

Monistat Gynazole-1

Cream

Vaginal Cream

Cream

Antican

Tablet

Cancap-VT

Tablet

Candid tab

Tablet

Canesten

Tablet

Gynostatum

Tablet

Surfaz-VT

Tablet

Vagibact

Cream

Monistat-Derm

Cream

Aloe Vesta

Cream

Baza Antifungal

Cream

Miranel AF

Liquid

Mitrazol

Powder

Zeasorb-AF

Powder

Canesten

Cream

Mycelex

Cream

Nizoral A-D

Shampoo

Xolegel

Gel

Mycostatin Topical

Powder and cream

Nyamyc

Powder

Mycostatin

Tablets

Nilstat

Tablets, troches, oral suspension

Mycostatin Pastilles

Pastilles, lozenges

Bio-Statin

Powder

Fluconazole

Diflucan

Tablet

Ketoconazole

Nizoral

Tablet

Itraconazole

Sporanox

Capsule

Voriconazole

Vfend

Lyophilized powder for solution for IV infusion, film-coated tablets for oral administration and as a powder for oral suspension

Butaconazole

Clotrimazole

Skin

Azole antifungals

Miconazole

Clotrimazole

Ketoconazole

Polyene

Gastrointestinal Candidiasis Medications

Esophageal Candidiasis

Polyenes

Azole antifungals

Nystatin

Nystatin


Hani et al.

(Table 2) Contd….

Type of candidiasis

Class

Generic name

Brand name

Dosage form

Echinocandins

Micafungin

Mycamine

IV Injection

Anidulafungin

Eraxis

IV Injection

Caspofungin

Cancidas

IV Injection

Amphotericin- B

Amphocin

IV Injection

Fungizone

IV Injection

Itraconazole

Onmel

Tablet

Miconazole

Miranel AF

Liquid

Polyenes

Nail

Table 3.

Azole antifungals

Treatment recommendations by IDSA for mucocutaneous Candida infections.

Disorder Oropharyngeal candidosis

Drug

Dosage

Amphotericin B (oral suspension/tablets)

0.5 (to 2.4) g/day

Nystatin (oral suspension)

6 × 100,000 units/day

Fluconazole

50-200 mg/day

Itraconazole (oral solution)

100-200 mg/day

Posaconazole

100 mg/day

Laryngitis

As for oropharyngeal

Oesophagitis

Fluconazole

200-400 mg/day

Itraconazole (oral solution)

2 × 200 mg/day

D-AMB (i.v.)

0.5-0.7 mg/kg/day

L-AMB (i.v.)

1-3 mg/kg/day

Anidulafungin

100 mg/day

Caspofungin

50 mg/day

Micafungin

150 mg/day

Voriconazole

400 mg/day

Clotrimazole (suppository)

-

Fluconazole

150 mg/day

Fluconazole

50-200 mg/day

Itraconazole

100-200 mg/day

Fluconazole

50-400 mg/day

Itraconazole

100-400 mg/day

Posaconazole

100-400 mg/day

Vaginal candidosis

Skin/nails infections

Chronic mucocutaneous candidosis (CMC)

growth as well as modulate human immune response thereby making it a new possibility towards antifungal therapy. The different strains of lactobacillus proved to be effective against C. albicans are Lactobacillus reuteri, Bifidobacterium animalis, Lactobacillus acidophilus, Lactobacillus casei GG, or Lactobacillus fermentum L23 and LF5, Lactoba-

cillus casei etc. [36]. The probiotic bacteria such as Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei GG and Bifidobacterium animalis have shown prophylactic action against mucosal and systemic candidiasis and through immunologic and non-immunologic responses, they protected mice from candidiasis [37].



49

Fig. (4). Mechanism of action of antifungal agents.

To study the prevalence of oral candidiasis, Hatakka et al., using probiotics, carried out a randomized controlled trial in the elderly. About 32% reduction was found in the prevalence of oral Candida in the probiotic group, whereas it increased in the control group (28%-34%). It was found that probiotics intervention reduced the risk of high yeast counts by 75% with a 56% decrease in hypo-salivation. Therefore, it was concluded that probiotics could be effective towards controlling oral Candida as well as hypo salivation [38]. A randomized study to determine the effectiveness of orally supplemented probiotics towards prevention of gastrointestinal Candida species colonization in preterm, very low birth weight neonates in a neonatal ICU were evaluated. From results it was a reported that there was significant reduction in the incidence and the intensity of enteric colonization of Candida species [39]. Kumar et al., highlighted that 10-20% of bloodstream infections in pediatric ICU are due to candidiasis and thus there is significant increase in morbidity, mortality and duration of hospital stay. Candida species enteric colonization still remains one of the most alarming risk factors for invasive candidiasis. Localized defensive action is either diminished or altered; thereby facilitating Candida overgrowth and leading to candidiasis. Systemic anti-fungal agents are proven to be effective towards reduction in colonization as well as invasive fungal infections, but with some associated major side effects. Hence, for rapid restoration of the intestinal flora, probiotics provides the necessary tools for Candida reduction. Though studies depicted significant effect of probiotics towards prevention and colonization of Candida in neonates, their role in preventing invasive infections is yet unclear [40]. A pilot study carried out by Vicariotto et al., in order to determine the effectiveness of association of two probiotic strains viz. Lactobacillus fermentum LF10 (DSM 19187) and Lactobacillus acidophilus LA02 (DSM 21717). The strains were formulated into slow release effervescent vaginal tablet (ActiCand 30) and its effect on women suffering from vulvovaginal candidiasis was studied. Results indicated that ActiCand 30 not only cures Candida infections at high per-

centage in women but also provides longer physiological defense on the account of vaginal mucosal adhesion and colonization. Further, it also mediates the two types of barrier effects i.e. formation of an anaerobic environment by release of CO2 and by colonization and adhesion to the vaginal epithelium by the probiotics L. acidophilus LA02 and L. fermentum LF10, respectively [21]. Kohler et al., investigated the potential of two probiotics Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 in controlling C. albicans infections. In vitro results reflected that at lower pH, lactic acid plays a major role in suppressing fungal growth. Post staining observations revealed that C. albicans cells lost their metabolic activity and were eventually killed. Increased expression of stress-related genes and lower expression of genes involved in fluconazole’s resistance explains augmented eradication of Candida which was confirmed by Transcriptome analysis [41]. Bohbot et al., have conducted an open randomized pilot study on 20 childbearing age healthy women in order to assess vaginal impact on oral administration of completely freeze-dried Lactobacillus casei rhamnosus LCR 35 culture. It was concluded from the study that at minimum dose of 2*108 CFU/day of LCR 35 brings the nugent score to normal in healthy childbearing age women. The preventive or culture impact of the orally administered strain on various vaginal disorders such as bacterial vaginosis or vulvovaginal candidiasis can be specified by Phase-III clinical trials [42]. A practitioner survey of complementary and alternative medicine (CAM) in recurrent vulvovaginal candidiasis (RVVC) was conducted by Watson et al., which included lactobacillus, oral and vaginal yoghurt, vinegar, garlic, Chinese medicine and tea-tree oil. Amongst these, Lactobacillus was found to be most commonly recommended. In other non-pharmacological management tips, they recommended dietary changes as well as use of cotton undergarments. From the survey researchers concluded that CAM is popular with patients and many clinicians actively recommend its use in RVVC despite limited supporting evidence [43].


Ishijima et al., developed a non-pathogenic oral probiotic microorganism (Streptococcus salivarius K12), an innovative therapeutic option for candidiasis, and evaluated its ability to curb C. albicans growth in vitro. Its therapeutic activity was also assessed in-vivo on experimental model of oral candidiasis. By inhibiting the process of invasion of C. albicans into mucous surfaces S. salivarius K12 protected the experimental animal from severe candidiasis and hence proved it an effective probiotic [44]. In an approach, Matsubara et al. evaluated oral colonization by C. albicans in experimental murine immunosuppressed DBA/2 and probiotic bacteria (Lactobacillus acidophilus and Lactobacillus rhamnosus) treated animal groups. They reported that in comparison with animal group (untreated), a significant reduction in C. albicans colonization was observed on the oral mucosa of animal group treated with probiotics. Moreover, L. rhamnosus treated animal group reflected significantly higher reduction in yeast colonization with respect to that of group treated with nystatin [45]. A prospective double blinded, randomized controlled trial was conducted by Kumar et al. to evaluate efficacy of probiotics in prevention of Candida colonization in a paediatric intensive care unit of Indian hospital and concluded that in critically ill children treated with broad spectrum antibiotics (to reduce candiduria and gastrointestinal Candida colonization), probiotics supplementation could be a potential beneficial strategy [46]. 6. RECENT PROGRESS IN THE DEVELOPMENT OF VACCINES AGAINST CANDIDIASIS A number of protective and highly immunogenic vaccine formulations have been developed against candidiasis in the last decade [47]. Vaccine can be defined as a biological preparation which improves immunity against a particular disease. Vaccines comprise of an agent that resembles a disease causing microorganism, synthesized either using killed or weakened form of the microbe or one of its surface protein or its toxins, which stimulates the immune system of the body to recognize the agent as antigen and to destroy it [48]. Liu et al. have shown that heat-killed Saccharomyces (HKY) is a protective vaccine against aspergillosis and coccidioidomycosis. To test the hypothesis that the efficacy of HKYinduced protection may be due to the cross-reactive antigens in the cell walls of the different fungi, they studied the effect of HKY against systemic candidiasis. Male CD-1 mice were given different regimens of HKY subcutaneously prior to intravenous challenge with C. albicans. They found that HKY protects mice from infection by C. albicans in a doseand regimen-dependent manner [49]. C. albicans mannan extracts encapsulated in liposomes were used previously to stimulate mice to produce antibodies protective against candidiasis. Subsequently, mannan-protein conjugates without liposomes as vaccine candidates were tested. Mannan extracts were coupled to bovine serum albumin where isolated conjugates consisted of carbohydrate and protein in a ratio of 0.7-1. It has been reported that the conjugate vaccine can induce protective antibody responses against experimental disseminated candidiasis and Candida vaginal infection [50]. Lack of C5 is known to aggravate candidal infection. Conjugated-mannan is 100% protective in C5-deficient mice against disseminated candidiasis; thus

Hani et al.

conjugate vaccines seem to be promising in eradication of diverse C. albicans infections [51]. An optimal inhibitor of two protective monoclonal antibodies i.e. β-(1→2)-linked mannose trisaccharide epitope, which is specific for phosphomannan of cell wall of C. albicans was used to develop a synthetic conjugate vaccine by Lipinski et al. They concluded that in combating against C. albicans infections, antibody mediated immunity plays an important role and hence suggested that synthetic conjugate vaccine possibly has therapeutic potential [52]. A study on disseminated candidiasis pathogenesis observed that antibodies specific for C. albicans cell surface β1, 2-mannotriose [β-(Man)(3)] protect mice. A selfadjuvanting vaccine was obtained by adding tetanus toxoid to glycopeptide conjugate which enhances robust antibody responses without additional adjuvant against disseminated candidiasis [53]. De Bernardis et al. designed and studied a novel vaccine (PEV7), comprised of influenza virosomes in which a truncated, recombinant aspartyl proteinase-2 of C. albicans was incorporated. Following intramuscular immunization in mouse and rat, the aforesaid vaccines generated a potent serum antibody response. Following the intravaginal administration of PEV7 in rats, anti-Sap2 IgG and IgA were detected in vaginal fluid and induced significant, safe and long lasting protection against Candida vaginitis [54]. To develop both active and passive immunization strategies against candidiasis recombinant N-terminus of Hyr1p (rHyr1p-N) is a novel target as it was reported to have efficacy to reduce the fungal burden of disseminated candidiasis in both immunocompromised and immuncompetant mice, using alum; an FDA approved adjuvant [55]. Using an antigen bearing dual delivery system i.e. fibrin cross- linked plasma beads and C. albicans cytosolic proteins (Cp) as antigen, a prophylactic vaccine was developed against systemic candidiasis. It has been found that the preparation comprising of liposomized Cp entrapped in plasma beads exhibited superior protection in the immunized mice in comparison with other antigen delivery systems [56]. In another approach, significant protection was achieved in murine models against vaginal candidiasis and disseminated candidiasis by a vaccine conjugated with a protein laminaran (algal glucan) linked to a carrier protein diphtheria toxoid [57, 58]. Laminaran vaccine was found to be a promising candidate and effective against fungi, which contains glucan in cell wall [59]. The candidal vaccine furthest along the developmental pathway is based on the agglutinin-like sequence (Als) family of proteins from C. albicans. Vaccination with the recombinant N-termini of the candidal surface adhesins Als1p or Als3p (rAls1p-N or rAls3p-N) protected mice from lethal disseminated candidiasis, and also reduced fungal burden in a vaginitis model and a steroid-treated oropharyngeal candidiasis model [60]. Shibasaki et al. developed an oral vaccine against candidiasis using yeast molecular display system. They have generated surface Enolase 1 (Eno1p) antigen engineered Saccharomyces cerevisiae cells for oral delivery, that had shown extended survival rate in C. albicans challenged mice. Furthermore, vaccine produced using this molecular display


technology was found promising in the prevention of a range of fungal infections and it also overcomes the protein purification requirement [61]. In a study adjuvant consequences interceded by Physalisalkekengi L. fruit polysaccharide in DNA vaccine (pDHSP90C) were assessed in mice. Results depicted that polysaccharide notably enhanced serum concentration of interleukin-2, interleukin-4 and precise antibody titers in DNA vaccine immunized mice. In addition, just few colony forming units (CFU) were traced in the kidneys of polysaccharide-DNA vaccine immunized mice group with significantly higher survival rate post Candida albican challenging, as compared to the only DNA vaccine immunized group. Thus, researchers concluded the aforesaid polysaccharide as a promising adjuvant in enhancing the efficacy of DNA vaccines [62]. Such studies continue to build upon the fundamental knowledge of protective immunity against Candida, which will ultimately lead to ease, advancement in developing novel potential and clinically efficient vaccines against candidiasis.


[6]

[7]

[8] [9] [10]

[11] [12]

[13]

7. CONCLUSION Candidiasis can be treated based on the type of candidiasis and severity of infection. Understanding in depth the host defense mechanism against Candida albicans will be helpful in the development of vaccines against candidiasis. Commonly used antifungal agents for oral and vaginal candidiasis are nystatin, Amphotericin-B and azole derivatives. As probiotic bacteria are capable of inhibiting the growth of pathogens and thereby modulating human immune response as discussed in the article they necessitate research attention. Further, they would offer innovative prospects in candidiasis and antifungal therapy in the near future. Moreover, progress in development of clinically useful vaccines is swiftly evolving day by day which may proffer an ideal remedy and paramount way out to invasive candidiasis and associated tribulations.

[14] [15]

[16] [17]

[18]

[19]

CONFLICT OF INTEREST

[20]

The author(s) confirm that this article content has no conflict of interest.

[21]

ACKNOWLEDGEMENTS Declared none.

[22] [23]

REFERENCES [1] [2]

[3] [4]

[5]

Tortora, G.J.; Funke, B.R.; Case, C.L. Microbiology: An introduction, 10th ed.; The Benjamin/Cummings Publishing Co. Inc.: Redwood City, CA, 1995, 601-2 and 758-9. Walsh, T.J.; Dixon, D.M. In Baron's Medical Microbiology; Samuel Baron, Ed.; Galveston (TX): University of Texas Medical Branch at Galveston: Galveston, Texas, 1996. Eckert, L.O.; Lentz, G.M. In Comprehensive Gynecology; Lentz, G.M.; Lobo, R.A.; Gershenson, D.M.; Katz, V.L., Ed.; 6th ed.; Mosby Elsevier: Philadelphia, 2012. James, W.D.; Berger, T.G.; Elston, D.M.; Odom, R.B. Andrews' Diseases of the Skin: clinical Dermatology, 10th ed.; Saunders Elsevier: Philadelphia, 2006, 308-311. Kourkoumpetis, T.; Manolakaki, D.; Velmahos, G.; Chang, Y.; Alam, H.B.; De Moya, M.M.; Sailhamer, E.A.; Mylonakis, E. Can-

[24]

[25]

[26]

51

dida infection and colonization among non-trauma emergency surgery patients. Virulence, 2010, 1(5), 359-66. van der Meer, J.W.; van de Veerdonk, F.L.; Joosten, L.A.; Kullberg, B.J.; Netea, M.G. Severe Candida spp. infections: new insights into natural immunity. Int. J. Antimicrob. Agents, 2010, 36(2), 58-62. Mohamed, S.P.; Muzzammil, S.; Pramod, K.T. Buccoadhesive dosage form containing antifungal agent for treating oropharyngeal candidiasis: A review. Current Drug Therapy, 2011, 6, 55-64. Antifungal chemotherapy in patients with acquired immuno deficiency syndrome. British Society for Antimicrobial Chemotherapy Working Party. Lancet, 1992, 340(8820), 648-51. Greenspan, D.; John, S.G. HIV-related oral disease. Lancet, 1996, 348, 729-734. Chakravarthi, S.; Haleagrahara N. A comprehensive review of the occurrence and management of systemic candidiasis as an opportunistic infection. Microbiology J., 2011, 1(1), 1-7. Guzel, MAB. The epidemiology, pathogenesis, and diagnosis of vulvovaginal candidosis: A mycological perspective. Critical Reviews in Microbiology, 2011, 37(3), 250-61. Li, F.Q.; Ma, C.F.; Shi, L.N.; Lu, J.F.; Wang, Y.; Huang, M.; Kong, Q.Q. Diagnostic value of immunoglobulin G antibodies against Candida enolase and fructose-bisphosphate aldolase for candidemia. BMC Infect. Dis., 2013, 13, 253. Smeekens, S.P.; Ng, A.; Kumar, V.; Johnson, M.D.; Plantinga, T.S.; van Diemen, C.; Arts, P.; Verwiel, E.T.; Gresnigt, M.S.; Fransen, K.; van Sommeren, S.; Oosting, M.; Cheng, S.C.; Joosten, L.A.; Hoischen, A.; Kullberg, B.J.; Scott, W.K.; Perfect, J.R.; van der Meer, J.W.; Wijmenga, C.; Netea, M.G.; Xavier, R.J. Functional genomics identifies type-I interferon pathway as central for host defense against C. albicans. Nat. Commun., 2013, 4, 1342. Gauglitz, G.G.; Callenberg, H.; Weindl, G.; Korting, H.C. Host defense against C. albicans and the role of pattern-recognition receptors. Acta Derm. Venereol., 2012, 92(3), 291-8. Costa, S.; de Lourdes Branco, C. Evaluation of molybdenum culture medium as selective and differential for yeasts. J. Pathol. Bacteriol., 1964, 87, 428-431. Odds, F.; Bernaerts, R. CHROMagar Candida; a new differential isolation medium for presumptive identification of clinically important Candida species. J. Clin. Microbiol., 1994, 32, 1923-1029. Ghelardi, E.; Pichierri, G.; Castagna, B.; Barnini, S.; Tavanti, A.; Campa, M. Efficacy of chromogenic Candida agar for isolation and presumptive identification of pathogenic yeast species. Eur. J. Clin. Microbiol. Infect. Dis., 2008, 14, 141-147. Horvath, L.L; Hospenthal, D.R.; Murray, C.K.; Dooley, D.P. Direct isolation of Candida spp. from blood cultures on chromogenic medium CHROMagar Candida. J. Clin. Microbiol., 2003, 41, 26292632. Ellepola, A.N.; Morrison, J.C. Laboratory diagnosis of invasive candidiasis. The J. Microbiol., 2005, 43, 65-84. Myers, R.S. Immunizing and antimicrobial agents. MEDCH, 2006, 401, 2-3. Vicariotto, F.; Del Piano, M.; Mogna, L.; Mogna, G. Effectiveness of the association of two probiotic strains formulated in a slow release vaginal product, in women affected by vulvovaginal candidiasis: A pilot study. J. Clin. Gastroenterol., 2012, 46, S73-80. Hasenclever, H.F.; Mitchell, W.O. Observation of two antigenic groups in Candida albicans. J. Bacteriol., 1961, 82, 570-573. Merz, W.G.; Evans, G.L.; Shadomy, S.; Anderson, S.; Kaufman, L.; Kozinn, P.J.; Mackenzie, D.W.; Protzman, W.P.; Remington, J.S. Laboratory evaluation of serological tests for systemic candidiasis: A cooperative study. J. Clin. Microbiol., 1977, 5, 596-603. Corrales, I.; Gimenez, E.; Aguilar, G.; Delgado, C.; Puig, J.; Izquierdo, A.; Belda, J.; Navarro, D. Detection of fungal DNA in peritoneal fluids by a PCR DNA low-density microarray system and quantitation of serum (1-3)-β-D-glucan in the diagnosis of peritoneal candidiasis. Med. Mycol., 2014, pii: myu075 (Epub ahead of print). Levesque, E.; El Anbassi, S.; Sitterle, E.; Foulet, F.; Merle, J.C.; Botterel, F. Contribution of (1,3)-beta-D-glucan (BG) to the diagnosis of invasive candidiasis after liver transplantation. J. Clin. Microbiol., 2014, pii: JCM.03018-14 (Epub ahead of print). Kautzky, S.; Staudinger, T.; Prester, E. Invasive Candida infections in patients of a medical intensive care unit: Attempt of improving diagnosis by quantifying the colonization. Wien KlinWochenschr., 2014 (Epub ahead of print).

52 Infectious Disorders – Drug Targets, 2015, Vol. 15, No. 1 [27]

[28]

[29] [30]

[31]

[32]

[33]

[34] [35] [36]

[37]

[38]

[39]

[40] [41]

[42]

[43]

Ardizzoni, A.; Posteraro, B.; Baschieri, M.C.; Bugli, F.; SaezRoson, A.; Manca, L.; Cacaci, M.; Paroni Sterbini, F.; De Waure, C.; Sevilla, M.J.; Peppoloni, S.; Sanguinetti, M.; Moragues, M.D.; Blasi, E. An antibody reactivity-based assay for diagnosis of invasive candidiasis using protein array. Int. J. Immunopathol. Pharmacol., 2014, 27(3), 403-12. Lewis, L.E.; Bain, J.M.; Lowes, C.; Gillespie, C.; Rudkin, F.M.; Gow, N.A.; Erwig, L.P. Stage specific assessment of C. albicans phagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants. PLoS Pathog., 2012, 8(3), e1002578. van de Veerdonk, F.L.; Kullberg, B.J.; Netea, M.G. Adjunctive immunotherapy with recombinant cytokines for the treatment of disseminated candidiasis. Clin. Microbiol. Infect., 2012, 18(2), 112-9. Glocker, E.; Grimbacher, B. Chronic mucocutaneous candidiasis and congenital susceptibility to Candida. Curr. Opin. Allergy Clin. Immunol., 2010, 10(6), 542-50. Kullberg, B.J.; Netea, M.G.; Vonk, A.G.; van der Meer, J.W. Modulation of neutrophil function in host defense against disseminated C. albicans infection in mice. FEMS Immunol. Med. Microbiol., 1999, 26(3-4), 299-307. Ruhnke, M.; Rickerts, V.; Cornely, O.A.; Buchheidt, D.; Glockner, A.; Heinz, W.; Hohl, R.; Horre, R.; Karthaus, M.; Kujath, P.; Willinger, B.; Presterl, E.; Rath, P.; Ritter, J.; Glasmacher, A.; LassFlorl, C.; Groll, A.H. Diagnosis and therapy of Candida infections: Joint recommendations of the German Speaking Mycological Society and the Paul-Ehrlich-Society for Chemotherapy. Mycoses, 2011, 54(4), 279-310. Pappas, P.G.; Kauffman, C.A.; Andes, D.; Benjamin, D.K.; Calandra, T.F.; Edwards, J.E.; Filler, S.G.; Fisher, J.F.; Kullberg, B.J.; Ostrosky-Zeichner, L.; Reboli, A.C.; Rex, J.H.; Walsh, T.J.; Sobel, J.D. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin. Infect. Dis., 2009, 48(5), 503-35. Fuller, R. History and development of probiotics. Probiotics, 1992, 1-8. Ardatskaia, M.D.; Minushkin, O.N. Probiotics in the treatment of functional intestinal diseases. Eksp. Klin. Gastroenterol., 2012, 3, 106-13. Mailander-Sanchez, D.; Wagener, J.; Schaller, M. Potential role of probiotic bacteria in the treatment and prevention of localized candidosis. Mycoses, 2012, 55(1), 17-26. Wagner, R.D.; Pierson, C.; Warner, T.; Dohnalek, M.; Farmer, J.; Roberts, L.; Hilty, M.; Balish, E. Biotherapeutic effects of probiotic bacteria on candidiasis in immunodeficient mice. Infect. Immun., 1997, 10, 4165-4173. Hatakka, K.; Ahola, A.J.; Yli-Knuuttila, H.; Richardson, M.; Poussa, T.; Meurman, J.H.; Korpela, R. Probiotics reduce the prevalence of oral Candida in the elderly-A randomized controlled trial. JDR, 2007, 86(2), 125-130. Manzoni, P.; Mostert, M.; Leonessa, M.L.; Priolo, C.; Farina, D.; Monetti, C.; Latino, M.A.; Gomirato, G. Oral supplementation with Lactobacillus casei subspecies rhamnosus prevents enteric colonization by Candida species in preterm neonates: A randomized study. Clin. Infect. Dis., 2006, 42(12), 1735-42. Kumar, S.; Singhi, S. Role of probiotics in prevention of Candida infection in critically ill children. Mycoses, 2013, 56(3), 204-11. Kohler, G.A.; Assefa, S.; Reid, G. Probiotic interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the opportunistic fungal pathogen C. albicans. Infect. Dis. Obstet. Gynecol., 2012, 2012, 636474. Bohbot, J.M.; Cardot, J.M. Vaginal impact of the oral administration of total freeze-dried culture of LCR 35 in healthy women. Infect. Dis. Obstet. Gynecol., 2012, 2012, 503648. Watson, C.J.; Pirotta, M.; Myers, S.P. Use of complementary and alternative medicine in recurrent vulvovaginal candidiasis-results of a practitioner survey. Complement Ther. Med., 2012, 20(4), 218-21.

Received: November 09, 2014

Revised: February 12, 2015

Accepted: February 16, 2015

Hani et al. [44]

[45]

[46]

[47] [48] [49] [50]

[51] [52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60] [61]

[62]

Ishijima, S.A.; Hayama, K.; Burton, J.P.; Reid, G.; Okada, M.; Matsushita, Y.; Abe, S. Effect of Streptococcus salivarius K12 on the in vitro growth of C. albicans and its protective effect in an oral candidiasis model. Appl. Environ. Microbiol., 2012, 78(7), 2190-9. Matsubara, V.H.; Silva, E.G.; Paula, C.R.; Ishikawa, K.H.; Nakamae, A.E. Treatment with probiotics in experimental oral colonization by C. albicans in murine model (DBA/2). Oral Dis., 2012, 18(3), 260-4. Kumar, S.; Bansal, A.; Chakrabarti, A.; Singhi, S. Evaluation of efficacy of probiotics in prevention of Candida colonization in a PICU-a randomized controlled trial. Crit. Care Med., 2013, 41(2), 565-72. Cassone, A.; Casadevall, A. Recent progress in vaccines against fungal diseases. Curr. Opin. Microbiol., 2012, 15(4), 427-33. http://en.wikipedia.org/wiki/Vaccine. Liu, M.; Clemons, K.V.; Johansen, M.E.; Martinez, M.; Chen, V.; Stevens, D.A. Saccharomyces as a vaccine against systemic candidiasis. Immunol. Invest., 2012, 41(8), 847-55. Han, Y.; Ulrich, M.A.; Cutler, J.E. C. albicans mannan extractprotein conjugates induce a protective immune response against experimental candidiasis. J. Infect. Dis., 1999, 179(6), 1477-84. Han, Y.; Rhew, K.Y. Comparison of two Candida mannan vaccines: The role of complement in protection against disseminated candidiasis. Arch Pharm. Res., 2012, 35(11), 2021-7. Lipinski, T.; Wu, X.; Sadowska, J.; Kreiter, E.; Yasui, Y.; Cheriaparambil, S.; Rennie, R.; Bundle, D.R. A β-mannan trisaccharide conjugate vaccine aids clearance of C. albicans in immunocompromised rabbits. Vaccine, 2012, 30(44), 6263-9. Xin, H.; Cartmell, J.; Bailey, J.J.; Dziadek, S.; Bundle, D.R.; Cutler, J.E. Self-adjuvanting glycopeptide conjugate vaccine against disseminated candidiasis. PLoS One, 2012, 7(4), e35106. De Bernardis, F.; Amacker, M.; Arancia, S.; Sandini, S.; Gremion, C.; Zurbriggen, R.; Moser, C.; Cassone, A. A virosomal vaccine against candidal vaginitis: Immunogenicity, efficacy and safety profile in animal models. Vaccine, 2012, 30(30), 4490-8. Luo, G.; Ibrahim, A.S.; French, S.W.; Edwards, J.E.; Fu, Y. Active and passive immunization with rHyr1p-N protects mice against hematogenously disseminated candidiasis. PLoS One, 2011, 6(10), e25909. Ahmad, E.; Fatima, M.T.; Saleemuddin, M.; Owais, M. Plasma beads loaded with C. albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice. Vaccine, 2012, 30(48), 6851-8. Pietrella, D.; Rachini, A.; Torosantucci, A.; Chiani, P.; Brown, A.J.; Bistoni, F.; Costantino, P.; Mosci, P.; d'Enfert, C.; Rappuoli, R.; Cassone, A.; Vecchiarelli, A. A beta-glucan-conjugate vaccine and anti-betaglucan antibodies are effective against murine vaginal candidiasis as assessed by a novel in- vivo imaging technique. Vaccine, 2010, 28, 1717-25. Torosantucci, A.; Bromuro, C.; Chiani, P.; De Bernardis, F.; Berti, F.; Galli, C.; Norelli, F.; Bellucci, C.; Polonelli, L.; Costantino, P.; Rappuoli, R.; Cassone, A. A novel glyco-conjugate vaccine against fungal pathogens. J. Exp. Med., 2005, 202, 597-606. Bromuro, C.; Romano, M.; Chiani, P.; Berti, F.; Tontini, M.; Proietti, D.; Mori, E.; Torosantucci, A.; Costantino, P.; Rappuoli, R.; Cassone, A. Beta- glucan-CRM197 conjugates as candidate antifungal vaccines. Vaccine, 2010, 28, 2615-23. [60]Spellberg, B. Vaccines for invasive fungal infections. F1000 Medicine Reports, 2011, 3, 1-8. Shibasaki, S.; Aoki, W.; Nomura, T.; Miyoshi, A.; Tafuku, S.; Sewaki, T.; Ueda, M. An oral vaccine against candidiasis generated by a yeast molecular display system. Pathog. Dis., 2013, 69(3), 262-268. Yang, H.; Han, S.; Zhao, D.; Wang, G. Adjuvant effect of polysaccharide from fruits of Physalisalkekengi L. in DNA vaccine against systemic candidiasis. Carbohydr. Polym., 2014, 109, 77-84.