The incidence of human fungal infection has dramatically increased over the past three decades. This may be attributed to the HIV/AIDS epidemic, the increased use of indwelling medical devices, increase in broad spectrum antibiotics use, use of immunosuppressive therapy, and the increase in the number of people with advanced age (Silva et al., 2012). Candida albicans remain the leading cause of fungemia amongst the Candida species, however non-albicans Candida species incidence are on the rise since 1990 (Tavanti et al., 2005).
Candida species cause 8 - 10% of nosocomial blood stream infections (BSI) and are the fourth leading cause of BSI in the United State. Data from ARTEMIS DISK Antifungal Surveillance program (1997 - 2003) showed an increase in the number of Candida species isolated. The specimens analysed were collected from patient with invasive candidiasis (IC) from 127 institutions in 39 countries. The isolation rate of C. parapsilosis increased from 4.2% to 7.3% over a period of 6.5-years. C.parapsilosis was the second most prevalent Candida species globally (Pfaller et al. 2011), especially in Latin America, Asia and some European country. It is the leading cause of candidemia in many neonatal ICU, even exceeding C .albicans (Huang et al., 1999; Levy et al., 1998; da Silva et al.; 2001). Recent studies indicate almost five to six fold increase in the incidence of C. parapsilosis infection in the neonate, accounting for 67% of candidemia in neonatal ICU (Rodriguez et al., 2006). C. parapsilosis can carry on in units or patients for years .REF.
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Ashford was the first person to isolate C. parapsilosis from a stool of a patient with diarrhoea in 1928 in Puerto Rico. He described it as a species of Monilia that was a non-maltose fermenter. He named the species Monilia parapsilosis, to differentiate it from Monilia psilosis (now known as Candida albicans). It was initially considered to be non-pathogenic until in 1940 when it was described as the causative organism of a fatal endocarditis in a 48years old intravenous drug abusing male (Trofa et al. 2008). Prior to 2005, C. parapsilosis was divided into three groups, I, II and III because of genotypic heterogeneity. Further genetic studies showed the distinct differences in the genes of the three closely related "group'' that led to the renaming of group II and III to C. orthopsilosis and C. metapsilosis respectively (Tavanti et al., 2005). Group I is called C.parapsilosis sensu stricto. The authors also postulated the evolution of the C. parapsilosis sensu lato/group/complex and that the species were not novel yeast. They postulated that the progenitor of both C. parapsilosis and C. orthopsilosis was C. metapsilosis (Tavanti et al., 2005).
The increase in the incidence of nosocomial blood stream infections caused by C.parapsilosis has stimulated interest in this group. Various studies looking at the differences in geographic distribution, antifungal susceptibility and pathogenesis have emerged. C. orthopsilosis (6.1%) and C. metapsilosis (1.8 %) accounted for less than 10% of infections caused by C .parapsilosis group globally. Uneven geographic distribution exist, in Italy the incidence was 8 % C. orthopsilosis and 5% C. metapsilosis while in South Africa both species accounted for only 0.7% each. (Lockhart et al., 2008). C. orthopsilosis was the 5th (0.020 per 1000 admission) cause of fungemia in a study conducted at different tertiary hospital around Spain, with majority of patients admitted in the general ward (63%) and were mostly elderly patients (Perman et al.,2012)
Figure 1: Percentages of C. parapsilosis isolates that were C. orthopsilosis by year (P = 0.01 for an increasing trend over time in the proportion of all isolates that were C. orthopsilosis).
Source: (Lockhart et al., 2008).
Species distribution difference by age group was also observed in patients with fungemia, none of the neonate had candidemia caused by C. orthopsilosis or C. metapsilosis. The percentage of C. orthopsilosis and C. metapsilosis increased with increasing age and was highest in the elderly group (CantoÂ´n et al., 2011).
Table 1: Species distribution by age group.
Source: CantoÂ´n et al., 2011.
C. parapsilosis is a normal skin commensal (Trofa et al., 2008) with the potential to cause superficial and systemic diseases .REF. In addition it is found ubiquitous in nature (Posteraro et al., 2004). It is the Candida species that is often isolated from the hand of the health care workers (HCW), subsequently leading to catheter related blood stream infection (BSI) as a result of central venous catheter manipulation. Bonassolli et al. showed a high colonization rate (51%) by C. parapsilosis compared to other yeast on the hands of healthy individuals including HCW (Bonassolli et al., 2004). HCW are considered to be the potential reservoirs for horizontal nosocomial transmission to susceptible patients. Patients do not need prior colonization of the mucosal surfaces with C. parapsilosis to be infected as the infection can be acquired exogenously (Zancopé-Oliveira et al., 2001). C. parapsilosis is as less virulent yeast compared to C. albicans and C. tropicalis (Almirante et al., 2006; Weems JJ Jr. 1992). It is known for causing infections in neonate especially those with low birth weight, infants, transplant recipient; patient receiving total parental nutrition (TPN) and patient in intensive care unit especially if they have indwelling devices in-situ or are receiving lipid based solution (Nguyen et al., 2011; Silva et al., 2012; ). Outbreaks or sporadic cases have been reported which are connected with contaminated fluids, monitoring equipment e.g. blood pressure machines. C. parapsilosis is clearly an opportunistic pathogen that is well adapted to the host niche.
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Although most cases are nosocomial infections, outpatient cases have been reported; Almirante and colleagues reported 5% (5 of 78 cases) community acquired C.parapsilosis infections (Almirante et al., 2006). However the study did not look into the risk factors associated with C.parapsilosis infection in this group of patient e.g. prior antibiotic use, transplant recipient, indwelling medical devises or malignancy. In another study conducted in Maryland and Connecticut in United State C. parapsilosis was responsible for 16% (57 of 356 cases) of community-onset candidemia. Patients had regular interaction with the health care facilities and recognized risk features for candidemia, including central venous catheter in-situ, prior use of systemic antibiotics and immunosuppressive agents were found. History of hospitalization in the past 90 days was also documented to be a risk factor in this group of patients. Interestingly there were patients with no previous hospitalization history (Sofair et al., 2006).
Check TROFA: RE: rate of death in neonates high
Phenotypic markers of pathogenesis: Virulence factors
A combination of virulence factors contribute to C. parapsilosis pathogenesis. The consequences of invasive C. parapsilosis can be devastating especially in immunosuppressed hosts. To be able to control the rise in C. parapsilosis infections it is crucial to understand its virulence factors. Most of the understanding of the putative virulence factors of C.parapsilosis was through studies that compared it with C. albicans. These virulence factors will be discussed below.
Adhesion and biofilm formation
Adherence to host tissue and plastic surfaces e.g. to a catheter by C. parapsilosis is the initial step to colonization which may subsequently end as invasive candida infection. (el-Azizi and Khardori .1999). Adhesins are surface proteins which partake in cell adherence; they are different for the different Candida species.
Duncan and his colleagues showed no correlation between adherence and strain pathogenicity in C. parapsilosis outbreak. (Duncan et al. 2004)
C. parapsilosis has the propensity to form biofilms on the surface and the lumen of catheters and other indwelling medical devises. Biofilms are highly organized population of microorganisms that are attached to the surface and to one another and are walled within an extracellular matrix. (Trofa et al., 2008). Biofilm formation contributes to pathogenesis by allowing the yeast to invade the host tissues. Steps in biofilm formation include adhesion, morphogenic conversion, polysaccharide production, induction of drug resistance, maturation and dispersion (Ramage et al. 2001, Ramage et al, 2005). C. albicans biofilm formation involves the dimorphic switch from the yeast to hyphal growth and consists of two layers, the thin basal yeast layer and the thicker hyphal layer. In contrast, C.parapsilosis does not form true hyphae but form pseudohyphae when attached to plastic surfaces. The biofilm formed in C. parapsilosis are a combination of pseudohyphae and the yeast cell and enormous quantity of carbohydrates ((Ding and Butler. 2007; Ding et al.2012). The biofilm formed is structurally less complex than in C. albicans (Trofa et al. 2008). Microorganisms found in biofilm tend to have unique characteristics compared to the rest of the freely floating counterparts (Kojic and Darouiche. 2004). These characteristics include resistance to antifungal drugs (Pfaller et al. 2006) and immune evasion (Ramage et al. 2001.)
Correlation between biofilm formation and pathogenicity has been shown in several studies: The study of Kuhn et al. (Kuhn et al. 2004), which looked at the pathogenicity of C.parapsilosis outbreak strain versus nonoutbreak strains revealed the significant difference in biofilm formation. The amount of biofilm formed by outbreak strain was much higher. C. parapsilosis sensu stricto (Group 1) was found to form biofilm more than C. orthopsilosis and C. metapsilosis (Song et al. 2005). This group is more associated with bloodstream infection and transmission through the hand of health care workers. This attribute (i.e. biofilm formation) may be the reason why C.parapsilosis is the predominant species that is isolated in fungemia and in outbreaks, and also the reason why infection can persist. Biofilm formation relationship to invasiveness. More invasive( those from normally sterile sites e.g. such as catheters and blood) isolates produce more biofilm .The degree of biofilm formation was shown to be associated with the geographic and anatomical origin of the isolates. Isolates from blood and cerebrospinal fluid were common producer of biofilm as compared to isolates from other sites e.g. nails and mucosa. Of note is the significant difference between biofilm formation by blood and catheter isolates, with the former producing more biofilm. Greater number of isolates obtained from Hungary and Argentina produced biofilm compared to isolates from Italy (Tavanti et al. 2010). ? Hungary and Argentina Latin America
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The molecular basis of biofilm formation has been extensively studied in C. albicans. Earlier studies revealed the importance played by morphogenesis in C. albicans biofilm formation, whereby hyphae are crucial foundations for structural integrity. From these studies different genes which encoded regulatory proteins for biofilm development were identified. However there is paucity of studies with regard to C. parapsilosis biofilm regulatory signals. BCR1 (Biofilm and Cell wall Regulator) has been shown to be important regulator in biofilm development in C. albicans. Ding and Butler showed the importance of BCR1 for biofilm development in C. parapsilosis as well, by demonstrating a reduction in biofilm mass and depth after deletion of the BCR1. The difference between the depths of biofilm formed by the wild-type strain and the biofilm formed by the C. parapsilosis bcr1 knockout was > 80Âµm (Ding and Butler. 2007). Therefore there is biofilm formation defect in C. parapsilosis bcr1 knockout. This suggests that this type of mutation renders C.parapsilosis less virulent and susceptible to antifungal therapy and may need to be exploited further as target for antifungal agents.
C. parapsilosis secrete extracellular hydrolytic enzymes which contribute to its pathogenesis. The enzymes are produced constitutively or are induced, leading to the host cell membrane architecture derangement and subsequent malfunctioning (Júnior et al. 2011). These enzymes; phospholipases, lipases, haemolysin and aspartyl proteinases have different roles that facilitate invasion and destruction of the host tissues.
Phospholipase is an enzyme that hydrolyses phospholipids, which are the major component of the cell membrane. It degrades the cell membrane component and cause direct tissue damage and cell lysis (Mahmoud. 2000).
Júnior et al. investigated the in vitro activity of phospholipase amongst the clinical isolates of Candida species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis). Only 15.4% (2 out of 13 isolates) of C. parapsilosis were phospholipase producers, which was the lowest amongst the 4 species (Júnior et al. 2011). This is in contrast with the study by Ge et al., where a significantly high number of their isolates were phospholipase producers 90.5% of C. parapsilosis and 91.7% of C. metapsilosis (Ge et al., 2011).
Still need to check putative gene encoding phospholipase in C.parapsilosis and compare mutant strains and wild type in terms of phospholipase production.
Lipases are enzymes that hydrolyse and synthesise of triacylglycerol (Sonia et al., 2011; Trofa et al., 2008). Four genes encoding for lipases in C. parapsilosis have been recognized, CpLIP 1 - 4 (Nguyen et al., 2011). CpLIP 2 encode for an active lipase protein (Trofa et al. 2008). Secreted extracellular lipases are multifunctional enzyme with the following putative roles :( 1) Digest lipids for nutrient acquisition, which may explain the preference of C. parapsilosis for lipid- rich total parenteral nutrition that is administered to low-birth infants. Lipase contributes to the capacity of C. parapsilosis to thrive in lipid-rich media (Gácser et al. 2007). (2) Immune evasion: lipase negative mutants were shown to be easily phagocytosed by macrophages compared to the wild type. This finding suggests that lipase is capable of protecting C. parapsilosis against the host immune system mechanisms (Gácser et al. 2007). (3) Play a role in biofilm formation, as shown by the decreased biofilm formation by lipase negative mutant when compared to the wild type C. parapsilosis (Gácser et al. 2007). (4) Involved in host tissue damage during C. parapsilosis infection and the use of lipase inhibitors can combat tissue damage (Gácser et al. 2007).
Secreted aspartyl proteinases (Saps)
Secreted aspartyl proteinases (Saps) are one of the secreted enzymes that have a role in the pathogenesis of C. parapsilosis. C. parapsilosis possess three SAP genes, SAPP1 to SAPP3 which encode Sapp1p to Sapp3p isoenzyme. Of the three types of isoenzyme Sapp1p has been well enzymologically characterised (Trofa et al. 2008). The three Saps isoenzyme possess the ability to acquire nitrogen sources for nutrients by breaking down the host proteins (HorvaÂ´th et al. 2012). The host proteins that are digested include proteins that form the structure of host tissues and those which participate in the immune system e.g. cytokines, complement (Fisher et al. 2011). Lipases are also involved in tissue adhesion and invade the host by disrupting the integrity of the host cell surface structure (HorvaÂ´th et al. 2012).
HorvaÂ´th and colleague recently studied the role of Sapp1 isoenzyme in the host-pathogen interaction of C.parapsilosis. They identified the 2 copies of SAPP1 gene (SAPP1a and SAPP1b) in the genome of C. parapsilosis which had indistinguishable DNA sequences. Sapp1p was found to evade the immune system by degrading the components of the complement system of the host. In addition they were found to equip the organism with the advantage of surviving in the macrophages by inhibiting phagolysososome fusion (HorvaÂ´th et al. 2012).
The quantity of Saps produced is not proportional to its virulence. The quantity of Sapp production from C. parapsilosis isolates causing fungemia was less impressive when compared to C.parapsilosis from skin isolates. However the fungemia causing isolates were found to be more virulent than skin isolate in neutropenic mice (De Bernardis et al. 1999). De Bernardis and colleagues also documented that C. parapsilosis isolates from skin have better capability to cause rat vaginitis than their fungemia- causing counterpart. This supports the author's hypothesis that isolates which produce more Sapp were mostly causing superficial rather than systemic invasion. (De Bernardis et al. 1999). Saps in C. albicans were found to be more crucial for adhesion than invasion of mice's gut mucosa as the mutant strains revealed no difference in virulence with the wild type C. albicans (Kretschmar et al. 2002). Extrapolating from the above information could explain why the fungemia- causing isolates of C.parapsilosis were able to cause systemic infection despite their low Sapp production.
Pepstatin is a low molecular weight, potent inhibitor specific for acid proteases.
Iron has been shown to be important for growth of Candida species (Fisher at al. 2011), and for initiating infection in the host (Malcok et al. 2009). Human iron is not readily available for use by pathogens. Pathogens need mechanism that assists in the acquisition of this essential element which they acquire from iron-containing molecules, mostly from haemoglobin or haemin. The enzyme haemolysin is used to source out elemental iron from haemoglobin or haemin (Sonia et al., 2011; Fisher et al. 2011; Malcok et al. 2009). Haemolysins lyse the erythrocytes resulting in the liberation of the elemental iron and therefore favours proliferation in the host serum. Haemolysis enables the fungi to invade the host tissue and cause infection (Franca et al. 2009). The activity of haemolysins is enhanced in the presence of glucose.
Earlier studies revealed the absence of haemolysins in C.parapsilosis despite the use of glucose supplemented media (Lou et al. 2001). However Bonassoli et al showed the presence of haemolysins in almost all the isolates that were tested (25 of the 26 isolates), with 61.5 % of the isolates producing total haemolysis and 34.6 producing partial haemolysis (Bonassoli et al. 2004). Of note is that the above two studies used sheep blood agar with different glucose concentration, 3% glucose was used by Lou et al vs. 7% glucose used by Bonassoli et al. As mentioned earlier the activity of haemolysins is enhanced in the presence of glucose. However this does not account for the difference observed in these studies, as C.parapsilosis isolates from Malcok and colleague study demonstrated the haemolytic activity with the use of 3% glucose-enriched media (Malcok et al. 2009). Genetic variability between the three species of C.parapsilosis complex may explain the variability in the haemolytic activity. Both studies did not speciate the isolates.
C.parapsilosis isolates show the least haemolytic activity (6.8%) when compared with other Candida species; with the highest haemolytic activity (49.6%) observed with C. albicans isolates (Malcok et al. 2009). Haemolytic activity is therefore directly correlated with virulence. Isolate from tracheal aspirate showed more haemolytic activity than blood isolates (Franca et al.), while no variation was demonstrated
Molecular Epidemiology Technique
Identification of Candida species can be achieved in the routine diagnostic laboratory using commercially available kits which are based on assimilation tests; but cannot distinguish between the three species of C.parapsilosis complex. The three species of C. parapsilosis are indistinguishable morphologically and physiologically, however they can be distinguished into three distinct species by the use of molecular techniques (Dizbay et al. 2008). In epidemiological studies the application of molecular techniques facilitates the following activities: disease surveillances; outbreak investigation in order to show the clonal relationship among the isolates (Dizbay et al., 2008); to identifying the transmission pattern, risk factors and appropriate infection control measures to be instituted (Dizbay et al., 2008); to characterise host-pathogen interaction; provide better understanding of disease pathogenesis at molecular level (REF); to distinguish microevolutionary changes (Enger et al. 2001) and to determine appropriate antifungal therapy (Merhendi et al. 2006). There is no "gold standard" for typing C. parapsilosis therefore to understand the epidemiology of C. parapsilosis at molecular level several methods are used (van Asbeck et al. 2009).
DNA fingerprinting methods
Restriction fragment length polymorphism (RFLP)
This method is the first DNA-base fingerprinting method that was implemented to study the relatedness of the pathogenic fungi. The procedure involves extraction of DNA and digestion by restriction endonuclease enzymes, the fragment are then separated by electrophoresis on agarose gel. This is followed by analysis of the banding pattern (Soll, 2000). It was through the use of RLFP in the earlier studies that C. parapsilosis was divided into three groups (1, 11, and 111) and five subtypes; more subtypes have subsequently been discovered. The most common RFLP subtype is subtype VII-1 (van Asbeck et al. 2009).
Deresinski and colleague were able to identify the origin of a C.parapsilosis isolated from six clinical specimens from five patients. No clinical indication of C.parapsilosis infection was seen on the patients. The RFLP pattern resulting from EcoRi and HindIII digestion of the six clinical isolates were identical to each other and to that of C. parapsilosis cultured from Hanks' balanced salt solution (HBSS) used in the laboratory during specimen processing. The use of the RFLP technique was able to reveal the relatedness and the origin (i.e. the HBSS) of the cluster of C.parapsilosis isolated. The C. parapsilosis genotype isolated in the study had not been previously identified (Deresinski et al. 1995). The findings saved patients from unnecessary medical intervention and also highlight the fact that laboratory personnel can contaminate clinical specimen and lead to pseudoepidemic. Molecular typing therefore played a role in the investigation of the ''outbreak''. The source of HBSS contamination was not identified but could have been from the manufactures, hands of the laboratory personnel or the surrounding surfaces in the laboratory.
Tavanti et al, based speciation of C. parapsilosis group
The SADH gene has been the basis of sequencing analysis or Ban1-RLFP group since the new species were described by Tavanti and colleagues in 2005 (Tavanti et al. 2005). The use of Ban1 was found unreliable for identifying C. metapsilosis, instead suggested the use of Nla111 (Hossein M et al. 2010)
The 3 groups are unrelated at species level.
Randomly amplified polymorphic DNA (RAPD) profile
"This technique is based on the amplification of random fragments of DNA" (Lathar et al. 2010). The DNA sequence of the target gene is not known as the primers will bind randomly in the sequence. Lehman et al. (1992) used RADP to study the genotypic relatedness of medically important Candida species; the RADP analysis differentiated the homogenous C. parapsilosis into 3 distinct groups.
Sequence based methods
Dendrogram: phylogenetic tree showing the relatedness among the isolates which was constructed based on genetic similarities.calculate similarity index S ab
Has been shown to be reproducible and reliable in id candida spp
Used to confirm species id and assess genetic relatedness
C. parapsilosis, shows too little sequence diversity to be typeable by MLST
Strain typing by sequencing of several housekeeping genes in a microbial species (multilocus sequence typing; MLST) has rapidly developed as a reliable technology for epidemiological studies of infectious disease (5, 23). The method involves determination of DNA sequence polymorphisms between isolates with a set of fragments of five to seven genes, which are ideally under neutral selective pressure and chromosomally dispersed. The data obtained are highly reproducible, amenable to statistical analyses to quantify similarities and putative genetic relationships between isolates, and able to be stored in a single central database for global internet access.
Among fungal diseases, deep-seated Candida infections are the most commonly encountered opportunistic problems that affect seriously immunocompromised or debilitated hosts. Superficial Candida infections are responsible for considerable morbidity among neonates, the elderly, and patients with AIDS (oral infections) and among women of childbearing age (vulvovaginal infections). Candida albicans accounts for the majority of these infections, but other Candida species, particularly C. glabrata, C. parapsilosis, and C. tropicalis, are by no means uncommon causes, with some authorities documenting a rise in prevalence of the latter at the expense of C. albicans. There is therefore a clear need for strain typing of these species for epidemiological purposes. MLST technology has been developed for Candida albicans (3, 4, 22) and C. glabrata (7). MLST could not be used for C. parapsilosis because of the paucity of allelic polymorphisms in this species (20). The MLST approach provides for definition of population structures within a species and can reveal differences in geographical origins, anatomical sources, and other properties between clades of closely related isolates (2, 3, 7, 19, 21). J Clin Microbiol. 2005 November; 43(11): 5593-5600
Tavanti et al. BMC Microbiology 2010, 10:203Arianna Tavanti , Lambert AM Hensgens , Selene Mogavero , László Majoros , Sonia Senesi , Mario Campa. Genotypic and phenotypic properties of Candida parapsilosis sensu strictu strains isolated from different geographic regions and body sites
The mechanisms of bioï¬lm resistance to antimicrobial agents - Candida bioï¬lms and their role in infection. TRENDS in Microbiology Vol.11 No.1 January 2003. L. Julia Douglas. (FILE IN FLASH DISC. CANDIDA BIOFILM.2003)
Journal med micro 2010 vol59 no4Â 414-420. Hossein M et al
ITS and the 26S rRNA gene sequence are well documented known targets for most of the taxonomic studies in nearly all microorganism. They are the basis of routine and research id of fungal pathogens and recognizing new species
The ITS molecule contains several regions of highly conserved sequence useful for obtaining proper sequence alignments, but with sufficient sequence variability in other regions of the molecule that can serve as markers for of species-species RFLP (Hossein et al 2006. Japanes journal of med.mycology)
conserved 18S-ribosomal DNA (rDNA) sequences shared by most fungi
A variety of methods are utilized for DNA strain subtyping of Candida spp. because no 'gold standard' exists.
The universal fungal oligonucleotide primer pair, ITS3 and ITS4, amplifies portions of the 5.8S ad 28S rDNA subunits, and the ITS2 region. Although rRNA genes are highly conserved, the ITS regions are distinctive.
pulsed-field gel electrophoresis (PFGE) represents the gold standard for typing, but is also one of the most lengthy and expensive, while simple sequence repeats (SSRs) is based on polymerase chain reaction (PCR) amplification and is, therefore, faster.
MLST multilocus sequence typing
rRNA gene sequencing
Echinocandins are lipopeptide antifungals which inhibit 1, 3-ß-D-glucan synthase, the enzyme that is responsible for synthesis of the integral part of fungal cell wall 1, 3-ß-D-glucan. Douglas et al identified FKS gene, which encode for the subunits of the enzyme 1, 3-ß-D-glucan synthase to be the target gene for Echinocandins (Douglas et al. 1997). The enzyme complex consists of two subunits the Fks and Rho; the former is the catalytic subunit of the enzyme (Perlin. 2007) and the latter regulate a number of cellular processes (Kondoh et al., 1997). Fks is coded by three genes related to each other: fks1, fks2 and fks3 (Kondoh et al., 1997).
These drugs include caspofungin, anidulafungin and micafungin; have been proven to be effective against medically important yeast and moulds (Denning. 2003). However reduced susceptibility has been observed in Candida spp, more so in C. parapsilosis and C. guilliermondii; which show high MIC (Axner-Elings et al. 2011; REF)
Reduced susceptibility is due to mutation of FKS gene (Perlin. 2007). Two regions on FKS gene called "hot-spot 1(HS1)'' and ''hot-spot 2 (HS2)''were identified through genetic studies by Park and colleagues (Park et al. 2005) and mutation in one or both regions led to reduced Echinocandin susceptibility. The intrinsic reduced susceptibility displayed by C. parapsilosis, C. orthopsilosis, and C. metapsilosis was proved to be due to a naturally occurring amino acid substitution of Proline at amino acid position 660 by Alanine at the hot-spot 1 region of FKS. Higher MIC (4- 21 fold higher than wild type strains) were also observed in C. albicans and C. glabrata with amino acid substitution in the highly conserved region of Fks1/Fks2 (Garcia-Effron et al. 2008). ''In addition C. orthopsilosis and C. metapsilosis have isoleucine-to-valine substitution in the hot spot 2 region of Fks1 which is not associated with drug susceptibility'' (Riccombeni et al.,2012). The finding support that mutation of FKS gene is indeed attributed to be the cause of reduced Echinocandins susceptibility in Candida species. Although the naturally occurring FKS gene mutation in C. parapsilosis result in high MIC for echinocandins, the sensitivity of glucan synthase to this drugs was not remarkable reduced. This was in contrast with other Candida species with acquired FKS mutation; they displayed reduced glucan synthase sensitivity and were associated with clinical failure (Beyda et al., 2012).
The higher MIC for the C. parapsilosis group was also established in a study in Portugal, isolates included clinical specimen from various anatomical sites and environmental specimens. C. parapsilosis sensu stricto was isolated in 91.4 % of the isolates and 38% were from blood. C. orthopsilosis and C. metapsilosis were isolated in less than 10% of the isolates and none were from blood. This report showed higher caspofungin and anidulafungin MIC for C. parapsilosis sensu stricto, 38% were nonsusceptible with MIC ranging from 4 to 32mg lË‰Â¹, however C. metapsilosis and C. orthopsilosis isolates were all susceptible for caspofungin and anidulafungin with MIC less than 2 mg lË‰Â¹ (Silva et al. 2009). The higher MIC by the C.parapsilosis group was also established in the Brazilian study by Bonfietti et al. The caspofungin MIC range for C. parapsilosis and C. orthopsilosis was 0.25 -2mg lË‰Â¹ and 0.25mg lË‰Â¹ C. metapsilosis (Bonfietti et al. 2012). The findings of the Spanish multicentre study were in agreement with the above study, all C. metapsilosis and C. orthopsilosis were susceptible to the three Echinocandins (caspofungin, anidulafungin and micafungin) tested, with MIC ranging from 0.003 to 2mg lË‰Â¹, conversely C. parapsilosis sensu stricto MIC were much higher; anidulafungin (0.016 - 8mg lË‰Â¹), caspofungin (0.008- 8mg lË‰Â¹), and micafungin 0.008 - 16mg lË‰Â¹) (Canton et al. 2011). The antifungal susceptibility profile of the three species is clearly discrepant, with C. parapsilosis sensu stricto more resistant than the two related species. Looking at the data it may seem appropriate to know the predominant species in our setting in order to institute appropriate empiric treatment for at risk patients with suspected fungemia e.g. low birth weight neonates admitted to the neonatal ICU. However the clinical efficacy of the three echinocandins in treating candidemia associated with C.parapsilosis group was proved to as good as that observed with other susceptible Candida species(.?PFALLER 2008, 2010.) The risk for clinical failure was minimal. Despite the evidence the Infectious Disease Society of America guidelines do not recommend the use of echinocandins as the first-line therapy for C.parapsilosis candidemia.
Molecular techniques and in vitro susceptibility testing used together have the potential to detect mechanisms that confer resistance to echinocandin and strains of Candida species with potential resistance to echinocandin.
Azoles are frequently used for treatment and prophylaxis of Candida infections and other yeast. They inhibit ergosterol synthesis, the major part of cell membrane by targeting 14âˆ-demethylase. This enzyme is produced by ERG11 gene REF. The extensive use of Azole has been associated with an increase in resistance in C. parapsilosis. REF.
Acquired resistance to Azole drugs is linked to the following mechanisms:
(1) Modification in the quantity and quality of the target enzyme, 14âˆ-demethylase. Exposure to azole drugs has been linked to development of resistance through point mutation in the active region of the ERG11 gene leading to reduced affinity to azoles; however the extent of the effect was depended on the azole in question. Fluconazole and voriconazole are more affected than posaconazole and itraconazole. The latter two have long side chains that have the capacity to bind to another area of the enzyme, and hence they retained their activity against the isolates.
(2) The active efflux of the drug out of the cell through efflux transporters; (3)
We found that after incubation with fluconazole or voriconazole, strains became resistant to both azoles but not to posaconazole, although susceptibility to this azole decreased, whereas the strain incubated with posaconazole displayed resistance to the three azoles. The resistant strains obtained after exposure to fluconazole and to voriconazole have increased expression of the transcription factor MRR1, the major facilitator transporter MDR1, and several reductases and oxidoreductases. Interestingly, and similarly to what has been described in C. albicans, upregulation of MRR1 and MDR1 is correlated with point mutations in MRR1 in the resistant strains. The resistant strain obtained after exposure to posaconazole shows upregulation of two transcription factors (UPC2 and NDT8) and increased expression of 13 genes involved in ergosterol biosynthesis. This is the first study addressing global molecular mechanisms underlying azole resistance in C. parapsilosis; the results suggest that similarly to C. albicans, tolerance to azoles involves the activation of efflux pumps and/or increased ergosterol synthesis.