Antifungal susceptibility and virulence determinants profile of candida species isolated from patients with candidemia

Candida is the most prevalent fungal bloodstream infection (BSI) with a high mortality rate among hospitalized patients. Another concern facing physicians is rising global incidence of drug-resistant Candida. This study aimed to characterize the prevalence, antifungal susceptibility, biofilm formation, and virulence genes (HWP1, ALS1, SAP2) of different Candida spp. isolated from patients with candidemia. 52 isolates of Candida spp. were identified from blood cultures by chromogenic Candida agar and confirmed by the VITEK 2 system. Isolates were tested for antifungal susceptibility by disk diffusion and VITEK 2 system. Biofilm formation and investigated genes were detected by the Congo red method and conventional PCR, respectively. Candida spp. caused 2.3% of detected BSIs, of which 32.7% were caused by Candida albicans (C. albicans) and 67.3% by non-albicans Candida (NAC), with the predominance of C. tropicalis (25%), followed by C. parapsilosis (17.3%), and C. krusei (13.5%). The susceptibility rates to fluconazole, voriconazole, caspofungin, micafungin, amphotericin B, and flucytosine were 64.7%, 76.5%, 100.0%, 100%, 100.0%, and 100.0% in C. albicans, while 53.6%, 71.4%, 91.4%, 91.4%, 94.3%, and 94.3% in NAC, respectively. Biofilm production, HWP1, ALS1, and SAP2 were detected in 70.6%, 82.4%, 76.5%, and 52.9% of C. albicans and 74.3%, 85.7%, 80.0%, and 48.6% of NAC, respectively. There is remarkable shift to NAC BSIs and high azole resistance. Antifungal stewardship and analysis of risk factors associated with this shift are needed.


Isolation of Candida
Blood culture bottles which alarm positive were subcultured on Sabouraud dextrose agar (SDA) medium supplemented with chloramphenicol (0.5 g/l) (Oxoid, UK) then incubated for 24-48 h at 37 °C.Colonies were then identified by standard methods (colony morphology, Gram stain and germ tube) 7 .Identifying Candida spp. in at least one positive blood culture from patients having symptoms and signs of infection was considered BSI with Candida 8 .A patient with multiple episodes of candidemia had one specimen included.Blood cultures from patients having incomplete records were excluded.Nutrient broth supplemented with 20% glycerol was used to preserve confirmed Candida isolates at -80 °C9 .

Identification of Candida spp.
According to manufacturer guidance, HiCrome™ Candida Differential Agar (Himedia, India) was used to facilitate provisional rapid differentiation of candida spp.within 48 h of incubation at 37 °C based on colony morphology and color.VITEK 2 compact System (bioMerieux, France) was then used to confirm the results using identification cards YST.

Antifungal susceptibility testing
Following the Clinical and Laboratory Standards Institute 10 , disk diffusion method was used to assess the antifungal susceptibility of candida isolates then confirmed by VITEK 2 system using sensitivity cards AST-Y08.The utilized antifungal disks (LIOFILCHEM, Italy) included fluconazole (FLU) 100 µg, voriconazole (VO) 1 µg and caspofungin (CAS, 5 μg).The reference strains C. albicans ATCC 90028, was employed as quality control.

Detection of biofilm formation with Congo red agar method
Candida isolates producing black colonies on Congo red agar during a 48-h incubation at 37 °C, were considered biofilm forming isolate 11 .

Genotypic detection of virulence genes (HWP1, ALS1 and SAP2) by conventional multiplex PCR
Using the QIAamp DNA Mini Kit 50 tests (Qiagen, Germany, cat.no.56304), Candida DNA was extracted and purified as per manufacturer's instructions.With the use of a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, USA), the amount and quality of the DNA were examined.The sequences of used primers and PCR conditions were followed as previously described by 12,13 (Table 1).The PCR amplification was done using Table 1.Primers and PCR conditions used for PCR for detection of investigated genes.
HWP1 and ALS1 genes were significantly (P value < 0.05) higher among biofilm forming C. albicans and NAC isolates compared to non-biofilm producing isolates.However, SAP2 gene was higher among biofilm forming C. albicans and NAC isolates but with no statistically significant difference (P value > 0.05) (Table 4).

Discussion
In hospital settings, candidemia continues to be the most common invasive fungal infection with a high rate of morbidity and mortality.Studies that directly compare the epidemiology and treatment approaches of different countries are rare in real life and have the potential to provide more focused insights on enhancing clinical outcomes 14 .
In the current study, 2.3% of BSIs was caused by candida spp.This is nearly matched with previous studies of Alkharashi et al. 8 in KSA (2.8%) and El-Mahallawy et al. 6 in Egypt (3.1%).Higher prevalence of candidemia were reported among pediatric patients by studies presented by Karaağaç et al. 15 in Turkey (12.9%),Khairat et al. 16 in Figure 1.Distribution of isolated candida spp.among patients with candidemia.

Egypt (17.3%
).An Egyptian study performed by Reda et al. 3 documented that 1.6% of BSIs in adults and 10.8% in children were caused by Candida spp.
The exact species distribution among patient with candidemia display considerable geographical, hospitalto-hospital, and even unit-to-unit diversity because of risk factors and practices.Despite C. albicans is still the most frequently isolated species, a progressive shift to NAC spp. is recently reported in most parts of the world 17 .Furthermore, species distribution might be affected by the kinds of antifungal drugs empirically used.Fluconazole use has been shown to increase the risk of C. glabrata and C. krusei infections, whereas caspofungin increases the risk of C. parapsilosis, C. glabrata, and C. krusei infections 18 .
Regarding distribution of NAC in this study, there is predominance of C. tropicalis 25% followed by C. parapsilosis 17.3% and C. krusei 13.5%.This is in line with previous studies 3,29,30 .In contrast, in studies in Israel and Nordic countries C. glabrata was the principal NAC causing candidemia 31,32 .In a study conducted in Egypt, C. krusei was the most predominant NAC (28%) followed by C. parapsilosis (20%) and C. tropicalis (16%) among ICU patients 33 .In several studies C parapsilosis was the most frequent NAC in children 26,34,35 .
In the present study, age had significant effect on Candida spp.distribution among patients.In previous studies analyzing species distribution in association with patient age, they observed that C. glabrata was the most prevalent isolate in elderly patients, while C. parapsilosis was more frequent in younger patients.C. albicans and C. tropicalis were isolated from all age groups 36,37 .
Further studies with a larger number of isolates, preferably multicenter studies, should be performed to confirm our findings and retrieve more statistically significant results.Also further researches were needed to discuss the association of Candida spp.virulence determinants (biofilm capacity and their related genes) with the clinical course and prognosis of affected patients.Additionally, relation between prophylaxis with antifungal drugs and the prevalence of Candida spp.should be investigated in future researches.

Conclusion
There is remarkable rising incidence of NAC BSIs and high rates of fluconazole resistance highlighting the need for continuous candidemia surveillance, antifungal stewardship to maintain antifungal efficacy and analysis of risk factors associated with shift to NAC candidemia.Future research into the molecular mechanisms underlying azole resistance is recommended.

Table 2 .
Demographic and clinical characteristics of patients with candidemia.

Table 3 .
Antifungal susceptibility and virulence determinants of candida isolates.Significant values are in [italics].*Comparison between C. albicans and Total NAC.---C.kruzei has intrinsic resistance to fluconazole.

Table 4 .
Association between investigated genes and biofilm formation among candida isolates.a Comparison of biofilm and non-biofilm forming isolates of C. albicans.b Comparison of biofilm and non-biofilm forming isolates of Non-albicans candida.
b resistance in NAC and C. albicans were 64.3% and 50.0%respectively.This agreed with another Egyptian study conducted at Cairo University on pediatric patients in which they found high resistance rate to fluconazole among C. albicans (38.9%) and NAC (44.0%) causing BSIs 16 .Another study in ICU at Ain Shams University Hospital, Egypt, showed that all identified candida spp., including C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis had high resistance to voriconazole and fluconazole (38.4% and 38.5%; 21.5% and 100.0%; 100.0% and 40.0%; 12.5% and 25.0%; and 10.0% and 20.0%, respectively 33 .In Solomon et al., study 38 , all Candida isolates were susceptible to caspofungin, micafungin, amphotericin B and voriconazole.While C. parapsilosis and C. auris showed complete resistance to fluconazole and C. tropicalis was resistant to flucytosine.