Introduction

Invasive fungal infections in immunocompromised individuals have been commonly linked to candida bloodstream infections (BSIs) in hospital settings. Candida is the fourth most prevalent cause of nosocomial BSIs in the US, the third detected cause of fungal sepsis in Europe and had a 37% mortality rate within 30-day duration1.

An increased risk of candidemia in immunocompromised individuals has been linked to a number of variables, such as broad-spectrum antibiotics, chemotherapy, neutropenia and invasive interventions. Blood cultures are the most reliable method for detection of candidemia. Nevertheless, physicians need to base their diagnosis on clinical picture and the existence of risk factors due to false negative results, long time required for diagnosis and the potential harmful effects of delayed or ineffective antifungal therapy2.

Despite the fact that C. albicans is still regarded as a significant pathogen of candidemia, an ongoing shift from C. albicans to non-albicans Candida (NAC) spp. was reported by several countries. C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata are responsible for over 90% of cases of candidemia. More unusual species including C. guilliermondii, C. lusitaniae, and C. kefyr have recently been known to induce candidemia, which poses a new risk to hospitalized patients' health3.

Candida infections often get therapeutic failure, mostly as a result of antifungal resistance that is caused by several mechanisms including biofilm production as biofilm-producing strains show significant increased resistance to antifungal drugs and host immunity4.

Numerous genes are reported to promote biofilm formation including hyphal wall protein1 (HWP1), agglutinin-like sequence (ALS1) and ALS3 genes. Also, release of extracellular hydrolytic enzymes make candida more virulent. For example, the release of aspartic proteinases (Saps), a family of ten enzymes, promotes cell wall proteins cleavage that facilitates the penetration of deeper epithelial layers and adhesion to host tissues. Furthermore, fungus release of phospholipases in order to break down phospholipids, a significant part of the cell membrane, may help in host tissue invasion. The expression of the SAP1, SAP2, SAP3, SAP4, SAP5, SAP6, PLB1, PLB2, and LIP1-10 genes controls the synthesis of these enzymes5.

Patients’ underlying medical conditions have an impact on Candida spp. distribution and their antifungal susceptibility profile, which differs geographically. Consequently, choosing an empirical antifungal therapy for candidemia requires an understanding of the local pathogenic spectrum and changes in susceptibility6.

Our aim was to detect prevalence, antifungal susceptibility, biofilm formation and virulence genes (HWP1, ALS1 and SAP2) of different Candida spp. isolated from patients with candidemia.

Methodology

Study design, setting and subjects

This cross-sectional study was conducted in the Faculty of Medicine and National liver institute, Menoufia University during the period from June 2022 to January 2024 and included patients from different Departments (ICU, oncology, dialysis, pediatric and transplantation unit). Patients were subjected to full history taking and clinical examination. Each patient provided written informed consent and the study was performed according to the Declaration of Helsinki. The study protocol has been approved by the Ethical Committee, Faculty of Medicine, Menoufia University (ANET 14-2). Demographic and clinical data including primary illness and risk factors at the time when blood culture was positive were all collected.

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 Candida8. 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 Institute10, 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 isolate11.

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 by12,13 (Table 1). The PCR amplification was done using pre-programmed thermal cycler (Biometra, Germany). Electrophoresis for 20 min was done using agarose gel 2% (EGY technology) that was stained with the dye ethidium bromide (Sigma, USA). The gene products were seen using a UV trans-illuminator and 100 bp DNA ladder (Cleaver scientific, UK).

Table 1 Primers and PCR conditions used for PCR for detection of investigated genes.
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Ethical approval

This experiment was approved by the National Liver Disease Institute's Research Ethics Committee (ANET 14-2). The Declaration of Helsinki's essential principles and practices were followed throughout the research.

Consent to participate

Every participant gave their written authorization after being informed of the study's objectives and any potential negative side effects.

Results:

Of all detected BSIs, 2.3% (n = 52/2272) were caused by candida. C. albicans represented 32.7% (n = 17/52) while NAC constituted the majority 67.3% (n = 35/52) of isolated candida spp. with the predominance of C. tropicalis 25% (n = 13/52) followed by C. parapsilosis 17.3% (n = 9/52) and C. krusei 13.5% (n = 7/52) (Figs. 1 and 2A).

Figure 1
figure 1

Distribution of isolated candida spp. among patients with candidemia.

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Figure 2
figure 2

(A) Identification of candida spp. by Chromogenic Candida agar. 1, C. albicans; 2, C. krusei; 3, C. parapsilosis; 4, C. tropicalis. (B) Detection of biofilm production by congo red agar method: black colonies for biofilm producing isolates while red colonies for non-biofilm producing isolates.

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Among our patients, the potential risk factors for candidemia included antibiotic therapy (82.7%), invasive procedure exposure (75.0%), prolonged hospital stay (> 7 days) and malignancy (69%). Sex, risk factors and ward of isolation didn’t significantly (P value > 0.05) affect distribution of C. albicans and NAC. However, most of isolated NAC was from elderly (57.1%) and very young patients (22.9%) while most of isolated C. albicans (70.6%) was from patients aged 10–60 years old with highly statistically significant difference (P value = 0.001). most of isolated Candida spp. were from ICU (38.5%) and oncology unit (28.8%) (Table 2).

Table 2 Demographic and clinical characteristics of patients with candidemia.
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C. albicans showed higher susceptibility to antifungal drugs and lower biofilm formation compared to NAC but with no statistically significant difference (P value > 0.05). Susceptibility rate to fluconazole, voriconazole, caspofungin, micafungin, amphotericin B and flucytosine was 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. C. glabrata was more frequently resistant to the azole antifungals. The majority of C. albicans (70.6%) and NAC (74.3%) were biofilm producers. HWP1, ALS1 and SAP2 were detected in 82.4%, 76.5% and 52.9% of C. albicans and 85.7%, 80.0% and 48.6% of NAC respectively (Table 3, Figs. 2B and 3).

Table 3 Antifungal susceptibility and virulence determinants of candida isolates.
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Table 4 Association between investigated genes and biofilm formation among candida isolates.
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Figure 3
figure 3

Gel electrophoresis showing the amplified product of the HWP1 (572 bp), ASL1 (318 bp) and SAP2 (178 bp) genes. Lane 1: 100 bp DNA ladder (Cleaver Scientific, UK). Lane 2, 3, 4, 5 and 7: HWP1 positive isolate; Lanes 2, 3 and 6: ALS1 positive isolates; Lane 5 and 6: SAP2 positive isolates; Lane 8: negative control.

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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 outcomes14.

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 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, hospital-to-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 world17. 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 infections18.

In agreement with our results, previous studies in different countries reported C. albicans to be the primary cause of candidemia8,15,16,19,20,21. However, higher rates of C. albicans isolation among patients with candidemia were detected in other studies in KSA (51.3%)22, Turkey (39.42%)23 and USA (67%)24. In accordance, two different Italian studies in a large Italian University and tertiary hospitals found that 43.63% and 61.2% of candidemia caused by C. albicans respectively25,26. In contrast, El-Mahallawy et al.6, Muderris et al.27 and Treviño-Rangel et al.28 found that C. tropicalis (36.7%), C. parapsilosis (49.1%) and C. tropicalis (52.8%) respectively were the predominant species followed by C. albicans.

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 studies3,29,30. In contrast, in studies in Israel and Nordic countries C. glabrata was the principal NAC causing candidemia31,32. 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 children26,34,35.

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 groups36,37.

Yardimci et al.24 reported that micafungin sensitivity was the highest (97.4%) while fluconazole showed lowest sensitivity (66.1%) in 236 isolates which is consistent with this study. Similarly, an Egyptian study6, reported that resistance to fluconazole and voriconazole was 58.3% and 16.7% respectively among Candida BSIs. Fluconazole 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 BSIs16. 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%, respectively33. 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.

Our data revealed that the majority of isolates (> 70%) were biofilm producers and positive for biofilm related genes (HWP1 and ALS1). In agreement, Brunetti et al.23 stated that 69.28% of Candida BSIs were biofilm producers with the highest intrinsic production among C. albicans and C. tropicalis. Additionally, Treviño-Rangel et al.28 discovered that all the strains (89 isolates) produced biofilm with the highest biofilm density among C. tropicalis.

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.