Molecular analysis of ERG11 gene in azole resistant Candida Species among immunocompromised patients

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Salem, Mervat E. Mashaly, Douaa R. El-deeb, Salah A. Agha, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7526278/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Candida species are opportunistic pathogens that can cause life-threatening infections, especially in immunocompromised patients. Azole antifungals are widely used for treatment, but resistance is increasing and often due to mutations in the ERG11 gene that compromises their efficacy . Objectives: To determine the prevalence of azole-resistant Candida spp . among immunocompromised patients and identify mutations in the ERG11 gene associated with resistance. Methods: In this cross-sectional study, clinical samples were processed from immunocompromised patients at two University Hospitals in Egypt. Identification of Candida spp . was done using culture, chromogenic media, and VITEK 2 system. Susceptibility to fluconazole and voriconazole was assessed using broth microdilution method. Resistant isolates were subjected to PCR amplification and Sanger sequencing of ERG11 hot spot regions . Results: Out of 1404 eligible samples, 90 (6.4%) yielded Candida spp ., predominantly C. tropicalis (44.4%) and C. albicans (41.1%). Resistance to fluconazole and voriconazole was observed in 14.4% and 13.3% of isolates, respectively. ERG11 sequencing revealed multiple mutations. Common missense mutations included Y132F (50%) and S154F (33.3%). A novel mutation (M306R) was identified in two C. tropicalis isolates. Conclusion: Azole resistance among Candida isolates is notable in immunocompromised patients. The present study highlights both known and novel ERG11 mutations, emphasizing the need for routine molecular surveillance to guide antifungal therapy . Health sciences/Diseases Biological sciences/Microbiology Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Introduction Candida species generally constitute commensal flora of the oral cavity, gastrointestinal tract and genital tract of healthy hosts, but they are also opportunistic pathogens that have the potential to cause numerous superficial and systemic infections. Candida infections in most people are usually asymptomatic in immunocompetent subjects. However, depletion in immune system or changes in microbiota balance, coupled with various virulence factors, can facilitate the spread of Candida which often cause fatal infection 1 . Azoles are the most common and preferred antifungal drug class in clinical practice due to its inexpensiveness, limited toxicity and potentiality for oral administration. However, the widespread use of azoles for curative and prophylactic purposes, especially in developing countries, in addition to their fungistatic mode of action against Candida has led to a remarkable rise in azoles resistance among Candida spp 2 . Azoles, such as fluconazole and voriconazole, inhibit the cytochrome P450 14α–demethylase enzyme, Erg11p which is encoded by ERG11 gene, thus inhibit the process of ergosterol biosynthesis with accumulation of toxic sterol precursors that lead to cell membrane instability and impaired fungal growth 3 . Point mutations in the coding region of the ERG11 gene represent one of the main molecular mechanisms that impact susceptibility to azoles. These mutations lead to amino acid substitutions which alter the structure of Erg11p protein, and render binding of the azoles to the active site of the enzyme less efficient; thus, reducing azole susceptibility 4 . There is a challenge to successful treatment of azole resistant Candida infections. Thus, determining the local prevalence of resistant Candida isolates and understanding the underlying resistance mechanisms will help in effective treatment and better outcome for patients 5 . In our locality, there are limited studies for detection of ERG11 gene mutations in azoles resistant Candida isolates. Therefore, the aim of this study was molecular analysis of ERG11 gene in azole-resistant Candida species isolated from immunocompromised patients to detect mutations in this gene that could be associated with azoles resistance. Subjects and methods Study design This cross-sectional study was conducted from September 2021 to March 2022 at Mansoura University Hospital and Kafr Elsheikh University Hospital ensuring a representative cohort of patients across different clinical settings. Ethical approval Approval was obtained from the Mansoura Institutional Research Board (MFM-IRB; code number MDP.19.11.33). The procedures used in this study adhered to the principles of the Declaration of Helsinki. Written informed consent was obtained from all adult participants. For minors, informed consent for participation was obtained from a parent or legal guardian prior to sample collection. Inclusion criteria Samples were included if collected from immunocompromised patients, such as neonates admitted to intensive care units, patients with haematological or solid malignancies, transplant recipients, and individuals receiving chemotherapy, radiotherapy, long-term corticosteroids, or other immunosuppressive therapies. Patients with chronic illnesses associated with impaired immunity, such as uncontrolled diabetes mellitus or chronic kidney disease, were also included. Exclusion criteria Samples collected from immunocompetent patients as well as samples which revealed polymicrobial, bacterial or non-yeast fungal growth was excluded from the study. Species identification and antifungal susceptibility testing All clinical samples were aerobically cultured on various suitable culture media at 37°C for 24 hours, then examined macroscopically for colony morphology and microscopically after Gram staining. Candida isolates were further purified by subculture on Sabouraud’s Dextrose Agar ( Thermo Scientific Oxoid, UK ) supplemented with chloramphenicol and gentamicin. Further identification to species level was performed by; germ tube test; Candida Differential Agar ( HiMedia, Mumbai, India ); then for some isolates, species identification was confirmed automatically using VITEK 2 system for yeast identification ( bioMérieux, Marcy l'Etoile, France ) according to the manufacturer’s instructions. Antifungal susceptibility testing to fluconazole and voriconazole was done by broth microdilution method according to M27 guideline 6 . For selected isolates, AST was confirmed automatically using VITEK 2 system ( bioMérieux, Marcy l'Etoile, France ). Candida species isolates that revealed resistance to fluconazole and/or voriconazole were submitted to the following molecular analysis of ERG11 gene. DNA extraction: performed using FavorPrep Fungi/Yeast Genomic DNA Extraction Mini Kit ( Favorgen Biotech Corp., Taiwan ), according to manufacturer’s instructions. PCR amplification of ERG11 gene Conventional PCR for amplification of Hot spot region 1 (HS1) and hot spot region 2 (HS2) of ERG11 gene, all primers were manufactured by ( Thermo Fisher Scientific, USA) , as shown in table 1. Table 1: Primer sequences of ERG11 gene. Candida species Primer sequence (5’ to 3’) Amplicon size C. albicans F : ATG GCA TAT GCA TTC TGA GAG TT 748 bp R : TCC AGT TTT CGG TAA AGG GGTT C. tropicalis F : GGT TTG GTT CTG CTG CTA GT 834 bp R : AGT CAC CAC CCT TTT CTT TC C. parapsilosis F : ACA ACA TCA TTT GGC AAC TAC TTT 789 bp R : CCT GAT CAG TCA TTT TAA CAC CATC F : forward, R : reverse, bp: base pair. PCR was performed in a total volume of 50 mL consisting of; 25 mL of a ready-to-use DreamTaq PCR Master Mix solution (2X) ( Thermo Fisher Scientific, USA ), 1 mL of each primer (10 pmol/mL); 8 mL of extracted DNA; then completed up to 50 mL with nuclease free water. It was performed using a thermal cycler ( Applied Biosystems, Thermo Fisher Scientific, USA ) with the following steps; initial denaturation at 95°C for 4 min; followed by 45 cycles (DNA denaturation at 95°C for 10 secs, primer annealing for 60 secs; at 58°C for C. albicans , at 60°C for C. tropicalis and 57°C for C. parapsiliosis ; followed by extension at 72°C for 90 secs), then a final extension at 72°C for 5 min. Evaluation of the ampilified PCR products by agarose gel electrophoresis was done to ensure the existence of single sharp intact bands of amplified DNA. The resulting PCR amplicons were purified using QIA-quick PCR Purification Kit (Qiagen, Germany). Sanger sequencing Sanger sequencing of ERG11 gene was performed through the following steps: Cycle sequencing, using BigDye Terminator version 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA) . Sequencing reaction was performed in a total volume of 20 μL in 2 Eppendorf tubes (one for forward primer and one for reverse primer). The reaction mix composed of; 4 μL of Big Dye Terminator Ready Reaction Mix, 4 μL of 5X Sequencing buffer, 1 μL of purified template (20 ng/μL), and 1μL of forward or reverse primer (3.2 pmol/ μL), then completed up to 20 μL with nuclease free water. Tubes were placed in the thermal cycler and cycle sequencing was performed as following; initial denaturation at 96 ◦ C for 1 min; followed by 25 cycles (DNA denaturation at 96 ◦ C for 10 sec, primer annealing at 55 ◦ C for 5 sec, and extension at 60 ◦ C for 4 min) then hold at 4 ◦ C until ready to purify. Finally, a sequencing cleanup, using BigDye X Terminator Purification Kit (Thermo Fisher Scientific, USA) , and Capillary electrophoresis was performed by Applied Biosystems 3500 Genetic Analyzer ( Thermo Fisher Scientific, USA ). Bioinformatics and statistical analysis Data analysis was conducted using Unipro UGENE version 50.0 for sequence alignment and bioinformatics analysis. Sample sequences were aligned against the following reference strains: Candida albicans ATCC MYA-2876 (GenBank accession no. NC_032093.1), Candida parapsilosis ATCC 22019 (GenBank accession no. GQ302972.1), and Candida tropicalis ATCC 750 (GenBank accession no. M23673.1). The data were analyzed using the IBM SPSS Statistics software version 22.0 ( IBM Corp., Armonk, NY, USA ). Results Out of 1958 different non-duplicate clinical samples, 1404 (71.1%) samples were selected for being withdrawn from clinically diagnosed and laboratory confirmed immunocompromised patients. Microbiological culture of these selected samples revealed that 90/1404 (6.4%) samples were identified as pure Candida growth which represented the target isolates of this study. They were 55 (61.1%) males versus 35 (38.9%) females, their age ranged from 1 to 84 years. 73 (81.1%) of them were pediatric age group, while 17 (18.9%) were adult (Figure 1). Forty-nine (54.4%) were blood samples, 18 (20%) urine samples, 12 (13.3%) central venous catheter, 6 (6.7%) broncho-alveolar lavage, 4 (4.4%) peritoneal fluid, and one (1.1%) pleural fluid sample. As shown in table 2, there was a significant decrease of absolute lymphocytic count among cases of non-albicans Candida (NAC) compared to cases of Candida albicans ( P=0.04 ). History of antifungal prophylaxis was significantly higher among NAC cases compared to Candida albicans cases ( P<0.05 ). Otherwise, there was no statistically significant difference regarding other characteristics. Table 2: Characteristics of the studied cases in relation to isolated Candida spp. Characteristic Total (n=90) Candida albicans (n=37) Non-albicans Candida (n=53) P-value Age (years) Median (Min-Max) 6.0 (1-84) 12.0 (1-84) 5.0 (1-80) 0.13 Age ≤ 1 year Age ≥ 60 years 9 (10%) 10 (11.1%) 4 (44.4%) 6 (60.0%) 5 (55.6%) 4 (40.0%) 0.66 Sex Male Female 55 (61.1%) 35 (38.9%) 20 (36.4%) 17 (48.6%) 35 (63.6%) 18 (51.4%) 0.25 ICU admission 69 (76.7%) 28 (40.6%) 41 (59.4%) 0.47 History of prior Surgery 17 (18.9%) 8 (47.1%) 9 (52.9%) 0.58 History of antibiotic intake 87 (96.7%) 36 (41.4%) 51 (58.6%) 1.0 History of antifungal prophylaxis 28 (31.1%) 7 (25.0%) 21 (75.0%) 0.04 * Underlying co-morbidities: Liver cell failure 15 (16.7%) 9 (60.0%) 6 (40.0%) 0.1 Diabetes mellitus 13 (14.4%) 6 (46.2%) 7 (53.8%) 0.69 Renal failure 12 (13.3%) 3 (25.0%) 9 (75.0%) 0.22 Encephalopathy 8 (8.9%) 4 (50.0%) 4 (50.0%) 0.71 Pulmonary diseases 6 (6.7%) 2 (33.3%) 4 (66.7%) 1.0 Hematological diseases 8 (8.9%) 2 (25.0%) 6 (75.0%) 0.46 Solid tumors 2 (2.2%) 0 (0.0%) 2 (100.0%) 0.51 1ry immunodeficiency 2 (2.2%) 1 (50.0%) 1 (50.0%) 1.0 Medical indwelling devices Multiple devices Peripheral venous catheter only 76 (84.4%) 14 (15.6%) 30 (39.5%) 7 (50.0%) 46 (60.5%) 7 (50.0%) 0.46 Total leucocytic count (×10 9 /L) 11 (×10 9 /L) 16 (17.8%) 40 (44.4%) 34 (37.8%) 5 (31.3%) 16 (40.0%) 15 (47.1%) 11(68.8%) 24 (60.0%) 18 (52.9%) 0.56 Absolute Neutrophil count (×10 9 /L) Median (Min-Max) * 6.8 (0.93-32.25) 8.9 (1.1-32.25) 6.5 (0.93-28.1) 0.25 Absolute Lymphocyte count (×10 9 /L) Median (Min-Max) * 0.66 (0.24-1.93) 0.71 (0.42-1.93) 0.58 (0.24-1.24) <0.05 * Anemia N (%) 82 (91.1%) 32 (39.0%) 50 (61.0%) 0.26 Thrombocytopenia N (%) 45 (50.0%) 16 (35.6%) 29 (64.4%) 0.28 * Significant There was statistically significant higher sensitivity to fluconazole & voriconazole among Candida albicans compared to NAC isolates, whereas there was significant higher resistance to fluconazole and voriconazole in NAC compared to Candida albicans isolates ( P=0.03 ) (Table 3). Table 3: Fluconazole and voriconazole susceptibility pattern among isolated Candida species (n=90) Candida species (n) Sensitive to Fluconazole & Voriconazole (n= 73) (81.1%) SDD ** to Fluconazole, Resistant to Voriconazole (n= 1) (1.1%) Resistant to Fluconazole only (n= 5) (5.6%) Resistant to Voriconazole only (n= 3) (3.3%) Resistant to Fluconazole & Voriconazole (n= 8) (8.9%) P-value Candida albicans (37) 36 (97.3%) 0 0 0 1 (2.7%) 0.03* Non-albicans Candida (53) 37 (69.8%) 1 (1.9%) 5 (9.4%) 3 (5.7%) 7 (13.2%) C. tropicalis (40) 30 (75.0%) 1 (2.5%) 0 2 (5.0%) 7 (17.5%) NC C. parapsilosis (8) 7 (87.5%) 0 0 1 (12.5%) 0 C. krusei (5) 0 0 5 (100%) 0 0 * Significant, ** Susceptible dose dependent . PCR amplification of HS1 and HS2 regions of the ERG11 gene yielded clear species-specific bands. Ten isolates of C. tropicalis produced the expected 834 bp fragment, one isolate of C. parapsilosis produced a 789 bp fragment, and one isolate of C. albicans yielded a 748 bp fragment (Figure 2). Nine different nucleotide mutations were detected; they were four silent mutations, (T225C, G264A, T591C and T783C), and five missense mutations with subsequent amino acid substitutions, T348A (D116E), A395T (Y132F), A428G (K143R), C461T (S154F) (Figure 3) , which were located in ERG11 hot spot region I, Interestingly, a novel missense mutation, T917G (M306R) located in between ERG11 hot spot regions II & III was revealed in this study for the first time (Table 4). Table 4: ERG11 gene nucleotide mutations and corresponding amino acid substitutions detected among azole resistant Candida isolates (n=12). Nucleotide mutation Number of isolates showing this mutation Mutation type Corresponding amino acid substitution / Hot spot region T225C 6 Silent - G264A 5 Silent - T348A 1 Missense D116E / I Asp → Glu A395T 6 Missense Y132F / I Tyr → Phe A428G 2 Missense K143R / I Lys → Arg C461T 4 Missense S154F / I Ser → Phe T591C 1 Silent - T783C 2 Silent - T917G 2 Missense M306R * Met → Arg * New substitution Among the twelve-azole resistant Candida spp . Y132F mutation was the most frequent (50%) missense mutation, detected in six C. tropicalis isolates, followed by S154F mutation (33.3%), detected in four C. tropicalis isolates, then K143R mutation (16.6%), detected in one C. albicans isolate and one C. tropicalis isolate, while D116E mutation (8.3%) was detected in only one C. albicans isolate. Interestingly, the newly detected M306R mutation (8.3%) was detected in two C. tropicalis isolates (Table 5). Table 5: Missense mutations among azole resistant Candida species isolates (n=12). Missense mutation Isolates showing this mutation N (%) Candida species (n=12) Candida albicans N (%) Non-albicans Candida N (%) D116E 1 (8.3%) 1 (100%) 0 Y132F 6 (50%) 0 6 (100%) K143R 2 (16.6%) 1 (50%) 1 (50%) S154F 4 (33.3%) 0 4 (100%) M306R 2 (16.6%) 0 2 (100%) Discussion The increasing incidence of antifungal resistance has become a global concern, limiting treatment options and complicating the management of fungal infections. Among these, azole-resistant Candida species represent a growing threat in clinical practice 7 . In the present study, the prevalence of Candida infection among immunocompromised patients was 90/1404 (6.4%). A similiar rate (6.3%) was reported by El-Mashad et al. among cancer patients, with 95.3% of cases attributed to Candida species 8 . In contrast, El-Mahallawy et al . and Ghrenassia et al. reported lower prevalence rates of Candida infections, at 3.1% and 0.7% respectively 9,10 . These differences highlight that the prevalence of Candida infections is strongly influenced by the patient population, underlying risk factors, and clinical setting. Most Candida -infected cases in this study occurred in pediatric patients (81.1%), while adults accounted for 18.9%. Similarly, Furlaneto et al. (2011) reported a higher prevalence of Candida bloodstream infections in neonates (35%) compared with older age groups (22.5%). Non-albicans Candida (NAC) species were more frequently isolated than Candida albicans (58.9% vs. 41.1%). Similar observations were reported by El-Mashad et al., Liu et al., and Abouzeid et al., who also documented higher isolation rates of NAC species 8,11,12 . This shift toward NAC dominance has been increasingly recognized worldwide and is of clinical concern, as many NAC species possess inherently higher levels of antifungal resistance, particularly to azoles 13 . Our findings indicate that Candida albicans remained more susceptible to fluconazole and voriconazole, whereas NAC isolates showed higher resistance. This resistance may be linked to prior antifungal prophylaxis and lower lymphocyte counts in NAC cases 14,15 . Similar findings were reported by El-Mashad et al., whereas El-Mahallawy et al. and Khairat et al. observed higher resistance rates. Differences across studies may reflect variations in patient populations, antifungal exposure, and species distribution 8,9,16 . The most frequently detected missense mutations were Y132F (6/12, 50%) and S154F (4/12, 33.3%), followed by K143R (2/12, 16.6%). Consistent with these findings, previous studies have also identified Y132F and S154F as the predominant mutations in fluconazole-resistant Candida isolates 17-19 . By contrast, the least common mutation was D116E (1/12, 8.3%), detected in a single C. albicans isolate with MIC values of 16 μg/mL for fluconazole and 1 μg/mL for voriconazole. Importantly, this mutation was not found alone but co-occurred with K143R. Similarly, Xiang et al. found the D116E substitution in both azole-susceptible and azole-resistant isolates, indicating that this substitution alone is unlikely to confer resistance 20 . Interestingly, this current study revealed a novel missense point mutation (T917G/M306R), located in between hot spot region II and hot spot region III of ERG11 gene. This novel mutation was identified in two azole-resistant C. tropicalis isolates (2/12, 16.6%). The first was recovered from a bronchoalveolar lavage sample of a diabetic ICU patient and showed MIC values of 32 μg/mL for fluconazole and 2 μg/mL for voriconazole. The second was isolated from the blood of an ICU patient with bone marrow failure and exhibited MIC values of 8 μg/mL for fluconazole and 2 μg/mL for voriconazole. Notably, this mutation co-occurred with other missense mutations (Y132F and S154F). Therefore, additional studies are needed to determine whether this mutation is specifically associated with azole resistance or may also be present in azole-susceptible strains. In addition, four silent point mutations were identified (T225C, G264A, T591C, and T783C) in our isolates. Notably, three of these (T225C, G264A, and T783C) have been previously reported in Azole-resistant C. tropicalis clinical isolates 21 . T225C and G264A mutations have also been observed in Candida isolates from Egypt 22 . Similarly, Corzo-Leon et al. reported T591C as a common silent SNP in all C. parapsilosis isolates analyzed by ERG11 sequencing 23 . The recurrence of these mutations across different studies suggests that they may represent conserved genetic changes rather than random variations. These previously mentioned silent mutations, being synonymous mutations and therefore cannot be linked to azole resistance. However, recent studies suggest that synonymous mutations can influence the nucleic acid sequence and its structural properties. They may affect RNA secondary structure, stability, folding, and regulatory elements such as ribosome binding sites or splicing signals. These changes can alter codon usage, impact protein expression levels, and even modify substrate specificity, indicating that synonymous mutations are not entirely silent. Over time, the accumulation of such mutations, together with other factors, could ultimately affect the function of the target enzyme 22 . Conclusion Azole resistance among Candida species isolated from immunocompromised patients represents a significant clinical challenge. In this study, we identified a notable prevalence of azole-resistant Candida isolates, particularly among non-albicans Candida species. Molecular analysis revealed several known missense mutations in the ERG11 gene such as Y132F and S154F associated with resistance. Importantly, we also detected a novel mutation, M306R, in C. tropicalis , which may contribute to resistance but requires further functional validation. These findings underscore the importance of routine antifungal susceptibility testing and molecular surveillance to guide effective antifungal therapy and prevent treatment failure in vulnerable patient populations. Declarations Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ contributions: Elshaer M. wrote the main manuscript text. Salem A. H., and Elshaer M. carried out study analysis and prepared figures and tables. Salem A. H., and Eldegwi M. contributed to samples ordering and data collection. Elshaer M., Eldegwi M., Mashaly M. E., El-deeb D. R., and Agha S. A. revised the final draft. All authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Ethics declarations Conflict of interest: The authors have no competing financial or personal interests to declare. Ethical approval: Approval was obtained from the Mansoura Institutional Research Board (MFM-IRB; code number MDP.19.11.33). The procedures used in this study adhered to the principles of the Declaration of Helsinki. Written informed consent was obtained from all adult participants. For minors, informed consent for participation was obtained from a parent and/or legal guardian prior to sample collection. Data availability: All data supporting this article will be made available by the corresponding author to any qualified researcher upon request. References Bongomin, F., Gago, S., Oladele, R. O. & Denning, D. W. Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. Journal of fungi (Basel, Switzerland) 3 , doi:10.3390/jof3040057 (2017). Huang, D. et al. 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Candida tropicalis antifungal cross-resistance is related to different azole target (Erg11p) modifications. Antimicrobial agents and chemotherapy 57 , 4769-4781, doi:10.1128/aac.00477-13 (2013). El-Kholy, M. A., Helaly, G. F., El Ghazzawi, E. F., El-Sawaf, G. & Shawky, S. M. J. B. J. o. M. Analysis of CDR1 and MDR1 gene expression and ERG11 substitutions in clinical Candida tropicalis isolates from Alexandria, Egypt. 54 , 2609-2615 (2023). Corzo-Leon, D. E., Peacock, M., Rodriguez-Zulueta, P., Salazar-Tamayo, G. J. & MacCallum, D. M. General hospital outbreak of invasive candidiasis due to azole-resistant Candida parapsilosis associated with an Erg11 Y132F mutation. Medical mycology 59 , 664-671, doi:10.1093/mmy/myaa098 (2021). Additional Declarations No competing interests reported. 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Mashaly","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Mervat","middleName":"E.","lastName":"Mashaly","suffix":""},{"id":524652445,"identity":"ad112754-cac7-4ecc-9046-c1b522b7abf2","order_by":2,"name":"Douaa R. El-deeb","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Douaa","middleName":"R.","lastName":"El-deeb","suffix":""},{"id":524652446,"identity":"14247c8a-275e-4afc-803e-a7c9f4e68e70","order_by":3,"name":"Salah A. Agha","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Salah","middleName":"A.","lastName":"Agha","suffix":""},{"id":524652447,"identity":"40456d35-3f2f-41d0-843a-0976ab0d95e8","order_by":4,"name":"Marwa Eldegwi","email":"","orcid":"","institution":"Kafr Elsheikh University","correspondingAuthor":false,"prefix":"","firstName":"Marwa","middleName":"","lastName":"Eldegwi","suffix":""},{"id":524652448,"identity":"abd043e1-fe0a-40c7-8fbe-7c9ddea21971","order_by":5,"name":"Mohammed Elshaer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYFAC5gZmEMUvf/gAVCSBkBZGiBbJGWwwpcRqMbjBY0CcFt32xubPBTU2+ZKze75u+LmjjoGfPceA4cMv3FrMzhxsk55xLM2yX+bstpu9Zw4zSPa8MWCc2YdHy43ENmYetsMGkg25227wth0AujDHgJm3B4+W+w+bP/P8+29gcCDn2c2/bXUM9iAtf/FpucHYIA003ABoONtt3jZmBgMJoBaGH/j8ktgmzduXbCDZc8zstmzbYR6JM88KDvY24NFy/PDhzzzf7Az42Zuf3XzbVifH35688cGPP7i1YAAeEHGAsY0ELVBAii2jYBSMglEw3AEAGiZZd9obfAcAAAAASUVORK5CYII=","orcid":"","institution":"Mansoura University","correspondingAuthor":true,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Elshaer","suffix":""}],"badges":[],"createdAt":"2025-09-03 11:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7526278/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7526278/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93011051,"identity":"2153ea0e-41a8-4f7f-bde5-7d98bb181552","added_by":"auto","created_at":"2025-10-08 07:16:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1566617,"visible":true,"origin":"","legend":"","description":"","filename":"FinalPaperAmal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7526278/v1/2f106c66d453e669fe0e544d.docx"},{"id":93011049,"identity":"6bf16455-e052-4355-bc9b-cdd6c281f368","added_by":"auto","created_at":"2025-10-08 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07:16:20","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94402,"visible":true,"origin":"","legend":"","description":"","filename":"b13004374af44c8fb4f6a927e957ed541structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7526278/v1/2c7e132236345721c1f350b7.xml"},{"id":93011061,"identity":"6c02052c-dbc3-4d74-bbd7-3a482f4d7944","added_by":"auto","created_at":"2025-10-08 07:16:21","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106030,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7526278/v1/6995d4f8192a0b885ba2261a.html"},{"id":93013255,"identity":"25308632-b788-49b1-abc0-9ad761e43c89","added_by":"auto","created_at":"2025-10-08 07:24:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19287,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of \u003cem\u003eCandida\u003c/em\u003especies among pediatric and adult cases.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7526278/v1/5f786c142a9cddfeef530179.png"},{"id":93011053,"identity":"306cd0b3-dada-4e6d-89f1-1dbb0789f133","added_by":"auto","created_at":"2025-10-08 07:16:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337862,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis of PCR products.\u003c/p\u003e\n\u003cp\u003e(Lanes 1, 4, 5, 6, 7, 8, 9, 10, 11, \u0026amp; 12: correspond to \u003cem\u003eC. tropicalis \u003c/em\u003eisolates (834 bp).\u003c/p\u003e\n\u003cp\u003eLane 2: corresponds to \u003cem\u003eC. parapsilosis\u003c/em\u003e isolate (789 bp).\u003c/p\u003e\n\u003cp\u003eLane 3: corresponds to \u003cem\u003eC. albicans\u003c/em\u003e isolate (748 bp).\u003c/p\u003e\n\u003cp\u003eLane 13: Ladder (100-1200 bp).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7526278/v1/d53d721bddd99a13b838dbdd.png"},{"id":93013256,"identity":"edda4b34-7d42-4ed6-a27b-50fcb4dbfba4","added_by":"auto","created_at":"2025-10-08 07:24:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":644154,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide mutations of the \u003cem\u003eERG11\u003c/em\u003e gene of \u003cem\u003eCandida spp\u003c/em\u003e. with corresponding amino acid substitutions. The left panel shows nucleotide sequences’ alignment; where the upper line refers to the reference sequence, while the lower line refers to the mutated sequence. The middle panel shows chromatogram files of mutant strains. The right panel shows amino acid sequences’ alignment with missense mutations. Detected mutations: T348A (D116E), A395T (Y132F), A428G (K143R), C461T (S154F), and T917G (M306R).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7526278/v1/a644417c15ce6dd842f973cf.png"},{"id":96927264,"identity":"613f34ea-4af2-4aad-ad61-5ac403101ef5","added_by":"auto","created_at":"2025-11-27 14:27:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2220302,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7526278/v1/8d552610-275c-4b9f-8cdf-d7ac0f479fa5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular analysis of ERG11 gene in azole resistant Candida Species among immunocompromised patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eCandida\u003c/em\u003e species generally constitute commensal flora of the oral cavity, gastrointestinal tract and genital tract of healthy hosts, but they are also opportunistic pathogens that have the potential to cause numerous superficial and systemic infections. \u003cem\u003eCandida\u003c/em\u003e infections in most people are usually asymptomatic in immunocompetent subjects. However, depletion in immune system or changes in microbiota balance, coupled with various virulence factors, can facilitate the spread of \u003cem\u003eCandida\u003c/em\u003e which often cause fatal infection \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAzoles are the most common and preferred antifungal drug class in clinical practice due to its inexpensiveness, limited toxicity and potentiality for oral administration. However, the widespread use of azoles for curative and prophylactic purposes, especially in developing countries, in addition to their fungistatic mode of action against \u003cem\u003eCandida\u003c/em\u003e has led to a remarkable rise in azoles resistance among \u003cem\u003eCandida spp\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAzoles, such as fluconazole and voriconazole, inhibit the cytochrome P450 14α\u0026ndash;demethylase enzyme, Erg11p which is encoded by \u003cem\u003eERG11\u003c/em\u003e gene, thus inhibit the process of ergosterol biosynthesis with accumulation of toxic sterol precursors that lead to cell membrane instability and impaired fungal growth \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Point mutations in the coding region of the \u003cem\u003eERG11\u003c/em\u003e gene represent one of the main molecular mechanisms that impact susceptibility to azoles. These mutations lead to amino acid substitutions which alter the structure of Erg11p protein, and render binding of the azoles to the active site of the enzyme less efficient; thus, reducing azole susceptibility \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThere is a challenge to successful treatment of azole resistant \u003cem\u003eCandida\u003c/em\u003e infections. Thus, determining the local prevalence of resistant \u003cem\u003eCandida\u003c/em\u003e isolates and understanding the underlying resistance mechanisms will help in effective treatment and better outcome for patients \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In our locality, there are limited studies for detection of \u003cem\u003eERG11\u003c/em\u003e gene mutations in azoles resistant \u003cem\u003eCandida\u003c/em\u003e isolates. Therefore, the aim of this study was molecular analysis of \u003cem\u003eERG11\u003c/em\u003e gene in azole-resistant \u003cem\u003eCandida\u003c/em\u003e species isolated from immunocompromised patients to detect mutations in this gene that could be associated with azoles resistance.\u003c/p\u003e"},{"header":"Subjects and methods","content":"\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis cross-sectional study was conducted from September 2021 to March 2022 at Mansoura University Hospital and Kafr Elsheikh University Hospital ensuring a representative cohort of patients across different clinical settings.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval was obtained from the Mansoura Institutional Research Board (MFM-IRB; code number MDP.19.11.33). The procedures used in this study adhered to the principles of the Declaration of Helsinki. Written informed consent was obtained from all adult participants. For minors, informed consent for participation was obtained from a parent or legal guardian prior to sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were included if collected from immunocompromised patients, such as neonates admitted to intensive care units, patients with haematological or solid malignancies, transplant recipients, and individuals receiving chemotherapy, radiotherapy, long-term corticosteroids, or other immunosuppressive therapies. Patients with chronic illnesses associated with impaired immunity, such as uncontrolled diabetes mellitus or chronic kidney disease, were also included.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples collected from immunocompetent patients as well as samples which revealed polymicrobial, bacterial or non-yeast fungal growth was excluded from the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecies identification and antifungal susceptibility testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll clinical samples were aerobically cultured on various suitable culture media at 37\u0026deg;C for 24 hours, then examined macroscopically for colony morphology and microscopically after Gram staining. \u003cem\u003eCandida\u003c/em\u003e isolates were further purified by subculture on Sabouraud\u0026rsquo;s Dextrose Agar (\u003cem\u003eThermo Scientific Oxoid, UK\u003c/em\u003e) supplemented with chloramphenicol and gentamicin. Further identification to species level was performed by; germ tube test; \u003cem\u003eCandida\u003c/em\u003e Differential Agar (\u003cem\u003eHiMedia, Mumbai, India\u003c/em\u003e);\u0026nbsp;then for some isolates, species\u0026nbsp;identification\u0026nbsp;was confirmed automatically using VITEK 2 system for yeast identification\u0026nbsp;(\u003cem\u003ebioM\u0026eacute;rieux, Marcy l\u0026apos;Etoile, France\u003c/em\u003e)\u0026nbsp;according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003eAntifungal susceptibility testing to fluconazole and voriconazole was done by broth microdilution method according to M27 guideline \u003csup\u003e6\u003c/sup\u003e.\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eFor selected isolates, AST was confirmed automatically using VITEK 2 system (\u003cem\u003ebioM\u0026eacute;rieux, Marcy l\u0026apos;Etoile, France\u003c/em\u003e). \u003cem\u003eCandida\u003c/em\u003e species isolates that revealed resistance to fluconazole and/or voriconazole were submitted to the following molecular analysis of \u003cem\u003eERG11\u003c/em\u003e gene. DNA extraction: performed using FavorPrep Fungi/Yeast Genomic DNA Extraction Mini Kit\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003e\u003cem\u003eFavorgen Biotech Corp., Taiwan\u003c/em\u003e\u003cstrong\u003e),\u0026nbsp;\u003c/strong\u003eaccording to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR amplification of \u003cem\u003eERG11\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConventional PCR for amplification of\u003cem\u003e\u0026nbsp;\u003c/em\u003eHot spot region 1 (HS1) and hot spot region 2 (HS2) of \u003cem\u003eERG11\u003c/em\u003e gene, all primers were manufactured by (\u003cem\u003eThermo Fisher Scientific, USA)\u003c/em\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eas shown in table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Primer sequences of \u003cem\u003eERG11\u003c/em\u003e gene. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"625\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 367px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer sequence (5\u0026rsquo; to 3\u0026rsquo;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmplicon size\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 367px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e: ATG GCA TAT GCA TTC TGA GAG TT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e748 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 367px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eR\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e: TCC AGT TTT CGG TAA AGG GGTT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. tropicalis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 367px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e: GGT TTG GTT CTG CTG CTA GT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e834 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 367px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eR\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e: AGT CAC CAC CCT TTT CTT TC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. parapsilosis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 367px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e: ACA ACA TCA TTT GGC AAC TAC TTT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e789 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 367px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eR\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e: CCT GAT CAG TCA TTT TAA CAC CATC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e forward, \u003cstrong\u003e\u003cem\u003eR\u003c/em\u003e:\u003c/strong\u003e reverse, \u003cstrong\u003ebp:\u003c/strong\u003e base pair.\u003c/p\u003e\n\u003cp\u003ePCR was performed in a total volume of 50\u0026nbsp;mL consisting of; 25\u0026nbsp;mL of a ready-to-use DreamTaq PCR Master Mix solution (2X) (\u003cem\u003eThermo Fisher Scientific, USA\u003c/em\u003e), 1\u0026nbsp;mL of each primer (10 pmol/mL); 8\u0026nbsp;mL of extracted DNA; then completed up to 50\u0026nbsp;mL with nuclease free water. It was performed using a thermal cycler (\u003cem\u003eApplied Biosystems, Thermo Fisher Scientific, USA\u003c/em\u003e) with the following steps; initial denaturation at 95\u0026deg;C for 4 min; followed by 45 cycles (DNA denaturation at 95\u0026deg;C for 10 secs, primer annealing for 60 secs; at \u0026nbsp;58\u0026deg;C for \u003cem\u003eC. albicans\u003c/em\u003e, \u0026nbsp;at 60\u0026deg;C for \u003cem\u003eC. tropicalis\u0026nbsp;\u003c/em\u003eand 57\u0026deg;C for \u003cem\u003eC. parapsiliosis\u003c/em\u003e; followed by extension at 72\u0026deg;C for 90 secs), then a final extension at 72\u0026deg;C for 5 min. Evaluation of the ampilified PCR products by agarose gel electrophoresis was done to ensure the existence of single sharp intact bands of amplified DNA. The resulting PCR amplicons were purified using QIA-quick PCR Purification Kit \u003cem\u003e(Qiagen, Germany).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSanger\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSanger sequencing of \u003cem\u003eERG11\u003c/em\u003e gene was performed through the following steps: Cycle sequencing, using BigDye Terminator version 3.1 Cycle Sequencing Kit\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003e(Thermo Fisher Scientific, USA)\u003c/em\u003e. Sequencing reaction was performed in a total volume of 20 \u0026mu;L in 2 Eppendorf tubes (one for forward primer and one for reverse primer). The reaction mix composed of; 4 \u0026mu;L of Big Dye Terminator Ready Reaction Mix, 4 \u0026mu;L of 5X Sequencing buffer, 1 \u0026mu;L of purified template (20 ng/\u0026mu;L), and 1\u0026mu;L of forward or reverse primer (3.2 pmol/ \u0026mu;L), then completed up to 20 \u0026mu;L with nuclease free water. Tubes were placed in the thermal cycler and cycle sequencing was performed as following; initial denaturation at 96\u003csup\u003e◦\u003c/sup\u003eC for 1 min; followed by 25 cycles (DNA denaturation at 96\u003csup\u003e◦\u003c/sup\u003eC for 10 sec, primer annealing at 55\u003csup\u003e◦\u003c/sup\u003eC for 5 sec, and extension at 60\u003csup\u003e◦\u003c/sup\u003eC for 4 min) then hold at 4\u003csup\u003e◦\u003c/sup\u003eC until ready to purify. Finally, a sequencing cleanup, using BigDye X Terminator Purification Kit\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003e(Thermo Fisher Scientific, USA)\u003c/em\u003e,\u0026nbsp;and\u0026nbsp;Capillary electrophoresis\u0026nbsp;was performed by Applied Biosystems 3500 Genetic Analyzer (\u003cem\u003eThermo\u0026nbsp;\u003c/em\u003e\u003cem\u003eFisher Scientific, USA\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analysis was conducted using Unipro UGENE version 50.0 for sequence alignment and bioinformatics analysis. Sample sequences were aligned against the following reference strains: \u003cem\u003eCandida albicans\u003c/em\u003e ATCC MYA-2876 (GenBank accession no. NC_032093.1), \u003cem\u003eCandida parapsilosis\u003c/em\u003e ATCC 22019 (GenBank accession no. GQ302972.1), and \u003cem\u003eCandida tropicalis\u003c/em\u003e ATCC 750 (GenBank accession no. M23673.1). The data were analyzed using the IBM SPSS Statistics software version 22.0 (\u003cem\u003eIBM Corp., Armonk, NY, USA\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOut of 1958 different\u0026nbsp;non-duplicate\u0026nbsp;clinical\u0026nbsp;samples, 1404\u0026nbsp;(71.1%) samples were selected for being withdrawn from clinically diagnosed and laboratory confirmed immunocompromised patients. Microbiological culture of these selected samples revealed that 90/1404 (6.4%) samples were identified as pure \u003cem\u003eCandida\u0026nbsp;\u003c/em\u003egrowth which represented the target isolates of this study. They were 55 (61.1%) males versus 35 (38.9%) females, their age ranged from 1 to 84 years. 73 (81.1%) of them were pediatric age group, while 17 (18.9%) were adult (Figure 1). Forty-nine (54.4%) were blood samples, 18 (20%) urine samples, 12 (13.3%) central venous catheter, 6 (6.7%) broncho-alveolar lavage, 4 (4.4%) peritoneal fluid, and one (1.1%) pleural fluid sample.\u003c/p\u003e\n\u003cp\u003eAs shown in table 2, there was a significant decrease of absolute lymphocytic count among cases of \u003cem\u003enon-albicans Candida\u003c/em\u003e (NAC) compared to cases of \u003cem\u003eCandida albicans\u003c/em\u003e (\u003cem\u003eP=0.04\u003c/em\u003e). History of antifungal prophylaxis was significantly higher among NAC cases compared to \u003cem\u003eCandida albicans\u003c/em\u003e cases (\u003cem\u003eP\u0026lt;0.05\u003c/em\u003e). Otherwise, there was no statistically significant difference regarding other characteristics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u0026nbsp;\u003c/strong\u003eCharacteristics of the studied cases in relation to isolated \u003cem\u003eCandida\u003c/em\u003e spp.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"670\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n=90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida albicans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n=37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eNon-albicans\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eCandida\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n=53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cem\u003eP-value\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u0026nbsp;\u003c/strong\u003eMedian (Min-Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e6.0 (1-84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e12.0 (1-84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e5.0 (1-80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Age \u0026le; 1 year\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Age \u0026ge; 60 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e9 (10%)\u003c/p\u003e\n \u003cp\u003e10 (11.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e4 (44.4%)\u003c/p\u003e\n \u003cp\u003e6 (60.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e5 (55.6%)\u003c/p\u003e\n \u003cp\u003e4 (40.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Male\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e55 (61.1%)\u003c/p\u003e\n \u003cp\u003e35 (38.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20 (36.4%)\u003c/p\u003e\n \u003cp\u003e17 (48.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e35 (63.6%)\u003c/p\u003e\n \u003cp\u003e18 (51.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eICU admission\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e69 (76.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e28 (40.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e41 (59.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.47\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistory of prior Surgery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e17 (18.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e8 (47.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e9 (52.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.58\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistory of antibiotic intake\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e87 (96.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e36 (41.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e51 (58.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e1.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistory of antifungal prophylaxis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e28 (31.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e7 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e21 (75.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 670px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnderlying co-morbidities:\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Liver cell failure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e15 (16.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e9 (60.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e6 (40.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.1 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Diabetes mellitus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e13 (14.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e6 (46.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e7 (53.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.69\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Renal failure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e12 (13.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e3 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e9 (75.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.22\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Encephalopathy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e8 (8.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e4 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e4 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.71\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Pulmonary diseases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e6 (6.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e2 (33.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e4 (66.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e1.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Hematological diseases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e8 (8.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e2 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e6 (75.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Solid tumors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e2 (2.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e0 (0.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e2 (100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.51\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;1ry immunodeficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e2 (2.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e1 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e1 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e1.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedical indwelling devices\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Multiple devices\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Peripheral venous catheter only\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e76 (84.4%)\u003c/p\u003e\n \u003cp\u003e14 (15.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e30 (39.5%)\u003c/p\u003e\n \u003cp\u003e7 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e46 (60.5%)\u003c/p\u003e\n \u003cp\u003e7 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal leucocytic count (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u0026nbsp;\u003c/strong\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026lt; 4 (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;4-11 (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;\u0026gt; 11 (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e16 (17.8%)\u003c/p\u003e\n \u003cp\u003e40 (44.4%)\u003c/p\u003e\n \u003cp\u003e34 (37.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5 (31.3%)\u003c/p\u003e\n \u003cp\u003e16 (40.0%)\u003c/p\u003e\n \u003cp\u003e15 (47.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11(68.8%)\u003c/p\u003e\n \u003cp\u003e24 (60.0%)\u003c/p\u003e\n \u003cp\u003e18 (52.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.56\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute Neutrophil count\u0026nbsp;\u003c/strong\u003e(\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMedian (Min-Max) *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e6.8 (0.93-32.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e8.9 (1.1-32.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e6.5 (0.93-28.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute Lymphocyte count\u0026nbsp;\u003c/strong\u003e(\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u0026nbsp;Median (Min-Max) *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e0.66 (0.24-1.93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e0.71 (0.42-1.93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e0.58 (0.24-1.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnemia\u0026nbsp;\u003c/strong\u003eN (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e82 (91.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e32 (39.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e50 (61.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.26\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThrombocytopenia\u0026nbsp;\u003c/strong\u003e N (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e45 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e16 (35.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e29 (64.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.28\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003e Significant\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere was statistically significant higher sensitivity to fluconazole \u0026amp; voriconazole among \u003cem\u003eCandida\u003c/em\u003e albicans compared to NAC isolates, whereas there was significant higher resistance to fluconazole and voriconazole in NAC compared to \u003cem\u003eCandida\u003c/em\u003e albicans isolates (\u003cem\u003eP=0.03\u003c/em\u003e) (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003eFluconazole and voriconazole susceptibility pattern among isolated \u003cem\u003eCandida\u003c/em\u003e species (n=90)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"723\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitive to Fluconazole \u0026amp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVoriconazole\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n= 73)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(81.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSDD\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e**\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;to Fluconazole, Resistant to Voriconazole\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n= 1)\u003c/p\u003e\n \u003cp\u003e(1.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResistant\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eto Fluconazole only\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n= 5)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(5.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResistant\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eto Voriconazole only\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n= 3)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(3.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResistant to Fluconazole \u0026amp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVoriconazole\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n= 8)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(8.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida albicans\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(37)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e36 (97.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1 (2.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.03*\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eNon-albicans Candida\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(53)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e37 (69.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1 (1.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e5 (9.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3 (5.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e7 (13.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. tropicalis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(40)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e30 (75.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1 (2.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2 (5.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e7 (17.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. parapsilosis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(8)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e7 (87.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1 (12.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. krusei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e5 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003e Significant, \u003csup\u003e**\u003c/sup\u003e Susceptible dose dependent .\u003c/p\u003e\n\u003cp\u003ePCR amplification of HS1 and HS2 regions of the ERG11 gene yielded clear species-specific bands. Ten isolates of \u003cem\u003eC. tropicalis\u003c/em\u003e produced the expected 834 bp fragment, one isolate of \u003cem\u003eC. parapsilosis\u003c/em\u003e produced a 789 bp fragment, and one isolate of \u003cem\u003eC. albicans\u003c/em\u003e yielded a 748 bp fragment (Figure 2).\u003c/p\u003e\n\u003cp\u003eNine different nucleotide mutations were detected; they were four silent mutations, (T225C, G264A, T591C and T783C), and five missense mutations with subsequent amino acid substitutions, T348A (D116E), A395T (Y132F), A428G (K143R), C461T (S154F)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Figure 3)\u003cem\u003e,\u0026nbsp;\u003c/em\u003ewhich were located in \u003cem\u003eERG11\u003c/em\u003e hot spot region I, Interestingly, \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ea novel missense mutation, T917G (M306R) located in between\u003cem\u003e\u0026nbsp;ERG11\u003c/em\u003e hot spot regions II \u0026amp; III was revealed in this study for the first time (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4:\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eERG11\u003c/em\u003e gene nucleotide mutations and corresponding amino acid substitutions detected among azole resistant \u003cem\u003eCandida\u003c/em\u003e isolates (n=12).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"643\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNucleotide mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of isolates showing this mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutation type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCorresponding amino acid substitution / Hot spot region\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT225C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eSilent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eG264A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eSilent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT348A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eMissense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eD116E / I\u003c/p\u003e\n \u003cp\u003eAsp \u0026rarr; Glu\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eA395T\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eMissense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eY132F / I\u003c/p\u003e\n \u003cp\u003eTyr \u0026rarr; Phe \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eA428G\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eMissense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eK143R / I\u003c/p\u003e\n \u003cp\u003eLys \u0026rarr; Arg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC461T\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eMissense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eS154F / I\u003c/p\u003e\n \u003cp\u003eSer \u0026rarr; Phe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT591C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eSilent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT783C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eSilent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT917G\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eMissense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eM306R\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eMet \u0026rarr; Arg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNew substitution\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the twelve-azole resistant \u003cem\u003eCandida spp\u003c/em\u003e. Y132F mutation was the most frequent (50%) missense mutation, detected in six \u003cem\u003eC. tropicalis\u003c/em\u003e isolates, followed by S154F mutation (33.3%), detected in four C. \u003cem\u003etropicalis\u0026nbsp;\u003c/em\u003eisolates, then K143R mutation (16.6%), detected in one \u003cem\u003eC. albicans\u003c/em\u003e isolate and one C. \u003cem\u003etropicalis\u0026nbsp;\u003c/em\u003eisolate, while D116E mutation (8.3%) was detected in only one \u003cem\u003eC. albicans\u003c/em\u003e isolate. Interestingly, the newly detected M306R mutation (8.3%) was detected in two C. \u003cem\u003etropicalis\u0026nbsp;\u003c/em\u003eisolates (Table 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5:\u0026nbsp;\u003c/strong\u003eMissense mutations among azole resistant \u003cem\u003eCandida\u0026nbsp;\u003c/em\u003especies isolates (n=12).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"622\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMissense mutation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIsolates showing this mutation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 336px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;species (n=12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida albicans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eNon-albicans Candida\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD116E\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e1 (8.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e1 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eY132F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e6 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK143R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e2 (16.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e1 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e1 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS154F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e4 (33.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e4 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eM306R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e2 (16.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e2 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe increasing incidence of antifungal resistance has become a global concern, limiting treatment options and complicating the management of fungal infections. Among these, azole-resistant \u003cem\u003eCandida\u003c/em\u003e species represent a growing threat in clinical practice \u003csup\u003e7\u003c/sup\u003e. In the present study, the prevalence of \u003cem\u003eCandida\u0026nbsp;\u003c/em\u003einfection among immunocompromised patients was 90/1404 (6.4%). A similiar rate (6.3%) was reported by\u003cem\u003e\u0026nbsp;El-Mashad et al.\u0026nbsp;\u003c/em\u003eamong cancer patients, with 95.3% of cases attributed to \u003cem\u003eCandida\u003c/em\u003e species\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e. In contrast, \u003cem\u003eEl-Mahallawy et al\u003c/em\u003e. and \u003cem\u003eGhrenassia et al.\u003c/em\u003e reported lower prevalence rates of \u003cem\u003eCandida\u003c/em\u003e infections, at 3.1% and 0.7% respectively\u0026nbsp;\u003csup\u003e9,10\u003c/sup\u003e.\u0026nbsp;These differences highlight that the prevalence of \u003cem\u003eCandida\u003c/em\u003e infections is strongly influenced by the patient population, underlying risk factors, and clinical setting.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Most \u003cem\u003eCandida\u003c/em\u003e-infected cases in this study occurred in pediatric patients (81.1%), while adults accounted for 18.9%. Similarly, \u003cem\u003eFurlaneto et al.\u003c/em\u003e (2011) reported a higher prevalence of \u003cem\u003eCandida\u003c/em\u003e bloodstream infections in neonates (35%) compared with older age groups (22.5%). \u003cem\u003eNon-albicans\u003c/em\u003e \u003cem\u003eCandida\u003c/em\u003e (NAC) species were more frequently isolated than \u003cem\u003eCandida albicans\u003c/em\u003e (58.9% vs. 41.1%). Similar observations were reported by \u003cem\u003eEl-Mashad et al., Liu et al.,\u003c/em\u003e and \u003cem\u003eAbouzeid et al.,\u003c/em\u003e who also documented higher isolation rates of NAC species \u003csup\u003e8,11,12\u003c/sup\u003e. This shift toward NAC dominance has been increasingly recognized worldwide and is of clinical concern, as many NAC species possess inherently higher levels of antifungal resistance, particularly to azoles \u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur findings indicate that \u003cem\u003eCandida\u003c/em\u003e \u003cem\u003ealbicans\u003c/em\u003e remained more susceptible to fluconazole and voriconazole, whereas NAC isolates showed higher resistance. This resistance may be linked to prior antifungal prophylaxis and lower lymphocyte counts in NAC cases \u003csup\u003e14,15\u003c/sup\u003e. Similar findings were reported by \u003cem\u003eEl-Mashad et al.,\u0026nbsp;\u003c/em\u003ewhereas\u003cem\u003e\u0026nbsp;El-Mahallawy et al.\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Khairat et al.\u003c/em\u003e observed higher resistance rates. Differences across studies may reflect variations in patient populations, antifungal exposure, and species distribution \u003csup\u003e8,9,16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The most frequently detected missense mutations were Y132F (6/12, 50%) and S154F (4/12, 33.3%), followed by K143R (2/12, 16.6%). Consistent with these findings, previous studies have also identified Y132F and S154F as the predominant mutations in fluconazole-resistant \u003cem\u003eCandida\u003c/em\u003e isolates \u003csup\u003e17-19\u003c/sup\u003e. By contrast, the least common mutation was D116E (1/12, 8.3%), detected in a single \u003cem\u003eC. albicans\u003c/em\u003e isolate with MIC values of 16 μg/mL for fluconazole and 1 μg/mL for voriconazole. Importantly, this mutation was not found alone but co-occurred with K143R. Similarly, \u003cem\u003eXiang et al.\u003c/em\u003e found the D116E substitution in both azole-susceptible and azole-resistant isolates, indicating that this substitution alone is unlikely to confer resistance \u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eInterestingly, this current study revealed a novel missense point mutation (T917G/M306R), located in between hot spot region II and hot spot region III of \u003cem\u003eERG11\u003c/em\u003e gene.\u0026nbsp;This novel mutation was identified in two azole-resistant \u003cem\u003eC. tropicalis\u003c/em\u003e isolates (2/12, 16.6%). The first was recovered from a bronchoalveolar lavage sample of a diabetic ICU patient and showed MIC values of 32 μg/mL for fluconazole and 2 μg/mL for voriconazole. The second was isolated from the blood of an ICU patient with bone marrow failure and exhibited MIC values of 8 μg/mL for fluconazole and 2 μg/mL for voriconazole. Notably, this mutation co-occurred with other missense mutations (Y132F and S154F). Therefore, additional studies are needed to determine whether this mutation is specifically associated with azole resistance or may also be present in azole-susceptible strains.\u0026nbsp;In addition, four silent point mutations were identified (T225C, G264A, T591C, and T783C) in our isolates. Notably, three of these (T225C, G264A, and T783C) have been previously reported in Azole-resistant \u003cem\u003eC. tropicalis\u003c/em\u003e clinical isolates\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eT225C and G264A mutations have also been observed in \u003cem\u003eCandida\u003c/em\u003e isolates from Egypt\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e. Similarly, \u003cem\u003eCorzo-Leon et al.\u003c/em\u003e reported T591C as a common silent SNP in all \u003cem\u003eC. parapsilosis\u003c/em\u003e isolates analyzed by \u003cem\u003eERG11\u003c/em\u003e sequencing\u0026nbsp;\u003csup\u003e23\u003c/sup\u003e. The recurrence of these mutations across different studies suggests that they may represent conserved genetic changes rather than random variations. These previously mentioned silent mutations, being synonymous mutations and therefore cannot be linked to azole resistance. However, recent studies suggest that synonymous mutations can influence the nucleic acid sequence and its structural properties. They may affect RNA secondary structure, stability, folding, and regulatory elements such as ribosome binding sites or splicing signals. These changes can alter codon usage, impact protein expression levels, and even modify substrate specificity, indicating that synonymous mutations are not entirely silent. Over time, the accumulation of such mutations, together with other factors, could ultimately affect the function of the target enzyme \u003csup\u003e22\u003c/sup\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAzole resistance among \u003cem\u003eCandida\u003c/em\u003e species isolated from immunocompromised patients represents a significant clinical challenge. In this study, we identified a notable prevalence of azole-resistant \u003cem\u003eCandida\u003c/em\u003e isolates, particularly among non-albicans \u003cem\u003eCandida\u003c/em\u003e species. Molecular analysis revealed several known missense mutations in the \u003cem\u003eERG11\u003c/em\u003e gene such as Y132F and S154F associated with resistance. Importantly, we also detected a novel mutation, M306R, in \u003cem\u003eC. tropicalis\u003c/em\u003e, which may contribute to resistance but requires further functional validation. These findings underscore the importance of routine antifungal susceptibility testing and molecular surveillance to guide effective antifungal therapy and prevent treatment failure in vulnerable patient populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;\u003c/strong\u003eElshaer M. wrote the main manuscript text. Salem A. H., and Elshaer M. carried out study analysis and prepared figures and tables. Salem A. H., and Eldegwi M. contributed to samples ordering and data collection. Elshaer M., Eldegwi M., Mashaly M. E., El-deeb D. R., and Agha S. A. revised the final draft. All authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors have no competing financial or personal interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003eApproval was obtained from the Mansoura Institutional Research Board (MFM-IRB; code number MDP.19.11.33). The procedures used in this study adhered to the principles of the Declaration of Helsinki. Written informed consent was obtained from all adult participants. For minors, informed consent for participation was obtained from a parent and/or legal guardian prior to sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eAll data supporting this article will be made available by the corresponding author to any qualified researcher upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBongomin, F., Gago, S., Oladele, R. O. \u0026amp; Denning, D. W. 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Q.\u003cem\u003e et al.\u003c/em\u003e Molecular mechanisms of azole resistance in Candida bloodstream isolates. \u003cem\u003eBMC infectious diseases\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 63, doi:10.1186/s12879-019-3672-5 (2019).\u003c/li\u003e\n\u003cli\u003eXiang, M. J.\u003cem\u003e et al.\u003c/em\u003e Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans. \u003cem\u003eFEMS yeast research\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 386-393, doi:10.1111/1567-1364.12042 (2013).\u003c/li\u003e\n\u003cli\u003eForastiero, A.\u003cem\u003e et al.\u003c/em\u003e Candida tropicalis antifungal cross-resistance is related to different azole target (Erg11p) modifications. \u003cem\u003eAntimicrobial agents and chemotherapy\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 4769-4781, doi:10.1128/aac.00477-13 (2013).\u003c/li\u003e\n\u003cli\u003eEl-Kholy, M. A., Helaly, G. F., El Ghazzawi, E. F., El-Sawaf, G. \u0026amp; Shawky, S. M. J. B. J. o. M. Analysis of CDR1 and MDR1 gene expression and ERG11 substitutions in clinical Candida tropicalis isolates from Alexandria, Egypt. \u003cstrong\u003e54\u003c/strong\u003e, 2609-2615 (2023).\u003c/li\u003e\n\u003cli\u003eCorzo-Leon, D. E., Peacock, M., Rodriguez-Zulueta, P., Salazar-Tamayo, G. J. \u0026amp; MacCallum, D. M. General hospital outbreak of invasive candidiasis due to azole-resistant Candida parapsilosis associated with an Erg11 Y132F mutation. \u003cem\u003eMedical mycology\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 664-671, doi:10.1093/mmy/myaa098 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7526278/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7526278/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eCandida\u003c/em\u003e species are opportunistic pathogens that can cause life-threatening infections, especially in immunocompromised patients. Azole antifungals are widely used for treatment, but resistance is increasing and often due to mutations in the \u003cem\u003eERG11\u003c/em\u003egene that compromises their efficacy\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eTo determine the prevalence of azole-resistant \u003cem\u003eCandida\u003c/em\u003e \u003cem\u003espp\u003c/em\u003e. among immunocompromised patients and identify mutations in the \u003cem\u003eERG11\u003c/em\u003e gene associated with resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn this cross-sectional study, clinical samples were processed from immunocompromised patients at two University Hospitals in Egypt. Identification of \u003cem\u003eCandida\u003c/em\u003e \u003cem\u003espp\u003c/em\u003e. was done using culture, chromogenic media, and VITEK 2 system. Susceptibility to fluconazole and voriconazole was assessed using broth microdilution method. Resistant isolates were subjected to PCR amplification and Sanger sequencing of \u003cem\u003eERG11\u003c/em\u003e hot spot regions\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eOut of 1404 eligible samples, 90 (6.4%) yielded \u003cem\u003eCandida\u003c/em\u003e \u003cem\u003espp\u003c/em\u003e., predominantly \u003cem\u003eC. tropicalis\u003c/em\u003e(44.4%) and \u003cem\u003eC. albicans\u003c/em\u003e (41.1%). Resistance to fluconazole and voriconazole was observed in 14.4% and 13.3% of isolates, respectively. \u003cem\u003eERG11\u003c/em\u003esequencing revealed multiple mutations. Common missense mutations included Y132F (50%) and S154F (33.3%). A novel mutation (M306R) was identified in two \u003cem\u003eC. tropicalis\u003c/em\u003e isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eAzole resistance among \u003cem\u003eCandida\u003c/em\u003eisolates is notable in immunocompromised patients. The present study highlights both known and novel \u003cem\u003eERG11\u003c/em\u003e mutations, emphasizing the need for routine molecular surveillance to guide antifungal therapy\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Molecular analysis of ERG11 gene in azole resistant Candida Species among immunocompromised patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 07:16:16","doi":"10.21203/rs.3.rs-7526278/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ded7e5d6-c77d-4712-96af-0c472f14a557","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55767789,"name":"Health sciences/Diseases"},{"id":55767790,"name":"Biological sciences/Microbiology"},{"id":55767791,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-12-26T20:11:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 07:16:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7526278","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7526278","identity":"rs-7526278","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}