Fluconazole Resistance Research Articles

Virulence Traits and Azole Resistance in Korean Candida auris Isolates.

We analyzed the virulence traits and azole resistance mechanisms of 104 Candida auris isolates collected from 13 Korean hospitals from 1996 to 2022. Of these 104 isolates, 96 (5 blood and 91 ear isolates) belonged to clade II, and 8 (6 blood and 2 other isolates) belonged to clade I. Fluconazole resistance (minimum inhibitory concentration ≥32 mg/L) was observed in 68.8% of clade II and 25.0% of clade I isolates. All 104 isolates were susceptible to amphotericin B and three echinocandins. In 2022, six clade I isolates indicated the first nosocomial C. auris cluster in Korea. Clade II C. auris isolates exhibited reduced thermotolerance at 42 °C, with diminished in vitro competitive growth and lower virulence in the Galleria mellonella model compared to non-clade II isolates. Of the 66 fluconazole-resistant clade II isolates, several amino acid substitutions were identified: Erg11p in 14 (21.2%), Tac1Ap in 2 (3.0%), Tac1Bp in 62 (93.9%), and Tac1Bp F214S in 33 (50.0%). Although there were a limited number of non-clade II isolates studied, our results suggest that clade II C. auris isolates from Korean hospitals might display lower virulence traits than non-clade II isolates, and their primary fluconazole resistance mechanism is linked to Tac1Bp mutations.

Mutations in TAC1 and ERG11 are major drivers of triazole antifungal resistance in clinical isolates of Candida parapsilosis

ObjectivesThe aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates. MethodsSequences of CpERG11 and CpTAC1 were determined for 35 resistant Candida parapsilosis clinical isolates. A plasmid-based CRISPR-Cas9 system was used to introduce mutations leading to amino acid substitution in CpTac1 and CpErg11. Triazole susceptibility was determined by broth microdilution and E-test. Differential expression of genes mediated by CpTAC1 mutation was determined by RNA sequencing, and relative expression of individual transporter genes was assessed with RT-qPCR. ResultsSix isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11Y132F substitution. When introduced into susceptible isolates, this CpERG11 mutation led to a 4- to 8-fold increase in fluconazole minimum inhibitory concentrations (MIC; 0.125 μg/mL vs. 0.5 μg/mL, 0.125 μg/mL vs. 0.5 μg/mL, and 0.5 μg/mL vs. 4 μg/mL). When introduced into a susceptible isolate, the CpTAC1 mutation leading to the G650E substitution resulted in an 8-fold increase in fluconazole MIC (0.25 μg/mL vs. 2 μg/mL), whereas correction of this mutation in resistant isolates led to a 16-fold reduction in MIC (32 μg/mL vs. 2 μg/mL). CpCDR1, CpCDR1B, and CpCDR1C were overexpressed in the presence CpTac1G650E. Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 μg/mL vs. 8 μg/mL). DiscussionThese results define the specific contribution made by the Y132F substitution in CpERG11 and demonstrate a role for activating mutations in CpTAC1 in triazole resistance in C. parapsilosis.

Characterization and Susceptibility Pattern of Candida Species from Various Clinical Samples in a Rural Tertiary Care Hospital

Fungi infections are becoming more prevalent and burdensome on a global scale leading to an important concern for immunocompromised patients. Hospitals often become infected with serious, invasive Candida infections. Higher frequency of Non-albicans Candida (NAC) species are found in the hospital setting, and some of these fungi can become opportunistic. Pathogens after a change in the host environment trigger them to move from a commensal to a pathogenic phase. Various clinical symptoms of Candida species, which are common human commensals, range from mucocutaneous overgrowth to bloodstream infections. In many hospitals, phenotypic methods are still considered the gold standard method for identification. Among the 112 isolates, Candida albicans (n=47; 52.64%) was noted as a significant etiology isolated from clinical samples. Further, C. albicans accounted the principal etiology in urine (n=28; 31.36%), and vaginal swab (n=13; 14.56%), followed by C. tropicalis (urine: n=15; 16.8% and vaginal swab: n=5; 5.6%). In blood C. pelliculosa (n=14; 15.68%) was found to be predominant followed by C. tropicalis (n=11; 12.32%). Antifungal susceptibility pattern was performed for (n=51) samples by VITEK AST and 100% susceptibility (voriconazole, and micafungin) was recorded in C. tropicalis and C. albicans. Whereas, fluconazole resistance was observed in C. tropicalis (n=3; 15%), and C. pelliculosa (n=1; 11.11%) and amphotericin B resistance in C. tropicalis (n=1; 5%) and C. albicans (n=1; 9.1%).

Candida auris: a global pathogen that has taken root in Colombia

Candida auris has been recognized as an emerging multidrug-resistant pathogen with a significant public health burden, causing cases of invasive infection and colonization due to its persistence on inanimate surfaces, ability to colonize skin of some patients, and high transmissibility in healthcare settings. The first sporadic report of the isolation of this species from the ear canal of a patient in Asia was in 2009 and reports from other regions of the world soon followed. However, it was not until 2015 that global epidemiological alerts were communicated as a result of an increasing number of reports of invasive infections caused by C. auris in several countries. Colombia was soon added to this list in 2016 after an unusual increase in the number of C. haemulonii isolates was reported, later confirmed as C. auris. Since the issuing of a national alert by the Colombian National Institute of Health together with the Ministry of Health in 2016, the number of cases reported reached over 2,000 by 2022. Colombian isolates have not shown pan resistance to available antifungals, unlike C. auris strains reported in other regions of the world, which leaves patients in Colombia with therapeutic options for these infections. However, increasing fluconazole resistance is being observed. Whole-genome sequencing of Colombian C. auris isolates has enhanced molecular epidemiological data, grouping Colombian isolates in clade IV together with other South American isolates.

Expression of ERG11, ERG3, MDR1 and CDR1 genes in Candida tropicalis

Drug resistance to azoles is a growing problem in the Candida genus. To analyze molecularly the genes responsible for fluconazole resistance in Candida tropicalis strains. Nineteen strains, with and without exposure to fluconazole, were selected for this study. The expression of MDR1, CDR1, ERG11, and ERG3 genes was analyzed in sensitive, dose-dependent sensitive, and resistant strains exposed to different concentrations of the antifungal drug. MDR1, ERG11 and ERG3 genes were significantly overexpressed in the different sensitivity groups. CDR1 gene expression was not statistically significant among the studied groups. Seven of the eight fluconazole-resistant strains showed overexpression of one or more of the analyzed genes. In some dose-dependent sensitive strains, we found overexpression of CDR1, ERG11, and ERG3. The frequency of overexpression of ERG11 and ERG3 genes indicates that they are related to resistance. However, the finding of dose-dependent resistant/sensitive strains without overexpression of these genes suggests that they are not exclusive to this phenomenon. More basic research is needed to study other potentially involved genes in the resistance mechanism to fluconazole.

Analysis of CDR1 and MDR1 Gene Expression and ERG11 Substitutions in Clinical Candida tropicalis Isolates from Alexandria, Egypt

IntroductionCandida tropicalis is a common non-albicans Candida (NAC) species that causes numerous fungal infections. Increasing antifungal resistance to azoles in NAC is becoming a major health problem worldwide; however, in Egypt, almost no data is available regarding fluconazole resistance mechanisms in C. tropicalis. The current study aims to investigate two possible important molecular mechanisms involved in fluconazole resistance in C. tropicalis isolates.MaterialsFifty-four clinical C. tropicalis isolates were included. Identification and antifungal susceptibility profiles of the isolates were carried out using the VITEK 2 compact system. The molecular investigation of fluconazole resistance included the expression of the CDR1 and MDR1 genes by quantitative real-time RT-PCR as well as the sequence analysis of the ERG11 gene.ResultsAntifungal susceptibility testing identified 30 fluconazole-non-susceptible isolates. Statistically, CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher than that in fluconazole-susceptible isolates, with MDR1 gene expression levels that were similar in both non-susceptible and susceptible isolates. Sequence analysis of the ERG11 gene of 26 fluconazole-resistant isolates identified two missense mutations: A395T (Y132F) and G1390A (G464S).ConclusionsThis study has highlighted the role of overexpression of the CDR1 gene and ERG11 gene mutations in fluconazole non-susceptibility. Further studies in Egypt are required to investigate other possible molecular mechanisms involved in azole resistance.

European candidaemia is characterised by notable differential epidemiology and susceptibility pattern: Results from the ECMM Candida III study

The objectives of this study were to assess Candida spp. distribution and antifungal resistance of candidaemia across Europe. Isolates were collected as part of the third ECMM Candida European multicentre observational study, conducted from 01 to 07-07-2018 to 31-03-2022. Each centre (maximum number/country determined by population size) included ∼10 consecutive cases. Isolates were referred to central laboratories and identified by morphology and MALDI-TOF, supplemented by ITS-sequencing when needed. EUCAST MICs were determined for five antifungals. fks sequencing was performed for echinocandin resistant isolates. The 399 isolates from 41 centres in 17 countries included C. albicans (47.1%), C. glabrata (22.3%), C. parapsilosis (15.0%), C. tropicalis (6.3%), C. dubliniensis and C. krusei (2.3% each) and other species (4.8%). Austria had the highest C. albicans proportion (77%), Czech Republic, France and UK the highest C. glabrata proportions (25-33%) while Italy and Turkey had the highest C. parapsilosis proportions (24-26%). All isolates were amphotericin B susceptible. Fluconazole resistance was found in 4% C. tropicalis, 12% C. glabrata (from six countries across Europe), 17% C. parapsilosis (from Greece, Italy, and Turkey) and 20% other Candida spp. Four isolates were anidulafungin and micafungin resistant/non-wild-type and five resistant to micafungin only. Three/3 and 2/5 of these were sequenced and harboured fks-alterations including a novel L657W in C. parapsilosis. The epidemiology varied among centres and countries. Acquired echinocandin resistance was rare but included differential susceptibility to anidulafungin and micafungin, and resistant C. parapsilosis. Fluconazole and voriconazole cross-resistance was common in C. glabrata and C. parapsilosis but with different geographical prevalence.

Biofilm Formation and Aspartyl Proteinase Activity and Their Association with Azole Resistance Among Candida albicans Causing Vulvovaginal Candidiasis, Egypt.

Candida albicans (C. albicans) is a major cause of vulvovaginal candidiasis (VVC), a condition that is commonly treated with azole agents. Biofilm formation and aspartyl proteinase production are important virulence factors that could be linked to azole resistance in C. albicans impeding therapy. To find out the association of both factors with azole resistance among C. albicans isolated from VVC cases in Egyptian nonpregnant women of childbearing age. In a cross-sectional study, C. albicans was isolated from nonpregnant females diagnosed clinically as having VVC during a 1-year study period. Susceptibility to azole agents was tested using the disc diffusion method. Biofilm formation and aspartyl proteinase production were assessed phenotypically. Additionally, two biofilm-related genes (ALS1 and HWP1) and three proteinase genes (SAP2, SAP4, and SAP6) were screened for using polymerase chain reaction (PCR). Among 204 C. albicans isolates, azole resistance ratios were as follows: voriconazole (30.4%), itraconazole (17.6%), fluconazole (11.3%) and econazole (6.4%). Biofilm-producing capacity was detected in 63.2% of isolates, and 63.2% were proteinase producers. The frequencies of ALS1 and HWP1 were 69.6% and 74.5%, respectively, while SAP2, SAP4, and SAP6 were 69.2%, 88.7%, and 64.7%, respectively. Biofilm formation was significantly associated with azole resistance (P < 0.001 for each tested azole agent) as was proteinase production (P < 0.001 for fluconazole, voriconazole, and econazole resistance and P = 0.047 for itraconazole). Among nonpregnant Egyptian women of childbearing age, azole resistance in C. albicans causing VVC is significantly associated with biofilm formation and proteinase production. The development of new therapeutic agents that can target these factors is warranted.

COVID-19-associated candidiasis and the emerging concern of Candida auris infections.

The incidence of COVID-19-associated candidiasis (CAC) is increasing, resulting in a grave outcome among hospitalized patients with COVID-19. The most alarming condition is the increasing incidence of multi-drug resistant Candida auris infections among patients with COVID-19 worldwide. The therapeutic strategy towards CAC caused by common Candida species, such as Candida albicans, Candida tropicalis, and Candida glabrata, is similar to the pre-pandemic era. For non-critically ill patients or those with a low risk of azole resistance, fluconazole remains the drug of choice for candidemia. For critically ill patients, those with a history of recent azole exposure or with a high risk of fluconazole resistance, echinocandins are recommended as the first-line therapy. Several novel therapeutic agents alone or in combination with traditional antifungal agents for candidiasis are potential options in the future. However, for multidrug-resistant C. auris infection, only echinocandins are effective. Infection prevention and control policies, including strict isolation of the patients carrying C. auris and regular screening of non-affected patients, are suggested to prevent the spread of C. auris among patients with COVID-19. Whole-genome sequencing may be used to understand the epidemiology of healthcare-associated candidiasis and to better control and prevent these infections.

A successful management of fungal peritonitis caused by Rhodotorula glutinis in CAPD patient, coincident with onychomycosis by Penicillium sp: Case report.

Rhodotorula glutinis is a rare cause of fungal peritonitis in peritoneal dialysis (PD) patients. The combination treatment between aggressive PD catheter replacement and adequate antifungal treatment is crucial in managing peritonitis due to fungal infection and onychomycosis. A 37-year-old man PD patient presented with cloudy effluent, abdominal pain, and black debris in the lumen of his PD catheter. Twelve days before admission, the patient traveled for 10 days to a high-temperature country, Saudi Arabia, for purpose of accomplishing haj. From the physical examinations, there was an onychomycosis in his right toenail. The result of the dialysate cell count confirmed the evidence of peritonitis (i.e., cell count of 187 cells per µL, however with polymorphonuclear 31%). The dialysate culture indicated R glutinis, with no growth of bacteria. Fungal culture of his toenail scrapings was obtained and the result was Penicillium sp. Based on the high clinical suspicion of fungal peritonitis, fluconazole intraperitoneal (IP) was immediately given on the first day in addition to empirical antibiotics, that is, cefazolin and gentamycin IP. His peritoneal catheter was simultaneously removed and reinserted on the 4th day of treatment. Since there was a sign of fluconazole resistance, fluconazole IP was switched into oral voriconazole, without any antimicrobial treatment intraperitoneally. After 21 days of voriconazole, oral itraconazole was given until 3 months for his onychomycosis. Clinical improvement was seen on the effluent where the leucocyte count falls below 100 cells after 21 days of giving voriconazole. This case report suggests the need for comprehensive evaluations of the risk for fungal infection in continuous ambulatory PD patients, especially those who live in a tropical country.

Yeasts from blood cultures in the wake of the COVID-19 pandemic in a tertiary care hospital: Shift in species epidemiology, steady low antifungal resistance and full in vitro ibrexafungerp activity.

Several institutions reported a rise not only in fungemia incidence but also in the number of cases caused by Candida auris or fluconazole-resistant C. parapsilosis during the COVID-19 pandemic. Since the pandemic broke out in early 2020, we studied its impact on fungemia incidence, species epidemiology, potential patient-to-patient transmission, and antifungal resistance in 166 incident yeast isolates collected from January 2020 to December 2022. Isolates were molecularly identified, and their antifungal susceptibilities to amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp were studied following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method, and genotyped. The fungemia incidence (episodes per 1000 admissions) tended to decrease over time (2020=1.60, 2021=1.36, 2022=1.16); P>.05). Species distribution was C. albicans (50.6%, n=84), C. parapsilosis (18.7%, n=31), C. glabrata (12.0%, n=20), C. tropicalis (11.4%, n=19), C. krusei (3.0%, n=5), other Candida spp. (1.2%, n=2), and non-Candida yeasts (3.0%, n=5). The highest and lowest proportions of C. albicans and C. parapsilosis were detected in 2020. The proportion of isolates between 2020 and 2022 decreased in C. albicans (60.3% vs. 36.7%) and increased in C. parapsilosis (10.3% vs. 28.6%; P<.05) and C. tropicalis (8.8% vs. 16.3%; P>.05). Only three C. albicans intra-ward clusters involving two patients each were detected, and the percentages of patients involved in intra-ward clusters reached 9.8% and 8.0% in 2020 and 2021, respectively, suggesting that clonal spreading was not uncontrolled. Fluconazole resistance (5%) exhibited a decreasing trend (P>.05) over time (2020=7.6%; 2021=4.2%; and 2022=2.1%). Ibrexafungerp showed high in vitro activity.

Genotyping and susceptibility testing uncovers large azole-resistant Candida tropicalis clade in Alexandria, Egypt

Candida tropicalis is an emerging medically relevant Candida species. The yeast primarily causes opportunistic infections in intensive care units and is highly prevalent in tropical countries. The genetic diversity within this species is high, and nosocomial transmission has been reported. C. tropicalis genotyping of isolates from low- and middle-income countries is underrepresented when compared with that from high-income countries. Also, in Egypt, only limited genotyping has been conducted for C. tropicalis isolates, while antifungal resistance seems to increase, especially against azoles. Antifungal susceptibility testing was performed on 64 C. tropicalis isolates from ICU patients collected from multiple hospitals in Alexandria, Egypt. Genotyping by means of short tandem repeat (STR) and whole genome sequencing (WGS) single nucleotide polymorphism (SNP) analysis was performed. Using antifungal susceptibility testing, fluconazole resistance was observed in 24 isolates (38%), of which 23 harboured an ERG11 G464S substitution, previously shown to cause resistance in Candida albicans. STR genotyping showed that these 23 isolates were related, forming a distinct resistant clade. WGS SNP analysis subsequently confirmed this genetic relationship, although isolates within this clade differed in at least 429 SNPs, suggesting that these were independently introduced. Overall, STR and WGS SNP analysis of this collection indicates limited C. tropicalis nosocomial transmission in Alexandria, while the presence of this large azole-resistant C. tropicalis clade within this city hampers the treatment of intensive care unit patients.

Molecular and Clinical Features of Fluconazole Non-susceptible Candida albicans Bloodstream Isolates Recovered in Korean Multicenter Surveillance Studies.

Acquired fluconazole resistance (FR) in bloodstream infection (BSI) isolates of Candida albicans is rare. We investigated the FR mechanisms and clinical features of 14 fluconazole non-susceptible (FNS; FR and fluconazole-susceptible dose-dependent) BSI isolates of C. albicans recovered from Korean multicenter surveillance studies during 2006-2021. Mutations causing amino acid substitutions (AASs) in the drug-target gene ERG11 and the FR-associated transcription factor genes TAC1, MRR1, and UPC2 of the 14 FNS isolates were compared with those of 12 fluconazole-susceptible isolates. Of the 14 FNS isolates, eight and seven had Erg11p (K143R, F145L, or G464S) and Tac1p (T225A, R673L, A736T, or A736V) AASs, respectively, which were previously described in FR isolates. Novel Erg11p, Tac1p, and Mrr1p AASs were observed in two, four, and one FNS isolates, respectively. Combined Erg11p and Tac1p AASs were observed in seven FNS isolates. None of the FR-associated Upc2p AASs were detected. Of the 14 patients, only one had previous azole exposure, and the 30-day mortality rate was 57.1% (8/14). Our data show that Erg11p and Tac1p AASs are likely to contribute to FR in C. albicans BSI isolates in Korea and that most FNS C. albicans BSIs develop without azole exposure.

Involvement of AoMdr1 in the Regulation of the Fluconazole Resistance, Mycelial Fusion, Conidiation, and Trap Formation of Arthrobotrys oligospora.

Multidrug resistance (Mdr) proteins are critical proteins for maintenance of drug resistance in fungi. Mdr1 has been extensively studied in Candida albicans; its role in other fungi is largely unknown. In this study, we identified a homologous protein of Mdr (AoMdr1) in the nematode-trapping (NT) fungus Arthrobotrys oligospora. It was found that the deletion of Aomdr1 resulted in a significant reduction in the number of hyphal septa and nuclei as well as increased sensitivity to fluconazole and resistance to hyperosmotic stress and SDS. The deletion of Aomdr1 also led to a remarkable increase in the numbers of traps and mycelial loops in the traps. Notably, AoMdr1 was able to regulate mycelial fusion under low-nutrient conditions, but not under nutrient-rich conditions. AoMdr1 was also involved in secondary metabolism, and its deletion caused an increase in arthrobotrisins (specific compounds produced by NT fungi). These results suggest that AoMdr1 plays a crucial role in the fluconazole resistance, mycelial fusion, conidiation, trap formation, and secondary metabolism of A. oligospora. Our study contributes to the understanding of the critical role of Mdr proteins in mycelial growth and the development of NT fungi.

Coregulation of extracellular vesicle production and fluconazole susceptibility in Cryptococcus neoformans.

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

Comparative Analysis of Two Candida parapsilosis Isolates Originating from the Same Patient Harbouring the Y132F and R398I Mutations in the ERG11 Gene.

This work presents a comparative analysis of two clinical isolates of C. parapsilosis, isolated from haemoculture (HC) and central venous catheter (CVC). Both strains harboured Y132F and R398I mutations in the gene ERG11 associated with resistance to fluconazole (FLC). Differences between the HC and CVC isolates were addressed in terms of virulence, resistance to FLC, and lipid distribution. Expression of the ERG6 and ERG9 genes, lipid analysis, fatty acid composition, and lipase activity were assessed via qPCR, thin-layer chromatography/high-performance liquid chromatography, gas chromatography, and spectrophotometry, respectively. Regulation of the ERG6 and ERG9 genes did not prove any impact on FLC resistance. Analysis of lipid metabolism showed a higher accumulation of lanosterol in both the isolates regardless of FLC presence. Additionally, a decreased level of triacylglycerols (TAG) with an impact on the composition of total fatty acids (FA) was observed for both isolates. The direct impact of the ERG11 mutations on lipid/FA analysis has not been confirmed. The higher lipase activity observed for C. parapsilosis HC isolate could be correlated with the significantly decreased level of TAG. The very close relatedness between both the isolates suggests that one isolate was derived from another after the initial infection of the host.

Fluconazole resistance in non-albicans Candida species in the United States, 2012-2021

Background:Candida spp can cause a variety of infections known as candidiasis, ranging from severe invasive infections to superficial mucosal infections of the mouth and vagina. Fluconazole, a triazole antifungal, is commonly prescribed to treat candidiasis but increasing fluconazole resistance is a growing concern for several Candida spp. Although C. albicans has historically been the most common cause of candidiasis, other species are increasingly common and antifungal resistance is more prevalent in these non-albicans species, including C. glabrata, C. parapsilosis, and C. tropicalis, which were the focus of this analysis. Methods: We used the PINC AI healthcare data (PHD) database to examine fluconazole resistance for inpatient isolates between 2012 and 2021 from 187 US acute-care hospitals with at least 1 Candida spp culture with a fluconazole susceptibility result over the entire period. We calculated annual percentage fluconazole resistance for C. glabrata, C. tropicalis, and C. parapsilosis isolates using the clinical laboratory interpretation for resistance. Results: We identified 4,264 C. glabrata, 2,482 C. parapsilosis, and 2,283 C. tropicalis isolates between 2012 and 2021 with susceptibility results. The percentage of C. glabrata isolates resistant to fluconazole doubled between 2020 and 2021 (14.6% vs 29.3%) (Fig. 1a). The percentage of C. parapsilosis isolates resistant to fluconazole steadily increased since 2017 (Fig. 1b), with an 82% increase in 2021 compared with 2020 (3.8% in 2020 vs 6.9% in 2021). Fluconazole resistance among C. tropicalis isolates varied over the years, with a 0.3% decrease in 2021 from 2020 (Fig. 1c). Of hospitals reporting at least 1 result each year 2020–2021, 44% observed an increase in the proportion of C. glabrata isolates resistant to fluconazole in 2021 compared to 2020. Conclusions: Our analysis highlights a concerning increase in fluconazole resistance among C. glabrata and C. parapsilosis isolates in 2021 compared with previous years. Further investigation of the observed increases in fluconazole resistance among these Candida spp could provide further insight on potential drivers of resistance or limitations in reported results from large databases. More analyses are needed to understand rates, sites of Candida infections, and risk factors (eg, antifungal exposure) associated with resistance.Disclosures: None

Fluconazole Resistance in C. Tropicalis by Broth Microdilution Method and to compare the relative gene expression of erg11 gene in both Fluconazole Resistant and Sensitive C Tropiocalis

Aim: To evaluate fluconazole resistance among C. tropicalis by broth micro dilution methodand relative expression of ergosterol(ERG11) gene in fluconazole resistant and sensitive Candida tropicalis. Methods: The study design was comparative study conducted inMicrobiology Department of UHS.A sum up of 66 confirmed isolates of Candidatropicalis were attained from Jinnah Hospital, Lahore. The fluconazoleresistance of the isolates was determined by broth microdilution methodand the relative gene expression of ERG11 gene was analyzed by real time PCR, in Microbiology Department of UHS. Results: The antibiotic susceptibility testing of C.tropicalis showed fifty one fluconazole sensitive, eight susceptible dose dependent and seven fluconazole resistantC. tropicalis. Relative gene expression ofERG11gene showed anincrease expression about 2.0 fold in C. tropicalis resistant isolates than C. tropicalis susceptible isolates. Globally, C. albicanswas the most frequently isolated species but now there is a decline in trendwith increase figures for non-albicans Candida species. Thesenon-candidaalbicans are now emerging as resistant species and cause serious ailments. This study showed seven fluconazole resistant C. tropicalis among sixty six isolates by broth microdilution method. Further on molecular testing by relative gene expression of ERG11 gene it was noticed that these resistant isolates have 2.0 fold increased mRNA expression levels of this gene as compared to sensitive strains. Conclusion: C. tropicalis was found to be resistant to fluconazole. Ergosterol expression was markedly raised in fluconazole resistant C. tropicalis in contrasted to drug sensitive C. tropicalis. Keywords: C. tropicalis, fluconazole resistance antifungal susceptibility testing, non-albicans Candida, broth microdilution method, relative gene expression.

Fluconazole Resistance and Virulence in In Vitro Induced-Fluconazole Resistant Strains and in Clinical Fluconazole Resistant Strain of Cryptococcus deuterogattii.

Neuromeningeal cryptococcosis is a life-threatening infection of the central nervous system, caused by encapsulated yeast belonging to the Cryptococcus neoformans and Cryptococcus gattii species complexes. Recent data showed that virulence and antifungal resistance are variable for yeasts belonging to the C. gattii species complex. There is an increase in resistance to fluconazole for yeasts of the C. gattii species complex and the virulence is variable according to the genotype. In the present study, (i) we explored and compared the mechanisms of resistance to fluconazole between C. deuterogattii clinically resistant strains and induced fluconazole-resistant strains by exposure to fluconazole in vitro, and (ii) we studied their virulence in the Galleria mellonella study model. We demonstrated that the fluconazole resistance mechanisms involved were different between clinically resistant strains and induced resistant strains. We also demonstrated that fluconazole-induced resistant strains are less virulent when compared to the original susceptible strains. On the contrary, the clinically resistant strain tested maintains its virulence compared to fluconazole-susceptible strains of the same sequence type.

A 3-year study of Candida infections among patients with malignancy: etiologic agents and antifungal susceptibility profile.

Opportunistic fungal infections by Candida species arise among cancer patients due to the weakened immune system following extensive chemotherapy. Prophylaxis with antifungal agents have developed the resistance of Candida spp. to antifungals. Accurate identification of yeasts and susceptibility patterns are main concerns that can directly effect on the treatment of patients. Over a period of three years, 325 cancer patients suspected to Candida infections were included in the current investigation. The clinical isolates were molecularly identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). All strains, were examined for in vitro susceptibility to the amphotericin B, itraconazole, fluconazole, and anidulafungin according to the CLSI M27 document. Seventy-four cancer patients had Candida infections (22.7%). Candida albicans was the most common species (83.8%). Antifungal susceptibility results indicated that 100% of the Candida isolates were sensitive to amphotericin B; however, 17.6%, 9.4%, and 5.4% of clinical isolates were resistant to anidulafungin, fluconazole, and itraconazole, respectively. The findings of the present work shows a warning increase in resistance to echinocandins. Since all fluconazole resistance isolates were obtained from candidemia, we recommend amphotericin B as the first line therapy for this potentially fatal infection.