Candida Haemulonii Species Complex Research Articles

Mechanism of azole resistance in Candida vulturna, an emerging multidrug resistant pathogen related with Candida haeumulonii and Candida auris.

Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.

Cell Aggregation Capability of Clinical Isolates from Candida auris and Candida haemulonii Species Complex.

The opportunistic fungal pathogens belonging to the Candida haemulonii complex and the phylogenetically related species Candida auris are well-known for causing infections that are difficult to treat due to their multidrug-resistance profiles. Candida auris is even more worrisome due to its ability to cause outbreaks in healthcare settings. These emerging yeasts produce a wide range of virulence factors that facilitate the development of the infectious process. In recent years, the aggregative phenotype has been receiving attention, as it is mainly associated with defects in cellular division and its possible involvement in helping the fungus to escape from the host immune responses. In the present study, we initially investigated the aggregation ability of 18 clinical isolates belonging to the C. haemulonii species complex (C. haemulonii sensu stricto, C. duobushaemulonii, and C. haemulonii var. vulnera) and C. auris. Subsequently, we evaluated the effects of physicochemical factors on fungal aggregation competence. The results demonstrated that cell-to-cell aggregation was a typically time-dependent event, in which almost all studied fungal isolates of both the C. haemulonii species complex and C. auris exhibited high aggregation after 2 h of incubation at 37 °C. Interestingly, the fungal cells forming the aggregates remained viable. The aggregation of all isolates was not impacted by pH, temperature, β-mercaptoethanol (a protein-denaturing agent), or EDTA (a chelator agent). Conversely, proteinase K, trypsin, and sodium dodecyl sulfate (SDS) significantly diminished the fungal aggregation. Collectively, our results demonstrated that the aggregation ability of these opportunistic yeast pathogens is time-dependent, and surface proteins and hydrophobic interactions seem to mediate cell aggregation since the presence of proteases and anionic detergents affected the aggregation capability. However, further studies are necessary to better elucidate the molecular aspects of this intriguing phenomenon.

Candida vulturna Outbreak Caused by Cluster of Multidrug-Resistant Strains, China.

Candida vulturna belongs to the Candida haemulonii species complex and is phylogenetically related to C. auris. We report a C. vulturna outbreak among persons in Shanxi Province, China, during 2019-2022. Isolates were resistant to multiple antifungal drugs and exhibited enhanced adhesion and biofilm formation properties.

Candida haemulonii Species Complex: A Mini-review.

Candida haemulonii species complex (CHSC) are emerging multidrug-resistant yeast pathogens able to cause life-threatening human infections in at-risk populations for invasive candidiasis worldwide. A recent laboratory survey conducted in 12 medical centers found that prevalence rates of Candida haemulonii complex isolates rose from 0.9 to 1.7% along the period between 2008 and 2019. We present a mini-review addressing recent aspects of the epidemiology, diagnosis and therapy of infections due to CHSC.

Extracellular Vesicles from Candida haemulonii var. vulnera Modulate Macrophage Oxidative Burst

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (1010 particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O2•- and H2O2 levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2-PGE2 pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets.

Candida khanbhai sp. nov., a new clinically relevant yeast within the Candida haemulonii species complex.

Invasive fungal infections caused by non-albicans Candida species are increasingly reported. Recent advances in diagnostic and molecular tools enabled better identification and detection of emerging pathogenic yeasts. The Candida haemulonii species complex accommodates several rare and recently described pathogenic species, C. duobushaemulonii, C. pseudohaemulonii, C. vulturna, and the most notorious example is the outbreak-causing multi-drug resistant member C. auris. Here, we describe a new clinically relevant yeast isolated from geographically distinct regions, representing the proposed novel species C. khanbhai, a member of the C. haemulonii species complex. Moreover, several members of the C. haemulonii species complex were observed to be invalidly described, including the clinically relevant species C. auris and C. vulturna. Hence, the opportunity was taken to correct this here, formally validating the names of C. auris, C. chanthaburiensis, C. konsanensis, C. metrosideri, C. ohialehuae, and C. vulturna.

Candida haemulonii species complex: emerging fungal pathogens of the Metschnikowiaceae clade.

Candida species represent the most common fungal pathogens of humans, causing not only superficial infections but also life-threatening invasive infections, especially in immunocompromised individuals. While Candida albicans remains the most frequent cause of candidiasis, infections caused by non-albicans Candida species have been increasingly reported in clinical settings over the past two decades. Recently, species of the Metschnikowiaceae clade including the "superbug" Candida auris and other members of the Candida haemulonii species complex have attracted significant attention due to their multidrug resistance and high rates of transmission in clinical settings. In this review, we summarize the epidemiology, biology, virulence, and drug resistance of the C. haemulonii species complex and discuss potential reasons for the recent increase in prevalence of infections caused by non-albicans species in clinical settings.

S4.3d Mechanism of fluconazole resistance in Candida vulturna, a member of the Candida haemulonii complex of multidrug - resistant yeasts

Emergent species of the Candida genus, September 22, 2022, 10:30 AM - 12:00 PM Objetives Candida vulturna is an emerging yeast firstly isolated in 2016 from flowers and from a blood sample in Malaysia. It is a member of the Candida haemulonii species complex. This complex has 9 species within which C. haemulonii (including the vulnera variety), C. pseudohemulonii, C. duobushaemulonii, C. auris, and now C. vulturna are considered human pathogens. Like the other members of the haemulonii complex, the identification of C. vulturna is difficult. Due to the scarcity of strains to study, the susceptibility pattern of this species is unknown. The objectives of this work were to study C. vulturna strains isolated from clinical samples from different cities of Colombia, to study their susceptibility patterns, and to establish their mechanisms of azole resistance.MethodsFive C. vulturna strains isolated from urine (n = 1) and blood (n = 4) in three different cities in Colombia were studied. Strains were identified as C. duobushaemulonii (n = 4) and C. haemulonii (n = 1) by means of VITEK2 (software version 9.02) and as C. pseudohaemulonii (n = 5) by using Bruker's MALDI-TOF in Colombia. Upon arrival in Argentina, isolates were molecularly identified by sequencing the ITS regions (rDNA). Antifungal susceptibility testing was performed following the CLSI´s M27 4th ed document. Amphotericin B, anidulafungin, caspofungin, 5-flucytosine, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole were tested. In addition, ERG11 genes of the five strains were amplified and sequenced.ResultsThe sequences of the ITS regions were analyzed and the 5 strains were identified as C. vulturna (100% homology). All the strains presented low echinocandins MICs (0.06-0.12 μg/ml), 5-flucytosine (0.06-0.12 μg/ml) and triazoles with the exception of fluconazole (isavuconazole: <0.015 -0.12 μg/ml, itraconazole: 0.06-0.50 μg/ml, posaconazole: <0.015-0.015 μg/ml and voriconazole: 0.03-0.50 μg/ml). In the case of amphotericin B and fluconazole, 4 of the 5 strains presented high MICs for both antifungals (>8 μg/ml and >64 μg/ml), while the remaining one presented MIC values = 1 μg/ml for the polyene and for the azole (cross amphotericin B and fluconazole high MICs). The strain with low fluconazole and amphotericina B MICs was the one isolated from urine. ERG11 sequences analysis showed that the 4 strains with high fluconazole MICs (blood isolates) harbor five amino acid substitutions: S119A, P135S, D180E, K266S, N326K (using C. albicans numbering). Only the P135 residue is highly conserved among ERG11 from different species (Fig. 1, box shows the substitution). In addition, this substitution is present in the Erg11p of fluconazole-resistant Debaryomyces hansenii and the P135 residue is located near the Erg11p´s Heme group where azole drugs bind (Fig. 2: Saccharomyces cereviciae Erg11p complexed with voriconazole 3D structure. In yellow the region between Y131 and P135 residues. In grey voriconazole binding the Heme group).ConclusionThis emerging multi-resistant pathogen may be underdiagnosed by the methods commonly used in clinical laboratories. The only method capable of identifying this species is DNA sequencing. The strains studied presented very high MIC values for amphotericin B and fluconazole. The P135S substitution could be responsible for the high fluconazole MIC values observed in 4 of the 5 strains of C. vulturna.

The Threat Called Candida haemulonii Species Complex in Rio de Janeiro State, Brazil: Focus on Antifungal Resistance and Virulence Attributes.

Although considered rare, the emergent Candida haemulonii species complex, formed by C. haemulonii sensu stricto (Ch), C. duobushaemulonii (Cd) and C. haemulonii var. vulnera (Chv), is highlighted due to its profile of increased resistance to the available antifungal drugs. In the present work, 25 clinical isolates, recovered from human infections during 2011–2020 and biochemically identified by automated system as C. haemulonii, were initially assessed by molecular methods (amplification and sequencing of ITS1-5.8S-ITS2 gene) for precise species identification. Subsequently, the antifungal susceptibility of planktonic cells, biofilm formation and susceptibility of biofilms to antifungal drugs and the secretion of key molecules, such as hydrolytic enzymes, hemolysins and siderophores, were evaluated by classical methodologies. Our results revealed that 7 (28%) isolates were molecularly identified as Ch, 7 (28%) as Chv and 11 (44%) as Cd. Sixteen (64%) fungal isolates were recovered from blood. Regarding the antifungal susceptibility test, the planktonic cells were resistant to (i) fluconazole (100% of Ch and Chv, and 72.7% of Cd isolates), itraconazole and voriconazole (85.7% of Ch and Chv, and 72.7% of Cd isolates); (ii) no breakpoints were defined for posaconazole, but high MICs were observed for 85.7% of Ch and Chv, and 72.7% of Cd isolates; (iii) all isolates were resistant to amphotericin B; and (iv) all isolates were susceptible to echinocandins (except for one isolate of Cd) and to flucytosine (except for two isolates of Cd). Biofilm is a well-known virulence and resistant structure in Candida species, including the C. haemulonii complex. Herein, we showed that all isolates were able to form viable biofilms over a polystyrene surface. Moreover, the mature biofilms formed by the C. haemulonii species complex presented a higher antifungal-resistant profile than their planktonic counterparts. Secreted molecules associated with virulence were also detected in our fungal collection: 100% of the isolates yielded aspartic proteases, hemolysins and siderophores as well as phospholipase (92%), esterase (80%), phytase (80%), and caseinase (76%) activities. Our results reinforce the multidrug resistance profile of the C. haemulonii species complex, including Brazilian clinical isolates, as well as their ability to produce important virulence attributes such as biofilms and different classes of hydrolytic enzymes, hemolysins and siderophores, which typically present a strain-dependent profile.

The serine peptidase inhibitor N-ρ-tosyl-l-phenylalanine chloromethyl ketone (TPCK) affects the cell biology of Candida haemulonii species complex

Candida haemulonii species complex (C. haemulonii, C. haemulonii var. vulnera and Candida duobushaemulonii) is composed by emerging and multidrug-resistant (MDR) yeasts. Candidiasis, the disease caused by these species, is difficult to treat and culminates in clinical failures and patient death. It is well-known that Candida peptidases play important roles in the fungus–host interactions, and hence these enzymes are promising targets for developing new antifungal drugs. Recently, serine-type peptidases were described in clinical isolates of C. haemulonii complex with the ability to cleave relevant key host proteins. Herein, the effects of serine peptidase inhibitors (SPIs) on the cell biology of this fungal complex were evaluated. Initially, eight distinct SPIs (phenylmethylsulfonyl fluoride – PMSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride – AEBSF, N-α-tosyl-l-lysine chloromethyl ketone hydrochloride – TLCK, N-p-tosyl-l-phenylalanine chloromethyl ketone – TPCK, simeprevir, boceprevir, danoprevir and telaprevir) were tested on the fungal growth. TPCK showed the best efficacy in controlling cell proliferation, being selected for the following experiments. This SPI induced changes in the architecture of yeast cells, as observed by scanning electron microscopy, besides injuries at the plasma membrane and reduction in the ergosterol content. TPCK also diminished the ability of yeasts to adhere to abiotic (polystyrene and glass) and biotic (murine macrophages) surfaces in a typically concentration-dependent manner. In addition, the 24 h-treatment of the mature biofilm promoted a decrease in biomass, viability and extracellular matrix. Altogether, our results highlight that SPIs may be promising new therapeutic agents in the treatment of candidiasis caused by emergent, opportunistic and MDR species forming the C. haemulonii complex.

Insights into the Multi-Azole Resistance Profile in Candida haemulonii Species Complex

The Candida haemulonii complex (C. duobushaemulonii, C. haemulonii, and C. haemulonii var. vulnera) is composed of emerging, opportunistic human fungal pathogens able to cause invasive infections with high rates of clinical treatment failure. This fungal complex typically demonstrates resistance to first-line antifungals, including fluconazole. In the present work, we have investigated the azole resistance mechanisms expressed in Brazilian clinical isolates forming the C. haemulonii complex. Initially, 12 isolates were subjected to an antifungal susceptibility test, and azole cross-resistance was detected in almost all isolates (91.7%). In order to understand the azole resistance mechanistic basis, the efflux pump activity was assessed by rhodamine-6G. The C. haemulonii complex exhibited a significantly higher rhodamine-6G efflux than the other non-albicans Candida species tested (C. tropicalis, C. krusei, and C. lusitaneae). Notably, the efflux pump inhibitors (Phe-Arg and FK506) reversed the fluconazole and voricolazole resistance phenotypes in the C. haemulonii species complex. Expression analysis indicated that the efflux pump (ChCDR1, ChCDR2, and ChMDR1) and ERG11 genes were not modulated by either fluconazole or voriconazole treatments. Further, ERG11 gene sequencing revealed several mutations, some of which culminated in amino acid polymorphisms, as previously reported in azole-resistant Candida spp. Collectively, these data point out the relevance of drug efflux pumps in mediating azole resistance in the C. haemulonii complex, and mutations in ERG11p may contribute to this resistance profile.

Evaluation of the potential virulence of Candida haemulonii species complex and Candida auris isolates in Caenorhabditis elegans as an in vivo model and correlation to their biofilm production capacity

Candida haemulonii species complex (Can. haemulonii sensu stricto, Can. duobushaemulonii and Can. haemulonii var. vulnera) and related species (Can. auris and Can. pseudohaemulonii) have attracted attention due to reduced susceptibility to azoles and amphotericin B. Furthermore, attributes of potential virulence have been recognized in Can. haemulonii species complex and Can. auris, like the capability to form biofilm, which represent the most important risk factors for persistent candidemia. However, the relationship between biofilm production and impact on host mortality is still unclear. To evaluate the potential virulence of Can. haemulonii species complex and Can. auris isolates by correlating biofilm production and capacity to kill Caenorhabditis elegans as an in vivo model. In this study, virulence factors were characterized among a total of sixty-six Can. haemulonii species complex and Can. auris isolates to gain insight about virulence traits of these pathogenic yeasts by evaluating the in vitro biofilm production and potential pathogenicity for Cae. elegans, as an in vivo infection model. All clinical isolates tested were biofilm producer, inter- and intra-specific differences on the biofilm forming capacity by the strains were observed. Can. auris and Can. haemuolonii var. vulnera showed similar biofilm production, both higher than Can. haemulonii sensu stricto and Can. duobushaemulonii. Regarding the virulence of the Cae. elegans model, Can. haemulonii species complex and Can. auris isolates were capable of causing infection in Cae. elegans, and our data suggest that the high biofilm production by Can. haemulonii var. vulnera and Can. duobushaemulonii isolates may impact in the pathogenicity caused on Cae. elegans.

Increasing Prevalence of Multidrug-Resistant Candida haemulonii Species Complex among All Yeast Cultures Collected by a Reference Laboratory over the Past 11 Years.

There is worldwide concern with the increasing rates of infections due to multiresistant Candida isolates reported in tertiary medical centers. We checked for historical trends in terms of prevalence rates and antifungal susceptibility of the Candida haemulonii species complex in our yeast stock culture collected during the last 11 years. The isolates were identified by sequencing the rDNA internal transcribed spacer (ITS) region, and antifungal susceptibility tests for amphotericin B, voriconazole, fluconazole, anidulafungin, and 5-fluorocytosine were performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. A total of 49 isolates were identified as Candida haemulonii sensu stricto (n = 21), followed by C. haemulonii var. vulnera (n = 15) and C. duobushaemulonii (n = 13), including 38 isolates cultured from patients with deep-seated Candida infections. The prevalence of the C. haemulonii species complex increased from 0.9% (18 isolates among 1931) in the first period (December 2008 to June 2013) to 1.7% (31 isolates among 1868) in the second period (July 2014 to December 2019) of analysis (p = 0.047). All isolates tested exhibited high minimum inhibition concentrations for amphotericin B and fluconazole, but they remained susceptible to 5-fluorocytosine and anidulafungin. We were able to demonstrate the increased isolation of the multiresistant Candida haemulonii species complex in our culture collection, where most isolates were cultured from patients with deep-seated infections.

Pathogenicity Levels of Colombian Strains of Candida auris and Brazilian Strains of Candida haemulonii Species Complex in Both Murine and Galleria mellonella Experimental Models.

Candida auris and Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) are phylogenetically related species that share some physiological features and habits. In the present study, we compared the virulence of these yeast species using two different experimental models: (i) Galleria mellonella larvae to evaluate the survival rate, fungal burden, histopathology and phagocytosis index and (ii) BALB/c mice to evaluate the survival. In addition, the fungal capacity to form biofilm over an inert surface was analyzed. Our results showed that in both experimental models, the animal survival rate was lower when infected with C. auris strains than the C. haemulonii species complex. The hemocytes of G. mellonella showed a significantly reduced ability to phagocytize the most virulent strains forming the C. haemulonii species complex. Interestingly, for C. auris, it was impossible to measure the phagocytosis index due to a general lysis of the hemocytes. Moreover, it was observed a greater capability of biofilm formation by C. auris compared to C. haemulonii species complex. In conclusion, we observed that C. auris and C. haemulonii complex have different levels of pathogenicity in the experimental models employed in the present study.

Biofilm Formed by Candida haemulonii Species Complex: Structural Analysis and Extracellular Matrix Composition.

Candida haemulonii species complex (C. haemulonii, C. duobushaemulonii, and C. haemulonii var. vulnera) has emerged as opportunistic, multidrug-resistant yeasts able to cause fungemia. Previously, we showed that C. haemulonii complex formed biofilm on polystyrene. Biofilm is a well-known virulence attribute of Candida spp. directly associated with drug resistance. In the present study, the architecture and the main extracellular matrix (ECM) components forming the biofilm over polystyrene were investigated in clinical isolates of the C. haemulonii complex. We also evaluated the ability of these fungi to form biofilm on catheters used in medical arena. The results revealed that all fungi formed biofilms on polystyrene after 48 h at 37 °C. Microscopic analyses demonstrated a dense network of yeasts forming the biofilm structure, with water channels and ECM. Regarding ECM, proteins and carbohydrates were the main components, followed by nucleic acids and sterols. Mature biofilms were also detected on late bladder (siliconized latex), nasoenteric (polyurethane), and nasogastric (polyvinyl chloride) catheters, with the biomasses being significantly greater than on polystyrene. Collectively, our results demonstrated the ability of the C. haemulonii species complex to form biofilm on different types of inert surfaces, which is an incontestable virulence attribute associated with devices-related candidemia in hospitalized individuals.

Surface, adhesiveness and virulence aspects of Candida haemulonii species complex.

The emerging opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii [Ch], C. duobushaemulonii [Cd] and C. haemulonii var. vulnera[Chv]) are notable for their intrinsic antifungal resistance. Different clinical manifestations are associated with these fungal infections; however, little is known about their biology and potential virulence attributes. Herein, we evaluated some surface properties of 12 clinical isolates of Ch (n=5), Cd (n=4) and Chv (n=3) as well as their virulence on murine macrophages and Galleria mellonella larvae. Scanning electron microscopy demonstrated the presence of homogeneous populations among the species of the C. haemulonii complex, represented by oval yeasts with surface irregularities able to form aggregates. Cell surface hydrophobicity was isolate-specific, exhibiting high (16.7%), moderate (25.0%) and low (58.3%) hydrophobicity. The isolates had negative surface charge, except for one. Mannose/glucose- and N-acetylglucosamine-containing glycoconjugates were evidenced in considerable amounts in all isolates; however, the surface expression of sialic acid was poorly detected. Cd isolates presented significantly higher amounts of chitin than Ch and Chv. Membrane sterol and lipid bodies, containing neutral lipids, were quite similar among all fungi studied. All isolates adhered to inert surfaces in the order: polystyrene>poly-L-lysine-coated glass>glass. Likewise, they interacted with murine macrophages in a quite similar way. Regarding in vivo virulence, the C. haemulonii species complex were able to kill at least 80% of the larvae after 120hours. Our results evidenced the ability of C. haemulonii complex to produce potential surface-related virulence attributes, key components that actively participate in the infection process described in Candida spp.

The High-Quality Complete Genome Sequence of the Opportunistic Fungal Pathogen Candida vulturna CBS 14366T

Candida vulturna is a new member of the Candida haemulonii species complex that recently received much attention as it includes the emerging multidrug-resistant pathogen Candida auris. Here, we describe the high-quality genome sequence of C. vulturna type strain CBS 14366T to cover all genomes of pathogenic C. haemulonii species complex members.

Superficial candidosis by Candida duobushaemulonii: An emerging microorganism

The Candida haemulonii species complex and other phylogenetically related species, such as Candida auris, are emerging pathogens. The C. haemulonii complex comprises C. haemulonii, C. haemulonii var. vulnera, and Candida duobushaemulonii species. The correct identification of this fungal complex is clinically and epidemiologically relevant; however, conventional identification methods are currently inadequate. In this study, we report the molecular reidentification of two clinical isolates: isolate 553 that was recovered from a patient with total dystrophic onychomycosis and isolate 77–18 from a patient with mucocutaneous candidiasis. Both patients had diabetes mellitus as baseline disease. These isolates were initially identified as C. haemulonii by the VITEK® 2 system but were later determined to be C. duobushaemulonii based on the amplification and sequencing of a 115-bp fragment of the region of 26S rDNA. The isolates were resistant to fluconazole, and only isolate 553 was resistant to amphotericin B. Patients showed clinical and mycological cure after treatment with itraconazole. The clinical and microbiological data of these two cases of superficial candidiasis caused by C. duobushaemulonii, along with previous reports about infections with the C. haemulonii complex species, suggest that patients with diabetes mellitus are highly susceptible to infection with these fungi. Therefore, for diagnosing candidiasis in patients with diabetes mellitus, yeasts must be identified at the species level, and antifungal susceptibility testing must be carried out for adequate treatment.

Planktonic growth and biofilm formation profiles in Candida haemulonii species complex.

Candida haemulonii species complex have emerged as multidrug-resistant yeasts able to cause fungemia worldwide. However, very little is known regarding their physiology and virulence factors. In this context, planktonic growth and biofilm formation of Brazilian clinical isolates of Candida haemulonii (n = 5), Candida duobushaemulonii (n = 4), and Candida haemulonii var. vulnera (n = 3) were reported. Overall, the fungal planktonic growth curves in Sabouraud dextrose broth reached the exponential phase in 48h at 37°C. All the clinical isolates formed biofilm on polystyrene in a time-dependent event, as judged by the parameters evaluated: biomass (crystal violet staining), metabolic activity (XTT reduction), and extracellular matrix (safranin incorporation). No statistically significant differences were observed when the average measurements among the three Candida species were compared regarding both planktonic and biofilm lifestyles; however, typical isolate-specific differences were clearly noticed in fungal growth kinetics.

Identification and Antifungal Susceptibility Profiles of Candida haemulonii Species Complex Clinical Isolates from a Multicenter Study in China

Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera, and Candida duobushaemulonii) consists of emerging pathogens. Thirty-one isolates from 14 hospitals in China were studied for their species classification and antifungal susceptibilities. Performances of molecular (i.e., ribosomal DNA [rDNA] internal transcribed spacer [ITS] sequencing, D1/D2 sequencing, and ITS sequencer-based capillary gel electrophoresis [SCGE]) and phenotypic identification methods in species identification were compared. Twenty-six (83.9%) of 31 isolates were identified as C. haemulonii and 5 isolates were identified as C. duobushaemulonii by ITS sequencing as the reference method; results obtained by D1/D2 sequencing and ITS SCGE were concordant with those obtained by ITS sequencing for all (100%) of the isolates. All 31 isolates were identified as C. haemulonii by the Vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system (bioMérieux, France), whereas the Bruker MS system (Bruker Daltoniks, Germany) correctly provided species identification for 77.4% and 100% of isolates using cutoff scores for species of ≥2.0 and ≥1.70, respectively. The Vitek 2 compact (bioMérieux) only identified 9 (29%) of 31 isolates. All isolates showed high MICs for amphotericin B (range, 2 to >8 μg/ml) and fluconazole (≥128 μg/ml) but low MICs (≤0.5 μg/ml) for the echinocandins. Our results reinforce the need for MALDI-TOF MS and/or molecular differentiation of species within the C. haemulonii complex. The multiresistant antifungal susceptibility profile of these isolates represents a challenge to therapy.