Project description:Candida haemulonii complex species can be multidrug-resistant and cause infections such as candidemia. This study determined the genetic relationship between isolates from Brazil and the United States through whole-genome sequencing and performed antifungal susceptibility testing to investigate drug resistance. Contrary to what is widely described, most isolates were susceptible to azoles. However, an atypical susceptibility profile was found in 50% of Candida pseudohaemulonii strains, including resistance to the three echinocandins. Isolates from both countries formed distinct clusters with wide genetic diversity. Isolates from three hospitals in Brazil were clonal and involved in candidemia cases, pointing to the importance of improving hospital infection control measures and molecular identification.
Project description: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.
Project description: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.
Project description: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.
Project description: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.
Project description:The Candida haemulonii species complex is currently known as C. haemulonii groups I and II. Here we describe C. haemulonii group II as a new species, Candida duobushaemulonii sp. nov., and C. haemulonii var. vulnera as new a variety of C. haemulonii group I using phenotypic and molecular methods. These taxa and other relatives of C. haemulonii (i.e., Candida auris and Candida pseudohaemulonii) cannot be differentiated by the commercial methods now used for yeast identification. Four isolates (C. haemulonii var. vulnera) differed from the other isolates of C. haemulonii in the sequence of the internal transcribed spacer (ITS) regions of the nuclear rRNA gene operon. The new species and the new variety have a multiresistant antifungal profile, which includes high MICs of amphotericin B (geometric mean MIC, 1.18 mg/liter for C. haemulonii var. vulnera and 2 mg/liter for C. duobushaemulonii sp. nov) and cross-resistance to azole compounds. Identification of these species should be based on molecular methods, such as sequence analysis of ITS regions and matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Project description: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.
Project description:Leptospirosis is a zoonotic disease that is primarily transmitted through close contact with contaminated environments or infected animals. Brazil has the highest number of reported cases of leptospirosis in the Americas (approximately 4,000 annual cases). The purpose of this study is to identify the occupational groups with a higher risk of leptospirosis in Brazil from 2010 through 2015 among suspected cases reported to the national surveillance system. Confirmed and unconfirmed cases of leptospirosis with laboratory diagnosis, 20,193 and 59,034 respectively, were classified into 12 occupational groups. Confirmed cases were predominantly male (79.4%), between 25 and 59 years of age (68.3%), white (53.4%), illiterate or with incomplete primary education (51.1%), and participating in agricultural work (19.9%). After controlling for age, sex, race, and area of residency, the multivariate analysis identified that between confirmed and unconfirmed cases of leptospirosis reported to the Brazilian national surveillance system, five occupational groups are at higher risk for leptospirosis: garbage and recycling collectors (odds ratio [OR] = 4.10; 95% CI = 3.36-4.99); agricultural, forestry, and fishery workers (OR = 1.65; 95% CI = 1.49-1.84); prisoners (OR = 1.56; 95% CI = 1.04-2.35); building workers (OR = 1.36; 95% CI = 1.22-1.51); cleaners and mining workers (OR = 1.25; 95% CI = 1.07-1.45). This is the first nationwide study to examine leptospirosis risk by occupational group in Brazil using national surveillance data. Our results suggest that among suspected cases there was an increased risk among occupational groups with low income and low educational levels.
Project description:The recent emergence of a multidrug-resistant yeast, Candida auris, has drawn attention to the closely related species from the Candida haemulonii complex that include C. haemulonii, Candida duobushaemulonii, Candida pseudohaemulonii, and the recently identified Candida vulturna. Here, we used antifungal susceptibility testing and whole-genome sequencing (WGS) to investigate drug resistance and genetic diversity among isolates of C. haemulonii complex from different geographic areas in order to assess population structure and the extent of clonality among strains. Although most isolates of all four species were genetically distinct, we detected evidence of the in-hospital transmission of C. haemulonii and C. duobushaemulonii in one hospital in Panama, indicating that these species are also capable of causing outbreaks in healthcare settings. We also detected evidence of the rising azole resistance among isolates of C. haemulonii and C. duobushaemulonii in Colombia, Panama, and Venezuela linked to substitutions in ERG11 gene as well as amplification of this gene in C. haemulonii in isolates in Colombia suggesting the presence of evolutionary pressure for developing azole resistance in this region. Our results demonstrate that these species need to be monitored as possible causes of outbreaks of invasive infection.
Project description: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 haemuloniisensu 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.