Molecular epidemiology of Candida species isolated from urine at an intensive care unit

Candida spp. has been the leading microorganism isolated from the urine specimens of patients hospitalized at the Anesthesiology and Reanimation intensive care unit (ICU) of Dokuz Eylul University Hospital, Izmir, since 1998. This study was undertaken to investigate the clonal relationship of Candida urine isolates in order to find the mode of spread among the patients. Epidemiological surveillance of 38 Candida albicans, 15 Candida tropicalis and 12 Candida glabrata recovered from the urine specimens of patients who were hospitalized in the ICU between June 11, 2000 and October 15, 2001 was carried out by antifungal susceptibility testing and randomly amplified polymorphic DNA (RAPD) analysis. Two short primers [Cnd3 (5'-CCAGATGCAC-3') and Cnd4 (5'-ACGGTACACT-3')] were used for RAPD. None of the isolates had high minimal inhibitory concentration (MIC) values (>1 microg ml(-1)) against amphotericin B with MIC50 values of 0.5 microg ml(-1), 0.5 microg ml(-1) and 0.125 microg ml(-1) for C. albicans, C. tropicalis and C. glabrata isolates, respectively. However, three C. glabrata isolates were resistant and one C. albicans and five C. glabrata isolates were dose-dependent susceptible (D-DS) to fluconazole. Among C. albicans isolates 19 and 20 patterns were detected with primers Cnd3 and Cnd4, respectively. When primers Cnd3 and Cnd4 were evaluated together, three and four genotypes were identified for C. tropicalis and C. glabrata isolates, respectively. Our results suggest that the source of C. albicans isolates was mostly endogenous. It is difficult to interpret the mode of spread of C. tropicalis and C. glabrata urine isolates as we obtained insufficient banding patterns for these species.     

Identification of Candida species isolated from patients in intensive care unit and in vitro susceptibility to fluconazole for a 3-year period

Species level identification of Candida and antifungal susceptibility testing is not generally performed in routine laboratory practice. There is limited information about the distribution of Candida species and antifungal susceptibility in Turkey. In this study, we aimed at identifying Candida isolates to species level from various samples obtained from patients treated in an intensive care unit between 2002 and 2005 and to evaluate fluconazole susceptibilities of the isolates. A total of 320 Candida isolates obtained from 270 patients were identified by conventional methods and using API (Candida and/or 20C AUX) system. Antifungal susceptibility testing was performed by broth microdilution method. Candida albicans was isolated with the highest frequency (65.6%) followed by C. parapsilosis (11.3%), C. glabrata (8.8%) and C. tropicalis (7.8%). Of all the isolates, 92.9% revealed susceptibility to fluconazole. Susceptibility to fluconazole was highest for C. albicans followed by C. parapsilosis and C. glabrata. The MIC(90) values for C. albicans, C. parapsilosis, C. glabrata and C. tropicalis were 1, 2, 8 and 4 mug ml(-1) respectively. Fluconazole remains effective against both C. albicans and the majority of non-albicans Candida species. In this study, we determine the distribution of Candida species and evaluate the susceptibilities of the isolates, particularly for the azoles.     

Candida albicans strain carriage in patients and nursing staff of an intensive care unit: a study of morphotypes and resistotypes

Candida albicans carriage of patients and nursing staff of an intensive care unit (ICU) was studied over an 8-month period. Swabs were taken at weekly intervals from multiple sites from patients. None of the patients had clinical Candida infection at the time of the first sampling. The hands and mouth of the nursing staff were sampled at fortnightly intervals. Of the 68 patients investigated for varying periods, 37 (54%) yielded C. albicans from one or more body sites, resulting in the isolation of 269 strains. Nosocomial acquisition of C. albicans was recorded in seven (19%) patients. The frequency of C. albicans isolation increased with extended stay in ICU. Sixteen of the 180 samples taken from hands and mouth of nursing staff, yielded C. albicans, 12 of which came from the mouth. Morphotyping of 88 randomly selected strains of C. albicans originating from 31 patients yielded 34 morphotypes. There appeared to be no preference for any morphotype to colonize a particular anatomic site. Based on the susceptibility results, nine resistotypes were recognized. No correlation was apparent between any specific morphotype and resistotype patterns. The differences in morphotype and resistogram patterns of C. albicans isolates originating from same patients over a period of time suggest that some of the patients were colonized with more than one strain. Similarities in the morphotype and resistotype patterns of C. albicans strains isolated from patients and nursing staff tend to suggest possibility of exogenous acquisition.     

Genotype of Candida species associated with different conditions of vulvovaginal candidosis

It is unknown whether strains isolated from recurrent vulvovaginal candidosis (RVVC), vulvovaginal candidosis (VVC) and asymptomatic carriers are varying or not in genotyping. Candida isolates were genotyped to determine whether specific types were associated with RVVC and VVC. A total of 97 Candida isolates from RVVC patients (n = 43), from VVC patients (n = 47) and asymptomatic carriers (n = 7) were identified by germ tube test, chlamydospore test, CHROMagar Candida and API20 system and were genotyped by polymerase chain reaction (PCR) fingerprinting employing random amplification of polymorphic DNA (RAPD) assay with nine random primers screened. The RAPD identified distinct genotypes that were shared among isolates from most different individuals. Most of the 85 C. albicans strains failed to corroborate any obvious differences in the genotypic variability in the VVC, RVVC patient groups and carriers by clustering analysis. Only a few individuals were found to harbor a distinct genotype of C. albicans isolates associated with a specific disease condition.     

Anticandidal activity of Pseudomonas aeruginosa strains isolated from clinical specimens

Pseudomonads represent the major group of non-differentiating microorganisms that produce antibiotics. The antibiotic substances produced by this group of organisms are pyocyanin, pyrolnitrin and pseudomonic acid. This study was designed to investigate the in vivo and in vitro anticandidal activity of Pseudomonas aeruginosa strains against Candida species. Forty-four P. aeruginosa strains isolated from various specimens of intensive care patients were included in the study. All P. aeruginosa strains have pyocyanin pigment. Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258 and a clinical isolate of Candida tropicalis were used to measure the anticandidal activity of Pseudomonas strains by Kerr's method. The total inhibition rates obtained by using blood agar of C. albicans, C. parapsilosis, C. krusei and C. tropicalis were 41%, 34%, 34% and 25% respectively. When Sabouraud dextrose agar (SDA) was used, the rates were detected as 45%, 39%, 48% and 25% respectively. In the mouse model of concomitant subcutaneous infection with Candida species and P. aeruginosa no yeast were recovered from skin cultures despite 100% detection of P. aeruginosa. Pseudomonas aeruginosa strains isolated from intensive care patients showed anticandidal activity against the Candida species in the present study and this point may be important in the following and treatment of patients.     

肿瘤患者念珠菌血症的危险因素和病原体分析

  目的  本研究旨在确定念珠菌血症的危险因素, 评估肿瘤患者中近平滑假丝酵母菌和其他念珠菌之间可能存在的临床显著性差异。并对白假丝酵母菌血症和近平滑假丝酵母菌血症的临床特点及危险因素进行统计学分析, 旨在通过该类分析, 及时开展干预, 尽量避免易感因素, 进而改善真菌血症患者预后。  方法  回顾性分析郑州大学附属肿瘤医院2012年3月至2018年2月323例念珠菌血症患者的资料。分析患者发生念珠菌血症的危险因素, 同时对比分析近平滑假丝酵母菌和非近平滑假丝酵母菌、白假丝酵母菌和非白假丝酵母菌念珠菌血症的临床差异。通过微量肉汤稀释法对念珠菌常见抗真菌药物进行药敏试验和分析。  结果  念珠菌血症分离病原体中, 最常见的为近平滑假丝酵母菌37.15%（120/323）, 而白假丝酵母菌占34.37%。多变量回归分析结果表明, 下述因素与近平滑假丝酵母菌念珠菌血症的发生相关, 即肠外营养（P < 0.001）、粒缺（P < 0.001）、化疗（P < 0.001）和抗真菌药物的使用（P < 0.001）, 同时肠外营养为近平滑假丝酵母菌念珠菌血症发生的独立危险因素（OR=0.183, 95%CI:0.098~ 0.340;P < 0.001）。  结论  近平滑假丝酵母菌超越白假丝酵母菌为患者念珠菌血症主要病原体。通过评估患者念珠菌血症发生的可能危险因素, 以期加强和制定感染控制策略, 从而预防念珠菌血症的传播。      

Genetic variability of Candida albicans isolates in a university hospital in Hungary

The occurrence and genetic variability of Candida albicans isolates in a Hungarian hospital were examined. Among the 103 Candida isolates, 44 (42.7%) proved to be C. albicans species. Comparing with a previous study carried out in 2002, the percentage of infections caused by C. albicans decreased in Hungary in this period with an increasing incidence of non-albicans species, in accordance with the world-wide trend. The genetic variability of the isolates was examined using mitochondrial DNA (mtDNA), restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) analysis and electrophoretic karyotyping. The examined C. albicans isolates could be clustered into four groups based on their mtDNA profiles. The electrophoretic karyotypes of the isolates were mostly identical to that of the reference strain 1006, with the exception of mtDNA type II isolates. RAPD analy s is could be used to cluster the isolates into different groups, but this clustering was not in complete agreement with their assignment to mtDNA types. Population genetic analyses of the data indicated low amounts of recombination among these C. albicans strains. None of the isolates exhibited decreased susceptibilities to 5-fluorocytosine.     

Susceptibility of yeast isolates from defined German patient groups to 5-fluorocytosine.

Susceptibility and development of resistance to 5-fluorocytosine (5-FC) in Candida strains isolated from defined German groups of probands was investigated. 5-FC susceptibility was determined in a microdilution assay in yeast nitrogen base after 24 h of incubation at 37 degrees C. The range of 5-FC concentration investigated was between 0.015 and 16 micrograms ml-1. Isolates with a minimum inhibitory concentration (MIC) of > 16 micrograms ml-1 were regarded as 5-FC resistant. In total 335 Candida isolates were investigated, and 20 of them (6.0%) were found to be resistant. The Candida isolates were rather different with respect to their origin: out of 57 vaginal isolates from non-risk patients from Southern Germany 3.5% were 5-FC-resistant strains. One hundred and fifty-nine isolates from the urine of long-term intensive care patients from the whole of Germany showed 5-FC resistance (6.3%). Out of 74 isolates of different localization from intensive care patients of the University Clinics of Freiburg, 10.8% showed 5-FC resistance. Among 45 isolates from the oral cavity of HIV-positive patients from the Frankfurt region, no 5-FC-resistant strain was found. The epidemiology of 5-FC resistance is based mainly on the percentage of non-albicans isolates of the proband groups (C. tropicalis, C. krusei and others), and is less based on the frequency of Candida albicans serotype B isolates. In sequential observations with individual intensive care patients, no increase of 5-FC resistance in their Candida isolates could be observed with longer periods of hospitalization.     

Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus infected and non-infected patients and in a yeast culture collection

The incidence of Candida dubliniensis in oropharyngeal swabs of 132 human immunodeficiency virus (HIV)-positive and 89 HIV-negative patients was determined. The samples were plated onto CHROMagar Candida medium and 82 strains, presumptively identified as C. albicans or C. dubliniensis, were further investigated (temperature test, chlamydoconidia production, specific primer PCR). In addition, 487 collection strains (isolated from clinical samples and previously identified as C. albicans on the basis of a positive germ tube test) were screened in order to identify C. dubliniensis isolates. Two C. dubliniensis strains were isolated from two HIV-positive patients without oral candidiasis. Candida dubliniensis was not isolated from 89 HIV-negative patients nor was it identified among the collection strains.     

Etiology of fungaemia and catheter colonisation in Argentinean paediatric patients

Yeast strains obtained from blood cultures and catheters from intensive care units (ICU) and hospitalised oncology paediatrics were studied. Yeast were the first cause of catheter colonisation (51/627), and the third cause of bloodstream infection (44/6065). In catheter, the most frequent species were Candida albicans (34%), C. parapsilosis (27.7%) and C. tropicalis (15%). In blood, C. albicans (40.8%), C. parapsilosis (26.6%), C. tropicalis (15%). Malassezia furfur and Malassezia sympodialis were isolated from catheters from ICU patients. All isolates were susceptible to amphotericin B, 88.8% to itraconazole and 91.9% to fluconazole. Candida albicans and C. tropicalis strains resistant to fluconazole and itraconazol were detected. These results reveal a change in the predominant role of C. albicans as cause of candidemia in hospitalised children and the emergence of antifungal resistant species. These variations emphasise the importance of performing a permanent surveillance to observe and assess them.     

Prevalence of Candida spp. in hospitalized patients and their risk factors

A total of 133 Candida spp. strains originating from a group of 100 patients from Santa Casa de Misericórdia of Belo Horizonte, Brazil, between March 1995 and December 1996, were first identified and classified into six different species: Candida albicans (51%), C. tropicalis (33%), C. parapsilosis (8%), C. glabrata (5%), C. krusei (2%) and C. guilliermondii (1%). All C. albicans strains were serotyped and 55% of these were found to belong to serotype A and 45% belonging to serotype B. The medical records of each patient were examined to characterise and survey the main risk factors associated with them. Most of the patients were between 60- and 80-years-old, 53% were males and 47% were females. Most patients were from the intensive care unit (ICU). Only 10 patients were not exposed to antimicrobial agents and 72 patients were not prescribed antifungal agents. Forty patients showed no other clinical condition and all showed some underlying disease that justified hospitalization. Eighty-seven patients had undergone some invasive procedure and 31 patients had been submitted to two different procedures simultaneously.     

Microbial profile and drug resistance of Candida strains isolated from the blood of children: an 11-year study

The latest observations indicate a continuous increase in the frequency of fungal infections, particularly in hospital patients, accompanied by changes in both the profile of the isolated strains and their drug susceptibility. The objective of this study was to evaluate the trend in the incidence of candidaemia and susceptibility of antifungal drugs in the Polish Mother's Memorial Hospital over an 11-year period. Blood samples taken from the hospitalised children were sent to the Department of Clinical Microbiology for diagnostic purposes. A total of 195 Candida strains were isolated: 47.7% Candida albicans and 52.3% non- albicans . Candida parapsilosis was isolated in 65.7% of non- albicans strains. The prevalence of Candida spp. decreased from 16.9–20.5% in the years 1996–1997 to 3.1–2.1% in the years 2005–2006. In the years 2000–2005, non- albicans strains were more prevalent. All C. albicans strains were susceptible to amphotericin B, 2.94% of non -C. albicans strains were semisusceptible to amphotericin B, 98.92% of C. albicans and 85.29% of non- albicans strains were susceptible to 5-fluorocytosine. Candida spp. strains are predominant pathogens in fungaemia in children in our hospital. Over the last few years, C. albicans have been replaced by non- albicans strains (predominantly C. parapsilosis ), which exhibit a higher level of drug resistance. The number of Candida spp. isolated from blood decreased during the 11-year study.     

Four cases of Candida albicans infections with isolates developing pink colonies on CHROMagar Candida plates

Candida albicans, the most commonly isolated yeast species, is typically identified by its green colony-colour on CHROMagar Candida plates. We here report four cases of Candida albicans infections, in which the initial identification was non-albicans isolates due to a clear pink colour of the colonies on CHROMagar Candida plates. However, classical phenotypic criteria, biochemical assimilation pattern and molecular characterisation identified all four isolates as C. albicans isolates.     

Systemic neonatal candidosis: the karyotyping of Candida albicans strains isolated from neonates and health-workers

Candida albicans has become an important cause of nosocomial infections in neonatal intensive care units (NICUs). The aim of the present study was to compare C. albicans strains isolated from neonates (NN) suffering from systemic candidosis and from nurses in order to determine the relatedness between NN and health workers' strains. Thirty-one C. albicans strains were isolated from 18 NN admitted to the NICU of the neonatology service of Farhat Hached Hospital of Sousse, Tunisia and suffering from systemic candidosis, together with five strains recovered from nurses suffering from C. albicans onychomycosis. Two additional strains were tested, one from an adult patient who developed a systemic candidosis and the second from an adult with inguinal intertrigo. All strains were karyotyped by pulsed-field gel electrophoresis (PFGE) with a CHEF-DR II system. Analysis of PFGE patterns yielded by the 38 strains tested led to the identification of three pulsotypes that were designated I, II and III, and consisted of six chromosomal bands with a size ranging from 700 to >2500 kbp. The most widespread was the pulsotype I, which was shared by 17 NN and the five nurses' strains. The identity between NN and nurses' strains is very suggestive of a nosocomial acquisition from health-workers.     

Virulence attributes and genetic variability of oral Candida albicans and Candida tropicalis isolates

The wide spectrum of candidiasis and its clinical importance encourage the research with the purpose of clarifying the mechanisms of pathogenicity and identification of virulence factors of Candida sp. Therefore, the aim of this study was to verify the adhesion capacity, protease activity and genotypic diversity of oral C. albicans and C. tropicalis isolates. The adhesion ability to the extracellular matrix glycoproteins laminin and fibronectin was evaluated using the ELISA technique. The research of proteases was carried out in agar plate containing bovine albumin and through a quantitative method in buffer solution containing haemoglobin. Intra and interspecies polymorphisms was verified through random amplified polymorphic DNA (RAPD) technique. All C. albicans and C. tropicalis isolates binded to immobilised laminin and fibronectin. Ca33 and Ct13 isolates had relative adhesion index significantly higher than the other isolates for both glycoproteins (P < 0.001). Protease activity was observed in all isolates of C. albicans using either the semi-quantitative or quantitative assay. The protease activity of C. tropicalis was better detected through the quantitative assay. The genotypic diversity by RAPD revealed a heterogeneous population in both species. Nevertheless, C. tropicalis presented higher genetic variability than C. albicans strains.     

Susceptibility to antifungal agents of Candida species isolated from paediatric and adult patients with haematological diseases

Summary The susceptibility to six antifungals: amphotericin B (AMF), 5-fluorocytosine (5-F), miconazole (MIK), ketoconazole (KET), fluconazole (FLU) and itraconazole (ITR) was tested among 206 Candida spp. isolated from paediatric and adult patients with haematological malignancies. To determinate the susceptibility the commercial microdilution method Fungitest (Bio-Rad, France) was used. The strains were classified as susceptible, intermediate susceptible, or resistant on the base of the growth in following breakpoint concentrations of particular drugs: 2 and 8 microg ml(-1) for AMF, 2 and 32 microg ml(-1) for 5-F, 0.5 and 8 microg ml(-1) for MIK, 0.5 and 4 microg ml(-1) for KET and ITR, and 8 and 64 microg ml(-1) for FLU. The highest activity to overall species showed AMF (only one resistant strain) and 5-F (85% susceptible strains). Most of C. albicans isolates were susceptible to tested azoles. The percentages of C. albicans resistant to FLU, ITR, KET and MIK were 4, 11, 8, and 0.8%, respectively. The less susceptible to azoles were C. glabrata and C. krusei (14% and 44% isolates resistant to FLU). A non-albicans Candida isolated from adult patients receiving KET prophylaxis was more frequently resistant to FLU than isolates from patients without previous exposure to azoles (P < 0.05). We did not observe differences in the susceptibility of Candida strains isolated from children compared with those from adults.     

Evaluation of fluorescence in situ hybridisation (FISH) for the identification of Candida albicans in comparison with three phenotypic methods

Severe Candida infections are increasing and are associated with considerable morbidity and mortality. Rapid and accurate differentiation of Candida albicans from non-C. albicans species is essential for therapeutic decisions. We therefore developed a fluorescence in situ hybridisation (FISH) assay comprising previously described probes and a newly designed specific C. albicans probe/competitor probe combination. The FISH probes were first evaluated using 99 selected fungal strains covering 31 species, and a specificity between 96% and 100% and a sensitivity of 100%. The FISH assay was then applied to 110 clinical isolates in parallel with API32C, the chromogenic Candida ID agar, and determination of filamentous colony morphology. All tests produced highly reliable results. However, the Candida ID agar misidentified Candida dubliniensis as C. albicans. Determination of filamentous colony morphology allowed 100% reliable identification of C. albicans, but took 48 h. FISH allowed identification of clinical C. albicans isolates within 3 h with a sensitivity and specificity of 100%. FISH was additionally applied to 48 blood cultures showing yeasts in the Gram stain and correctly identified all 33 cases of C. albicans.     

Candida africana sp. nov., a new human pathogen or a variant of Candida albicans?

Atypical Candida strains were isolated from patients in Madagascar, Angola and Germany. These isolates were slow growing and were unable to produce chlamydospores. They had atypical carbohydrate assimilation profiles. All strains were unable to assimilate the amino sugars N-acteylglucosamine and glucosamine as well as the disaccharide trehalose and the organic acid DL-lactate. They were germ-tube-positive in serum, but only some of these organisms produced pseudohyphae after a long incubation. As shown by Fourier transform infrared spectroscopy the atypical Candida isolates clustered as a monophyletic group different from C. albicans and C. dubliniensis. All strains belonged to C. albicans serotype B. Considering all data presented here, this group of Candida strains differs from any other known member of the genus Candida. Therefore, it is suggested to represent a new species within the genus Candida for which the name Candida africana is proposed.     

Karyotyping of Candida albicans and Candida glabrata from patients with Candida sepsis

The aim of this study was to determine the relatedness of Candida strains from patients suffering from Candida septicaemia by typing of Candida isolates from blood cultures and different body sites by pulsed field gel electrophoresis (PFGE using a contour-clamped homogenous electric field, CHEF). We studied 17 isolates of Candida albicans and 10 isolates of Candida glabrata from six patients. Four patients suffered from a C. albicans septicaemia, one patient from a C. glabrata septicaemia, and one patient had a mixed septicaemia with C. albicans and C. glabrata. Eight isolates from blood cultures were compared with 19 isolates of other sites (stool six, urine four, genital swab four, tip of central venous catheter three, tracheal secretion one, sputum one). PFGE typing resulted in 10 different patterns, four with C. albicans and six with C. glabrata. Five of the six patients had strains of identical PFGE patterns in the blood and at other sites. Seven isolates of a 58-year-old female with a C. glabrata septicaemia fell into five different PFGE patterns. However, they showed minor differences only, which may be due to chromosomal rearrangements within a single strain. Thus it appears, that the colonizing Candida strains were identical to the circulating strains in the bloodstream in at least five of six patients.     

Karyotyping of Candida albicans and Candida glabrata from patients with Candida sepsis

The aim of this study was to determine the relatedness of Candida strains from patients suffering from Candida septicaemia by typing of Candida isolates from blood cultures and different body sites by pulsed field gel electrophoresis (PFGE using a contour-clamped homogenous electric field, CHEF). We studied 17 isolates of Candida albicans and 10 isolates of Candida glabrata from six patients. Four patients suffered from a C. albicans septicaemia, one patient from a C. glabrata septicaemia, and one patient had a mixed septicaemia with C. albicans and C. glabrata . Eight isolates from blood cultures were compared with 19 isolates of other sites (stool six, urine four, genital swab four, tip of central venous catheter three, tracheal secretion one, sputum one). PFGE typing resulted in 10 different patterns, four with C. albicans and six with C. glabrata . Five of the six patients had strains of identical PFGE patterns in the blood and at other sites. Seven isolates of a 58-year-old female with a C. glabrata septicaemia fell into five different PFGE patterns. However, they showed minor differences only, which may be due to chromosomal rearrangements within a single strain. Thus it appears, that the colonizing Candida strains were identical to the circulating strains in the bloodstream in at least five of six patients.