Evaluation of fungaemia infections in a Hungarian university hospital between 1996 and 2009

The incidence of Candida species causing bloodstream infections in the University Hospital of Szeged, Hungary, between 1996 and 2009, and the susceptibilities of these isolates to antifungal agents were evaluated.Automated blood culture systems (Vital, bioMérieux, Marcy-l'Etoile, France; and BACTEC 9120, Becton-Dickinson Diagnostic Systems, Sparks, USA) were used. The in vitro susceptibilities of the yeast isolates to antifungal agents were determined by the Etest method (AB Biodisk, Solna, Sweden).Bloodstream infections were caused by yeast strains in 231 cases during this period, and 226 Candida strains were cultured from 216 candidaemia patients. Bloodstream infections caused by multiple Candida spp. were diagnosed almost every year. Of the 216 patients, 67 were children; and 55 infants needed intensive care. In 2005, C. glabrata caused an increase in the incidence of invasive fungal infections in the Neonatal Intensive Care Unit. The PFGE analysis of 12 isolates distinguished 4 different karyotypes. The incidence of bloodstream infections caused by fungi did not change during the 14-year study period. The most frequent species cultured from blood samples were C. albicans and C. glabrata. The incidence of resistant isolates remained constant. The local trends of fungaemia must be monitored and compared with global reports.     

Candida Colonization in Urine Samples of ICU Patients: Determination of Etiology, Antifungal Susceptibility Testing and Evaluation of Associated Risk Factors

The presence of Candida in urine presents a therapeutic challenge for the physician as it is often asymptomatic, and management guidelines have not been clearly laid down on this issue. The presence of Candida in urine may represent contamination of clinical sample, actual colonization of the lower urinary tract or may be a true indicator of invasive infection of lower and/or upper urinary tract. In a clinical setting like the ICU, multiple risk factors for Candida colonization may be present in the same patient, thereby increasing the chances of candiduria, manifold. In the present study on 80 patients in ICU, high rate of Candida colonization (57.5%) was found in urine samples of ICU patients with C. tropicalis (57.3%) being the predominant species. We also isolated 8 strains of Trichosporon species, all of these presented as a mixed infection along with Candida species. Among the various risk factors studied, urinary catheterization and previous antibiotic therapy were identified as statistically significant (P value <0.05). The minimum inhibitory concentration of the isolates was determined for amphotericin B, fluconazole and itraconazole by E-test. Most of the isolates were susceptible to amphotericin B. The C. parapsilosis strains did not show any drug resistance; however, resistance to fluconazole was observed 18.6, 27.27, 50 and 25% in C. tropicalis, C. albicans, C. glabrata and Trichosporon species, respectively.     

Epidemiology, species distribution, antifungal susceptibility and outcome of nosocomial candidemia in a tertiary care hospital in Italy

Candida is an important cause of bloodstream infections (BSI), causing significant mortality and morbidity in health care settings. From January 2008 to December 2010 all consecutive patients who developed candidemia at San Martino University Hospital, Italy were enrolled in the study. A total of 348 episodes of candidaemia were identified during the study period (January 2008-December 2010), with an incidence of 1,73 episodes/1000 admissions. Globally, albicans and non-albicans species caused around 50% of the cases each. Non-albicans included Candida parapsilosis (28.4%), Candida glabrata (9.5%), Candida tropicalis (6.6%), and Candida krusei (2.6%). Out of 324 evaluable patients, 141 (43.5%) died within 30 days from the onset of candidemia. C. parapsilosis candidemia was associated with the lowest mortality rate (36.2%). In contrast, patients with C. krusei BSI had the highest mortality rate (55.5%) in this cohort. Regarding the crude mortality in the different units, patients in Internal Medicine wards had the highest mortality rate (54.1%), followed by patients in ICU and Hemato-Oncology wards (47.6%).This report shows that candidemia is a significant source of morbidity in Italy, with a substantial burden of disease, mortality, and likely high associated costs. Although our high rates of candidemia may be related to high rates of BSI in general in Italian public hospitals, reasons for these high rates are not clear and warrant further study. Determining factors associated with these high rates may lead to identifying measures that can help to prevent disease.     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Biofilm formation by and antifungal susceptibility of Candida isolates from urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC(50) of <1 mug/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

Sleep and temperature responses of inbred mice with Candida albicans-induced pyelonephritis

Human patients with renal disease frequently develop disturbed sleep and severe fatigue. To develop a model for studying factors that contribute to these symptoms, we characterized the sleep patterns of various strains of mice after acute challenge with the fungal organism Candida albicans. After intravenous administration to mice, C. albicans typically colonizes the kidney, producing acute pyelonephritis. Various strains of inbred mice demonstrate marked variation in the temperature and sleep responses that develop after challenge, with individual strains generally showing increased or reduced somnolence in association with fever or hypothermia, respectively. C. albicans-infected mice may be a useful model for identifying the genes and mechanisms that link sleep, temperature, fatigue, and the immune response.     

慢性阻塞性肺疾病并念珠菌性口炎患者病原学及危险因素分析

目的 探讨入院慢性阻塞性肺疾病(COPD)并念珠菌性口炎患者的病原学特征以及相关危险因素.方法 采用病例研究,对2007年4月1日至2011年1月31日入院的82例COPD并念珠菌性口炎患者和82例无念珠菌性口炎COPD患者进行匹配,应用SPSS 17.0统计软件行条件logistic回归模型分析COPD患者念珠菌性口炎发生的危险因素.结果 (1)从念珠菌性口炎患者假膜培养共分离出念珠菌83株,以白念珠菌(90.4％)为最多,其次为光滑念珠菌(3.6％)、克柔念珠菌(2.4％)、热带念珠菌(2.4％)、近平滑念珠菌(1.2％);(2)统计学分析结果显示全身应用、吸入糖皮质激素是入院患者口腔念珠菌感染的独立危险因素.结论 白色念珠菌是COPD患者口腔部念珠菌感染的主要病原菌,规范使用全身糖皮质激素、正确吸入糖皮质激素是预防COPD患者口腔念珠菌感染的关键.     

The relationship between O-chain expression and colonisation ability of Helicobacter pylori in a mouse model

The influence of lipopolysaccharide (LPS) O-polysaccharide chain production on the colonisation ability of Helicobacter pylori in four mouse models (NMRI, C57BL/6, CBA/Ca, and BALB/cA mice) was studied. H. pylori strains that produced smooth-form LPS (S-LPS) detectable in silver-stained electrophoretic gels colonised mice. In contrast, a laboratory-passaged strain G50 and the culture collection strain CCUG 17874 did not colonise mice; the former strain produced low amounts of O-chains only detectable in immunoblotting but not in silver-stained gels, whereas the latter produced rough-form LPS (R-LPS) without O-chains. Furthermore, a galE isogenic mutant, which produced R-LPS, did not colonise mice. However, after repeated broth culture, strains G50 and CCUG 17874 produced S-LPS detectable in silver-stained gels and were capable of colonising mice. Consistent with the production of O-chains, all colonising strains produced Lewis (Le) antigens, Le(x) and/or Le(y). Except for low expression of Le(y) by non-colonising G50, reflecting low production of O-chains, all other non-colonising strains and the galE mutant lacked expression of Le antigens consistent with their production of R-LPS. Lectin typing of strains supported these findings, and also showed that lectin types did not differ before and after colonisation. The low level of O-chain production and Le antigen expression by the non-colonising G50 may not be sufficient to aid colonisation. Examination of protein profiles of H. pylori strains before inoculation showed that protein expression was not significantly different between colonising and non-colonising strains. These results show that S-LPS production with O-chain expression is required by H. pylori for colonisation in a number of mouse models and that care should be taken with inoculating H. pylori strains that loss of O-chains does not occur during subculturing.     

Molecular and genetic aspects of resistance to azoles in Candida albicans

Fluconazole is one of the most useful drugs in the treatment of fungal systemic infections which frequently affect non immunocompetent individuals. However, the emergence of resistant strains in recent years may severely limit its usefulness in future. Although there are several described mechanisms involved in resistance to azoles, recent genetic studies demonstrate the role of specific genes in clinical resistance. Currently, the best characterized are the MDR1 and CDR1 genes, which code members of the MFS or ABC family of drug transporters, respectively. These proteins respond to the membrane potential (MFS) or hydrolyse ATP (ABC) thus promoting drug efflux and therefore reducing its intracellular accumulation. It has been shown that the mRNA from these genes is frequently increased in some Candida albicans resistant strains from patients receiving long term azole treatment. The development of molecular genetic tools in C. albicans is allowing characterization of their role in this and other important processes in the fungal cell.     

A comparison of phospholipase activity, cellular adherence and pathogenicity of yeasts

Phospholipase A and lysophospholipase activities were measured in the culture fluid and in the blastospores of Candida albicans. When phospholipase activity was measured in six yeasts (four strains of C. albicans and a single strain each of Candida parapsilosis and Saccharomyces cerevisiae) a correlation was found between this activity and two potential parameters of pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells and were most pathogenic in mice had the highest phospholipase activities. Non-pathogenic yeasts, including C. albicans isolates which did not adhere and did not kill mice, had lower phospholipase activities.     

Pseudomonas aeruginosa colonisation in an intensive therapy unit: role of cross infection and host factors.

Despite the sparsity of Pseudomonas aeruginosa in the environment colonisation and infection with this organism was found at several sites by selective culture in 20 out of 46 patients in an intensive therapy unit. Three patients developed Ps aeruginosa pneumonia. Serial serogrouping and phage typing identified multiple strains in the unit and in the same patient. Rectal carriage occurred in 16 patients but rectal strains did not subsequently appear in tracheal aspirates; strains varied in their affinity for the upper respiratory tract. Colonisation was not directly related to length of stay and was detected in 16 of those colonised within 24 hours of admission. In intubated patients, who were colonised more frequently than those not intubated, upper respiratory tract colonisation correlated strongly with low initial arterial pH values. Personnel were probably responsible for cross infection among patients when the unit was busy. Strain differences and the susceptibility of patients also influenced colonisation and infection. Elimination of major reservoirs of Ps aeruginosa and compliance with procedures to control cross infection remain essential if patients in hospital are to escape colonisation by the organism.     

Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.     

哈尔滨地区假丝酵母菌DNA异质性及药物敏感性分析

研究假丝酵母菌的DNA异质性及药物敏感性,为预防和监控院内假丝酵母菌感染奠定基础。将临床分离的假丝酵母菌菌株,用科玛嘉显色培养基鉴定菌种,经纸片扩散法进行药敏试验,应用随机扩增多态性DNA（RAPD）技术对这些菌株进行基因分型。结果显示：93株假丝酵母菌中白假丝酵母菌68株,非白假丝酵母菌25株,所有菌株对制霉菌素,两性霉素B两种药物的敏感率最高（100％）,酮康唑其次（70．9％）,氟康唑的敏感率最低（50．5％）,引物1和引物2将来源不同的68株白假丝酵母菌分别分成4型（A1、B1、C1、D1）和6型（A2、B2、C2、D2、E2、F2）。哈尔滨地区的假丝酵母菌感染以白假丝酵母菌为主,且主要为A1、B1型（引物1）或A2、B2型（引物2）;基因型与药敏谱无明显相关性。     

Requirement of heat-labile opsonins for maximal phagocytosis of Candida albicans.

The requirement for heat-labile opsonins for phagocytosis of 2 serologically distinct strains and a number of clinical isolates of Candida albicans was examined by a previously described radiometric technique. The results indicated that both strains and all 10 isolates of C. albicans examined required heat-labile opsonins for maximal phagocytosis. The data suggest that heat-labile opsonins play an important role in immunity to C. albicans. However, the possibility that some strains of C. albicans may not utilize heat-labile opsonins cannot be excluded.     

UV and X-ray sensitivity of Candida albicans laboratory strains and mutants having chromosomal alterations

Utilization of L-sorbose, D-arabinose or primary fluconazole resistance in Candida albicans are controlled by copy number of specific chromosomes. On the other hand, spontaneous morphological mutants have a wide range of chromosomal alterations. We have investigated the UV and X-ray sensitivity of these mutants, as well as C. albicans laboratory strains. While L-sorbose utilizing mutants had normal sensitivities, a large subclass of D-arabinose utilizing mutants was abnormally sensitive to UV. Spontaneous morphological mutants responded differently, an expected result because of the heterogeneous nature of their electrophoretic karyotypes. We suggest that the differences in UV and X-ray sensitivity are due to gene imbalance caused by some chromosomal alterations. In this respect, the radiation sensitivity is similar to other features impaired by changes in chromosomes, but is unlike the acquisition of the ability to utilize alternative nutrients or the acquisition of resistance to fluconazole. Our studies also revealed that strains of C. albicans heterozygous for the mating type loci exhibited the same X-ray sensitivity as homozygous or hemizygous strains, a finding which is in contrast to the properties of Saccharomyces cerevisiae, where heterozygous strains are more resistant. This feature of C. albicans strains may be indicative of an inefficient repair system that may be related to inefficiency of mating.     

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.     

Specific, high-affinity binding sites for human luteinizing hormone (hLH) and human chorionic gonadotrophin (hCG) in Candida species

The presence of specific binding sites for [125I]-labelled hLH and hCG is described in Candida species. Binding was present in three strains of Candida albicans, and in Candida tropicalis, and was greatest in microsomes, though binding was also present in cytosol fractions. hLH and hCG mutually competed for these binding sites. Other hormones did not bind and did not compete for hLH binding sites. Scatchard plots showed two classes of binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomes and cytosol. This is the first report of specific binding sites for mammalian peptide hormones in a yeast.     

Influence of incubation conditions on cell surface hydrophobicity of Candida species fungi

Cell surface hydrophobicity (CSH) is considered to be one of several virulence factors of Candida yeast-like fungi. The aim of the study was a measurment of hydrophobic properties of Candida sp. depending on growth conditions. A total of 139 strains of Candida (80 - C. albicans and 59 - C. non-albicans) were examined. The method of salt aggregation test (SAT) was used. The strains were cultured on three different media, in two variants of incubation temperature and time. The incubation temperature and microbiological medium affected CSH of just C. albicans strains. The influence of incubation time on CSH of examined species of Candida was not occurred. There was a strong correlation between CSH and species of Candida demonstrated in the study Hydrophobic properties were more frequent and stronger among strains of C. non-albicans than C. albicans species. The results of the study indicates that CSH of Candida spp. is a dynamic feature. The ability to change surface properties may play a role in pathogenesis of candidosis.     

The effect of azidothymidine on germ tube formation in Candida albicans

Candida albicans have a marked propensity to cause infections in AIDS patients. A virulent trait of C. albicans is the yeast-hypha transition (Y-->H) which is influenced, in vitro and in vivo, by several factors. Since azidothymidine (AZT) is used in HIV-positive patients, the effect, in vitro, of different concentrations of AZT on C. albicans Y-->H transition was evaluated. C. albicans isolated from HIV-negative and HIV-positive patients were used and strains of C. tropicalis isolated from HIV-positive patients were also tested. AZT concentrations from 0.01 microg/ml to 10 microg/ml did not have any influence on the Y-->H transition, whereas 100 microg/ml AZT significantly inhibited the germ tube formation. AZT did not influence the formation of pseudohyphae in C. tropicalis. It is suggested that C. albicans infection observed in HIV-positive patients was not influenced by AZT therapy, because at currently used dosages, the Y-->H transition was not expected to increase.