A case of recurrent episodes of Candida parapsilosis fungemia

Candida species is the fourth most commonly isolated organism in blood stream infections in the hospital setting. Patients with candidemia frequently succumb to this infection. For those that survive an initial candidemia, an increasing number of cases of breakthrough or recurrent candidemias have been reported in the literature. We report three episodes of C. parapsilosis fungemia in a cancer patient. Molecular testing was performed and confirmed that all these episodes occurring within an eight-month period were caused by the same organism. The incidence of recurrent candidemia is likely to increase in the near future. Studies and therapeutic interventions for patients at risk for recurrent candidemias are warranted.     

Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.     

Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Candida parapsilosis fungemia in neonates: genotyping results suggest healthcare workers hands as source, and review of published studies

An outbreak of Candida parapsilosis fungemia involving 17 neonatal intensive care unit (NICU) patients was studied. There were 14 blood culture and nine colonizing isolates from other sites available. The hands of NICU healthcare workers (HCW) yielded eight isolates. Screening of the isolates by random amplified polymorphic DNA (RAPD) method showed only three profiles. Typing by restriction fragment length polymorphism (RFLP) revealed all blood isolates were RFLP subtype VII-1. Among the nine infant colonizing isolates, there were four different RFLP subtypes; four of the isolates were subtype VII-1. Seven of the eight isolates from HCW were RFLP subtype VII-1. The majority of infant colonizers were not found in the blood, suggesting a possible direct spread of the epidemic subtype VII-1 strain from HCW hands to infant blood. The source of the infant colonizing strains is unclear, but non-VII-1 strains may be largely of maternal origin and VII-1 strains from HCW. These findings reinforce prior studies that have implicated HCW hands as the source of nosocomial, including neonatal, fungemia.     

侵袭性热带念珠菌感染流行病学及药物敏感性

全球范围内,随着抗肿瘤药物、免疫抑制剂和广谱抗菌药物的使用,真菌感染的发病率显著提高,其中念珠菌感染占到绝大多数。目前热带念珠菌已经成为非白念珠菌中最常见的病原菌。我国热带念珠菌的临床分离率及对氟康唑及伏立康唑的耐药率都明显高于世界平均水平。但是,相比于白念珠菌,关于热带念珠菌的研究及相关临床信息相对较少。该文就侵袭性热带念珠菌感染危险因素、流行病学以及药物敏感性进行全面的综述。     

328株念珠菌菌种分布及耐药性分析

目的调查医院念珠菌病原分布及对常用抗真菌药物的耐药性,为临床抗真菌治疗提供参考依据。方法回顾性调查我院2008年6月～2009年6月从住院患者分离到的328株念珠菌的种类、分布及耐药性。结果念珠菌种类主要为白念珠菌（68.6%）、热带念珠菌（13.4%）和光滑念珠菌（7.9%）;念珠菌来源以下呼吸道为主（82.3%）,其次为泌尿道（5.2%）、血液（4.0%）和肠道（3.4%）等;白念珠菌对氟康唑、伏立康唑、氟胞嘧啶及两性霉素B耐药性较低（0.0%～7.1%）。结论医院念珠菌感染以白念珠菌为主,感染部位以下呼吸道多见,氟康唑、伏立康唑、氟胞嘧啶及两性霉素B对白念珠菌具有较好的抗菌活性。对于念珠菌属开展耐药性监测有重要的临床意义。     

Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

北京朝阳医院深部真菌感染的临床分析和药敏试验

目的对院内临床深部真菌感染的标本进行分离鉴定和药敏试验,为临床感染性疾病提供病原学诊断和合理使用抗真菌药物的依据。方法采用念珠菌显色培养基进行鉴定。对真菌(丝状菌)用棉蓝染色直接镜检和培养相结合的方法进行鉴定。采用纸片扩散法进行药敏试验。结果450株真菌标本中,念珠菌396株,占88.0%;真菌(丝状菌)54株,占12.0%。分离菌株对药物的敏感性分别为氟康唑90.15%,两性霉素B97.98%,伊曲康唑91.67%,氟胞嘧啶89.1%。结论院内真菌感染以念珠菌最为多见,特别是白念珠菌和热带念珠菌。     

Species distribution and antifungal susceptibility among clinical isolates of Candida parapsilosis complex from India

Background

Candida parapsilosis is recognized as a species complex: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are three distinct but closely related species.

吸毒人群口腔念珠菌的药物敏感性分析

目的了解吸毒人群口腔念珠菌对常用抗真菌药物的敏感性,为临床治疗念珠菌病提供参考资料。方法采用美国CLSI推荐的微量稀释法测定75株念珠菌对4种常用抗真菌药物两性霉素B、5-氟胞嘧啶、氟康唑和酮康唑的药物敏感性。结果 75株吸毒人群口腔念珠菌对两性霉素B、5-氟胞嘧啶、氟康唑和酮康唑的耐药率分别为0%、4%、8%和13.3%(P0.01),对氟康唑和酮康唑的交叉耐药率为8%;非白念珠菌对氟康唑和酮康唑的耐药率高于白念珠菌(P0.05)。结论吸毒人群口腔念珠菌对两性霉素B和5-氟胞嘧啶的敏感性高于对氟康唑和酮康唑的敏感性。吸毒人群口腔念珠菌存在着对氟康唑、酮康唑和5-氟胞嘧啶的天然耐药株和对唑类药物的天然交叉耐药株,且非白念珠菌对氟康唑和酮康唑的耐药率及交叉耐药率高于白念珠菌。     

白念珠菌临床分离调查及基因分型研究

目的 了解白念珠菌临床分离情况,并探讨其药敏结果与基因分型的相关性.方法 回顾性分析本院2011年3～11月间临床分离白念珠菌分布及耐药性;随机选取232株,采用PCR方法扩增白念珠菌25S rDNA基因内含子区进行基因分型研究;采用ATB真菌药敏试剂条进行药敏分析;统计分析药敏结果与基因分型的相关性.结果 期间共检出酵母样真菌973例,占病原菌阳性样本数比率为15.7％ (973/6196);其中分离白念珠菌562株,占58％ (562/973),主要分布科室为呼吸科(39.1％)、老年科(13.2％)、ICU(7.7％)、神经内科(7.5％)、免疫科(6.0％)以及其他科室(26.5％);标本类型以下呼吸道为主(81.7％),其次为尿路(9.4％)、血液(1.8％)等.对氟胞嘧啶、两性霉素B、氟康唑、伊曲康唑及伏立康唑的耐药率分别为0.9％、0％、1.4％、1.6％和1.1％.随机选取的232株白念珠菌经PCR方法可分为3型:A型125株,B型96株,C型11株.各型在5种药物的耐药性上并无差异.结论 临床分离酵母样真菌以白念珠菌为主,感染部位以下呼吸道为主;临床分离株对5种抗真菌药物敏感度较高,主要基因型为A和B型,不同基因分型间药敏结果并无统计学差异.     

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from Sao Paulo State, Brazil

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 Sao Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 micro g mL(-1). For fluconazole, 22% occurred in the range 8-16 mug mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.     

新疆地区白念珠菌基因型分析及其体外药物敏感性研究

目的研究新疆地区汉族和维吾尔族患者来源的50株白念珠菌的基因型及其对两性霉素B、5-氟胞嘧啶、米卡芬净、伊曲康唑、氟康唑和咪康唑的体外敏感性。方法采用PCR法扩增白念珠菌rDNA 25S的Ⅰ类内含子包含区,根据扩增产物的大小判断基因型(A型为450 bp,B型为840 bp,C型为450 bp和840 bp)。采用CLSI M27-A液基微量稀释法测定50株白念珠菌对上述6种抗真菌药的体外敏感性。结果 50株菌分为3种基因型:A型30株,B型和C型各10株。所有菌株对两性霉素B、5-氟胞嘧啶、米卡芬净和咪康唑的MIC值较低,MIC范围依次为0.25～0.5μg/mL,0.125～0.5μg/mL,≤0.03μg/mL,0.25～8μg/mL;对伊曲康唑和氟康唑的MIC值较高,MIC范围分别为0.25～8μg/mL,0.5～64μg/mL。B型和C型对5-氟胞嘧啶的MIC值均为0.125μg/mL,对伊曲康唑和氟康唑的耐药率分别为84%、70%。不同族别来源的菌株基因型比较无显著差异(P>0.05),不同基因型菌株的抗真菌药物敏感性比较也无显著差异(P>0.05)。结论新疆地区白念珠菌分A,B,C三种基因型。汉...     

Electrophoretic karyotyping as a taxonomic tool in the genusSaccharomyces

Summary The electrophoretic karyotypes of strains of the ten species of the yeast genusSaccharomyces (sensu Vaughan-Martini & Martini 1992) were determined by the CHEF (contour-clamped homogeneous electric field) system of pulsed field gel electrophoresis. The number of bands was found to vary from 6 to 17 and the calculated molecular weights of haploid genomes ranged from 7.9 to 14.6 Mbp. The type strains ofS. exiguus and the four species of theSaccharomyces sensu stricto complex (S. bayanus, S. cerevisiae, S. paradoxus andS. pastorianus) have genomes comprised of chromosomes of all three size classes: light (< 500 kb), medium (500–1000 kb) and heavy (> 1,000 kb).Saccharomyces kluyveri DNA has only heavy bands, while the remaining species exhibit medium and heavy chromosomes. When more than one strain of each species was examined, it was seen that while the speciesS. bayanus, S. castellii, S. cerevisiae, S. kluyveri, S. paradoxus andS. pastorianus showed uniform karyotypes,S. dairensis, S. exiguus, S. servazzii andS. unisporus comprise heterogeneous taxa.     

Non-albicans Candida species isolated from plastic devices

In this work we point out an occurrence of non-albicans Candida species isolated from catheters, cannulas and drains. We detected eight non-albicans Candida species in 49 examined samples: C. parapsilosis (n = 26), C. tropicalis (n = 12), C. krusei (n = 4), C. claussenii, C. mesenterica (n = 2 for each), C. guilliermondii, C. kefyr and C. lusitaniae (n = 1 for each). Material examined from children hospitalized in intensive care units was the most frequent. Eight samples were from the oncology department, seven from the surgery department, six from the anaesthesiology department, four from the dialysis unit, two from the hematology department, one from the internal medicine department and one from the stomatology department. We examined cannula scraping 26 times (53%), catheter scraping 14 times (28.6%) and drain scraping nine times (18.4%). This revised version was published online in June 2006 with corrections to the Cover Date.     

Biochemical studies carried out on different groups ofCandida parapsilosis andCandida rhagii strains by comparing some cellular and mitochondrial activities

A comparative biochemical study was performed on some strains ofCandida rhagii and on strains belonging to different subgroups ofCandida parapsilosis. Measurements of alcohol dehydrogenase activity, resistance to drugs and occurrence of an alternative pathway enabled us to confirm the classification between several subgroups within theC. parapsilosis species.     

New antifungal agents that inhibit the growth of Candida Species: Dichlorinated 8-quinolinols

Five dichlorinated 8-quinolinols (2,5- 5,6-, 3,5-, 3,7-, and 4,5-dichloro-8-quinolinol) were tested against Candida albicans and C. Tropicalis in Sabouraud dextrosebroth with and without bovine serum. The 5,6-, 3,5-, and 3,7-dichloro-8-quinolinols proved to be more effective than the control, 5-fluorocytosine. In cytotoxicity tests employing baby hamster kidney (BHK) cells, all test agents proved to be more cytotoxic than the control. However, the minimum inhibitory concentration (MIC) of 3,5-dichloro-8-quinolinol to both fungi was only one tenth the cytotoxic dose,suggesting that the compound may be useful as a topical or systemic antifungal agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Interaction between Candida agglutinins and antifungal agents

Antifungal agents alter the function and morphology of Candida cell membranes and cell walls. We observed that brief (30 minute) exposure to either amphotericin B or clotrimazole inhibited the agglutination of Candida blastoconidia by murine bronchoalveolar lavage fluid. This inhibition required continuous drug presence. Neither amphotericin nor clotrimazole inhibited Candida agglutination by concanavalin A or pooled human serum. These results demonstrate that antifungal drugs can produce rapid changes in the surface characteristics of some fungi.     

Crystal structure of a carbonyl reductase from Candida parapsilosis with anti-Prelog stereospecificity

A novel short-chain (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) from Candida parapsilosis exhibits coenzyme specificity for NADPH over NADH. It catalyzes an anti-Prelog type reaction to reduce 2-hydroxyacetophenone into (S)-1-phenyl-1,2-ethanediol. The coding gene was overexpressed in Escherichia coli and the purified protein was crystallized. The crystal structure of the apo-form was solved to 2.7 Å resolution. This protein forms a homo-tetramer with a broken 2-2-2 symmetry. The overall fold of each SCR subunit is similar to that of the known structures of other homologous alcohol dehydrogenases, although the latter usually form tetramers with perfect 2-2-2 symmetries. Additionally, in the apo-SCR structure, the entrance of the NADPH pocket is blocked by a surface loop. In order to understand the structure–function relationship of SCR, we carried out a number of mutagenesis–enzymatic analyses based on the new structural information. First, mutations of the putative catalytic Ser-Tyr-Lys triad confirmed their functional role. Second, truncation of an N-terminal 31-residue peptide indicated its role in oligomerization, but not in catalytic activity. Similarly, a V270D point mutation rendered the SCR as a dimer, rather than a tetramer, without affecting the enzymatic activity. Moreover, the S67D/H68D double-point mutation inside the coenzyme-binding pocket resulted in a nearly 10-fold increase and a 20-fold decrease in the k_cat/K_M value when NADH and NADPH were used as cofactors, respectively, with k_cat remaining essentially the same. This latter result provides a new example of a protein engineering approach to modify the coenzyme specificity in SCR and short-chain dehydrogenases/reductases in general.     

Candida Parapsilosis peritonitis in patients on CAPD

Twenty-four episodes of C. parapsilosis peritonitis in 23 patients on continuous ambulatory peritoneal dialysis (CAPD) over 6 years were reviewed. Clinical manifestations and laboratory findings were similar to those of other pathogens. All started treatment with intravenous amphotericin B. In six cases it was attempted to maintain a peritoneal catheter in situ, but removal became essential to relieve fungal peritonitis. Of the patients who developed peritonitis, 15 episodes (62.5%) continued the CAPD program. Nine cases could not resume CAPD because of death in 4, patient preference in 2, and abdominal adhesion in 3. Antifungal treatment alone was ineffective in most cases. It was found that peritonitis developing after gram negative bacterial peritonitis and the use of fluconazole after catheter removal were associated with CAPD discontinuation. It was suggested that C. parapsilosis peritonitis in CAPD patients should be treated with rapid catheter removal, particularly those with fungal peritonitis who had prior gram negative peritonitis. This revised version was published online in June 2006 with corrections to the Cover Date.