Cryptococcal infection has become a public health challenge globally. However, information about cryptococcal infection in patients with hematological diseases remains relatively rare.

Objective To evaluate the clinical efficacy and safety of high-dose fluconazole (600-800 mg/d) used alone or combined with amphotericin B and/or flucytosine as salvage therapy for refractory cryptococcal meningitis (CM) in human immunodeficiency virus (HIV)-uninfected patients.Methods Twenty HIV-uninfected patients with refractory CM who were treated with high-dose fluconazole for over 1 week from September 2011 to August 2014 in Huashan Hospital,Fudan University were retrospectively reviewed.Clinical features including clinical characteristics,clinical responses,drugrelated adverse events,and prognosis were evaluated.Results Of the 20 patients enrolled in this study,11 patients had predisposing factors.Headache (20 cases),fever (14 cases),vomiting (14 cases) and meningeal irritation (13 cases) were commonly seen.Abnormalities of head computor tomography scan and/or magnetic resonance imaging were found in 19 cases.The median course for salvage therapy was 80 d (28-440 d).At the end of salvage therapy,17 patients achieved partial response and 2 patients achieved stable response,while 1 patient died.The overall efficacy rate was 85 ％.At the end of antifungal therapy,complete response was achieved in 9 patients,partial response in 8 patients,stable response in 1 patient and death in 2 patients were observed,and overall success rate was 85％.In addition,11 drugrelated adverse events were observed in 7 cases treated with high-dose fluconazole,including abnormal liver function,hemocytopenia,gastrointestinal reaction,etc.However,all these reactions were tolerable and none of the patients endured dosage reduction or drug discontinuance.Conclusion Salvage therapy with high-dose fluconazole may be appropriate for the treatment of refractory CM in HIV-uninfected patients with good efficacy and safety.

中枢神经系统隐球菌感染在中枢神经系统真菌感染中最为常见，且病死率长期以来居高不下，目前其发病机制尚不十分明确。因隐球菌主要通过血运播散，透过血-脑脊液屏障进入中枢神经系统，因此如何透过血-脑脊液屏障是其致病机制的研究热点。血-脑脊液屏障是介于血液和脑组织之间紧密连接的网状结构，由脑微血管内皮细胞、星形胶质细胞、周细胞共同组成。这种特殊结构允许疏水性物质，如O2、激素、CO2等扩散至脑内，却限制微生物及大分子物质通过，以此来调节血源性物质的通透性，维持中枢神经系统微环境的稳定。近年来随着对隐球菌透过血-脑脊液屏障机制研究的不断深入，已初步阐明其跨细胞膜转运机制、“特洛伊木马”机制及细胞旁扩散转运机制。现就其跨越血-脑脊液屏障的分子机制作一综述。

BACKGROUND:Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.METHODS:One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5' end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.RESULTS:For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.CONCLUSIONS:rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.

Dectin-2 is a C-type lectin receptor that can recognize critical structures of fungi and involve in the host immune response after pulmonary fungal infections. We aimed to investigate the association between Dectin-2 genetic polymorphisms and cryptococcosis among a series of human immunodeficiency virus (HIV)-uninfected Chinese patients. In this case control study, a total of 251 patients with cryptococcosis and 464 healthy controls were included. One tag-single nucleotide polymorphism (SNP) (rs11045418) located at 5'-flanking region of Dectin-2 gene was selected and genotyped in this study. Among 251 patients, there were 108 (43%) meningitis patients including 73 (67.7%) healthy ones, 74 (29.5%) pulmonary infected patients including 49 (66.2%) healthy ones, and 69 (27.5%) patients with both neural and pulmonary infection including 38 (55.1%) immunocompetent ones. One hundred and forty-three (74 plus 69) patients with pulmonary cryptococcosis and 177 (108 plus 69) patients with cryptococcal meningitis were compared with controls, respectively. Three samples from 143 pulmonary infected patients failed in genotyping. There was a significant difference between 86 immunocompetent pulmonary infected patients and controls in the overdominant model (C/T vs. T/T + C/C; OR, 0.59; 95%CI, 0.37-0.94; P, .026). Similar but not significant difference was found between the overall pulmonary infected patients and the controls in the overdominant model (OR, 0.77; 95%CI, 0.52-1.12; P, .17). No such difference was found between controls and patients with cryptococcal meningitis. Our study firstly showed a genetic association between Dectin-2 and pulmonary cryptococcosis.

Amphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cells in vitro was significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected with Cryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0; P < 0.05) and 30 min (5.2 versus 2.8; P < 0.05) after administration of verapamil or 45 min (6.0 versus 3.9; P < 0.05) and 60 min (5.4 versus 3.8; P < 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P < 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate.

Objective To investigate the association between genetic polymorphisms of Dectin-2 and pulmonary cryptococcosis.Methods A total of 134 non-human immunodeficiency virus (HIV)patients with pulmonary cryptococcosis and 464 healthy controls were included in this case control study.The peripheral leucocyte DNA was extracted and genotyping was performed by multiplex SNaPshot technology.The single nucleotide polymorphism (SNP)of rs11045418 located at 5′-flanking locus of Dectin-2 gene was genotyped.Patients without predisposing conditions were compared independently.The differences of gene polymorphism distributions compared between pulmonary patients and healthy control, and between patients without predisposing conditions and healthy control.All data were analyzed withχ2 tests.Results Among the total 134 patients,82 patients had no predisposing factors.Thirty two patients met the proven diagnosis criteria and 102 patients were probable pulmonary cryptococcosis.According to the site of infection, 72 patients had local infection in lungs and 62 patients had disseminated cryptococcosis.Three samples failed in genotyping,one of which was a patient without predisposing factor.Compared with the control group,there was a trend of increasing proportion of heterozygote rs11045418 CT in the 131 pulmonary cryptococcosis patients (59% vs 50%,P =0.069,OR=1.44,95%CI :0.97-2.13),and the heterozygote was significantly increased in 81 patients without predisposing conditions(64% vs 50%,P =0.017,OR= 1 .82,95 %CI :1 .11 -2.95 ).No significant difference of genotype distribution was found between the local and disseminated infection patients.Conclusion Our study shows that rs11045418 CT heterozygote in Dectin-2 is associated with the susceptibility of pulmonary cyrptococcosis among non-HIV-infected Chinese patients,which indicated that the change of Dectin-2 receptor may play a role in the pathogenesis of pulmonary cyrptococcosis.

患者男,41岁.因反复头痛27 d,发热13d于2012年4月27日入住复旦大学附属华山医院感染科.患者27 d前无明显诱因下感头痛、头晕,前额部阵发性隐痛,曾视物旋转,持续约2h后缓解.21 d前开始出现头部持续性胀痛,不剧烈,无发热,遂就诊于当地医院查颅脑CT未见异常.13d前开始出现发热,体温波动于36.3～38.0℃,无畏寒,头痛较前明显加剧,仍表现为头部持续性胀痛,无头晕,无视物模糊,无恶心、呕吐.再次就诊于当地医院,血WBC 10.18×109/L,中性粒细胞0.75,肝肾功能正常;脑脊液清,潘氏试验阴性,WBC 10×106/L、RBC 2×106/L,蛋白802mg/L,隐球菌涂片阴性.颅脑MRI示脑内多发腔隙性缺血灶.予抗病毒、脱水降颅压治疗后仍持续头痛,伴反复发热.复旦大学附属华山医院拟“中枢神经系统感染”收住入院.

Objective To evaluate the accuracy of ribosomal RNA gene (rDNA) molecular identification in identification of common pathogenic fungi.Methods Sixty pathogenic fungi including yeasts,filamentous fungi and Prototheca species were included in the study,among which 13 were reference strains and 47 were clinical/environmental isolates.Both phenotypic identification and rDNA molecular identification,including sequencing and alignment of internal transcribed spacer 1 (ITS1),internal transcribed spacer 2 (ITS2) and D1/D2 region on 5' end of 28S subunit rDNA (D1/D2) target regions were performed for all the 60 strains.The accuracy of single region,multiple regions molecular and phenotypic identification methods was compared.Results The highest accuracy of species-level identification for yeast isolates was yielded by D1/D2 molecular identification (20/23,87％),followed by combining ITS1 with ITS2 (19/23,82.6％),ITS2 alone(18/23,78.3％),and ITS1 alone (14/23,60.9％).While for filamentous fungal isolates,the highest species-level identification accuracy was yielded by combination of ITS1 and ITS2 molecular identification (16/22,72.7％),followed by ITS1 alone(15/22,68.2％),ITS2 alone(14/22,63.6％) and D1/D2 (13/22,59.1％).Combination of ITS1,ITS2 and D1/D2 molecular methods showed the best performance,which yielded 91.3 ％ (21/23) species-level identification for yeast isolates,and 72.7 ％ (16/22) for filamentous isolates.Conclusions rDNA molecular identification has high accuracy for identification of common pathogenic fungi,especially combination of ITS and D1/D2 regions,which might be a promising approach in the future.

Objective To describe the distributions of FCGR polymorphisms in human immunodeficiency virus (HIV)-uninfected patients with cryptococcosis,and to investigate the association of FCGR polymorphisms with the susceptibility to cryptococcosis.Methods The distributions of the four functional polymorphisms,including FCGR2A 131H/R,FCGR3A 158F/V,FCGR3B NA1/NA2,and FCGR2B 232I/T were compared between 198 cryptococcosis patients and 190 healthy controls.The polymorphisms distribution patterns were also compared between patients with central nervous system (CNS) infection and those without CNS infection.Genotyping of eight single nucleotide polymorphism (SNP) in FCGR were performed by multiplex SNaPshot technology using DNA extracted from blood samples.The comparison between patients and controls was performed by chi square test or Fisher exact test.Results Compared to healthy controls,the frequency of FCGR2B 232I/I increased (65％ vs 53％,x2 =4.27,P=0.039,OR=1.652,95％CI:1.02-2.67) and that of FCGR2B 232I/T decreased (27％ vs 40％,x2 =5.77,P=0.016.OR=0.542,95％CI:0.33-0.90) in patients with cryptococcal meningitis.Among immunocompetent patients,the frequency of FCGR2B 232I/I was also over-presented (69％ vs 53％,x2=4.53,P =0.033,OR=1.958,95％CI:1.05-3.66) and the FCGR2B 232I/T genotype was also less frequently observed (24％ vs 40％,x2=5.14,P=0.023,OR=0.467,95％CI:0.24-0.91) compared to healthy controls.There were 117 cases with CNS infection and 81 non-CNS infection cases.The genotype of FCGR2A 131R/Rwas over-presented (19％ vs 6％,x2 =6.48,P=0.011,OR=3.52,95％CI:1.27-9.73) and the FCGR2B 232I/T genotype was under-presented (27 ％ vs 46 ％,x2 =7.56,P =0.006,OR=0.431,95％CI:0.24-0.79) in patients with CNS infection compared with those without CNS infection.Furthermore,the frequency of FCGR2B 232I/I genotypes increased (69％ vs47％,x2 =5.47,P=0.019,OR=2.479,95％CI:1.15-5.34) and the frequency of FCGR2B 232I/T decreased (24％ vs 51％,x2 =8.66,P=0.003,OR=0.307,95％CI:0.14-0.68) in immunocompetent patients with CNS infection compared with those without CNS infection.Conclusions FCGR2A 131H/R and FCGR2B 232I/T are associated with the susceptibility to cryptococcal CNS infection,which suggests that FcγRⅡA and FcγRⅡB may contribute to the pathogenesis of cryptococcosis.