Objective To investigate the DNA typing, observe relationship between DNA fingerprinting patterns and serotypes of Cryptococcus neoformans, and find a suitable genotyping standard for Cryptococcus neoformans. Methods Three primers, including CN 1(GTG) 5, CN 2(GACA) 4 and CN 3(GATA) 4, were used to distinguish variations among strains of C.neoformans. Results The distinguishable fingerprinting bands for serotype of C.neoformans were yielded by primer CN 2. Using this primer, of 24 clinical and environmental isolates of serotype A of C.neoformans and 8 standard strains investigated by PCR, 20 strains produced complete identical fingerprinting patterns, the other 4 strains had different fingerprinting patterns. 2 strains of serotype B and C yielded indistinguishable fingerprinting patterns. Conclusions ①The majority of strains of serotype A had similar and stable fingerprinting patterns. ②Some strains of serotype B and C had an indistinguishable fingerprinting patterns. ③The same serotype strains from different sources may produce different DNA fingerprinting patterns. ④The DNA fingerprinting is a rapid, simple and feasible method for identifying Cryptococcus neoformans.

This paper reports 20 patients with cryptococcal meningitis,which were misdiagnosed before anti-fungal therapy,and the majority of them was diagnosed as tuberculous meningitis or viral meningitis.These patients were treated either with amphotericin B or fluconazale alone,or in combina- tion with flucytosine respectively,of the 15 patients could be evaluated,10 were cured(66.7%),2 im- proved(13.3%),3 died(20%),1 relapsed(10%).The mortality rate among the under age group and the aged group was significantly higher than that of the adult group(71.4% vs 23%).It is the authors' experience that in the early stage intrathecal amphotericin B combines with intravenous fluoconazole and changes to oral fluconazole or itraconazole in the later stage may be a valuable approach for cryptococcal meningitis.

The patient is an 11-year old boy, who was born with universe alopecia as well as dry and coarse skin. When he was 3 months old, photophobia was noticed, and since then, upper respiratory tract infection had occurred twice a month complicated by frequent diarrhea. He had short stature with slight conjunc- rival congestion, corneal vascularization, opacity, coarseness and poor vision. No abnormality was found in the teeth, sweating ability, or hearing. He had universal alopecia; his skin was dry and rough with generalized rhombus- or polygon-shaped scaly patches. Particularly thick brown scales were observed on the upper limbs. Moreover, there were spiny follicular papules on the abdomen and axillae, hyperkeratosis of palm and sole, and dystrophic nails. Hyperextensibility of proximal interphalangeal joints of the third, fourth and fifth fingers was noticed. He also suffered from mental retardation, the verbal intelligence quotient being 52, performance intelligence quotient lower than 40, full intelligence quotient lower than 40, but no abnormality was found in the heart, lung, liver or spleen. Histopathology of skin on the abdomen suggested a change characteristic of ichthyosis. Chromosome analysis revealed a karyotype of 46, XY. This is the first diagnosed case of ichthyosis follicularis with atrichia and photophobia syndrome in China.

Objective To analyze the clinical features,clinical prognosis and predictive factors of ulcerative colitis (UC) complicated with cytomegalovirus (CMV) infection.Methods From May 2004 to November 2014,120 hospitalized patients diagnosed as UC and screened for CMV infection were enrolled.A total of 31 patients with moderate to severe UC accompanied by CMV infection were screened out.Demographics,clinical features,endoscopic appearance and treatment of patients with UC complicated with CMV infection were analyzed,and compared with 60 moderate to severe UC patients without CMV infection at the same period of hospitalization.Mann Whitney U test was performed for statistical analysis.Logistic regression analysis was used to analyze risk factors of UC complicated with CMV infection.Results Among 120 patients with UC,29 were mild or in remission period,whose CMV screening tests were all negative.Ninety-one were moderate to severe,31 patients (34.1 ％) of them had CMV infection,and 20 of 31 patients were steroid-refractory.Among the 31 patients with UC complicated with CMV infection,median age was 39 years (22 years,51 years),median disease duration was 24.0 months (6.0 months,42.0 months) which was shorter than that of patients without CMV infection (36.0 months (13.5 months,84.0 months)),and the difference was statistically significant (U=639.5,P=0.015).A total of 23 patients (74.2％) had extensive colitis and 26 patients (83.9％) had history of severe colitis.A total of 29 patients (93.5％) had history of corticosteroids treatment,12 patients (38.7％) had history of immunosuppressive agents treatment,and six patients (19.4 ％) had history of infliximab treatment.Compared with UC patients without CMV infection,fever,abdominal pain and weight loss were more common in UC patients with CMV infection.Five CMV-infected patients had mild liver dysfunction.Endoscopic appearance was longitudinal ulceration,irregular ulceration,large deep ulceration,punchedout ulceration and worm-like ulceration.Among the 25 CMV-infected patients who were treated with corticosteroids,11 patients (44.0％) had no response.Among the 39 CMV-negative patients who were treated with corticosteroids,eight patients (20.5％) had no response.The rate of patients who needed rescue therapy of the former was higher than that of the latter,and the difference was statistically significant (x2 =4.026,P=0.045).The results of multivariate Logistic regression analysis showed that hemoglobin over 100 g/L(OR=0.144,95％ confidence interval (CI) 0.040 to 0.516,P=0.003) was a protective factor of CMV infection,however corticosteroids use within a month before the onset (OR=8.946,95％oCI 2.459 to 32.541,P=0.001) was a risk factor.Conclusions UC patients treated with corticosteroids and immunomodulator therapy may predispose UC patients to CMV infection,on the other hand,CMV infection can exacerbate the severity of UC.CMV infection should be screened and monitored in UC patients,and anti viral therapy should be taken in time in case of CMV infection.

Objective To investigate genetic relationships among five serotypes of two variants of Cryptococcus neoformans. Methods PCR mediated DGGE (denaturing gradient gel electrophoresis) and sequence analysis of 28S rDNA of C. neoformans were performed in ten reference strains, C. neoformans capsular-deficient strain CAP10, and nineteen clinical isolates from non-HIV patients. Results The results of DGGE and analysis of nucleotide sequences of 28S rDNA showed identical patterns and nucleotide sequences in the serotype A and D of C. neoformans var. neoformans, which were distinct from the serotye B and C of C. neoformans var. gattii. The patterns and sequences of serotype AD coincided with those of C. neoformans var. gattii. The patterns and nucleotide sequences of C. neoformans capsular-deficient strain CAP10 (serotype D) and serotype A and D were identical. Of the nineteen clinical isolates, seventeen had patterns of serotype A and D, and the others had patterns of serotype B and C. Conclusions PCR mediated DGGE integrated with sequence analysis of 28S rDNA is a valuable tool for the classification of C. neoformans. The clinical isolate of C. neoformans var. neoformans is predominant in Chinese non-HIV patients. Serotype AD is genetically close to C. neoformans var. gattii rather than C. neoformans var. neoformans. The data seem not to be in favor of previous study that serotype A, C. neoformans var. grubii, is a new variant of C. neoformans.

Objective To evaluate the role of Restriction fragment length polymorphism (RFLP) analysis in detection of the fragment of GEF1α/a gene which are both located at ct and a mating type loci in identification of species, varieties, genotypes and mating types of Cryptococcus neoformans species complex(Cryptococcus neoformans and Cryptococcus gattii). Methods The GEF1α/a gene was selected from 20 genes which both located at α and a mating type loci for RFLP analysis, according to the requirements of sequence similarities and primer design in PCR-RFLP analysis. Primer pair was designed from the conserved regions of GEF1α/a genes of distinct genotypes and mating types of reference strains to amplify a fragment of GEF1α/a gene from Cryptococcus neoformans and Cryptococcns gattii strains tested. Sequence alignment,restriction maps analysis, endonucleases selection and electrophoresis stimulation were conducted by using DNAMAN and Vector NTI software. EeoT14 Ⅰ and Hap Ⅱ endonucleases were selected for RFLP analysis of the GEF1α/a fragments amplified from 125 isolates of Cryptococcns neoformans and Cryptococcus gattii. Results An approximate 1 300 bp fragment was amplified from total 82 Cryptococcus neoformans and 43 Cryptoceccus gattii isolates. However, negative PCR results were found in the reference strains of Cryptococcus laurentii, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krnsei,Candida glabrata, Trichosporon asahii, Aspergillus fumigatns and Aspergillus flavus. RFLP analysis successfully identified the species, varieties, genotypes and mating types of total 125 isolates of Cryptococcus neoformans and Cryptococcns gattii tested in this study. Condusion PCR-RFLP analysis of the GEF1α/a fragment has the potential value in identification of species, varieties, genotypes and mating types of Cryptococeus neoformans species complex simultaneously and rapidly, and may be a useful tool in molecular epidemiological analysis.

Objective To investigate the molecular epidemiology of clinical cryptococcal isolates from China by analyzing the constituents and distributions of the varieties,genotypes and mating types (MAT)of them.Methods (1)PCR fingerprinting and PCR amplification were performed by using the minisatellite-specific core sequence of wild-type phage M13 as single primer.Genotypes of the 110 cryptococcal isolates from China were assigned by comparison with the reference strains of the 8 major molecular types loaded on gel.(2)Identification of the varieties and mating types was carried out by PCR using the specific primers of the varieties and mating types.Results Of the 110 clinical cryptococcal isolates,strains of Cryptococcus neoformans var.grubii with genetype VNⅠ and mating type MATα were the most representative ones(89.1%)followed by strains of C.neoformans var.gattii(8.2%)including isolates of genotype VG I,mating type MATα(7.3%)and genotype VGⅡ,mating type MATα(0.9%);AD hybrids with the genotype VNⅢ,mating type MAT-/α and genotype VN Ⅲ,mating type MATα/-(1.8%);and isolate of C.neoformans var.neoformans with the genotype VNⅣ and mating type MATa(0.9%).Conclusion Of the clinical isolates from China,all three varieties and AD hybrids are found.The vast majority(>99%) of strains possess the α allele in MAT locus and most of them are C.neoformans vat.grubii with the genotype VN I,which accord with the data of most studies of clinical molecular epidemiology in other geographic areas.However.no genotype of VNⅡ.VGⅢ and VGⅣ isolates are found in this study.

Objective To assess the subgenotypes of pathogenic Cryptococcus gattii isolates from China and to elucidate the epidemiological links between these domestic isolates and those from other parts of the world. Methods DNA was extracted from 9 clinical isolates of Ctyptococcus gattii from China. The partially variable regions of the three unlinked loci, namely IGS1, PLB1 and GEF1, were amplified and sequenced, and the bioinformation at these loci was obtained from GenBank for multi-locus sequences alignment and phylogenetic analysis. Results Of these 9 clinical isolates, 8 were genotype VG Ⅰ and mating type α with the same sequences at the tested regions as the reference strain WM276, which was a representative isolate of an independent subgenotype; 1 was of genotype VG Ⅱ and mating type α, which was the first report in China, with the tested sequences consistent with those of the referrence strain R272. Sequencing and phylogenetic analysis of GEF1 gene, which was located at mating type locus, successfully identified the genotypes and mating types of all the Cryptococcus gattii isolates involved here. Conclusions Multi-locus sequence analysis shows that causative Cryptococcus gattii isolates of genotype VG Ⅰ in China carry similar sequences at the tested loci in IGS1, PLB1 and GEF1 genes, to a widely distributed subgenotype in the world, and the sequences of the first VG Ⅱ genotype isolate from China resemble the less virulent subgenotype VG Ⅱ b found in Vancouver islands.

Objective To investigate the efficacy of autologous peripheral hematopoietic stem cell transplantation in the treatment of adult dermatomyositis. Methods A 21-year-old patient with dermatomyositis received autologous peripheral hematopoietic stem cell transplantation and was followed up for 6 years. Autologous peripheral hematopoietic stem cells were mobilized by recombinant human granulocyte colony stimulating factor (rhG-CSF) before the transplantation, and the conditioning regimens consisted of cyclophosphamide,methylprednisolone and cyclosporin. Rabbit anti-human T lymphocyte immunoglobulin began to be applied on day 3 after retransfer of stem cells. The improvement in symptoms, physical signs and biochemical indicators was observed, and hematopoietic restructuration and immunity resurrection were evaluated after the transplantation. Results After the transplantation, skin eruption greatly improved and gradually subsided. The muscle force of extremities restored from level Ⅳ before transplantation to level Ⅴ. The level of creatine kinase declined sharply after transplantation, but gradually returned to previous level. Leucocyte count began to decrease on the day of retransfer, and returned to the normal level on day 8. Immune function remained normal before and after the transplantation. Conclusion Autologous peripheral hematopoietic stem cell transplantation is an alternative treatment for severe and refractory dermatomyositis.

Objective To investigate the expression of μ-opioid system in the epidermis of patients with atopic dermatitis and its role in the pathogenesis of atopic dermatitis. Methods Thirty-two mice were equally divided into 4 groups, negative control group, pre-treatment group, naloxone group, and physiological saline group. Ovalbumin was used to sensitize mice in pretreatment group, naloxone group, and physiological saline group for 7 weeks, then, mice in naloxone group and physiological saline group were treated with intracutaneous naloxone or physiological saline solution for 1 week, respectively. Mice were killed in negative control group and pre-treatment group at the end of sensitization, and in naloxone group and physiological saline group after 1-week injection with naloxone or physiological saline, skin tissues were obtained from the back of killed mice and subjected to histological examination with HE staining and quantitative fluorescent PCR for the detection of mRNA expression of μ-opioid receptor (MOR) and its ligand (β-endorphin) in epidermis. The atopic dermatitis severity index of lesions and histological changes were assessed before and after the treatment. Results In comparison with the negative control mice, the epidermal expression level of MOR was signifieantly decreased (t = 2.549, P ＜ 0.05 ) in pre-treatment group, but increased in naloxone group and showed no statistical difference from the negative control group (t = 0.671, P ＞ 0.05). No significant difference was observed in the epidermal β-endorphin mRNA expression between negative control group and pre-treatment group or naloxone group (both P ＞ 0.05 ). The improvement of lesions could be visualized after treatment with naloxone (t = 8.338, P ＜ 0.01 ), which was concordant with the histological changes in naloxone group. Conchusions As an antagonist of MOR, naloxone can restore the expression of epidermal MOR in mice model for atopic dermatitis, and shows a certain efficacy in the treatment of atopic dermatitis, which proves that μ-opioid system is somewhat associated with the pathogenesis of atopic dermatitis.