Objective To develop a PCR method to detect common deep pathogenic fungi for the application in clinical settings.Methods A hot-start polymerase chain reaction (PCR)-based method was developed.A simplex PCR with a universal fungus-specific primer set was used to detect experimental and simulating clinical specimens.For specimens with positive results,a triplex PCR was further applied with the species-specific primers of Candida albicans,Aspergillus fumigatus and Cryptococcus neoformans.Results A 260 bp product was successfully amplified from all 78 strains of 55 fungal species in 9 fungal genera,but not amplified from any strain of other microbes and human cells.The detection system was of high specificity and sensitivity,and convenient and inexpensive for use.Conclusion The simplex and triplex PCR approach based on highly specific hot-start PCR might be useful for quick detection of deep fugal infection in routine clinical laboratory practice.

Objective To investigate the clinical and laboratory profiles of a case of infantile chronic mucocutaneous candidiasis due to malnutrition.Methods Skin specimens were collected and examined by KOH preparation,fungus culture,biochemical tests and molecular biological study.The pathogenic strain and antimicrobial susceptibility were defined.Results A 5 month-old-girl was presented with big head,and erythema,erosion and crust on her head,auricles,nostrils,oral cavity,neck and buttocks,and itching,for 1 month.Dystrophic hair and nails were observed.Candida albicans was isolated from specimens of the buttocks and Candida glabrata from the scalp.The abnormalities,CD16+ (56 6.5%),serum IgG (6.74 g/L),IgA (0.321 g/L),and albumin (18 g/L) were found with flow cytometry and immunoassays.Conclusion The patient is diagnosed as infantile chronic mucocutaneous candidiasis,who is immunocompromised due to protein malnutrition.

Objective To investigate the differential expression of secretory aspartyl proteinase in patients with vaginal C.albicans infection and in asymptomatic candidal carriers.Methods Secretory aspartyl proteinase expression was determined with reverse transcription-PCR in vaginal specimens taken from 10 asymptomatic candidal carriers,14 patients with vulvovaginal candidiasis (VVC) and 10 patients with recurrent VVC (RVVC).Results SAP2 and SAP4 to SAP6 subfamily were detected in both candidal carriers and patients with vaginal candidiasis.SAP1 and SAP3 transcripts were not observed in 10 asymptomatic candidal carriers.All 9 SAP genes were differently expressed in VVC and RVVC patients.SAP1 and SAP3 transcripts were frequently amplified in some patients.Conclusion It is shown that C.albicans infection is associated with differential expression of individual SAP genes,which may be involved in the pathogenesis of vaginal candidiasis.

Objective To determine whether Trichophyton rubrum isolated from different lesions in the same patient is of different strains. Methods DNA was extracted from the isolates, then subjected to a polymerase chain reaction-based typing which analyzed the number of repetitive elements in the non-transcribed spacer region of the ribosomal DNA gene repeats. Results Thirty-six strains of T. rubrum were isolated from 17 patients with fungal infection on multiple sites. All strains could be classified into 10 genotypes. The genotype distribution was unrelated to sites of infection. It happened to 8 of the 17 patients that multiple genotypes were involved in T. rubrum infection on different sites in the same body. Conclusion The study shows that multiple genotypes are involved in T. rubrum infection on different sites in the same patient, suggesting external sources of infection rather than infection from a different site in the same individual.

Objective To investigate the association of two microsatellite mar kers, D17S784 and D17S928, with psoriasis vulgaris. Methods Fluorescent multiple x PCR, genescaning and GenotypeTM software were employed to amplify the microsat ellite DNA markers and conduct genotyping. Results The significant association w as found between D17S784, D17S928 and psoriasis vulgaris (P

Objective To investigate the impact of mutations in the V2 domain of HIV-1 envelop glycoprotein (gp) 120 gene on the recognition of neutralizing antibodies (NAbs) specific to the other domains of gp120.MethodsHIV-1 pseudoviruses (JR-FL) containing wild type or V2-mutant gp120 monomers were constructed,and the neutralization of CD4-binding site-specific and CD4-induced NAbs to the HIV-1 pseudoviruses was observed.Enzyme linked immunosorbent assay(ELISA) was performed to evaluate the binding affinity of CD4-binding site-specific and CD4-induced NAbs to wild type or V2-mutant gp120.Results Neither CD4-binding site-specific nor CD4-induced NAbs could neutralize the wild type JR-FL pseudoviruses,but both of them could neutralize pseudoviruses containg the gp120 V2 mutant at a low concentration.There was no significant difference in the binding affinity to CD4-binding site-specific NAbs between the wild type and mutant gp120,while the ELISA binding curves of wild type and mutant gp120 against CD4-induced NAbs were separate,and the affinity of CD4-induced NAbs to the mutant gp120 (L175P) was notably higher than that to the wild type gp120.Conclusion The mutations in the V2 domain of HIV-1 gp120 may affect the antiviral activity of NAbs.

Objective To study the impact of HIV-1 V2 L175P mutation on the binding capability of anti-V3 neutralizing antibodies to HIV-1.Methods A series of eukaryotic cell expression plasmids were used to concatenate wild type and mutant env gene of HIV-1 and green fluorescent protein(GFP)gene.The recombinant plasmids were transfected into 293T cells to express HIV-1 gp120 protein on the surface of cells.The successfully transfected cells were screened by GFP florescence marker.Immunostaining and dual fluorescence flow cytometry were performed to test the binding affinity of several common V3 region specific neutralizing antibodies to wild type or mutant gp120 proteins.Results The mean fluorescence intensity(MFI)of mutant gp120-expressing 293T cells were significantly higher than that of negative control cells(expressing GFP).Flow cytometry showed that the curve for mutant gp120-expressing 293T cells was obviously different in shape and peak from that for the negative control,while most parts of the curve for the wild type gp120-expressing 293T cells overlapped with those for the negative control.Conclusion The V2 region mutation may increase the sensitivity of HIV-1 to the neutralization by V3 region specific antibodies.

Objective To study the impact of V2 mutations on neutralizing ability of HIV-1-specific neutralizing antibodies. Methods We tested the influence of L175P mutation to the neutralizing ability of V3-specific antibodies by pseudotype virus and the binding affinity of those V3-spesific antibodies to gpl20 monomer by ELISA. Results We found L175P mutation changed the neutralizing ability of V3-specific antibodies. However, L175P mutation showed no effects on the binding affinity of these antibodies to gpl20 monomer. Conclusion Our results revealed the L175P mutation at V2 loop changed the natural trimmer structure of gp120 and enhanced the neutralizing ability of V3-specific antibodies.

Objective To establish a diagnosis method for fungal infection using two-round PCR,and to evaluate its sensitivity in the detection of clinical specimens suspected to be infected with fungi.Methods A total of 29 specimens of clinical sputum and alveolar wash solution were collected from patients with suspicious fungal infection.All specimens uaderwent direct microscopy with 10%KOH,fungal culture,one-round PCR and two-round PCR.The fungal universal primer targeting ITS regions of rDNA was used in PCR.The detection rate for fungi was compared between these methods.Results The detection rate for fungi was 20.69%by direct microscopy,37.9%by fungal culture,17.2%by one-round PCR,and 48.3%by two-round PCR.More than one species of fungus were detected in 6.9%(2/29),3.4%(1/29)and 24.1%(7/29)of these specimens by fungal culture.one-round PCR and two-round PCR, respectively.There was a significant difference in the detection rate between two-and one-round PCR(x2=6.34,P＜0.05).With regard to the detection rate for more than one species of fungus,two-round PCR was significantly higher than one-round PCR and fungal culture(x2=4.09,6.30.bom P＜0.05).Conclusion Two-round PCR may help to improve the sensitivity of molecular diagnosis of fungus-infected specimens.

Objective To develop a murine model for infection by Cladosporium carrioni. Methods A total of 72 ICR mice were equally divided into 4 groups, group A (healthy mice inoculated by C. Carrioni suspension of 1 × 108 cfu conidia mL-1, group B (immune-suppressed mice inoculated by C. Carrioni sus pension of 1 × 108 cfu conidia mL-1), group C (immune-suppressed mice inoculated by C. Carrioni suspen-sion of 1 × 106 cfu conidia mL-1), group D (healthy mice inoculated by sodium chloride solution). C. Car-rioni suspension or sodium chloride solution was subcutaneously inoculated into foot pads of mice. On day 7, 30 and 60 after inoculation, 6 mice were killed in each group followed by the measurement of thickness of foot pads, pathology and mycology of skin samples taken from foot pads. Results In group A, B and C, there were swelling, blackening, ulceration and crusts at the inoculation site of all mice, with a morbidity of 100%. The thickness of foot pads in group A on day 30 was significantly higher than that on day 7 (2.40 ± 0.45 mm vs 2.85 ± 0.47 mm, P < 0.05), but lower than that on day 60 (1.64 ± 0.13 ram, P < 0.05). In group B, increased thickness of foot pads was observed on day 30 compared with that on day 7 and day 60 (2.19 ± 0.27 mm vs 1.80 ± 0.21 mm and 1.86 + 0.22 mm, respectively, both P < 0.05), which was the case with group C (1.98 ± 0.06 nun vs 1.51 ± 0.11 mm and 1.82 ± 0.09 mm, respectively, both P < 0.05). No significant changes occurred to the thickness of foot pads in group D from day 7 to day 60 (P > 0.05). Pathological changes in group A, B and C included necrosis, abscess and chronic granuloma formation; dark brown sclerotic bodies were observed on HE and PAS staining as well as on direct microscopy; cultures of tissue samples grew Cladosporium carrionii. The mice in group D remained uninfected. Conclusion Mouse model for chromoblastomycosis may be established by subcutaneous inoculation of Cladosporium carrionii suspension into foot pads of healthy or immuno-suppressed mice.