Gyu-Nam Park

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

Roles of human apolipoprotein E in the infectivity and replication of hepatitis C virus genotype 2a.

Hepatitis C virus (HCV) infection is associated with lipoproteins, and apolipoprotein E (apoE) plays an essential role in infectious HCV particles. Although the role of apoE in HCV infection is well known, its role in the replication of HCV remains unclear. The aims of this study were to determine the role of apoE in the RNA replication of major HCV genotypes 1b and 2a, and to determine whether this role is HCVgenotype-dependent using HCV genotype 1b replicon cells and HCV genotype 2a producing (HP) cells. HCV infection was blocked in Huh7.5 cells treated with low-density lipoproteins, very low-density lipoproteins, or apoE3. An apoE3-specific monoclonal antibody also efficiently neutralized HCV infectivity, and HCV infection was dramatically suppressed by the knockdown of apoE expression with an apoE-specific small interfering RNA, suggesting a requirement for apoE in infectious HCV particles. HCV RNA replication was not affected in HP cells treated with each apoE isoform or transfected with apoE-specific siRNAs. However, the knockdown of apoE expression suppressed RNA replication of HCV genotype 1b. The siRNA-mediated knockdown of apoE, apoA1, and apoB expression also suppressed the RNA replication of HCV genotype 1b, but not that of HCV genotype 2a. Taken together, these findings indicate that apoE plays an important role in HCV genotype 2a infection and in HCV genotype 1b RNA replication, but not in the replication of HCV genotype 2a. These results provide important information for the future development of HCV-genotypespecific anti-HCV agents.

Haemolytic differential identification of Arcanobacterium haemolyticum isolated from a patient with diabetic foot ulcers

Introduction: Arcanobacterium haemolyticum (formerly known as Corynebacterium haemolyticum) is the causative agent of sore throat and also causes skin and soft tissue infections in diabetes patients. A. haemolyticum is a Gram-positive, catalase-negative, β-haemolytic bacillus. A. haemolyticum poses a diagnostic challenge in the hospital laboratory because most coryneform bacilli are considered as normal flora or contaminants, and it is therefore difficult to differentiate from β-haemolytic streptococci by colony characteristics. Case presentation: A. haemolyticum was isolated from a diabetic patient with foot ulcers and the isolate was identified by using a VITEK-2 system, CAMP inhibition test, reverse CAMP test and a 23S rRNA gene sequence analysis. The isolated A. haemolyticum inhibited haemolysis of Staphylococcus aureus in the CAMP test and enhanced haemolysis of Streptococcus agalactiae in the reverse CAMP test. The diabetic patient was treated with teicoplanin and imipenem, and the ulcers healed within 2 weeks. Conclusion: The present study suggests that a haemolytic differential method using the CAMP inhibition and reverse CAMP tests can be useful for differentiating A. haemolyticum from β-haemolytic streptococci.