Identification and viability assessment of dermatophytes infecting nail based on quantitative PCR of dermatophyte actin (ACT) mRNA]

The diagnosis and choice of treatment for dermatophytoses are usually based on the result of microscopic observation of hyphal elements and culture. However, false negative cultures have sometimes been encountered and appropriate timing of discontinuation of treatment has not been formulated. In this study, we attempted the identification and viability assessment of dermatophytes based on the quantitative measurement of dermatophyte actin (ACT) mRNA. An internal fragment of the ACT, 725 to 762 bp, was isolated by PCR from the genomic DNA of dermatophytes and sequenced. ACT intron-based primers were dermatophyte species-specific and primer pairs crossing the intron were dermatophyte genus-specific. The LightCycler (LC) instrument, employing the two-step RT-PCR/fluorescent hybridization system, was used to quantify the actin mRNA (ACT) of dermatophytes. A 669 bp ACT cDNA fragment was used as a quantification standard. Several mg of samples were collected from skin scales or nail plates before and after the treatment using oral terbinafine. The results indicated that quantification of ACT mRNA correlated with the results of culture and KOH examination and that copy numbers of dermatophyte ACT mRNA per mg sample decreased with progression of the therapy. This method comprises a sensitive (1 fg), specific, rapid (< 4 h) and quantitative assessment of the viability and identification of dermatophytes in skin tissue.     

Tinea faciei caused by Microsporum canis in a newborn

A case of tinea faciei caused by Microsporum canis in a 14-day-old infant is reported. The incubation period was seen to be 1 week. This was a familial infection which also affected the infant's grandmother and their pet cat and dog. Topical treatment with clotrimazole controlled the infection in the baby. A review of 14 cases (including our own) of dermatophytosis in newborn infants reported in the Japanese literature showed that newborn infants might be infected by several of these agents.     

Downward deviation of progressive addition lenses in a myopia control trial

PURPOSE: To clarify how the downward deviation of progressive addition lenses (PALs) reduces their near-addition effect in schoolchildren participating in a myopia control trial. METHODS: Among 95 schoolchildren wearing PALs for 6 months (age range: 6-12 years; refractive error range: -6.00 to -1.25 D), facial images were captured with a digital still camera placed 60 cm in front of the eyes while he or she was looking ahead with natural head posture. The vertical deviations of PALs from their ideal position (mm) were evaluated by analysing these images. RESULTS: The mean (+/-SD) downward deviations of PALs for the right and left eyes were 3.7 +/- 2.3 and 3.7 +/- 2.0 mm, respectively, and the largest downward deviation was 10.2 mm. For simulations using the average downward deviation, the near-addition effect of PALs was reduced to 30 and 63% of the expected value at the 10 degrees and 20 degrees downward eye positions, respectively. CONCLUSIONS: The downward deviation of PALs is a significant factor in reducing their therapeutic effect for near-addition. To ensure the proper alignment of PALs in children, the conventional spectacle-frame-fitting procedure is not sufficient, and repeated confirmation using a testing method similar to that used in this study is required.     

Bacteriological, pharmacokinetic and clinical studies on clarithromycin in the pediatric field. Pediatric Study Group of Clarithromycin

Clarithromycin (TE-031, A-56268), a new macrolide antibiotic agent, was evaluated bacteriologically and clinically for its efficacy and safety in pediatrics by a study group organized with pediatricians from all over the country. A summary of the results of the evaluation is as follows. 1. Absorption and excretion Pharmacokinetics of TE-031 was examined by single oral administration of 10% granules and 50 mg tablets at doses of 1, 5, 10 and 15 mg/kg. There were no significant differences between 10% granules and 50 mg tablets, and between administrations before and after meal. Peaks and half-life periods of blood level of TE-031 given once at doses of 5, 10 and 15 mg/kg (10% granules) before meal were 1.58, 4.37 and 3.79 micrograms/ml, and 2.53, 3.17 and 2.20 hours, respectively, and the urinary excretion in 6 hours after the administration were about 20-30%. 2. Antibacterial effects TE-031 was proved to have excellent antibacterial effect, i.e., inhibiting growth over 80% of strains of Streptococcus pneumoniae and Streptococcus pyogenes at 0.10 micrograms/ml, Branhamella catarrhalis at 0.39 micrograms/ml, and Campylobacter jejuni at 0.78 micrograms/ml. Against Staphylococcus aureus, TE-031 showed very similar activity spectrum to EM, and EM resistant strains were also resistant to TE-031. 3. Clinical results A total of 764 cases was studied. Clinical effects of TE-031 were evaluated in 717 cases out of the 764, excluding drop-outs and cases which did not meet specified protocols. Clinically, efficacies of TE-031 were "excellent" in 265 cases and "good" in 161 cases out of 453 cases of Group A in which causal agents were identified, with an efficacy rate of 94.0%, and out of 264 cases of Group B in which pathogens were not detected, clinical effects of TE-031 were "excellent" in 115 cases and "good" in 124 cases, with an efficacy rate of 90.5%. In terms of clinical effects of TE-031 classified by diseases when Group A and B were combined, efficacy rates were 91.6% for upper respiratory tract infection (217/237), 90.0% for bacterial pneumonia (108/120), 97.4% for Mycoplasma pneumonia (111/114), 100% for Chlamydia pneumonia (4/4), 85.0% for pertussis (34/40), 100% for scarlet fever (16/16), 83.9% for skin and soft tissue infection (26/31), and 98.9% for Campylobacter enteritis (87/88).(ABSTRACT TRUNCATED AT 400 WORDS)     

Locus of a virus neutralization epitope on the Japanese encephalitis virus envelope protein determined by use of long PCR-based region-specific random mutagenesis

We prepared recombinant Japanese encephalitis (JE) virus populations possessing random mutations at the envelope (E) protein region by a long PCR-based method. Neutralization-resistant mutants were selected from these populations by application of JE-specific virus neutralizing monoclonal antibody (mAb) 503, which possessed a 51,200-fold neutralization titer. We classified the mutants into three groups, each bearing two amino acid alterations at the E protein region: 52, Gln-Arg, and 136, Lys-Glu; 136, Lys-Glu, and 275, Ser-Pro; and 126, Ile-Thr, and 136, Lys-Glu, respectively. Three different genetically engineered variants, each bearing a single mutation, 126, Ile-Thr; 136, Lys-Glu; and 275, Ser-Pro, respectively, showed partial but not complete recovery of reactivity to mAb 503. Our results indicate that the amino acid substitutions at amino acid positions 52, 126, 136, and 275 altered the structure of the neutralization epitope for mAb 503 on the E protein. All these mutations were clustered at the junction of domains I and II of the E protein and it is likely that the epitope for mAb 503 is composed of at least E(0)-e, D(0)-a, and k strands of the E protein. We also demonstrated the efficacy of the long PCR-based recombinant virus technique as a useful tool for the creation of a variety of mutants bearing random mutations at targeted areas of the virus genome.     

Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease

Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-kappaB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-kappaB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.