Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission.

The target molecules of antibodies against falciparum malaria remain largely unknown. Recently we have identified multiple proteins as targets of immunity against Plasmodium falciparum using African serum samples. To investigate whether potential targets of clinical immunity differ with transmission intensity, we assessed immune responses in residents of low malaria transmission region in Thailand. Malaria asymptomatic volunteers (Asy: n=19) and symptomatic patients (Sym: n=21) were enrolled into the study. Serum immunoreactivity to 186 wheat germ cell-free system (WGCFS)-synthesized recombinant P. falciparum asexual-blood stage proteins were determined by AlphaScreen, and subsequently compared between the study groups. Forty proteins were determined as immunoreactive with antibody responses to 35 proteins being higher in Asy group than in Sym group. Among the 35 proteins, antibodies to MSP3, MSPDBL1, RH2b, and MSP7 were significantly higher in Asy than Sym (unadjusted p<0.005) suggesting these antigens may have a protective role in clinical malaria. MSP3 reactivity remained significantly different between Asy and Sym groups even after multiple comparison adjustments (adjusted p=0.033). Interestingly, while our two preceding studies using African sera were conducted differently (e.g., cross-sectional vs. longitudinal design, observed clinical manifestation vs. functional activity), those studies similarly identified MSP3 and MSPDBL1 as potential targets of protective immunity. This study further provides a strong rationale for the application of WGCFS-based immunoprofiling to malaria vaccine candidate and biomarker discovery even in low or reduced malaria transmission settings.

Role of notochord cells and sclerotome-derived cells in vertebral column development in fugu, Takifugu rubripes: histological and gene expression analyses.

Despite the common structure of vertebrates, the development of the vertebral column differs widely between teleosts and tetrapods in several respects, including the ossification of the centrum and the function of the notochord. In contrast to tetrapods, vertebral development in teleosts is not fully understood, particularly for large fish with highly ossified bones. We therefore examined the histology and gene expression profile of vertebral development in fugu, Takifugu rubripes, a model organism for genomic research. Ossification of the fugu centrum is carried out by outer osteoblasts expressing col1a1, col2a1, and sparc, and the growing centra completely divide the notochord into double cone-shaped segments that function as intercentral joints. In this process, the notochord basal cells produce a thick notochord sheath exhibiting Alcian-blue-reactive cartilaginous properties and composing the intercentral ligament in cooperation with the external ligament connective tissue. Synthesis of the matrix by the basal cells was ascertained by an in vitro test. Expression of twist2 indicates that this connective tissue is descended from the embryonic sclerotome. Notochord basal cells express sox9, ihhb, shh, and col2a1a, suggesting that the signaling system involved in chondrocyte proliferation and matrix production also functions in notochord cells for notochord sheath formation. We further found that the notochord expression of both ntla and shh is maintained in the fugu vertebral column, whereas it is turned off after embryogenesis in zebrafish. Thus, our results demonstrate that, in contrast to zebrafish, a dynamic morphogenesis and molecular network continues to function in fugu until the establishment of the adult vertebral column.

Evaluation of a simple phenotypic method for the detection of carbapenemase-producing Enterobacteriaceae.

We investigated the performance of a phenotypic test, the Carbapenemase Detection Set (MAST-CDS), for the identification of carbapenemase-producing Enterobacteriaceae. Our results indicated that MAST-CDS is rapid, easily performed, simple to interpret, and highly sensitive for the identification of carbapenemase producers, particularly imipenemase producers.

Genetic and biological features of catheter-associated Malassezia furfur from hospitalized adults.

Malassezia furfur, an etiological agent of catheter-associated fungemia, requires long-chain fatty acids for in vitro growth. We examined the applicability of rDNA sequence analysis, autoaggregation testing in liquid culture, utilization of parenteral lipid emulsions, and phospholipase activity for discrimination of catheter-associated M. furfur strains. The rDNA sequence types of catheter-associated M. furfur strains were distinct from those of other isolates. All M. furfur isolates recovered from blood culture bottles and the tips of catheters from patients receiving fat emulsion therapy were type I-3. Only M. furfur isolate GIFU 01 from a blood culture bottle showed no autoaggregation in liquid culture. All strains of M. furfur examined grew well on Sabouraud's dextrose agar supplemented with Intralipid lipid emulsion as compared to individual Tweens (20, 40, 60, 80) and Cremophor EL. A high percentage of type I-3 M. furfur strains (80.0%) showed very high phospholipase activity compared to type I-1 and I-4 strains obtained from healthy skin of the same subjects or healthy control subjects (20.0% and 0.0%, respectively). The blood culture bottle isolate GIFU 01 showed very high lipolytic enzymes activity for Intralipid but no phospholipase activity. These results suggest that particular factors, such as non-autoaggregation and very high lipolytic enzyme activity for parenteral lipid emulsions, play important roles in the growth and pathogenicity of Malassezia-related sepsis.

[A study of culture-based easy identification system for Malassezia].

Most species of this genus are lipid-dependent yeasts, which colonize the seborrheic part of the skin, and they have been reported to be associated with pityriasis versicolor, Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis. Malassezia have been re-classified into 7 species based on molecular biological analysis of nuclear ribosomal DNA/RNA and new Malassezia species were reported. As members of the genus Malassezia share similar morphological and biochemical characteristics, it was thought to be difficult to differentiate between them based on phenotypic features. While molecular biological techniques are the most reliable methods for identification of Malassezia, they are not available in most clinical laboratories. We studied ( i ) development of an efficient isolation media and culture based easy identification system, ( ii ) the incidence of atypical biochemical features in Malassezia species and propose a culture-based easy identification system for clinically important Malassezia species, M. globosa, M. restricta, and M. furfur.

[Etiological bacterial level in enterohemorrhagic Escherichia coli infection feces and chromogenic culture medium CHROMagar STEC usefulness].

We report a case of enterohemorrhagic Escherichia coli (EHEC) infection in which EHEC was not detected by culture on DHL agar medium. The proportion of EHEC bacterial count to enterobacterial count in feces was 1.7%, and the detection probability by 5-colony angling was low (8.1%). The probability of angling detection using CHROMagar STEC, a chromogenic medium for detecting EHEC, was high (100%). An additional and collection test was done using E. coli bacterial solutions to which two main sera groups--O157 and O26 were added. The maximum detectable level in the bacterial solution with O157 was 10(3)-10(4) CFU/mL in DHL and 10(2) CFU/mL in CHROMagar STEC. Bacterial solution levels with O26 were 10(3) CFU/mL in DHL and 10(2) CFU/mL in CHROMagar STEC. Assuming that the EHEC bacterial amount in feces of those with EHEC infection is low, we speculated that CHROMagar STEC may be useful as on EHEC screening medium.

Human external ear canal as the specific reservoir of Malassezia slooffiae.

The incidence of Malassezia species recovered from the external ear canal was characterized using culture medium optimized for Malassezia spp., CHROMagar Malassezia. The results of this study indicated that in healthy individuals M. slooffiae was the dominant Malassezia species followed by M. restricta.

[Clostridium difficile TOX A/B II TEST evaluation].

We compared the performance of two commercial toxin detection kits, C. difficile toxin A/B (C. difficile TOX A/B II test; TOX A/B II) and C. difficile toxin A (Uniquick), for (i) detection using highly purified toxin A solution; (ii) cross-reactivity using culture supernatants of toxin A-positive and B-positive C. difficile, toxin A-negative and B-positive C. difficile, and toxin A-negative and B-negative C. difficile strains and other bacteria; and (iii) sensitivity and specificity using clinical specimens. Results indicated that TOX A/B II detected toxin A at concentrations of 0.35 ng/mL and Uniquick at concentrations of 0.7 ng/mL. Uniquick performance was specific for detecting toxin A alone, while TOX A/B II detected toxin A/B specifically. Kit performance was then evaluated using 99 fecal specimens--43 specimens from patients with toxin B-positive C. difficile and 56 from those without. Sensitivity of TOX A/B II vs Uniquick was 95.3% vs 76.7%, specificity 98.2% vs 98.2%, positive predictive 97.6% vs 97.1%, and negative predictive value 96.5% vs 84.6%. Findings thus indicate that TOX A/B II is a more suitable diagnostic aid for CDAD than Uniquick because it correlates well with toxin B-positive C. difficile culture results. Stool culture for C. difficile is also required, however.

[A study of otitis externa associated with Malassezia].

Malassezia-positive smears can be recognized from otitis externa, however, there are few references in the literature to the relation between Malassezia and otitis externa. Therefore, the bacterial and clinical characteristics of 72 cases (63 patients) with otitis externa were investigated at the Department of Otorhinolaryngology, Takinomiya General Hospital to analyze this. Thirty-seven cases were bacterial otitis externa, 20 cases were fungal otitis externa, and 15 cases were etiological agents unknown in this study. The causative organisms in fungal otitis externa were the genera Aspergillus (10 cases), Malassezia (5) and Candida (5), respectively. We suspected that 5 cases were caused by Malassezia because Malassezia cell counts were greater than 10 per field (x 400), and a large number of Malassezia were isolated from all cases. In these cases, many squamous epithelial cells were observed by direct examination, and cells from the middle or basal layer of the ear canal were also recognized in three cases. Therefore, accelerated turnover of epidermal cells of the ear canal was suggested. The main symptoms were itching and fullness in the ear, with observations of redness and erosion in objective deterioration, and we felt that these conditions were similar to seborrheic dermatitis (SD). In addition, these five cases were confirmed as fungus-related otitis externa by their improvement with antifungal agents.

Comparative study of ability for growth support and species differentiation by colony features on commercial chromogenic agar, Pourmedia Vi Candida and CHROMagar Candida.

Ability for growth support and species differentiation by colony features were compared on two commercial chromogenic agars, CHROMagar Candida and newly developed Pourmedia Vi Candida. Eleven strains (ten species) of standard strains and twenty-four isolates (five species) of clinical strains were tested. All isolates were grown on both agar plates in 48 hours at 35 degrees C, however, several species had not matured in 22 hours. Color of the colonies for each strain were stable on both agars. The results show that Pourmedia Vi Candida is equivalent to CHROMagar Candida in its ability to differentiate species as a primary culture plate.

Revised culture-based system for identification of Malassezia species.

Forty-six strains of Malassezia spp. with atypical biochemical features were isolated from 366 fresh clinical isolates from human subjects and dogs. Isolates obtained in this study included 2 (4.7%) lipid-dependent M. pachydermatis isolates; 1 (2.4%) precipitate-producing and 6 (14.6%) non-polyethoxylated castor oil (Cremophor EL)-assimilating M. furfur isolates; and 37 (34.3%) M. slooffiae isolates that were esculin hydrolyzing, 17 (15.7%) that were non-tolerant of growth at 40 degrees C, and 2 (1.9%) that assimilated polyethoxylated castor oil. Although their colony morphologies and sizes were characteristic on CHROMagar Malassezia medium (CHROM), all strains of M. furfur developed large pale pink and wrinkled colonies, and all strains of M. slooffiae developed small (<1 mm) pale pink colonies on CHROM. These atypical strains were distinguishable by the appearance of their colonies grown on CHROM. Three clinically important Malassezia species, M. globosa, M. restricta, and M. furfur, were correctly identified by their biochemical characteristics and colony morphologies. The results presented here indicate that our proposed identification system will be useful as a routine tool for the identification of clinically important Malassezia species in clinical laboratories.

Development of safety culture tube for molds and proposed procedure for collecting conidia or fixing strains to control fungal infection and allergy.

The conidia of filamentous fungi can be easily blown into the air and tend to be contaminants in the laboratory environment. We developed a new "safety culture tube for fungi" to prevent biohazards and a procedure for collecting conidia for passage or fixing strains was proposed.

Tween 40-based precipitate production observed on modified chromogenic agar and development of biological identification kit for Malassezia species.

We developed a simple identification kit for nine species of Malassezia (M. furfur, M. slooffiae, M. sympodialis, M. restricta, M. obtusa, M. globosa, M. pachydermatis, M. dermatis, and M. japonica) based on their biological features. This method utilizes Tween 40-based precipitate production on modified chromogenic agar (CHROMagar) Malassezia medium, growth on specific agars (Sabouraud's dextrose agar, Cremophor EL agar, Tween 60-esculin agar), and catalase reactions. This identification kit was verified with 11 type and reference strains of nine Malassezia species. An additional 26 clinical isolates were also successfully identified using the kit and the results were confirmed by molecular biological analysis.

Vital growth factors of Malassezia species on modified CHROMagar Candida.

A comparison of several media, i.e., potato dextrose agar with olive oil (Oil-PDA), modified Dixon agar (mDIX) and variations of Leeming and Notman agar (LNA) for the isolation and growth of Malassezia and Candida species was examined. Since LNA supported the highest growth of Malassezia species its key components, i.e., ox bile, glycerol monostearate, glycerol and Tween 60, were added to CHROMagar Candida. All 7 species of Malassezia grew well on this modified medium (LN-CHROM) after incubation for 4 days at 30 degrees C and development was equal to that observed on LNA. Colonies on LN-CHROM were smooth and from pink to dark purple in color. Furthermore, the use of LN-CHROM did not alter the colony characteristics of Candida species as compared to that found on CHROMagar Candida. The results of the present investigation indicate that the use of LN-CHROM would make possible the simultaneous isolation and identification of Malassezia and Candida species.

[Evaluation of a new chromogenic medium for isolation of MRSA].

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections, and accurate methods to detect such strains are needed. We tested the susceptibility of 3 kinds of MRSA isolation medium, MRSA Screen Agar, Oxacillin Resistance Screening Agar and CHROMagar MRSA. Both sensitivity and specificity of CHROMagar MRSA were 100%. The sensitivity and specificity of both MRSA Screen Agar and Oxacillin Resistance Screening Agar were 100% and 91.5%, respectively. It is suggested that CHROMagar MRSA is a useful medium to detect MRSA including mecA positive and oxacillin susceptible strains.