Koichi Makimura

Molecular epidemiology of dermatophytosis in Tehran, Iran, a clinical and microbial survey

In the framework of a survey on dermatophytoses, 14,619 clinical specimens taken from outpatients with symptoms suggestive of tinea and referred to a Medical Mycology laboratory in Tehran, Iran, were analyzed by direct microscopy and culture. In total, 777 dermatophyte strains recovered in culture were randomly identified by a formerly established RFLP analysis method based on the rDNA ITS regions. For confirmation of species identification, 160 isolates representing the likely entire species spectrum were subjected to ITS-sequencing. Infection was confirmed in 5,175 collected samples (35.4%) by direct microscopy and/or culture. Tinea pedis was the most prevalent type of infection (43.4%), followed by tinea unguium (21.3%), tinea cruris (20.7%), tinea corporis (9.4%), tinea manuum (4.2%), tinea capitis (0.8%) and tinea faciei (0.2%). Trichophyton interdigitale was the most common isolate (40.5%) followed by T. rubrum (34.75%), Epidermophyton floccosum (15.6%), Microsporum canis (3.9%), T. tonsurans (3.5 %) and M. gypseum (0.5%). Other species included M. ferrugineum, T. erinacei, T. violaceum, T. schoenleinii, and a very rare species T. eriotrephon (each one 0.25%). The two strains of T. eriotrephon isolated from tinea manuum and tinea faciei are the second and third reported cases worldwide. Application of DNA-based methods is an important aid in monitoring trends in dermatophytosis in the community.

The Definitive Diagnostic Process and Successful Treatment for ABPM Caused by Schizophyllum commune: A Report of Two Cases

BACKGROUNDnAlthough mucoid impaction of the bronchi (MIB) is a well-known manifestation in allergic bronchopulmonary mycosis (ABPM), when unknown samples or plural eumycetes are cultured from bronchial materials, several problems are encountered which can affect the definitive diagnostic process or successful treatment.nnnCASE SUMMARYnThe definitive diagnostic process of two patients [a 58-(Case 1) and a 70-(Case 2) year-old female] with MIB was: 1) to identify the existence of any allergic respiratory disorder, 2) to detect the fungi obtained from bronchial materials, with use of the 28S rDNA sequencing and analysis, 3) to investigate whether the detected fungus was a probable etiologic antigen, and 4) to make the final diagnosis based on the results of the inhalation examinations using the antigenic solution of the fungi. As a treatment strategy, bronchial toilet and low dose itraconazole therapy were planned according to the clinical manifestations of each patient.nnnDISCUSSIONnThe two patients with MIB were successfully diagnosed as ABPM caused by Schizophyllum commune (Sc-ABPM) accompanied with hyperattenuating mucoid impaction. The reliability of some allergological makers as a substitution for the bronchoprovocation test should be clarified in near future. Clinical manifestations demonstrated in our cases suggested that the allergic reaction such as eosinophilic bronchoalveolitis spreading around the mucus plug was a primary lesion underlying the Sc-ABPM. The success of the treatment for Sc-ABPM will be achieved by the strategy targeting to fundamental condition and by the control of the disease recurrence by means of effective environmental management.

Rapid real-time diagnostic PCR for Trichophyton rubrum and Trichophyton mentagrophytes in patients with tinea unguium and tinea pedis using specific fluorescent probes

BACKGROUNDnTrichophyton rubrum and Trichophyton mentagrophytes human-type (synonym, Trichophyton interdigitale (anthropophilic)) are major causative pathogens of tinea unguium. For suitable diagnosis and treatment, rapid and accurate identification of etiologic agents in clinical samples using reliable molecular based method is required.nnnOBJECTIVEnFor identification of organisms causing tinea unguium, we developed a new real-time polymerase chain reaction (PCR) with a pan-fungal primer set and probe, as well as specific primer sets and probes for T. rubrum and T. mentagrophytes human-type.nnnMETHODSnWe designed two sets of primers from the internal transcribed spacer 1 (ITS1) region of fungal ribosomal DNA (rDNA) and three quadruple fluorescent probes, one for detection wide range pathogenic fungi and two for classification of T. rubrum and T. mentagrophytes by specific binding to different sites in the ITS1 region. We investigated the specificity of these primer sets and probes using fungal genomic DNA, and also examined 42 clinical specimens with our real-time PCR.nnnRESULTSnThe primers and probes specifically detected T. rubrum, T. mentagrophytes, and a wide range of pathogenic fungi. The causative pathogens were identified in 42 nail and skin samples from 32 patients. The total time required for identification of fungal species in each clinical specimen was about 3h. The copy number of each fungal DNA in the clinical specimens was estimated from the intensity of fluorescence simultaneously.nnnCONCLUSIONnThis PCR system is one of the most rapid and sensitive methods available for diagnosing dermatophytosis, including tinea unguium and tinea pedis.

Discrimination of Trichophyton tonsurans and Trichophyton equinum by PCR-RFLP and by β -tubulin and Translation Elongation Factor 1- α sequencing

Trichophyton tonsurans and T. equinum are two closely related sister species of dermatophytes, but differ in their preferred hosts, i.e., humans or horses, respectively. Routine procedures for their identification depend on studies of their phenotypic, physiological and biochemical characteristics, which are laborious and may yield ambiguous results. Molecular methods using rDNA ITS also had been judged to be insufficiently discriminatory. In the present study two genetic markers were sequenced in addition to the ITS region, i.e., partial β-tubulin (BT2) and translation elongation factor 1-α (TEF1). The TEF1 locus revealed a consistent differences in a 13 bp indel and an additional SNP between the two species, along with a single base substitution in BT2 and one ITS1, enabling unambiguous distinction of the two taxa. RFLP targeting the ITS region was evaluated as a potential tool for routine screening of suspected isolates of T. tonsurans and T. equinum.

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 Sabourauds 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.

Characterization of Malassezia microbiota in the human external auditory canal and on the sole of the foot.

Of the fungal skin microbiota, the lipophilic yeast genus Malassezia predominates at all body sites. Of the members of this genus, M. globosa, M. restricta, and M. sympodialis are the most common on the face, limbs, and trunk. In the present study, the Malassezia microbiotas in the external auditory canal and on the sole of the foot were characterized. M. slooffiae was the most common species in both the external auditory canal and on the sole of the foot, followed by M. restricta. Principal component analysis further revealed that the Malassezia microbiota in the external auditory canal and on the sole of the foot constitute a different cluster from those on the scalp and cheek and in the nasal cavity. Additionally, five new Malassezia phylotypes were detected on the sole of the foot and in the external auditory canal. Our results suggest that a distinctive Malassezia microbiota is present in the external auditory canal and on the sole of the foot, although the clinical significance of this finding remains unknown.

Development of a Tightly Regulatable Copper-Mediated Gene Switch System in Dermatophytes

ABSTRACT Targeted gene deletion is now available for molecular genetic research of dermatophytes, and the physiological roles of several genes have been elucidated. However, this method cannot be applied to essential genes, which can be potential drug targets. To overcome this limitation, we have developed a conditional gene knockdown system using a copper-responsive promoter. The promoter sequence of the copper transporter gene CTR4 (P CTR4 ) and that of the copper efflux pump gene CRP1 (P CRP1 ) derived from Trichophyton rubrum were examined for their response to copper in Arthroderma vanbreuseghemii. P CTR4 was demonstrated to repress expression of a reporter gene in the presence of copper, while the activity of P CRP1 was induced by addition of copper. Importantly, P CTR4 regulated the gene expression more tightly. Furthermore, when P CTR4 was applied to regulate the expression of the endogenous genes ERG1 and TRP5, their conditional mutants exhibited decreased growth activity under the repressive conditions. These results suggest that the P CTR4 -based gene regulation system represents a powerful tool for identification and characterization of a broad range of genes, including essential genes, in dermatophytes.

Molecular characterization of fungal populations on the tongue dorsum of institutionalized elderly adults

OBJECTIVESnTo characterize the global composition of oral fungal populations in frail elderly adults and to investigate the relationship with their health status.nnnMATERIALS AND METHODSnWe investigated the fungal populations on the tongue dorsum in 291 institutionalized elderly adults by molecular PCR-based techniques using internal transcribed spacer regions of nuclear ribosomal DNA.nnnRESULTSnQuantitative PCR analysis showed that fungi were present on the tongue dorsum of 128 subjects at ≥10(4) u2003CFU per sample, and 35 of them exceeded 10(5) u2003CFU per sample. Length heterogeneity-PCR analysis and nucleotide sequence determinations showed that Candida albicans was most frequently detected in those subjects with fungi at ≥10(4) u2003CFU per sample (105 subjects), followed by Candida dubliniensis (78), Malassezia restricta (57), and Candida tropicalis (45). Statistical analysis revealed that those subjects with ≥10(5) u2003CFU of fungi other than C. albicans per sample had an increased risk of fever (≥7 febrile days per 12u2003months) compared with subjects with <10(5) u2003CFU per sample, after adjustment for other fever-associated confounding factors.nnnCONCLUSIONSnThese data demonstrate that the oral cavity of the elderly is inhabited by a diverse array of fungi not limited to typical Candida species and they suggest that the diversity in distribution is associated with health status.

Phenotypic characterization of Aspergillus niger and Candida albicans grown under simulated microgravity using a three‐dimensional clinostat

The living and working environments of spacecraft become progressively contaminated by a number of microorganisms. A large number of microorganisms, including pathogenic microorganisms, some of which are fungi, have been found in the cabins of space stations. However, it is not known how the characteristics of microorganisms change in the space environment. To predict how a microgravity environment might affect fungi, and thus how their characteristics could change on board spacecraft, strains of the pathogenic fungi Aspergillus niger and Candida albicans were subjected to on‐ground tests in a simulated microgravity environment produced by a three‐dimensional (3D) clinostat. These fungi were incubated and cultured in a 3D clinostat in a simulated microgravity environment. No positive or negative differences in morphology, asexual reproductive capability, or susceptibility to antifungal agents were observed in cultures grown under simulated microgravity compared to those grown in normal earth gravity (1 G). These results strongly suggest that a microgravity environment, such as that on board spacecraft, allows growth of potentially pathogenic fungi that can contaminate the living environment for astronauts in spacecraft in the same way as they contaminate residential areas on earth. They also suggest that these organisms pose a similar risk of opportunistic infections or allergies in astronauts as they do in people with compromised immunity on the ground and that treatment of fungal infections in space could be the same as on earth.

Role of fungal colonization for sensitization in asthma

Role of fungal colonization for sensitization in asthma H. Ogawa, M. Fujimura, Y. Takeuchi and K. Makimura Division of Pulmonary Medicine, Ishikawa-ken Saiseikai Kanazawa Hospital, Kanazawa, Japan, Respiratory Medicine, National Hospital Organization, Nanao National Hospital, Nano, Japan, Division of Respiratory Medicine and Clinical Allergy, Fujita Health University, Toyoake, Japan and Department of Molecular Biology and Gene Diagnosis, Institute of Medical Mycology and Genome Research Center, Graduate School of Medical Science, Teikyo University, Hachioji, Japan