Multilocus microsatellite typing for Cryptococcus neoformans var. grubii.

Fifteen randomly selected microsatellites (simple sequence repeats; SSRs), from the H99 Cryptococcus neoformans var. grubii (serotype A) genome, were sequenced, characterized and applied to sequence 87 clinical and environmental C. neoformans var. grubii isolates from 12 different countries based on Multilocus Microsatellite Typing (MLMT). Among the 15 SSR loci, three (designated CNG1, CNG2 and CNG3) were polymorphic, while the remaining 12 SSR loci showed no variations. The specific PCR primers of the polymorphic microsatellites, i.e., CNG1, CNG2 and CNG3, amplified those loci only from strains of C. neoformans (C. neoformans var. grubii, C. neoformans var. neoformans and the AD hybrid) but not from Cryptococcus gattii, suggesting a species-specific association. The three polymorphic microsatellites are useful markers for strain genotyping, population genetic analysis, epidemiological studies, and may be helpful for the diagnosis of cryptococcosis due to C. neoformans.

Molecular analysis and susceptibility profiling of Candida albicans isolates from immunocompromised patients in South India.

The genetic diversity and in vitro antifungal susceptibility profiles of 55 Candida albicans from immunocompromised patients were studied. PCR based analysis of the transposable intron in the 25S rDNA revealed 39 genotype A, 4 genotype B and 12 genotype C isolates. Serotype analysis categorized 52 isolates as serotype A and 3 as serotype B. All strains were susceptible to micafungin, 5-flucytosine and miconazole, whereas resistance against amphotericin B (3.6%), fluconazole (3.6%), itraconazole (7.3%) and voriconazole (5.5%) was observed. No association was seen between antifungal resistance and genotype/serotype status.

Marked renal damage in a child with hydronephrosis infected by Trichosporon asahii.

We describe a case of urinary infection caused by Trichosporon asahii, which drastically aggravated renal function in a left hydronephrotic kidney. Previous implantation of a nephrostomy tube and a ureteral J-stent and administration of broad-spectrum antibiotics seemed to have predisposed the non-granulocytopenic child to the urinary fungal infection.

The internal transcribed spacers and 5.8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans species complex.

Sequences of the internal transcribed spacer (ITS) region including the 5.8S rRNA gene delineated seven genotypes within the three varieties of Cryptococcus neoformans via specific combinations of eight nucleotide differences located at positions 10, 11, 15, 19, 108 (ITS1), 221 (5.8S), 298 and 346 (ITS2). The ITS types correlated to polymerase chain reaction fingerprint/random amplification of polymorphic DNA (RAPD) molecular types: with ITS type 1 (ATACTAGC)=C. neoformans var. grubii, molecular types VNI+VNII and the serotype A allele of the AD hybrid, VNIIIA; ITS type 2 (ATATAGGC)=the serotype D allele of the AD hybrid, VNIIIB, and C. neoformans var. neoformans, VNIV; and ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC)=VGI=RAPD type III, ITS type 4 (ACACTGAC)=VGII=RAPD type II, ITS type 5: (ACACTGGG)=VGIII=RAPD type I, ITS type 6 (ACACTGGC)=VGIV=RAPD type IV, all corresponding to C. neoformans var. gattii. Cloned sequences from serotype AD revealed that the hybrid serotype is diploid at the ITS1-5.8S-ITS2 locus carrying the ITS type 1 (ATACTAGC) and the ITS type 2 (ATATAGGC) alleles. ITS sequencing is a useful technique for genotyping the three C. neoformans varieties and for subtyping within C. neoformans var. gattii.

Immunomagnetic isolation of Cryptococcus neoformans by beads coated with anti-Cryptococcus serum.

Immunomagnetic separation (IMS) was utilized for the selective isolation of Cryptococcus neoformans from environmental sources, such as soils and pigeon droppings. Magnetic beads coated with anti-cryptococcal IgG (serotypes A and B) were used to isolate the fungus. In a modeled spiking experiment using C. neoformans serotype A strain and anti-serotype A antibody, the recovery rate of the cells was more than 47%. Specificity experiments using C. neoformans and Candida albicans showed that the beads, when coated with specific antibody for C. neoformans, were highly effective for the separation of C. neoformans strains from C. albicans (more than 97%). The IMS of serotype B cells with purified anti-serotype B antibody indicated a high specificity. When this IMS technique was applied to soils and pigeon droppings, C. neoformans cells were selectively isolated from 3 out of 8 samples, and C. neoformans DNAs were identified by PCR. Therefore C. neoformans cells were thus selectively isolated and the efficiency of the technique further confirmed.

Molecular epidemiology of invasive stomatitis due to Aspergillus flavus in patients with acute leukemia.

BACKGROUND: Invasive oral aspergillosis is a rare complication and only little information on the epidemiology of Aspergillus flavus infection is available. We present here the molecular analysis of the epidemiology of invasive stomatitis due to Aspergillus flavus in patients with acute leukemia. METHODS: During a 5-year period (1992-1996), six isolates of A. flavus were obtained from leukemic patients with invasive Aspergillus stomatitis. Random amplification of polymorphic DNA (RAPD) with three different PCR primers was carried out to investigate the DNA typing of the isolates. RESULTS: The molecular analysis using RAPD revealed that three isolates of A. flavus obtained in 1992 from three patients were of the same type, whereas each of the isolates from the other three patients had a distinct unique band, resulting in four groups of A. flavus. CONCLUSION: As the three patients with invasive oral aspergillosis detected in 1992 were infected by a single strain of A. flavus, the strain was suspected to have caused a nosocomial outbreak of invasive oral aspergillosis in the hematology unit.

Successful treatment of invasive stomatitis due to Exophiala dermatitidis in a patient with acute myeloid leukemia.

BACKGROUND: Although the most common orofacial fungal infection in immunocompromised patients is candidosis, infections caused by virulent molds, such as Aspergillus spp. and Furarium spp. are being recognized with increasing frequency. We report a case of oral Exophiala infection in a 39-year-old woman with acute myeloid leukemia. METHODS: Clinical records of the patient were reviewed and the following additional information was collected: histological and microbiological evidence; identification of the causative organism; in vitro antifungal susceptibility. RESULTS: The patient developed a necrotic ulcer surrounded by a violaceous rim in the gingiva during neutropenia. Exophiala dermatitidis was identified as the causative organism by histopathological examination and culture, and finally confirmed by sequencing of the ribosomal DNA internal transcribed space domain. In vitro, amphotericin B was found to show strong activity against the Exophiala isolate while itraconazole showed less activity. The patient was successfully treated with parenteral amphotericin B and oral itraconazole in combination with surgical removal of the fungi focus. CONCLUSION: Local excision with adequate antifungal agents can be used to treat immunocompromised patients with Exophiala stomatitis, based on early diagnosis.

Phylogenetic characterization of Histoplasma capsulatum strains based on ITS region sequences, including two new strains from Thai and Chinese patients in Japan.

Two strains of Histoplasma capsulatum var. capsulatum were isolated in Japan: one from a Thai AIDS patient and the other from a Chinese non-immunocompromised patient. The phylogenetical relationship among the two isolates and reference strains of H. capsulatum from other geographical populations was investigated. Random amplified polymorphic DNA (RAPD) analysis of the two H. capsulatum strains showed that they had RAPD band patterns similar to those of the reference Thai isolates and North American strains, although the patterns differed slightly from those of the reference strains. Phylogeny of thirty geographically diverse H. capsulatum isolates representing the three varieties, var. capsulatum, var. duboisii and var. farciminosum were evaluated using nucleotide sequences of the internal transcribed spacer (ITS) region (ITS1-5.8S rDNA -ITS2). We found that the ITS region contained sufficient information to resolve the phylogenetic relationship among the fungal isolates. An unrooted dendrogram constructed from the ITS sequences showed that thirty strains of H. capsulatum could be classified into eight geographic clades; Asia type (i), South America types A (ii) and B (iii), North American types 1 (iv) and 2 (v), H. duboisii types A (vi) and B (vii), and East Asia type (viii). Based on the ITS region sequence analysis, the two strains isolated from the Thai and Chinese patients in Japan were found to be distinct from Asia type (i) in which eight Thai, one Chinese, one English and one Indonesian isolate were included. Some extent of DNA polymorphism was observed between the North America type 1 isolates and the Thai and Chinese strains isolated in Japan. We believe that the Thai and Chinese isolates were unique and propose a new clade, East Asia type (viii) for the two strains. DNA sequence analysis of the ITS region provided useful information to understand the epidemiology and evolution of H. capsulatum.

First human case of nocardiosis caused by Nocardia pseudobrasiliensis in Japan.

Nocardia sp. IFM 0896, an actinomycete with biochemical characteristics that differed from Nocardia brasiliensis, was isolated from a 71-year-old Japanese man with a history of tuberculosis and cancer. Although the isolate was tentatively identified as N. brasiliensis, the morphological and physiological characteristics of strain IFM 0896 were different from those of N. brasiliensis IFM 0236T. The results of 16S rRNA gene sequence phylogenies and PCR-RFLP analysis of a heat shock protein revealed that Nocardia sp. IFM 0896 belongs to the species N. pseudobrasiliensis. This is the first clinical isolation report of N. pseudobrasiliensis in Japan.