Ayako Sano

An improved culture medium for detecting live yeast phase cells of Paracoccidioides brasiliensis

The plating efficiency of standard mycological media such as brain heart infusion (BHI) agar is poor for Paracoccidioides brasiliensis. We prepared a water-extract of yeast phase cells of P. brasiliensis and examined it for growth-enhancing activity for the fungus. The water-extract, when added to BHI agar to a concentration of 5%, improved the plating efficiency of the medium for the fungus to some extent, but the degree of improvement was considerably varied among P. brasiliensis isolates. By contrast, when the water-extract was added in combination with horse serum (4%), the plating efficiency was highly improved (to 94-99%) for all the P. brasiliensis isolates employed. The growth-enhancing factor(s) in the water-extract was heat-stable and heating at 120 degrees C for 15 min had little, if any, effect on growth-enhancing activity.

Novel Approach to Designing Primers for Identification and Distinction of the Human Pathogenic Fungi Coccidioides immitis and Coccidioides posadasii by PCR Amplification

ABSTRACT We developed a pair of primers that specifically identifies Coccidioides species, etiologic agents of the human fungal disease coccidioidomycosis. These primers could be used for distinguishing Coccidioides immitis and Coccidioides posadasii by simply comparing the amplicon sizes on an agarose gel.

A comparative study of four different staining methods for estimation of live yeast form cells ofParacoccidioides brasiliensis

A comparative study of four different staining methods for estimation of live yeast form cells ofParacoccidioides brasiliensis was carried out. The staining methods used were fluorescent staining, vital dye exclusion tests with erythrosin B and by Janus green and lactophenol cotton blue staining. Colony forming units (cfu) of the yeast form of eightP. brasiliensis isolates on brain heart infusion agar (BHIA) supplemented with 4% horse serum plus 5%P. brasiliensis cell extract (BHIA + HS + EXT) were examined for reliability of staining in determining the number of live fungal units in eight different isolates. Cfu on BHIA + HS + EXT plates showed an excellent plating efficiency over 96% in all isolates tested. The percentage of the live cells indicated by fluorescent staining (FL) or vital dye exclusion test with erythrosin B (EB) or Janus green (JG-1) was lower than that of cfu. By contrast, the percentage due to modified dye exclusion test with Janus green (JG-2) and that due to lactophenol cotton blue staining (LPCB) showed a close correration to that of cfu. Our results indicate that the modified dye exclusion test with Janus green and lactophenol cotton blue staining are useful for estimating cell viability of yeast form cells ofP. brasiliensis.

Fungistatic and fungicidal activities of murine polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis

Recently, a novel culture medium for detecting live yeast cells of Paracoccidioides brasiliensis was developed by Kurita et al. Using this culture medium, murine peritoneal polymorphonuclear leucocytes (PMN) were examined for fungistatic and fungicidal activities against P. brasiliensis yeast cells. The magnitude of the antifungal effect of PMN varied depending upon the fungal isolates used. PMN exhibited a killing effect on P. brasiliensis isolate Bt-4 in 2 h of coculture. In contrast, the other three fungal isolates employed were resistant to killing by PMN. However, PMN considerably suppressed the growth of isolates Tatu and Recife in a long-term assay (approximately 72 h). The growth of isolate Bt-9 was also suppressed by PMN during the first 24 h, but was found to be considerably promoted at 72 h of coculture. Interferon-gamma (IFN-gamma), but not tumour-necrosis factor-alpha, significantly augmented the antifungal activity of PMN. IFN-gamma-treated PMN exhibited a killing effect on isolates Tatu, Recife and Bt-9 after 24 h of coculture, and showed an enhanced killing effect on isolate Bt-4. Contact between PMN and fungal cells was required for PMN to exert the antifungal effect. Our results suggest that PMN, whether activated with cytokines or not, might play a critical role in host resistance in early infection with this fungus by buying time for development of more effective immunologic responses.

Diagnosis of histoplasmosis by detection of the internal transcribed spacer region of fungal rRNA gene from a paraffin-embedded skin sample from a dog in Japan.

The lesions of histoplasmosis in dogs in Japan differ from those in dogs in North America. Affected dogs in Japan have had multiple granulomatous or ulcerated foci in skin or gingiva and have not had pulmonary or gastrointestinal lesions. The present report introduces a polymerase chain reaction (PCR) diagnosis of canine histoplasmosis and the characteristic of disease in Japan. The surgically removed skin ulcerate samples from a 5-years-old female Shiba-inu native to Japan without traveling out of the country were evaluated. Tissue samples had many yeast-like organisms in the macrophages. DNA was extracted from paraffin-embedded tissue samples. A nested PCR technique was applied. The detected sequence of the internal transcribed spacer of ribosomal RNA gene had 99.7% in homology with Ajellomyces capsulatus (the teleomorph of Histoplasma capsulatum). Clinical manifestations, historical background of equine epizootic lymphangitis in Japan, and a human autochthonous case of histoplasmosis farciminosi indicated that this dog might have been infected with H. capsulatum var. farciminosum as a heteroecism.

Studies on the relationship between the estrous cycle of BALB/c mice and their resistance to Paracoccidioides brasiliensis infection

A relationship between the estrous cycle and non-specific host resistance to Paracoccidioides brasiliensis yeast cells was examined by using both sexes of adult BALB/c mice. They were divided into 6 groups, including a male group and females at proestrus, estrus, metestrus-I, metestrus-II and diestrus. The mice received yeast cells through three different inoculation routes; intravenous, intraperitoneal and intratracheal. In all of the inoculation routes, the clearance of the yeast cells was influenced by the estrous cycle. The female mice at estrus, which might have high blood estrogen levels, showed a marked clearance of the yeast cells from the blood, peritoneal cavity and lungs. These results suggested that non-specific host resistance to the yeast cells was enhanced by estrogen. All female groups inoculated by the three routes showed higher clearance of the yeast cells than the male group.

Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo Dasypus novemcinctus )

We studied three different isolates of Paracoccidioides brasiliensis obtained from the mesenteric lymph node (D3LY1), the spleen (D3S1) and the liver (D3LIV1) of the same armadillo ( Dasypus novemcinctus ).Pulmonal inflammatory area was evaluated by intravenous inoculation of 106 yeast cells of each isolates in young, male, ddY mice. Moreover, the partial sequence of GP43kDa gene of P. brasiliensis was analyzed. The lung inflammatory area was greater in animals inoculated with isolate D3S1. The partial sequence of GP43kDa gene indicated that isolate D3S1 is different from isolates D3LY1 and D3LIV1. This study suggested that the same armadillo might be susceptible to multiple P. brasiliensis isolates simultaneously.

Detection of Paracoccidioides brasiliensis gp43 Gene in Sputa by Loop-Mediated Isothermal Amplification Method

The fungus Paracoccidioides brasiliensis is the pathogen of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The loop‐mediated isothermal amplification method (LAMP) was used in this study to detect the presence of P. brasiliensis in sputa samples from patients with chronic PCM, suspected PCM, and a negative control. The target P. brasiliensis gp43 gene was amplified in less than 4 hr in 11 of 18 sputa samples tested. The LAMPmethod had the advantage of speed and simplicity compared with the classic diagnostic methods such as the histopathological test or biological material culture and did not require sophisticated technical apparatus. It would be an important aid in cases where immediate treatment would mean patient survival, especially in immune‐suppressed patients. J. Clin. Lab. Anal. 23:139–143 2009.

Molecular epidemiology of canine histoplasmosis in Japan

A recent case of canine histoplasmosis, the first confirmed case of disseminated infection accompanied by carcinoma in Japan, was diagnosed by clinical characteristics, histopathological examination, chest radiographs, ocular fundoscopy and molecular biological data. The clinical manifestations were not limited to cutaneous symptoms but were referable to disseminated infection, similar to human autochthonous cases. The partial sequences of the internal transcribed spacer (ITS1/2) regions of the ribosomal DNA genes of this and other Japanese canine histoplasmosis strains were 99-100% identical to the sequence AB211551 derived from a human isolate in Thailand, and showed a close relationship to the sequences derived from Japanese autochthonous systemic and cutaneous human cases. The phylogenetic analysis of 97 sequences of the ITS1/2 region disclosed six genotypes. The genotypes derived from Japanese autochthonous human and dog cases belonged to the cluster consisting of Histoplasma capsulatum var. capsulatum and H. capsulatum var. farciminosum sequences, indicating that these varieties might cause not only cutaneous but also systemic histoplasmosis, regardless of their host species. The current status of the 3 varieties of Histoplasma capsulatum according to the host species remains a subject of further investigation.

An atypical Paracoccidioides brasiliensis clinical isolate based on multiple gene analysis.

An atypical isolate of Paracoccidioides brasiliensis (IFM54648), recovered from the sputum of a Brazilian man, was not detected in immunodiffusion tests for paracoccidioidomycosis and in species-specific PCR for the major antigen 43-kDa glycoprotein coding gene (gp43). The mycological characteristics of the isolate were similar to those of a typical P. brasiliensis. A total of 8 genes were sequenced from IFM54648, and the sequences were compared between the new isolate and other reference isolates and database sequences. We analyzed fragments of the gene sequences that code for gp43, the internal transcribed spacer regions of ribosomal RNA, the D1/D2 domains of the large subunit ribosomal RNA, glucan synthase, chitin synthase, glyoxalase I mRNA, 70-kDa heat-shock protein mRNA and urease. The gene sequences were 98.9-100% identical between IFM54648 and Pb01 (another atypical isolate). When compared to the other typical isolates, the identities were generally lower than 98%. A phylogenetic tree constructed using gp43 sequences showed that IFM54648 clustered with Pb01 at a considerable distance from other isolates. Therefore, this isolate is likely related to Pb01, which has recently been shown to be genetically distinct from other isolates of this species.