Detection of circulating fungal DNA by polymerase chain reaction in a fatal case of Cunninghamella bertholletiae infection.

INTRODUCTION: Cunninghamella bertholletiae although rarely causing mucormycosis, is responsible for the highest mortality among mucormycetes. The diagnosis of mucormycosis is challenged by the absence of specific biomarkers. Herein, we report a fatal case of C. bertholletiae infection and detection of its DNA in the serum by polymerase chain reaction (PCR). PRESENTATION OF CASE: A 23-year-old male with refractory osteosarcoma was admitted with multiple lung metastases. He was on oral voriconazole prophylaxis after pulmonary aspergillosis. He suffered from fever during temporary neutropenia following chemotherapy and showed several neurological and respiratory symptoms. Despite liposomal-amphotericin B administration, the symptoms rapidly progressed, and he died five days after the onset of neurological symptoms.We retrospectively evaluated the filamentous fungus isolated after his death from gastric juices. Based on the sequence of the internal transcribed spacer (ITS) region we identified the fungal isolate as C. bertholletiae. A 146-bp portion of the D1/D2 region was quantified by quantitative-PCR using DNA extracted from the serum. C. bertholletiae DNA load in the serum was 18.0 copies/µL on the day of onset of neurological symptoms, with the highest (101.0 copies/µL) on the day of his death. DISCUSSION: Detection of circulating DNA of mucormycetes in the blood would greatly enhance the diagnosis of mucormycosis. Rapid diagnosis might alleviate mortality due to mucormycosis. CONCLUSION: The present case-report suggests that the quantification of C. bertholletiae DNA in the serum could be useful for the diagnosis and evaluation of mucormycosis pathogenesis in patients.

Usefulness of Gastric Aspirate Culture for Diagnosing Congenital Immunodeficiency in an Infant with Fungal Pneumonia Caused by Rasamsonia piperina.

Chronic granulomatous disease (CGD) is a type of primary immunodeficiency disease, which increases susceptibility to recurrent bacterial and fungal infections. Sputum and bronchoalveolar lavage fluid are often obtained using bronchoscopy from adult patients for pathogenic diagnosis, although this approach is much more invasive for infants. We report the case of a 2-month-old boy with CGD, in which gastric aspirate culture was used to diagnose fungal pneumonia. Rasamsonia piperina was isolated from the gastric aspirate, and the patient was successfully treated with micafungin based on the drug susceptibility test results for the fungal isolate. The acid tolerance test revealed that R. piperina could grow at pH 2, indicating high acid resistance. Although we can only report our experience with a single case, gastric aspirate culture may be a useful tool for detecting fungal respiratory pathogens in children with primary immunodeficiency. Detecting these pathogens may help improve outcomes, as early diagnosis and appropriate treatment are extremely important for immunocompromised patients with respiratory infections.

Sinusitis caused by Exserohilum rostratum after cord blood transplantation for myelodysplastic syndrome: A case report and literature review.

Invasive fungal disease is a serious infectious complication of allogeneic hematopoietic stem cell transplantation (HSCT). Exserohilum rostratum is a species causing phaeohyphomycosis, which rarely causes invasive disease in humans. We treated a case of sinusitis caused by E. rostratum after cord blood transplantation (CBT). A 60-year-old man with myelodysplastic syndrome, who had a medical history of an operation to correct deviation of the nasal septum, developed sinusitis caused by E. rostratum under prolonged profound neutropenia after a second CBT because of the graft rejection of the first transplantation. Liposomal amphotericin B improved the sinusitis. A literature review revealed nine reported cases of sinusitis caused by E. rostratum, including our case. Although five cases had severe neutropenia at onset (HSCT recipients, n = 2; aplastic anemia, n = 3), the remaining four had no preexisting immunosuppressive conditions. However, three of the four patients had preexisting nasal diseases with or without a history of surgery, as in our case. Excluding our case, the outcome was fatal in five neutropenic patients, whereas the four patients without neutropenia recovered. Although sinusitis caused by E. rostratum is rare, E. rostratum should be recognized as a possible pathogen causing sinusitis in highly immunosuppressed patients such as HSCT recipients. Preexisting nasal disease and/or nasal surgery could be risks for this infection.

Cryptococcus gattii genotype VGIIb infection in Japan.

This report describes a case of Cryptococcus gattii VGIIb infection of the pulmonary and central nervous systems in an immunocompetent Japanese man with a travel history, and it hypothesizes the place where he was infected with C. gattii using the genotype information.

Detection of Mucor velutinosus in a blood culture after autologous peripheral blood stem cell transplantation : a pediatric case report.

Filamentous fungi were detected in the blood culture of a one-year-old boy after autologous peripheral blood stem cell transplantation. The patient was suspected to have aspergillosis and received micafungin. Fungi were isolated on potato dextrose agar medium and incubated at 37℃ for 2-5 days. Grayish, cottony colonies formed. A slide culture showed a spherical sporangium at the tips of the sporangiophores. The fungus could have been a zygomycete. The zygomycete was isolated from three blood cultures. The antifungal drug was changed from micafungin to liposomal amphotericin B, which resulted in an improvement in the patient's symptoms. Growth was observed at 37℃, but not 42℃ in a growth temperature test. Gene sequence analysis identified the fungus as Mucor velutinosus. To the best of our knowledge, this is the first time M. velutinosus has been detected in Japan, and this case is very rare. Zygomycetes are known to be pathogens that cause fungal infections in immunodeficient patients such as those with leukemia. They are difficult to identify by culture and are identified at autopsy in many cases. Therefore, culture examinations should be performed for immunodeficient patients with the consideration of zygomycetes.

Antifungal susceptibility of Aspergillus fumigatus clinical isolates collected from various areas in Japan.

Azole resistance among clinical isolates of Aspergillus fumigatus is becoming a serious problem in Europe, but the status in Japan is not yet known in detail. The aim of this study was to determine the present status of azole resistance in A. fumigatus in Japan. We employed 171 clinical isolates of A. fumigatus sensu stricto collected from 1987 to 2008 at the Medical Mycology Research Center, Chiba University, Japan for azole resistance determination. Identification of all isolates were re-examined both from the aspect of morphology and molecular phylogeny. The antifungal susceptibility of these isolates was tested based on the CLSI M38-A2 broth microdilution method. In our collection, only 1 (0.6%) and 2 isolates (1.2%) showed elevated MIC to voriconazole and itraconazole, respectively. Our study disclosed that the frequency of azole resistance in A. fumigatus still remains low in this collection.

Exophiala dermatitidis pneumonia successfully treated with long-term itraconazole therapy.

Exophiala dermatitidis pneumonia is extremely rare. Here we report a case of E. dermatitidis pneumonia successfully treated with long-term itraconazole therapy. A 63-year-old woman without a remarkable medical history developed a dry and chest pain. Chest radiographs revealed consolidation in the middle lobe of the lung. Cytologic examination by bronchoscopy showed filamentous fungi and E. dermatitidis was detected in the bronchoalveolar lavage fluid. After 5 months of itraconazole therapy, her symptoms improved and the area of consolidation diminished. Two weeks after discontinuing the itraconazole therapy, the area of consolidation reappeared. Itraconazole therapy was restarted and continued for 7 months. The abnormal shadow observed on the chest X-ray gradually diminished. Over a 27-month follow-up with periodic examination, there was no relapse and the patient had a favorable clinical course.

Fatal fungemia with Scedosporium prolificans in a patient with acute myeloid leukemia.

Scedosporium prolificans (S. prolificans) is a type of mold, which rarely affects immunocompromised people. We treated a 71-year-old woman with acute myeloid leukemia (AML-M5a) with low-dose cytarabine, acralubicin, and filgrastim as the induction therapy. On day 7 after the initiation of chemotherapy, she became febrile and agranulocytic, and developed anal pain ; therefore, we discontinued the chemotherapy on day 8. Broad-spectrum antibiotics, micafungin, and then liposomal amphotericin B were ineffective. The serum concentration of ß-D-glucan was 525 pg/mL. She died of multiple organ failure on day 17. S. prolificans was detected from the blood culture on day 13. Physicians should consider Scedosporium spp. infection when principal antifungal agents are ineffective and fungal infection is strongly suspected.

Scedosporium aurantiacum brain abscess after near-drowning in a survivor of a tsunami in Japan.

Many victims of the tsunami that occurred following the Great East Japan Earthquake on March 11, 2011 developed systemic disorders owing to aspiration pneumonia. Herein, we report a case of tsunami lung wherein Scedosporium aurantiacum was detected in the respiratory tract. A magnetic resonance image of the patient's head confirmed multiple brain abscesses and lateral right ventricle enlargement. In this case report, we describe a potential refractory multidrug-resistant infection following a tsunami disaster.

Multiple Scedosporium apiospermum abscesses in a woman survivor of a tsunami in northeastern Japan: a case report.

INTRODUCTION: Scedosporium apiospermum is increasingly recognized as a cause of localized and disseminated mycotic infections in near-drowning victims. CASE PRESENTATION: We report the case of a 59-year-old Japanese woman who was a survivor of a tsunami in northeastern Japan and who had lung and brain abscesses caused by S. apiospermum. Initially, an aspergillus infection was suspected, so she was treated with micafungin. However, computed tomography scans of her chest revealed lung abscesses, and magnetic resonance images demonstrated multiple abscesses in her brain. S. apiospermum was cultured from her bronchoalveolar lavage fluid, and antimycotic therapy with voriconazole was initiated. Since she developed an increase in the frequency of premature ventricular contractions, an adverse drug reaction to the voriconazole was suspected. She was started on a treatment of a combination of low-dose voriconazole and liposomal amphotericin B. After combination therapy, further computed tomography scans of the chest and magnetic resonance images of her brain showed a demarcation of abscesses. CONCLUSIONS: Voriconazole appeared to have a successful record in treating scedosporiosis after a near drowning but, owing to several adverse effects, may possibly not be recommended. Thus, a combination treatment of low-dose voriconazole and liposomal amphotericin B may be a safe and effective treatment for an S. apiospermum infection. Even though a diagnosis of scedosporiosis may be difficult, a fast and correct etiological diagnosis could improve the patient's chance of recovery in any case.

Ochroconis calidifluminalis, a sibling of the neurotropic pathogen O. gallopava, isolated from hot spring.

Two strains resembling the neurotropic fungus Ochroconis gallopava were isolated from hot spring river water (IFM 54738 and IFM 54739). The isolates showed optimal growth at 42 degrees C, while the maximum growth temperature was 49 degrees C, thus having temperature relationships similar to those of O. gallopava. Colonies were light olive green, with a color change to dark reddish brown after several passages, which was also observed in O. gallopava. Conidia were indistinguishable from those of O. gallopava. The antifungal susceptibility profile of the isolates was also similar to that of O. gallopava, except for a lower susceptibility to micafungin. The two isolates had 100% homologous rRNA genes including the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit. The gene fragments, as O. gallopava, could be amplified with species-specific rDNA primers, and loop-mediated isothermal amplification designed for O. gallopava yielded positive results in the two isolates. However, homologies with O. gallopava in ITS and D1/D2 regions were 79.2 and 95.9%, respectively, widely exceeding generally accepted species boundaries. These differences were corroborated in virulence tested in experimental infection. The two isolates did not kill a mouse even until 28 days. However, mortalities of four O. gallopava strains ranged from 40 to 100%. The new isolates mainly affected the kidneys; whereas O. gallopava had a strong preference for the brain. We therefore propose a new species, Ochroconis calidifluminalis, for the two isolates.

[Penicillium marneffei isolated from a Thai AIDS patient with fungemia].

Penicillium marneffei was isolated from three blood cultures of a Thai woman with AIDS and then identified as such. The patient, 41 a year-old female from northeast Thailand came to Japan 10 years ago and married a Japanese man. She was reportedly the third patient infected with this fungal species in Japan, and considered to be the first case from whom the causative fungus was successfully cultured, which led to the diagnosis of penicilliosis marneffei. The colony of the isolate, which was cultured on Sabouraud dextrose agar at 25-27 degrees C, was initially white and pannose, gradually turned in color from yellow to yellow-green, and diffused a deep red pigment into the medium. Conidial heads were divergent, and chains of conidia were formed from phialides. Colonies of the isolate, which was cultured on brain-heart infusion agar at 35 degrees C, had a grayish white, membranous yeast-like form with fine plicae and microscopically consisted of short hyphae. Furthermore, 560 bases of the internal transcribed spacer (ITS) region of the ribosomal RNA gene including the 5.8S region (ITS1-5.8S-ITS2) (DDBJ accession number AB298970) were sequenced and allowed an unequivocal species identification.

An intrafamilial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) in a Japanese zoo.

An intra-familial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) housed in a Japanese zoo was studied. The family consisted of an adult couple and two offspring (a male and a female). The porcupettes, born in Japan, showed severe hair loss while the parent animals, imported from the USA. (male) and Canada (female), showed mild symptoms or were asymptomatic. Morphologically identical Tricophyton spp. isolates were recovered within seven days from quills of all animals on chloramphenicol-supplemented potato dextrose agar plates incubated at 37 degrees C. Two representative colonies from each animal were identified as Arthroderma benhamiae Americano-European race based on mating type (+) and the internal transcribed spacer (ITS) 1-5.5S-ITS 2 region of the rRNA gene sequences (AB236404-AB236408). The present cases constituted the second isolation of dermatophytes from porcupines. There were two different ITS types, i.e., the predominant one isolated from all animals and a secondary one recovered from only the mother porcupine. The sequences have never been recorded in Japan or in the GenBank database to the best of our knowledge. In addition, they were located at a cluster involving the type strain and mating strains of A. benhamiae Americano-European race and its F1 progeny. In contrast, 28 rodents (eight species) and three insectivora (1 species) exhibited in the petting zoo were negative for any dermatophytes as determined by culture.

Pathogenicity of Ochroconis gallopava isolated from hot springs in Japan and a review of published reports.

Four strains of Ochroconis gallopava from 3 out of 15 Japanese hot springs were isolated. Colonies of the hot spring isolates were uniformly floccose and dark olive green on the surface and dark brown on their reverse side on potato dextrose agar (PDA) plates, however, they became felty, flat, and brownish-black, and produced a reddish-brown pigment after several times of subculture at room temperature. Shapes and sizes of conidia of the four strains were individual, while the D1/D2 domain of the large subunit ribosomal RNA gene sequences showed 99.7% identity in the GenBank database. The DNA pattern of the hot spring isolates amplified by species specific loop mediated isothermal amplification method were as the same pattern as that of a clinical isolate. The minimum inhibitory concentrations of antifungal agents to O. gallopava isolated from the hot springs were ranged from 0.5 to 1 microg/ml in amphotericin B, 1 to 16 microg/ml in flucytosine, 0.125 to 0.25 microg/ml in itraconazole, 1 to 4 microg/ml in miconazole, 16 to 64 microg/ml in flconazole and 0.03 to 0.5 microg/ml in micafungin. The isolates had fatal outcome in experimentally infected mice intravenously with severe invasiveness to brains and kidneys. These findings suggested that O. gallopava habitats in hot springs could be one of sources for infection.

Rapid identification of Ochroconis gallopava by a loop-mediated isothermal amplification (LAMP) method.

Ochroconis gallopava is a species of dematiaceous fungi recognized as a causative agent of zoonotic and emerging fungal infections. It affects the central nervous system and respiratory tracts of humans, birds and cats. We designed O. gallopava species-specific primer sets to aid in its identification by a loop-mediated isothermal amplification (LAMP) method based on the D1/D2 domain of the LSU rDNA sequence. The LAMP method successfully detected the gene from both fungal DNA and experimentally infected brains and spleens of mice and will be helpful in the diagnosis of O. gallopava infection.