Recalcitrant primary subcutaneous phaeohyphomycosis due to Phialophora verrucosa.

Phialophora verrucosa has rarely been reported for causing phaeohyphomycosis, which tends to occur in immunocompromised individuals. The case of primary subcutaneous phaeohyphomycosis due to P. verrucosa in an otherwise healthy Chinese female is presented. The girl presented with asymptomatic skin lesions when she was only 16 year old. Histological examinations revealed multiple dematiceous hyphael elements in the dermis and subcutaneous tissues. Fungal cultures were identified as P. verrucosa repeatedly based on the morphological features and confirmed by the internal transcribed spacer region nucleotide sequencing. The infection was so extremely recalcitrant that prolonged systemic antifungal regimens for 12 years revealed limited effect. The skin lesions slowly progressed and caused marked disfigurement despite the encouraging results of in vitro susceptibility. However, no relevant side effects have been reported in the course, and the patient gave birth to a healthy baby while under the maintenance treatment of itraconazole and terbinafine. This case is special in terms of the early onset, the rare clinical aspect of the pathogen, the discrepancy between in vitro and in vivo antifungal activities and especially the prolonged and recalcitrant course in such an otherwise healthy host.

Aspergillus flavus myositis in a patient after liver transplantation.

We describe the first case of Aspergillus myositis caused by Aspergillus flavus in a liver transplant patient. The patient was a 43-year-old man who underwent liver transplantation because of end-stage hepatic cirrhosis. He experienced pain in his left calf two months after the operation. Nodules with weakness, swelling, and flaring were found in the calf two wk later. Color ultrasonic examination showed uneven resonance in the left gastrocnemius. Needle aspiration and biopsy of the muscle revealed septate hyphae consistent with Aspergillus species and focal necrosis of the muscle cells with inflammatory cell infiltration. A culture subsequently yielded A. flavus, confirming histopathologic diagnosis. Sequencing of the internal transcribed spacer region confirmed the morphologic identification. The patient was first given itraconazole 0.2 g twice daily for one wk and was then switched to terbinafine 0.25 g once a day. A three-month regimen of terbinafine therapy cured the infection, though the cultured fungus showed resistance to a number of antifungal agents. Aspergillus, a genus of ubiquitous molds, may cause invasive and even fatal disease in immunosuppressed patients.

[Population studies of Exophiala jeanselmeis by sequence analysis in the ribosomal DNA].

OBJECTIVE: To study the populations of Exophiala jeanselmei strains from diverse sources using rDNA sequences. METHODS: Tested strains included 20 isolates of Exophiala jeanselmei (type culture, reference culture, clinical and environmental isolates), 8 isolates of E. moniliae, 1 isolate of Exophiala werneckii, respectively. DNA was isolated with boiled-iced water method. Ribosomal DNA and its internal transcribed spacer regions were amplified and analyzed with sequencing. RESULTS: The ribosomal genes of E. jeanselmei varied greatly. Twenty strains of this species were divided into 7 types. The genetic distances of some strains in this species were quite long. CONCLUSION: The ribosomal RNA genes and their internal transcript sequences vary more widely than those of the other species in this genus.

[Application of molecular biology techniques in the identification of pathogenic fungi and the diagnosis of fungal infection].

With the increasing incidence and mortality of fungal infection, the requirements for strict diagnostic approaches became a very urgent issue. Because of the traditional detective techniques, such as culture, gave poor diagnostic outcomes, the molecular biological techniques are expected to develop the potential diagnostic approaches. During the past decades, we have carried out serial studies on the molecular properties of pathogenic fungi, and we would like to review as following. Firstly, we applied several molecular tools in classification and identification of pathogenic fungi. We performed random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and other techniques in studying the typing, to classify and identify the properties of Dermatophytes, Candida spp., Cryptococcus neoformans, Dematiaceous fungi, and Aspergillus spp. Interestingly, we found the same T. rubrum strain might infect different sites of the host, while a site-specificity displayed in T. mentagrophytes. This finding indicated the genetic discrepancies among the fungi. Beside, we also found that the E. dermatitis strains with different virulences possessed some discrepancies at gene level. We then developed a PCR-based molecular procedure to identify the novel species in Exophiala spp. As the applicable strategy, we also investigated the rDNA sequence properties in several fungi. And as a result, we submitted for the first time to GenBank the complete sequence of Aspergillus fumigatus rDNA/ITSI/ITSII, which provided the basis for designing the species-specific probes and for its further clinical applications. Secondly, we have tried to develop the molecular diagnostic approaches based on our DNA sequence data which were used for identification studies previously. By analyzing the DNA sequence of Aspergillus fumigatus rDNA/ITSI/ITSII, we developed a nested PCR method to detect Aspergillus fumigatus genes. Our preliminary results indicated that this PCR-based molecular approach has great importance in the diagnosis of invasive aspergillosis. We also designed the species-specific probes and then established several in situ hybridization procedures. We found these hybridization methods could get the positive rate up to 81% (13/16), which suggests that these methods have potential diagnostic value for invasive candidiasis and aspergillosis. Based on our experiences, we would conclude that the molecular biological techniques possess great value to investigate the biological properties of pathogenic fungi, and we are looking forward to see more and more molecular tools will be used in the pathogenic mechanisms of fungal infections and antifungal activity studies.

[Differentially expressed genes in immunosuppressive and healthy mice after intranasal inoculation of Aspergillus fumigatus].

OBJECTIVE: To screen out the differential expression genes between the immune suppressive host and healthy host after intranasal inoculation of Aspergillus fumigatus (A fumigatus). METHODS: Six immune suppressive mice and 6 healthy mice were inoculated with A, fumigatus intranasally. Three days after the inoculation the mice were killed and autopsied. The total RNA of the lungs was isolated. Gene chip technology was used to examine the differentially expressed genes. RESULTS: 66 differentially expressed genes were screened out of the 4098 genes. In the experimental group the genes encoding CD22, CD53, and 24p3 and the genes related to engulfment and digestion of macrophage or monocytes were up-regulated. The genes encoding surfactant associated protein C, immunoglobulin lambda chain and the genes related to pulmonary metabolism were down-regulated. The genes encoding IL18, CD8, and CD28 were not differentially expressed. CONCLUSION: The up-regulation of CD22 gene may be one of the ways by which the host defense is inhibited. CD53 may take part in the regulation of engulfment of phagocytes. 24p3 protein, an acute stress protein, protects the host from excessive inflammatory response.

Exophiala asiatica, a new species from a fatal case in China.

We describe a new species, Exophiala asiatica, isolated from an infection of the pharynx in a 20-year-old, immunocompetent woman in Nanjing, China. The infection was initiated by a fishbone prick in the pharynx, soon developed with facial nodules but subsequently seemed to have disappeared. Tonsil ulceration with progressive soreness of the pharynx was observed 3 years later. Dysphagia, headache and paralysis occurred four years after first signs of infection. Hyphae and yeast-like cells were detected in tissue and a black fungus was recovered repeatedly from pharynx tissue. Despite antifungal therapy for more than one year, the patient died of apparent cerebral dissemination of the etiologic agent. On the basis of morphology, nutritional physiology, ribosomal small subunit DNA and ITS sequence data the strain could not be matched with any existing species. A new species, Exophiala asiatica, is therefore proposed.