Diagnostic Mycology: Xtreme Challenges.

Developing any diagnostic assay that receives United States Food and Drug Administration (FDA) approval can be a slow and difficult process. FDA-approved assays for fungal diagnosis are generally few in number and are focused mainly on diagnosing candidiasis, which is caused by several species of Candida, in addition to a limited number of systemic mycotic agents. While all microbial diagnostic assays face challenges before they are FDA approved and reach the market, there are a number of challenges to fungal diagnostic assay development that have been difficult hurdles to overcome. These hurdles include template preparation, fungal morphology, how many fungi should be identified in a single assay (scope), taxonomy and nomenclature, discriminating colonizers from invasive infection, combining identification with antifungal susceptibility, and navigating the administrative hurdles required to integrate an assay into a clinical laboratory. Some of these challenges are easier to overcome than others, but all seem to be particularly difficult for fungal diagnostic assays.

Subcutaneous Mycoleptodiscus indicus infection in an immunosuppressed dog.

An 8-year-old dog presented with several dermal excoriations. Lesion cytology revealed pyogranulomatous inflammation with branching, septate hyphae. A mold identified as Mycoleptodiscus indicus by morphology and sequencing was cultured from fine-needle aspirates. This is the first report of a Mycoleptodiscus species as an etiologic agent in a dog.

Sequence-based identification of filamentous basidiomycetous fungi from clinical specimens: a cautionary note.

The species-level identification of sterile and/or arthroconidium-forming filamentous fungi presumed to be basidiomycetes based upon morphological or physiological features alone is usually not possible due to the limited amount of hyphal differentiation. Therefore, a reliable molecular approach capable of the unambiguous identification of clinical isolates is needed. One hundred sixty-eight presumptive basidiomycetes were screened by sequence analysis of the internal transcribed spacer (ITS) and D1/D2 ribosomal DNA regions in an effort to obtain a species identification. Through the use of this approach, identification of a basidiomycetous fungus to the species level was obtained for 167/168 of the isolates. However, comparison of the BLAST results for each isolate for both regions revealed that only 28.6% (48/168) of the isolates had the same species identification by use of both the ITS and the D1/D2 regions, regardless of the percent identity. At the less stringent genus-only level, the identities for only 48.8% (82/168) of the isolates agreed for both regions. Investigation of the causes for this low level of agreement revealed that 14% of the species lacked an ITS region deposit and 16% lacked a D1/D2 region deposit. Few GenBank deposits were found to be complete for either region, with only 8% of the isolates having a complete ITS region and 10% having a complete D1/D2 region. This study demonstrates that while sequence-based identification is a powerful tool for many fungi, sequence data derived from filamentous basidiomycetes should be interpreted carefully, particularly in the context of missing or incomplete GenBank data, and, whenever possible, should be evaluated in light of compatible morphological features.

Cerebral phaeohyphomycosis due to Rhinocladiella mackenziei (formerly Ramichloridium mackenziei): a taxonomic update and review of the literature.

Cerebral phaeohyphomycosis caused by Rhinocladiella mackenziei (formerly Ramichlo-ridium mackenziei) is extremely rare, and geographically limited to the Middle East. The fungus exclusively targets the brain and infections have a grave prognosis. Eighteen cases have been reported in the literature from 1983 to 2004 with almost 100% mortality. Our patient presented in February 2008 with a brain abscess while receiving chemotherapy for carcinoma of the breast. Diagnosis was by craniotomy and aspiration of the brain abscess. Direct microscopy showed dematiaceous fungal hyphae. R. mackenziei was recovered in culture and this identification was confirmed by molecular analysis. Examination of histopathological sections of tissue from the brain biopsy revealed moniliform hyphae characteristic for phaeohyphomycosis. The patient failed to respond to antifungal therapy with amphotericin B and voriconazole or amphotericin B and posaconazole and finally expired 64 days after diagnosis. In vitro antifungal susceptibility testing showed this isolate to be resistant to amphotericin B while susceptible to itraconazole, voriconazole, and posaconazole. Previously published antifungal susceptibility data indicate that although strains show variable susceptibility to amphotericin B, the organism is generally refractory to treatment with this agent. Similar outcomes are seen with the azole agents used alone or in combination with other drugs. Although no specific risk factors have been identified, the majority of cases have occurred in immunocompromised individuals. R. mackenziei is a highly virulent agent of serious cerebral phaeohyphomycosis, and should be considered in the differential diagnosis of central nervous system disease in the Middle East.

Beauveria keratitis and biopesticides: case histories and a random amplification of polymorphic DNA comparison.

PURPOSE: The purposes of this study were to describe 2 contact lens-associated Beauveria keratitis cases and to compare the isolates of 3 contact lens-associated Beauveria keratitis cases with Beauveria-based biopesticides using random amplification of polymorphic DNA (RAPD). METHODS: A 55-year-old diabetic woman from New Mexico and a 31-year-old healthy woman from southern Wisconsin developed soft contact lens-related corneal ulcers unresponsive to topical moxifloxacin and prednisolone acetate drops. Their corneal cultures grew B. bassiana. These isolates, an isolate from a third soft contact lens-related Beauveria keratitis case, and Beauveria-based biopesticides sold in the United States were analyzed using morphological features, DNA sequencing, and RAPD. A PubMed, Cochrane Library, OVID, UpToDate, and Google search using the term "Beauveria" found only 9 reported Beauveria keratitis infections. RESULTS: Patient 1 responded to topical natamycin, ketoconazole, and 200 mg oral ketoconazole twice daily before developing a secondary bacterial infection requiring penetrating keratoplasty. After subsequent cataract surgery, the best-corrected visual acuity was 20/20. Patient 2 was treated with topical natamycin, topical amphotericin, and 200 mg oral voriconazole twice daily for 1 month with residual scarring and a best-corrected visual acuity of 20/25. RAPD showed that all isolates were unrelated. CONCLUSIONS: Although earlier reported Beauveria keratitis cases occurred after corneal injury in patients who did not wear contact lenses, 3 recent patients wore soft contact lenses and denied trauma, mirroring a changing trend in microbial keratitis. RAPD analysis showed that the Beauveria isolates were unrelated to one another and to Beauveria-based biopesticides. In Patient 2, oral voriconazole worked well.

Fatal Actinomucor elegans var. kuwaitiensis infection following combat trauma.

We report the first case of invasive mucormycosis secondary to Actinomucor elegans infection. A severely injured soldier with a fatal A. elegans var. kuwaitiensis infection is described. The identification of this fungus was performed by classical and molecular methods, and this report documents the pathogenicity of the recently described variety Actinomucor elegans var. kuwaitiensis.

Cerebral aspergillosis caused by Aspergillus granulosus.

Disseminated disease by Aspergillus granulosus has been reported only once previously in a cardiac transplant recipient. We report a fatal central nervous system infection in a lung transplant recipient. Key features of this species in the section Usti include growth at 37 degrees C and large, randomly spaced aggregates of variably shaped Hülle cells.

Cutaneous infection caused by Macrophomina phaseolina in a child with acute myeloid leukemia.

We report a case of Macrophomina phaseolina skin infection in an immunocompromised child with acute myeloid leukemia, which was treated successfully with posaconazole without recurrence after a hematopoietic stem cell transplant. The fungus was identified by DNA sequencing using both the internal transcribed spacer and D1/D2 region of the 28S ribosomal DNA gene.

Disseminated Geosmithia argillacea infection in a German shepherd dog.

We report a systemic mycosis in a German Shepherd dog caused by Geosmithia argillacea. Although this etiologic agent microscopically resembles a Penicillium species, and is histopathologically compatible with members of the genus Aspergillus, morphologic features and molecular characterization clearly separate it from these genera. This appears to be the first report of disseminated disease by this species in humans or animals. In vitro antifungal susceptibility testing suggests resistance to amphotericin B and voriconazole and susceptibility to caspofungin, itraconazole, and posaconazole.

Postoperative intra-abdominal pressure and renal function after liver transplantation.

HYPOTHESIS: Intra-abdominal hypertension frequently threatens renal function early after orthotopic liver transplantation (OLT). DESIGN: A prospective study of consecutive patients who underwent OLT. SETTING: The intensive care unit of a National Health Service teaching hospital. PATIENTS AND MAIN OUTCOME MEASURES: The intra-abdominal pressure (IAP) of 108 consecutive patients who underwent OLT was postoperatively measured 3 times a day for 72 hours using the urinary bladder technique. Intra-abdominal hypertension was defined as an IAP of 25 mm Hg or higher. RESULTS: Thirty-four patients (31%) had a high IAP. Acute renal failure developed in 17 recipients (16%), 11 (65%) of whom had intra-abdominal hypertension (P<.01), with a mean +/- SD IAP of 27.9 +/- 9.9 mm Hg vs 18.6 +/- 5.2 mm Hg in those without acute renal failure (P<.001). The subjects with a high IAP were more frequently administered loop diuretics to maintain adequate diuresis (P<.001) and had a low mean arterial pressure on the day of surgery (P<.01), despite the fact that they were given more intravenous fluids (P<.01) and did not differ in the need for inotropic drugs. Logistic regression analysis showed that intraoperative transfusions of more than 15 U, respiratory failure, and intra-abdominal hypertension (P<.01) were independent risk factors for renal failure. The length of intensive care unit stay was similar in the patients with a normal and a high IAP, but mortality was higher among the latter (P =.02). CONCLUSIONS: Intra-abdominal hypertension is common after OLT and is significantly associated with renal failure, reduced urinary output, and intensive care unit mortality. It is, therefore, worth monitoring IAP in those undergoing OLT.