The 18S ribosomal RNA gene of Cryptococcus neoformans was amplified by polymerase chain reaction (PCR). The primers CPL1 and CPR4 were tested for their ability to amplify DNA from 30 strains of C. neoformans and 27 specimens of cerebrospinal fluid (CSF) from patients with cryptococcal meningitis. A 343 bp product was obtained and its specificity confirmed by Southern hybridization with an internal sequence (INSR4) probe. The sensitivity was 100 fg by Southern analysis and 1 pg using the PCR. Neither human nor a variety of other fungal and bacterial strains (n = 78) gave an amplified product. This PCR method can detect as few as 5 cells ml-l of C. neoformans in spiked-CSF following a simple processing procedure. The developed system of PCR was more sensitive than the culture method and revealed a very high specificity. The PCR was easy to perform and needed only 4 h for all processes from receiving the CSF to detection of a specific DNA band after agarose gel electrophoresis. This would provide another rapid laboratory method for the diagnosis of cryptococcal meningitis.

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We report the case of a 33 year old Thai female, who was married in Germany for eight years and used to travel to Thailand every year for several weeks. She presented with abdominal and back pain, prolonged fever, generalized lymphadenopathy, and a recent history of oral thrush. She was diagnosed HIV positive with initial CD4 counts of 18/μl and an HI virus load of 59.000 copies/ml. Antiviral therapy was installed with zidovudin, lamivudin, and efavirenz. Abdominal CT scans revealed greatly enlarged abdominal lymphnodes. Fine needle aspirates of cervical and retroperitoneal lymphnodes, sputum samples, blood samples, and a bone marrow biopsy were microscopically positive for Penicillium marneffei and grew P. marneffei. The isolates were sensitive to amphotericin B, flucytosine, itraconazole, and fluconazole. Both universal and specific fungal polymerase chain reaction assays were positive in various samples. Serum Aspergillus galactomannan antigen, which is known to crossreact with P. marneffei, was elevated and subsequently used for monitoring of therapy. With antifungal treatment (intravenous amphotericin B 0.6 mg/kg/d for two weeks, oral itraconazole 400 mg/d for 10 weeks and 200 mg/d as maintenance therapy), the fever declined in 6 days, the size of the enlarged lymphnodes gradually decreased in the CT scans, and the initial abdominal and back pain vanished.

A new carbazole alkaloid named clauraila E (1) together with 8 known compounds were isolated from the methanol extract of the roots of Clausena harmandiana. All compounds were evaluated for antifungal activity against Pythium insidiosum using disc diffusion assay. Pythium insidiosum is a fungus-like microorganism, for which antifungals available now are not effective. It was found that compounds 3, 6, 7 and 9 could inhibit the mycelia growth of P. insidiosum. The results show convincingly that they may be lead to compounds for the development of probiotic or novel antifungal drugs.

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