[Mycoserology--did we move on? Aspergillus]. / Mykoserologie--sind wir weitergekommen? Aspergillus.

Diseases caused by Aspergillus spp. are difficult to diagnose and thus require supplementary serological assays. This is the result of a selective review of the relevant literature with special regard to recent guidelines. In addition to conventional diagnostic tools (radiology, microscopy, culture) the measurement of the following serological markers is recommended, depending on the clinical type of aspergillosis: Invasive and chronic necrotising aspergillosis: Aspergillus-galactomannan antigen. Test format: EIA using the rat MAb EB-A2. Cut-off 0.5 (index). Monitoring of high risk patients: Twice weekly. Aspergillus-IgG (test format EIA) as confirmatory assay after recovery of the leukocyte function under therapy. Aspergilloma: Aspergillus IgG. Test format: EIA. Allergical aspergillosis: Aspergillus IgE. Test format: RAST. Galactomannan antigen detection rates high in the diagnosis of invasive aspergillosis. The evaluation of Aspergillus nucleic acid amplification assays is pending.

[Impact of antifungal prophylaxis on the gastrointestinal yeast colonisation in patients with haematological malignancies]. / Einfluss einer Antimykotika-Prophylaxe auf die gastrointestinale Besiedlung mit Sprosspilzen bei hämatologischen Patienten.

Patients with haematological malignancies are at high risk for developing invasive Candida infections. They are often colonised with Candida spp. in the gastrointestinal (GI) tract. In order to prevent infection, the prophylactic use of antifungal agents has been established. The widespread use of fluconazole may lead to the emergence of resistant Candida isolates. We studied the yeast colonisation of the GI tract in patients with haematological malignancies receiving antifungal prophylaxis (AP) in comparison with healthy controls. The study cohort included 46 neutropenic patients with 52 stool samples under 52 episodes of AP and 110 healthy controls. The patients received amphotericin B orally (n = 8), amphotericin B and fluconazole (n = 7), amphotericin B and itraconazole (n = 5), fluconazole orally (n = 15) and itraconazole orally (n = 17). Yeasts were cultured from the stool samples of 63.5% of the patients and 60% of the controls with a mean yeast load of 1.6 x 10(3) and 0.4 x 10(3) cfu g(-1), respectively (P = 0.045). Patients and controls had a low faecal yeast load of 10(3) to 10(4) cfu g(-1) in 19.3% and 37.3%, respectively (P = 0.021), and yeast overgrowth of >10(5) cfu g(-1) in 28.9% and 10.9%, respectively (P = 0.004). The rate of Candida albicans was 32.6% and 54.1% in the patients and controls, respectively (P = 0.021). The rates of fluconazole-resistant yeast species were higher in the patient group than in the control group: C. glabrata 20.9% vs. 11.7% (P = 0.168), C. krusei 25.6% vs. 4.7% (P = 0.001). Not a single patient under AP suffered from proven or probable invasive candidosis. In conclusion, oral AP in haematological patients resulted in a higher colonisation rate with fluconazole-resistant Candida species but efficiently prevented invasive candidosis.

Laschiatrion, a new antifungal agent from a Favolaschia species (Basidiomycetes) active against human pathogens.

Laschiatrion (1), a new antifungal antibiotic, was isolated from fermentations of Favolaschia sp. 87129. (1) exhibits broad in vitro activity against several human pathogens while no antibacterial and cytotoxic activities could be detected. The structure was elucidated by spectroscopic techniques. As to our knowledge laschiatrion possesses a new steroid skeleton.

Agar sublimation test for the in vitro determination of the antifungal activity of morpholine derivatives.

We studied the in vitro antifungal activities of a wide range of antimycotic agents, including amorolfine, terbinafine, naftifine, five morpholine derivatives, ciclopiroxolamine, bifonazole, clotrimazole, ketoconazole, itraconazole, fluconazole, voriconazole, flucytosine, amphotericin B, nystatin, and caspofungin, against Candida albicans and Trichophyton rubrum by conventional agar diffusion tests and by a novel sublimation method. For the sublimation method, 6 mm filter paper disks were soaked with defined amounts of antimycotic drugs, air dried, placed in the center of the lids of 9 cm Petri dishes, and incubated upside down with inoculated agar plates 10 mm above the disks. The conventional disk diffusion tests produced inhibition zones as previously described. The disk sublimation tests produced large inhibition zones with amorolfine, five amorolfine derivatives, and terbinafine, but with none of the other antifungal agents. Possible therapeutic advantages of agents, which are able to overcome air cavities in mycotic lesions, e.g. in onychomycosis, are discussed.

[Performance of the Candida mannan antigen detection in patients with fungemia]. / Performance des Candida-Mannan-Antigennachweises bei Patienten mit Fungamien.

For several years, the Platelia Candida mannan antigen enzyme immunoassay (Candida EIA) has been commercially available as a diagnostic test for invasive candidosis. We evaluated the Candida EIA with patients with proven fungemia caused by yeasts from which at least one serum sample was available. Fifty-nine patients with 121 serum samples were included in the study. Sixty-one different yeast strains were isolated from positive blood-cultures. The Candida EIA was positive (n = 35) or borderline positive (n = 8) in 43 of 59 patients with fungemia, resulting in an overall sensitivity of 73%. For the different yeast species, the following sensitivities were calculated: Candida albicans 30 of 39 (77%), Candida glabrata 7 of 11 (64%), Candida parapsilosis 1 of 3, Candida tropicalis 2 of 2, Candida kefyr 2 of 2, Candida lipolytica 0 of 1, Candida lusitaniae 1 of 1, Candida krusei 1 borderline positive of 1, Saccharomyces cerevisiae 1 of 1. In six patients the antigen levels over time were evaluable. In three cases the antigen was positive 3-4 days before the day the blood culture was drawn, in one case on the same day, and in two cases 2 and 5 days afterwards. In conclusion, the Candida EIA was suitable for the detection of fungemia due to the major facultatively pathogenic yeast species. The test was positive in about half of the patients before blood cultures became positive. In these cases, it contributed to an early diagnosis of invasive candidiasis.

[Antibody detection in patients with invasive aspergillosis]. / Antikorper-Nachweis bei invasiver Aspergillose.

The clinical significance of Aspergillus antibody assays for the diagnosis of invasive aspergillosis (IA) is unclear. In two studies, three different antibody assays were evaluated with patients suffering from proven IA: (i) a commercial haemagglutination test (HAT), (ii) a commercial enzyme immunoassay (EIA) for IgG, IgM, and IgA, and (iii) an experimental mitogillin enzyme immunoassay for IgG, IgM, and IgA. In the first study, 99 serum samples from 26 patients with IA and 22 serum samples from 22 control patients were tested with all the three tests. Ten of the 26 patients (38%) reacted positively in at least one antibody assay. The highest sensitivity was generated by the detection of IgG using the EIA formats (22 and 21%, respectively), the HAT had a sensitivity of 8%. IgM type antibodies were detected in only two patients; no IgA type antibodies were detected. The specificities of the IgG EIA and the HAT were 72 and 85%, respectively. Antibody detection was the single positive laboratory test in two patients with proven and probable IA. In the second study, antibody test results of 60 patients with proven IA were retrospectively evaluated. Fourteen patients (23%) tested positive in the EIA and/or in the HAT. Investigations of the antibody levels in individual immunocompromised patients over time revealed that IgG production started after a mean of 10.8 days after diagnosis of IA. To conclude, antibodies against Aspergillus were detected in 23% of patients with IA. The antibody production started in successfully treated immunosuppressed patients after a mean of 10.8 days after the onset of infection. In particular, the detection of IgG-antibodies with an EIA can be useful for the confirmation of the diagnosis of IA and for the monitoring of the treatment of IA.

Development and evaluation of a one-tube seminested PCR assay for the detection and identification of Penicillium marneffei.

A one-tube seminested polymerase chain reaction (PCR) assay was developed to detect and identify Penicillium marneffei DNA coding for 18S rRNA both from purified DNA and from clinical samples. DNA from 120 strains of organisms and 19 blood samples from AIDS patients was amplified with F3, CPL1 and PM primers. Under optimized conditions, these primers detected 100% specifically amplified products of 251 and 331 bp from all P. marneffei DNA preparations (47 strains) and from two blood samples of AIDS patients suspected to suffer from penicilliosis marneffei. The assay was sensitive to detect as little as 10 pg purified DNA, which is equivalent to 250 cells. This PCR assay might be useful as an alternative test, if a rapid diagnosis of penicilliosis marneffei is needed.

[Invasive aspergillosis: results of an 8-year study]. / Invasive Aspergillosen: Ergebnisse einer 8-Jahres-Studie.

We analysed retrospectively 90 cases of invasive aspergillosis (IA) which occurred at the University Hospital and the Thoraxklinik gGmbH Heidelberg between 1991 and 1998. 71 cases were histologically proven, 19 were probable diseases. There were 49 male and 41 female patients, with a mean age of 51.5 years (range 16 days to 80 years). Underlying diseases were: hematological malignancies in 52% (n = 47; 24 with acute leukemia), solid organ transplantation (n = 11; 9 liver, 1 kidney, 1 heart), solid cancer (n = 10), others (n = 21), and in one case no underlying disease was diagnosed. Only 54 cases (60%) were correctly diagnosed as IA during lifetime of the patients. In 59 cases (65%) only the lung was affected, 25 patients suffered from disseminated IA, in 6 patients only extrapulmonary lesions were present. 11 patients underwent lung surgery, 63 patients received antimycotic drugs (44 amphotericin B, 15 fluconazole, 4 itraconazole), 21 were not treated antimycotically. 68 patients (71%) died, from these 30 (36%) due to IA during remission of the underlying disease. The laboratory methods showed the following sensitivities, respectively: microscopy by calcofluor white staining 17%, culture 69%, Aspergillus-PCR from respiratory tract samples and biopsies 95%, galactomannan antigen detection by latex agglutination 28%, by enzyme immunoassay 59%, Aspergillus antibody detection 23%.

[Retrospective analysis of fluconazole efficacy in Candida-colonized, non-neutropenic, surgical patients in long-term intensive care]. / Retrospektive Analyse der Effektivität von Fluconazol bei nicht-neutropenischen, Candida-besiedelten chirurgischen Langzeit-Intensivpatienten.

BACKGROUND AND OBJECTIVE: The early clinical diagnosis of invasive candidiasis is difficult. Fluconazole, which has been available since the early 1990s, is a relatively atoxic intravenously applicable antimycotic agent. For this reason it has been widely used - possibly too much. The aim of this study was the retrospective critical evaluation of the efficacy of systemic antifungal chemotherapy in non-neutropenic, Candida-colonized, surgical patients in long-term intensive care. PATIENTS AND METHODS: 69 patients (54 men and 15 women, aged 55.8 [range 18-87] years) of 364 patients of the anaesthesiological intensive care unit (ICU) of the University Hospital of Heidelberg in 1991 and 1992 were selected for the study. None of the 69 patients was suffering from proven invasive candidiasis according to the gold-standard criteria of positive histology, blood culture, or isolation from a sterile compartment. However, 35 of the 69 patients were systemically treated with fluconazole (on average 295 mg per day for 10.2 days intravenously). 34 patients did not receive any antifungal therapy. Retrospectively we analysed the course of the disease in both groups of patients. Furthermore, 173 serum samples of these patients were available for investigations by Western blot for anti-Candida antibodies of the immune globulin classes M and G. RESULTS: Both groups, antimycotically treated and untreated patients, had similar characteristics at base-line: age, sex, underlying disease, severity of the disease (APACHE II Score), and also mortality (approximately 20 % in both groups). Only times in the ICU and on mechanical ventilation were significantly enhanced in fluconazole treated patients (p values 0.0004 each). Before therapy, the fluconazole patients had significantly more often yeasts in primarily non-sterile compartments (chi (2) test 0.05). The yeasts were partly eradicated by fluconazole (32/54, 59.3 %). Anti-Candida antibodies significantly correlated with higher age (anti 47 kDa antigen, p = 0.02), but not with other, clinically, diagnostically or prognostically relevant parameters. CONCLUSION: Fluconazole in non-neutropenic, Candida-colonized, surgical patients in long-term ICU care neither improved the clinical course nor the mortality rate among these patients. These observations indicate that there was a trend of overestimating the clinical significance of Candida in this group of patients. Fluconazole therapy may be significantly reduced in such patients.

Rate of transmission and endogenous origin of Candida albicans and Candida glabrata on adult intensive care units studied by pulsed field gel electrophoresis.

We determined the relative roles of endogenous origin and patient-to-patient transmission in Candida colonization of patients on adult intensive care units (ICU). A total of 48 Candida albicans and 18 Candida glabrata strains from various clinical samples of 28 long-term patients, hospitalized in two neurological ICUs between April and June 1999, were typed using pulsed field gel electrophoresis (PFGE). Three patients were co-colonized by both C. albicans and C. glabrata strains. Twenty-four C. albicans and 17 C. glabrata karyotypes were defined. The colonization was found to be polyclonal in six C. albicans and five C. glabrata patients. Twenty-six patients (93%) carried strains, which were not detected in other patients hospitalized at the same time, i.e. they were colonized by unique C. albicans and C. glabrata strains. Only two patients, who were hospitalized during the same period of time, although in different rooms of the same ICU, shared strains with an identical PFGE type, indicating possible patient-to-patient transmission. Patient-to-patient transmission of yeasts played a minor role on these ICUs.

Karyotyping of Candida albicans and Candida glabrata from patients with Candida sepsis.

The aim of this study was to determine the relatedness of Candida strains from patients suffering from Candida septicaemia by typing of Candida isolates from blood cultures and different body sites by pulsed field gel electrophoresis (PFGE using a contour-clamped homogenous electric field, CHEF). We studied 17 isolates of Candida albicans and 10 isolates of Candida glabrata from six patients. Four patients suffered from a C. albicans septicaemia, one patient from a C. glabrata septicaemia, and one patient had a mixed septicaemia with C. albicans and C. glabrata. Eight isolates from blood cultures were compared with 19 isolates of other sites (stool six, urine four, genital swab four, tip of central venous catheter three, tracheal secretion one, sputum one). PFGE typing resulted in 10 different patterns, four with C. albicans and six with C. glabrata. Five of the six patients had strains of identical PFGE patterns in the blood and at other sites. Seven isolates of a 58-year-old female with a C. glabrata septicaemia fell into five different PFGE patterns. However, they showed minor differences only, which may be due to chromosomal rearrangements within a single strain. Thus it appears, that the colonizing Candida strains were identical to the circulating strains in the bloodstream in at least five of six patients.

[Antimycotic therapy in otomycosis with tympanic membrane perforation]. / Antimykotische Therapie bei Otomykose mit Trommelfelldefekt.

Especially after prolonged antibiotic ototopic therapy otomycosis is not rare. An inoculation of fungi into the tympanic cavity however may have serious sequelae. Therefore an eradication of fungi from the external auditory canal is imperative before surgery. In addition to thorough cleaning of the outer ear canal antimycotic preparations are recommended in treating otomycosis. However, all of the commercially available ear drops contain ototoxic agents. In the case of defects of the tympanic membrane a damage of the inner ear may result. Alternatively, we suggest an aqueous solution of Miconazol 0,5%.

[Evaluation of a panfungal pcr assay for the diagnosis of pneumocystis carinii pneumonia (pcp)]. / Evaluation eines universellen Pilz-PCR-Assays zur Diagnostik der Pneumocystis carinii-Pneumonie (PCP).

We compared a universal fungal PCR assay with fluorescence microscopy for the diagnosis of Pneumocystis carinii pneumonia. 82 bronchoalveolar lavages (BALs) of 64 immunocompromised patients with atypical pneumonia and 50 BALs of 50 immunocompetent adults without lung disease were examined. 10 immunocompromised patients were clinically and/or histologically proven to suffer from PCP. For fluorescence microscopy, sensitivity and specificity in detecting P. carinii were 80.0% and 98.1%, for the PCR assay 100.0% and 96.2%, respectively. The PCR assay is a useful method for the diagnosis of PCP and is recommended as an additional test to microscopical methods.

Identification of contaminating fungal DNA sequences in Zymolyase.

When different preparations of Zymolyase were included in the pretreatment protocol of a panfungal PCR assay using a primer system for the 18S rRNA gene, an amplification product occurred in negative controls. The amplified fragment showed 100.0% sequence identity to the Saccharomyces sensu stricto complex and Kluyveromyces lodderae. Lyticase, lysing enzymes, and proteinase K appeared to be free from fungal DNA.

Disseminated Penicillium marneffei infection in an HIV-positive female from Thailand in Germany.

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/microliter 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 lymph nodes. Fine needle aspirates of cervical and retroperitoneal lymph nodes, 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 lymph nodes gradually decreased in the CT scans, and the initial abdominal and back pain vanished.

Antifungal activity of the new azole UK-109, 496 (voriconazole).

The in vitro activity of voriconazole fully includes Aspergillus, and also emerging moulds like Fusarium, Pseudallescheria boydii, and Penicillium marneffei. The minimal inhibitory concentrations of voriconazole for Candida krusei and Candida glabrata, which are resistant or less susceptible to fluconazole, promise clinical efficacy, although they are ten times higher (0.30-0.39 microgram/ml) than those for Candida albicans and other Candida spp. (0.001-0.05 microgram/ml). The endemic fungal pathogens Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, as well as Cryptococcus neoformans, and the dermatophytes are also fully susceptible to voriconazole. The zygomycetes and Sporothrix schenckii remain a problem. Voriconazole has been shown to be effective against invasive aspergillosis (IA) and fluconazole-resistant candidosis in animal models, when administered in doses between 2.5 and 45 mg/kg/day. The pharmacokinetics of voriconazole in man produced sustained high blood and tissue levels following oral and intravenous applications of 50 to 200 mg/day. Side effects included fully reversible mild to moderate visual disturbances (8 to 44%) and raised liver function enzymes (6 to 8%). In conclusion, voriconazole is highly active against Aspergillus and most other medically relevant fungi, it is applicable intravenously, and it appears to have an acceptable safety profile.

Disseminated Penicillium marneffei infection in an HIV-positive female from Thailand in Germany.

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.

Antifungal activity of the new azole UK-109, 496 (voriconazole).

The in vitro activity of voriconazole fully includes Aspergillus, and also emerging moulds like Fusarium, Pseudallescheria boydii, and Penicillium marneffei. The minimal inhibitory concentrations of voriconazole for Candida krusei and Candida glabrata, which are resistant or less susceptible to fluconazole, promise clinical efficacy, although they are ten times higher (0.30-0.39 µg/ml) than those for Candida albicans and other Candida spp. (0.001-0.05 µg/ml). The endemic fungal pathogens Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, as well as Cryptococcus neoformans, and the dermatophytes are also fully susceptible to voriconazole. The zygomycetes and Sporothrix schenckii remain a problem. Voriconazole has been shown to be effective against invasive aspergillosis (IA) and fluconazole-resistant candidosis in animal models, when administered in doses between 2.5 and 45 mg/kg/day. The pharmacokinetics of voriconazole in man produced sustained high blood and tissue levels following oral and intravenous applications of 50 to 200 mg/day. Side effects included fully reversible mild to moderate visual disturbances (8 to 44 %) and raised liver function enzymes (6 to 8 %). In conclusion, voriconazole is highly active against Aspergillus and most other medically relevant fungi, it is applicable intravenously, and it appears to have an acceptable safety profile.