Cryptococcus neoformans in the Seminal Fluid of an AIDS Patient. A Contribution to the Clinical Course of Cryptococcosis: Cryptococcus neofomans im Sperma eines AIDS-Patienten. Ein Beitrag zum Minischen Verlauf der Cryptococcose

In a 33-year-old HIV-positive homosexual male suffering from unexplained headache, cryptococcosis was diagnosed in a progressive secondary stage. After treatment with the standard combination therapy of amphotericin B + flucytosine for 34 d, the patient was clinically symptom-free and discharged, upon his own request, from the hospital. He remained under ambulatory mycological control. After an interval of 65 d during which the urine had been free from Cryptococcus neoformans (Cr.n.), the fungus could not be isolated from urine but 3 X 10(5) CFUs/ml were found in the seminal fluid. Andrologically, teratospermia and hyposemia were present. There were no clinical signs in the genitourinary tract including the prostate. The significance of ecological niches for Cr.n. colonization of the genitourinary tract after antimycotic therapy is discussed. In such cases, in addition to cultural examination of urine for Cr.n. by the membrane filtration technique (MFT) and Staib agar, an additional cultural examination of seminal fluid is recommended. It is also proposed to pay more attention to Cr.n. in andrological examinations. Special regard should be given to a possible occurrence of Cr.n. in the seminal fluid of AIDS patients. In cytology of the seminal fluid, use of the Giemsa stain is unsuitable for the purpose of Cr.n. detection. For this reason, it should be supplemented by PAS staining.     

Staib Agar Supplemented with a Triple Antibiotic Combination for the Detection of Cryptococcus neoformtans in Clinical Specimens: Staib-Agar mit einer Dreifach-Antibiotika-Kombination für den Nachweis von Cryptococcus neoformans in klinischem Untersuchungsmaterial

It was demonstrated that the in vitro growth of a mucoid Escherichia coli strain from the urine of an AIDS patient could disturb the concurrent growth of Cryptococcus neoformans and the development of its brown colour effect (BCE) on Staib agar (syn. Guizotia abyssinica creatinine agar, bird seed agar, niger seed agar etc.) supplemented with penicillin + streptomycin. Owing to the supplementation with the triple antibiotic combination of penicillin + streptomycin + gentamicin and the resulting inhibition of E. coli growth, the formation of an intense BCE of the Cr. neoformans colonies after 3 d at 26 degrees C could be observed. On the same medium supplemented with this triple antibiotic combination 40 Cr. neoformans strains tested showed growth with an intense BCE after 3 d at 26 degrees C; but on Emmons' neutral Sabouraud's dextrose agar (NSDA) supplemented with the same triple antibiotic combination, inhibition of growth was found. For the examination of clinical specimens for Cr. neoformans contaminated with gram-negative rod-like bacteria, Staib agar supplemented with this triple antibiotic combination is proposed. Various antibiotic supplements to primary recovery media for fungi are discussed and ecological interrelations of bacteria and fungi are emphasized.     

Mycological-diagnostic assessment of the efficacy of amphotericin B + flucytosine to control Cryptococcus neoformans in AIDS patients

Abstract: Optimal diagnostic data (microscopy, culture and serology) on 15 cases of Cr. neoformans infection in AIDS patients served as a basis for the preliminary subdivision of the stages of cryptococcosis: Primary (minor involvement of the lungs only) and secondary (hematogenous dissemination with involvement of various organs) stages. Cr. neoformans counts in body fluids and antigen titres in serum and CSF proved to be useful criteria to assess the stage of infection. The efficacy of the combination therapy with amphotericin B + flucytosine seems to be related to the stage of infection. From the mycological point of view, the standard combination of 0.3–0.5 mg/kg BW/d amphotericin B and 150 mg/kg BW/d flucytosine proved to be effective. Data on sensitivity of the agent to amphotericin B and flucytosine are presented in a summarized form. The subjects of duration of therapy, relapse and resistance to flucytosine are commented by means of examples. It should be the aim of an optimal therapy of cryptococcosis to diagnose this mycotic disease in its primary stage. Zusammenfassung: Anhand von 15 Fällen wird versucht, die Cr. neoformans-Infektion AIDS-Kranker in das Primärstadium (alleiniger und geringer Befall der Lunge) und das Sekundärstadium (hämatogene Dissemination mit Befall verschiedener Organe) einzuteilen. Die Keimzahl von Cr. neoformans in Körperflüssigkeiten und der Cr. neoformans-Antigentiter in Serum und Liquor dienten hierbei als die beiden wesentlichen Kriterien zur Einschätzung des Stadiums der Infektion und seiner Therapierbarkeit. Aus mykologischer Sicht erwies sich die Standardkombination von Amphotericin B (0,3–0,5 mg/kg KG täglich) + Flucytosin (150 mg/kg KG täglich) als wirksam. Anhand von Beispielen wird zu den Themen: Dauer der Therapie, Rezidiv und Flucytosin-Resistenz Stellung genommen. über die Empfindlichkeit der Cr. neoformans-Stämme gegen Amphotericin B und Flucytosin (Ancotil ) wird zusammenfassend berichtet. Voraussetzung für eine optimale Therapierbarkeit der Kryptokokkose scheint die Diagnostik dieser Mykose in ihrem Primärstadium zu sein.     

Cryptococcuria as manifestation of disseminated cryptococcosis: Staib agar as a selective identification medium

We conducted a retrospective study of 58 cases of cryptococcosis (1986-2008) with urine test positive for Cryptococcus sp, in Mycology Laboratory, Santa Casa-Hospital Complex, Porto Alegre, RS, Brazil. The diagnosis of cryptococcuria was based on microscopic examination and culture of urinary sediment. Cryptococcus was isolated from other clinical specimens such as blood, cerebrospinal fluid, ascitic and pleural fluids, respiratory secretions, biopsies of skin, nasal and bone marrow. Cryptocccus neoformans was present in 55 cases and Cryptocccus gattii in three cases. Males predominated (79.3%); age ranged from 12 to 86 years. Acquired Immune Deficiency Syndrome (AIDS) were present in 60.3%, 31.1% did not have AIDS and 5.2% were apparently immunocompetent patients. The most frequent signs and symptoms were headache (53.4%) and fever (51.7%). The most widely used medication was the amphotericin B (43 patients). The mortality rate was 45%. We conclude that the mycological examination of the urine can be an alternative simple, non-invasive and useful in diagnosis of disseminated cryptococcosis, especially when used in conjunction with techniques for demonstration of the capsule (nigrosine) and/or production of melanin in special culture media (Staib agar).     

Persistence of Cryptococcus neoformans in seminal fluid and urine under itraconazole treatment. The urogenital tract (prostate) as a niche for Cryptococcus neoformans

The open questions of the persistence of Cryptococcus neoformans in the urogenital tract under antimycotic treatment can be examined under optimal mycological-diagnostic conditions only. The example of a case of cryptococcosis in an AIDS patient diagnosed and treated with itraconazole in the early secondary stage of cryptococcosis is used to discuss the problems of the persistence of Cr. neoformans involvement in the urogenital tract (prostate). Data from a ten-week follow-up study are presented and discussed. The observations made have shown that itraconazole is effective in all regions of the body, with the exception of the urogenital tract. In addition to clinical examinations, cases treated with itraconazole should be finally subjected to cultural examination of prostatic secretion and/or seminal fluid, to exclude the possible presence of a symptom-free involvement of the prostate by Cr. neoformans.     

The green colour effect (GCE) of the killer strain Cryptococcus laurentii CBS 139 on Staib agar

Attention is drawn to the observation that the type strain Cryptococcus laurentii CBS 139, producing killer toxins (mycocins) directed at Cr. neoformans var. gattii, causes a green colour effect (GCE) on Staib agar (Guizotia abyssinica creatinine agar) in combination with an intense assimilation of creatinine. Five (9.6%) out of 52 strains of Cr. laurentii of various origin, showed a GCE and intense creatinine assimilation. Further research must show if all Cr. laurentii strains, characterized by a GCE similar to that of the strain CBS 139, are also capable of producing killer toxins. For further ecological and epidemiological research on strains producing killer toxins directed against species of the genus Cryptococcus, it is proposed to use Staib agar as differential culture medium indicating both colour effects, i.e. the GCE and the brown colour effect (BCE).     

Chlamydospore formation on Staib agar. Observations made before Candida dubliniensis was described

When routinely using Staib agar to detect Cryptococcus neoformans in AIDS patients by the brown colour effect of its colonies, rough-looking colonies of a questionable variety of Candida albicans were also found. Microscopically, these colonies consisted of pseudohyphae with abundant masses of chlamydospores. However, the colonies of C. albicans were smooth-edged and formed by round-oval blastospores only. Such observations were made during the mycological supervision of 36 cryptococcosis cases during the 1987-94 period. All these questionable cultures of Candida spp. were discarded. However, because the corresponding photographs of and records on such strains were found to be identical with those recently published by molecular biologists under the title 'Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis' [Staib, P. & Morschh?user, J. (1999) Mycoses 42, 521-524], the present communication presents a report on such observations in a representative and exemplary case of an AIDS patient.     

Flucytosine and cryptococcosis: which in vitro test is the best predictor of outcome?

The combination of flucytosine and amphotericin B is first choice treatment for active cryptococcosis. Because of innate or acquired resistance of Cryptococcus neoformans to flucytosine, in vitro testing is mandatory. Yeast nitrogen base (YNB) at pH 7.0 is the recommended medium for the broth microdilution test (NCCLS M27-A) and for the E-test. In order to verify if minimum inhibitory concentrations (MICs) were able to predict treatment outcome, the susceptibility of 24 isolates from 21 patients treated with flucytosine alone or in combination was tested by the broth microdilution, agar dilution and E-test using YNB either at pH 7.0 or at pH 5.4. Only those MICs obtained on YNB pH 5.4 proved to correlate with treatment outcome. The present study suggests that in vitro susceptibility to flucytosine of C. neoformans isolates should be evaluated on YNB pH 5.4 and the test should be standardized accordingly.     

Aspergillus Findings in AIDS Patients Suffering from Cryptococcosis Aspergillus-Befunde während des Cryptococcose-Verlaufs bei AIDS-Patienten

Because of the known pathogenicity of Cryptococcus neoformans and of aspergilli depending on defined but different immunodeficiencies of the host, the evaluation of their simultaneous cultural detection in specimens of the respiratory tract of AIDS patients is of epidemiological, diagnostic, pathogenetic and therapeutic interest. In 10 out of 15 AIDS patients the following species of the genus Aspergillus could be isolated either once or repeatedly during the course of Cr. neoformans infections in specimens from the respiratory tract: A. fumigatus, A. flavus, A. nidulans, A. niger and A. glaucus. From the 10 different case reports the conclusion can be drawn that the cultural findings of aspergilli in specimens from the respiratory tract during the course of cryptococcosis were of no pathogenic significance. Rather, they should be interpreted as inhaled and not yet germinated conidia still capable of growth lacking primary pathogenicity for the AIDS patient. The significance of serodiagnosis for the evaluation of the simultaneous occurrence of Cr. neoformans and Aspergillus spp. is demonstrated and discussed.     

Disseminated cryptococcosis in a patient with AIDS

Summary. We report the case of a 31-year-old AIDS patient who had generalized cryptococcal disease with involvement of the lungs, bone marrow, gastrointestinal tract and skin as well as chorioretinitis which may have been due also to Cryptococcus neoformans infection. Initially there was no involvement of the central nervous system. The patient recovered after 43 days' treatment with fluconazole and amphotericin B and flucytosine initially. Four months later he presented with a cryptococcal splenic abscess despite secondary prevention with fluconazole and twice-weekly liposomal amphotericin B. After spleaectomy the patient was discharged in good health. One year later he died of cryptococcal meningitis
Zusammenfassung. Es wird über einen 31 Jahre alten AIDS-Patienten mit generalisierter Cryptococcose berichtet. Befallen waren Lunge, Knochenmark, Gastrointestinaltrakt und die Haut; vermutlich war auch eine Chorioretinitis Cryptococcus neoformans -bedingt. Das Zentralnervensystem war anfänglich nicht betroffen. Der Patient erholte sich unter 43 d Fluconazol-Behandlung nach anfänglicher Amphotericin B-Flucytosin-Kombinationstherapie. 4 Monate später zeigte sich trotz Sekundärprophylaxe mit Fluconazol und wöchentlich zweimaliger Gabe von liposomalem Amphotericin B ein Cryptococcus -bedingter Milzabszeß. Nach Splenektomie wurde der Patient in gutem Gesundheitszustand entlassen. Ein Jahr später verstarb er an Cryptococcus -Meningitis.     

The Effect of Cilofungin (LY 121019) in Combination with Amphotericin B or Flucytosine Against Candida Species: Die Wirkung von Cilofungin (LY 121019) in Kombination mit Amphotericin B oder Flucytosin auf Candida-Arten

Cilofungin was combined with amphotericin B or flucytosine to determine if synergistic inhibition or killing occurred against 50 strains of various Candida species. Synergistic inhibition of growth occurred only once with amphotericin B and cilofungin and only 2 times with flucytosine and cilofungin. Synergistic killing occurred in 5 strains with the amphotericin B-cilofungin combination and in 7 strains with the flucytosine-cilofungin combination. Antagonism occurred frequently with both the amphotericin B-cilofungin and the flucytosine-cilofungin combinations.     

The Effect of Cilofungin (LY 121019) in Combination with Amphotericin B or Flucytosine AgainstCandidaSpecies

Cilofungin was combined with amphotericin B or flucytosine to determine if synergistic inhibition or killing occurred against 50 strains of various Candida species. Synergistic inhibition of growth occurred only once with amphotericin B and cilofungin and only 2 times with flucytosine and cilofungin. Synergistic killing occurred in 5 strains with the amphotericin B-cilofungin combination and in 7 strains with the flucytosine-cilofungin combination. Antagonism occurred frequently with both the amphotericin B-cilofungin and the flucytosine-cilofungin combinations.     

Comparison of Broth Dilution and Semisolid Agar Dilution for In Vitro Susceptibility Testing of Cryptococcus neoformans

Summary

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution.

By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively.

Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.     

In vitro combination between antifungals and diphenyl diselenide against Cryptococcus species

Cryptococcus species are an encapsulated fungal pathogen that cause cryptococcal meningitis. There are limited therapeutic options for this infection. The management includes the use of different antifungals such as amphotericin B, flucytosine, or fluconazole, either alone or in combination. However, numerous therapeutic failures, as well as the limited effectiveness of such therapeutics, have been described. Diphenyl diselenide is a chemically synthesised molecule with was found to have antimicrobial activity. In this study, we evaluated the antifungal activities of fluconazole, amphotericin B and flucytosine, in combination with diphenyl diselenide against 30 clinical isolates of Cryptococcus spp. using CLSI M27‐A3 method and the checkerboard microdilution technique. Our results show that the combination of flucytosine and diphenyl diselenide displayed 100% of synergism. However, when we analysed (PhSe)₂ plus AMB or FLZ we observed around 70% of indifference. Our results suggest that the combination of diphenyl diselenide with other antifungal agents deserves attention as a new option for the development of alternative therapies for cryptococcosis.     

Antifungal susceptibilities of Cryptococcus neoformans cerebrospinal fluid isolates from AIDS patients in Kenya

Poor susceptibility of Cryptococcus neoformans to fluconazole (FLC) is a matter of concern among clinicians in Africa. The emergence of resistance to FLC was recently reported in Kenya, but it is not known whether it is widespread. Thus, there is need for more antifungal drug susceptibility studies in Kenya. The aim of this study was to measure the in vitro antifungal drug susceptibilities of incident C. neoformans isolates from acquired immunodeficiency syndrome patients in Kenya. Antifungal susceptibility testing was performed in 67 C. neoformans isolates by broth microdilution method as outlined in the Clinical and Laboratory Standards Institute document M27-A3 using FLC, amphotericin B (AMB), voriconazole (VOR), ravuconazole (RAV) and flucytosine (5-FC). Isolates were grown on l-canavanine glycine bromothymol blue medium for serotype identification. Six per cent of the isolates were identified as C. neoformans var. gattii serotype B or C and 94% as C. neoformans var. neoformans. All isolates tested were susceptible to AMB, VOR and RAV (100%), and high susceptibilities were seen to FLC (97%), and 5-FC (90%). Only 3% and 10% of the isolates' susceptibility to FLC and 5-FC, respectively, was dose-dependent or intermediate. These results demonstrate high susceptibilities of incident C. neoformans isolates to FLC and AMB, antifungals used for treatment of cryptococcal meningitis in Kenya.     

In vitro susceptibility of Cryptococcus neoformans clinical isolates from Egypt to seven antifungal drugs

The in vitro susceptibility of 29 clinical isolates of Cryptococcus neoformans to fluconazole, miconazole, itraconazole, ketoconazole, flucytosine, nystatin and amphotericin B was tested by broth and colorimetric microdilution methods. Most of the isolates showed uniform patterns of susceptibility to the used antifungal agents. Only three isolates exhibited resistance [fourfold or greater rise in the minimum inhibitory concentrations (MICs)] to the tested antifungal drugs. The MIC50 and MIC90 were 0.5-8 mg l(-1) for 5-flucytosine, 0.2-8.25 mg l(-1) for nystatin, 0.5-16 mg l(-1) for fluconazole and 0.2-12.5 mg l(-1) for miconazole. However, MIC50 and MIC90 were in narrow range for the clinical yeast isolates in both methods used and showed 0.5-1 mg l(-1) for amphotericin B and 0.016-0.25 mg l(-1) for both ketoconazole and itraconazole. The combination of fluconazole plus flucytosine showed greater synergistic and fungicidal activity compared with that of fluconazole plus amphotericin B or the use of individual drugs.     

Phenotypic variation and antifungal susceptibility patterns of Candida albicans strains isolated from neutropenic patients

The aim of this study was to investigate the relationship between phenotypes of Candida albicans strains isolated from clinical specimens and the susceptibility of the strains to three antifungal agents, fluconazole, amphotericin B and flucytosine. Oropharyngeal, gastrointestinal and urogenital tract specimens were collected from 122 neutropenic patients who had received no previous prophylactic treatment. Each of 122 C. albicans strains recovered was found to express one of the six phenotypes: smooth, fuzzy, irregular, star, ring and stipple. The mean minimum inhibitory concentrations (MICs) of fluconazole was consistently higher for C. albicans strains expressing the stipple phenotype. The mean MICs for the six phenotypes of C. albicans strains ranged between 1.22 and 7.94 micrograms ml-1 for fluconazole, 0.99 and 2.55 micrograms ml-1 for amphotericin B and 1.23 and 1.83 micrograms ml-1 for flucytosine. The antifungal susceptibility of the stipple phenotype requires attention, especially in patients who are clinically unresponsive to fluconazole chemotherapy or in cases of life-threatening C. albicans infections of immunocompromised hosts. Long-term use of fluconazole may explain the outcome of the resistant stipple phenotype.     

Ground red hot pepper agar in the isolation and presumptive identification of Cryptococcus neoformans

The study compared ground red hot pepper agar (GRHP) and Guizotia abyssinica creatinine agar (GACA), a medium routinely used for isolation of Cryptococcus neoformans. In order to confirm the capacity of GRHP to support the Cr. neoformans growth and pigment production, 15 strains were inoculated onto GRHP and GACA. No significant differences in the growth and pigmentation of the tested strains on the two media were noted. As heavily contaminated specimens, 50 samples of pigeon droppings were examined by plating on GRHP and GACA, which resulted in the isolation of 14 and nine Cr. neoformans strains, respectively. The results indicate that GRHP, as a result of its superior selectivity and significant reduction of contaminant growth, provides better conditions than GACA for isolation and presumptive identification of Cr. neoformans from heavily contaminated specimens.     

Prevalence and phenotypic evaluation of Candida dubliniensis in pregnant women with vulvovaginal candidosis in a university hospital in Ankara

Candida dubliniensis is very similar to Candida albicans in terms of genotypic and phenotypic characteristics. As the hormonal milieu of the vagina during pregnancy, characterised by a lack of maternal cell-mediated immunity, enhances Candida colonisation and serves as a risk factor for symptomatic expression, investigation into the isolation of C. dubliniensis in vaginal discharges of pregnant women with vulvovaginal candidosis was made. A total of 77 Candida isolates obtained from 60 patients positive for vulvovaginal candidosis collected from 218 pregnant women were investigated for C. dubliniensis subsistence. In total 41 Candida species phenotypically identified as C. albicans on the basis of a positive germ tube test and carbohydrate assimilation tests were screened for the presence of C. dubliniensis. Phenotypic tests for differentiation of C. dubliniensis from C. albicans, such as growth at 42 and 45 degrees C on Sabouraud dextrose agar, appearance on CHROMagar and colony morphology on Cornmeal-Tween-80 agar and Staib agar were carried out. Only one strain (2.43%) was phenotypically identified as C. dubliniensis. According to our study, a combination of at least five phenotypic methods is necessary for an exact diagnosis of C. dubliniensis. Large-scale studies of pregnant women are required to discover the aetiological importance of this yeast.     

In vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and Cryptococcus gattii to five antifungal drugs

The in vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and C . gattii to five antifungal drugs (amphotericin B, flucytosine, fluconazole, itraconazole and ketoconazole) were determined using the Etest method. None of the Malaysian isolates was resistant to amphotericin B and ketoconazole. Isolates resistant to flucytosine, fluconazole and itraconazole were observed in this study. Minimum inhibition concentrations (MICs) of > or = 32 microg ml(-1) against flucytosine, > or = 64 microg ml(-1) against fluconazole and > or = 1 microg ml(-1) against itraconazole were noted in four (8.3%), two (4.2%) and one (2.1%) isolates respectively. There was no significant difference in the MICs for both Cryptococcus species (P > 0.05), indicating that C. gattii was as susceptible as var. grubii to all the antifungal drugs tested. No significant difference in the MICs for both Cryptococcus species collected from 1980 to 1990 and 2002 to 2004 were observed (P > 0.05).