Isolates Of Cryptococcus Neoformans Research Articles

Effect of hydrogen peroxide on growth of Candida, Cryptococcus, and other yeasts in simulated blood culture bottles

The addition of hydrogen peroxide to blood contained in liquid culture medium increased the dissolved-O2 partial pressure in direct proportion to the volume injected. The effect of hydrogen peroxide on the growth of subcultured clinical isolates of Candida albicans, Cryptococcus neoformans, Torulopsis glabrata, and other yeasts and on the growth of blood culture isolates of representative pathogenic bacteria was compared with its effect on their growth in vented and unvented stationary bottles. C. albicans and C. neoformans grew significantly better in bottles to which hydrogen peroxide had been added than in vented or unvented bottles. The advantage of hydrogen peroxide over venting was most marked with several slowly growing strains. Similar results were obtained in shaker cultures with strains of C. albicans which were inoculated directly from positive blood cultures. The effect of hydrogen peroxide tended to diminish during serial passage. T. glabrata grew less well when hydrogen peroxide was added, perhaps because of the absence of oxidase. The growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis was not significantly inhibited or augmented by the addition of hydrogen peroxide. The growth of Escherichia coli was inhibited slightly. The value of the addition of hydrogen peroxide to blood cultures to improve the isolation of yeasts needs to be established by a clinical trial which would compare this method with established methods.

Sexual compatibility of clinical and environmental isolates of Cryptococcus neoformans.

In vitro study to determine the sexual compatibility of 26 Cryptococcus neoformans isolates was conducted on sunflower (Helianthus annus) seed agar. Among 26 isolates examined, 14 were originated from animals, 6 from men and 6 from environmental substrates; 17 isolates were obtained in Japan and 9 in India. These isolates were crossed with each of the 2 mating types (α and a) of Filobasidiella neoformans var. neoformans or F. neoformans var. bacillispora. All the isolates produced basidiospores, basidia, dikaryotic hyphae with clamp connections when paired with the “a” mating type of F. neoformans var. neoformans. We could not find any isolate showing sexual reproduction with “α” mating type of either variety of F. neoformans. Furthermore, none of the isolates exhibited self-fertile behavior. Our results indicate that the “α” mating type occurs more frequently in both clinical and natural isolates of C. neoformans originated from Japan and India.

Collaborative investigation of variables in susceptibility testing of yeasts

A multicenter study was performed to evaluate the effect of medium, incubation time (24 and 48 h), and temperature (30 and 35 degrees C) on intra- and interlaboratory variations in MICs of flucytosine, amphotericin B, and ketoconazole for yeasts. Testing was performed on coded isolates of Candida species (11 strains) and Cryptococcus neoformans (2 strains) by using a standard macrodilution protocol 11 laboratories. Four chemically defined media buffered to pH 7.0 with morpholinepropanesulfonic acid were evaluated, including buffered yeast nitrogen base, synthetic amino acid medium-fungal, RPMI 1640 medium, and high-resolution antifungal assay medium. Intralaboratory variability was less than or equal to fourfold for 97% of the replicate sets of data. The highest level of interlaboratory agreement, irrespective of antifungal agent or incubation conditions, was observed with RPMI 1640 medium. Intralaboratory variability was less than or equal to fourfold for 93% of the determinations with ketoconazole and 100% with flucytosine tested in RPMI 1640 medium at 35 degrees C for 24 h. Variability in amphotericin B results was less than or equal to fourfold for 81% of the determinations in RPMI 1640 medium at 35 degrees C for 48 h. The rank order of MICs within each antifungal test group was similar among the various laboratories and was generally in agreement with the reference rank order regardless of the test medium that we used.

Itraconazole Therapy for Cryptococcal Meningitis and Cryptococcosis

We studied the efficacy of itraconazole, a new oral triazole, in 33 patients (32 were immunocompromised) with cryptococcosis. Diagnoses included cryptococcal meningitis (24 patients), cryptococcemia (19 patients), cryptococcuria (4 patients), osteomyelitis (1 patient), pulmonary cryptococcosis (1 patient), and soft-tissue cryptococcosis (2 patients). Twenty-six patients had the acquired immunodeficiency syndrome, and 4 were transplant recipients. Therapy (200 mg two times per day) was monitored by clinical response, culture, and cryptococcal antigen testing. Cryptococcemia was abolished in 10 (100%) of 10 assessable patients; clinical abnormalities also cleared. Thirteen (65%) of 20 assessable patients with cryptococcal meningitis had complete responses (clinical resolution and negative cultures), 5 (25%) had partial responses, and therapy failed in 2 (10%). Ten (71%) of 14 patients with the acquired immunodeficiency syndrome who had meningitis and were treated with itraconazole as their sole therapy had complete responses, 3 (21%) had partial responses, and therapy failed in 1 (7%). Partial responses or failures were all associated with the failure of previous therapy, severe disease, low serum itraconazole concentrations, or a resistant organism. Noncompliance was associated with relapse (4 patients). Meningitis recrudesced in 3 (20%) of 15 patients who responded to therapy. All 4 patients with pulmonary cryptococcosis, soft-tissue cryptococcosis, or osteomyelitis responded to therapy (100%). Cryptococcuria was abolished in 3 (60%) of 5 assessable patients. The median survival of the 20 patients with the acquired immunodeficiency syndrome who had meningitis exceeded 10.5 months at this writing. Overall results compare favorably with amphotericin B therapy with or without flucytosine. Forty of 44 isolates of Cryptococcus neoformans were susceptible in vitro to itraconazole (minimum inhibitory concentration less than or equal to 3.13 mg/L), 3 were borderline (minimum inhibitory concentration, 6.25 mg/L), and 1 was resistant (minimum inhibitory concentration, 12.5 mg/L). As itraconazole does not penetrate cerebrospinal fluid, the meningitis results are noteworthy and suggest that meningeal and parenchymal penetration is critical. Itraconazole is promising for the treatment of cryptococcosis in patients with and without the acquired immunodeficiency syndrome.

Isolation of saprophytic Cryptococcus neoformans from Puerto Rico: distribution and variety.

Until the present decade, no studies had been conducted in Puerto Rico on the saprophytic distribution and variety of Cryptococcus neoformans. Samples (522) of pigeon droppings from 14 western towns were tested for the presence of C. neoformans. The yeast was recovered from 24.7% (129 isolates) of the samples, representing 10 of the 14 towns studied. All environmental isolates were identified as C. neoformans var. neoformans using canavanine-glycine-bromthymol blue (CGB) agar. The yeast was isolated from 79.4% of the samples in one town, Isabela. The average number of yeast cells isolated from sites within this municipality was 5.1 x 10(5) per gram of pigeon droppings. This was 2.6 times the average number of yeast cells of C. neoformans isolated from sites in other towns. In addition, the yeast was isolated from four patients with the acquired immune deficiency syndrome (AIDS), each of whom died of cryptococcal meningitis. Each of these poorly encapsulated isolates was identified as C. neoformans var. neoformans using CGB agar. The results of this investigation demonstrate that C. neoformans var. neoformans is prevalent in Puerto Rico.

Biochemical serogrouping of clinical isolates of Cryptococcus neoformans.

Three hundred twenty-three clinical isolates of Cryptococcus neoformans of diverse geographic origins were biochemically serogrouped using glycine-cycloheximide-phenol red agar (GCP), the same medium less cycloheximide (GOP), and glycine-L-canavanine bromothymol blue agar (CGB). Twenty isolates gave positive reactions on all three media typical of the B and C serotypes. Three were from the Peoples' Republic of China; three each were from Michigan (two patients) and Louisiana; two each were from California, Georgia, and Virginia; and one each was from Alabama, Florida, North Carolina, Oklahoma, and Tennessee. Two hundred seventy-six isolates were identified as belonging to the A/D serogroup; 272 were of American origin and four were from China. Twenty-seven isolates were biochemically ungroupable. Evaluations of the reactions on all three media were open to subjective interpretations. Utilization of glycine was the most frequent atypical variable; 36 of 276 (13%) A/D isolates utilized glycine while being inhibited by either GCP or CGB or both. Significant differences between A/D and B/C serogroups in terms of susceptibility to 5-fluorocytosine but not to amphotericin B were observed; B/C serogroup isolates appeared to be less susceptible to 5-fluorocytosine in vitro than were the A/D serogroup isolates. These results provided new evidence on the distribution of B/C serogroup isolates of C. neoformans in America and demonstrate the difficulties of using biochemical tests for serotyping purposes. They also offer a possible explanation for the apparent more refractory therapeutic responses of infections caused by B and C serotypes to conventional antifungal chemotherapy.

Absence of fungistatic antagonism between flucytosine and cytarabine in vitro and in vivo.

Determination of the MIC of flucytosine for 16 wild isolates of Cryptococcus neoformans and Candida spp. in YNBG medium containing 10 mg/l cytosine demonstrated antagonism of fungistatic activity compared with that determined in YNBG alone. Determination of the MIC in YNBG containing 10 mg/l cytarabine showed no change in activity for 14 of the strains, an increase for one and a decrease for another. Determination of serum flucytosine concentrations in a patient receiving cytarabine simultaneously revealed therapeutic levels. Fluctuations in serum flucytosine concentrations were observed in samples taken before, during and after concurrent cytarabine therapy but these may have resulted from unstable renal function rather than in-vivo inactivation of flucytosine by cytarabine. These data do not support significant antagonism of the fungistatic activity of flucytosine by cytarabine.

Poorly encapsulated Cryptococcus neoformans from patients with AIDS. I: Preliminary observations.

Ten isolates of Cryptococcus neoformans recovered from individual patients with the acquired immunodeficiency syndrome (AIDS) were studied in conjunction with three other isolates from non-AIDS patients. On primary culture nine out of ten of the AIDS isolates grew as nonmucoid, dry, pasty colonies resembling those produced by "diphtheroids." One isolate formed moist colonies. In contrast, the three non-AIDS Cryptococcus neoformans isolates produced highly mucoid colonies. India ink mounts of primary cultures of the ten AIDS isolates after 48 hours incubation in 5% CO2 showed markedly smaller capsules than the three non-AIDS isolates. India ink preparations of fresh cerebrospinal fluid of two specimens from AIDS patients in which capsule sizes were systematically determined showed a few moderately encapsulated cells dispersed among numerous others that were poorly encapsulated. This observation was confirmed in mucicarmine-stained smears of cerebrospinal fluid. Poorly encapsulated Cryptococcus neoformans seem to be associated with infections in AIDS patients.

Serologic grouping and sexual compatibility of airborne Cryptococcus neoformans.

Through the use of Anderson air samples, 214 isolates of Cryptococcus neoformans were cultured from the air in a vacant tower in a large complex of buildings in Oklahoma City. The tower contained hundreds of pigeons, a massive amount of droppings, nests with eggs and young, dying and dead pigeons. All isolates were serotype A-D and self-sterile for the production of basidiospores. Among these 193 were of the "alpha" mating type, producing basidiospores when paired with "alpha" mating type. No isolates of "alpha" mating type were found. The findings imply that the infectious particles of C. neoformans in nature are relatively small, nonencapsulated yeast cells and not basidiospores.

Pseudohyphal forms of Cryptococcus neoformans: decreased survival in vivo.

Three pseudohyphal isolates of Cryptococcus neoformans were inoculated intracranially into mice. Four weeks post-inoculation the animals showed no symptoms of disease and the number of viable cells per brain decreased to zero. Possible roles of pseudohyphal forms of C. neoformans in the immunology and pathogenesis of cryptococcosis are discussed.

Synthesis of Tryptophol and Indolelactic Acid by Cryptococcus neoformans

SUMMARYTryptophol (TOL) and indolelactic acid (ILA) were synthesized by isolates of Cryptococcus neoformans in a synthetic medium containing tryptophan as the sole nitrogen source. The compounds were produced in maximal amounts at 48 to 72 h of growth at 37 C. Trace amounts of indolepyruvic acid (IPA) were found in acidic extracts, indicating that IPA is an intermediary in the conversion of tryptophan to TOL and ILA. Of all the Cryptococcus species tested, only isolates of C. neoformans synthesized both indole compounds. Thus, the two products—which can be readily isolated and identified by gas-liquid chromatography—could serve as markers for distinguishing C. neoformans from other species of Cryptococcus.

Synthesis of Tryptophol and Indolelactic Acid byCryptococcus Neoformans

Tryptophol (TOL) and indolelactic acid (ILA) were synthesized by isolates of Cryptococcus neoformans in a synthetic medium containing tryptophan as the sole nitrogen source. The compounds were prod...

Rapid presumptive identification of Cryptococcus neoformans

Carbohydrate-containing extracts were prepared from mature yeast colonies grown on Sabouraud dextrose agar by mixing a 0.001-ml loopful of yeast cells for 30 s in phenolized saline and removing the cells by centrifugation. Extracts were prepared from 54 Cryptococcus neoformans isolates, 29 isolates of other Cryptococcus species, 16 isolates of Candida species, 2 Rhodotorula, 2 Torulopsis, and 1 Saccharomyces species. Initially the carbohydrate content of each extract was estimated (Molisch method) and adjusted to 1, 5, and 10 microgram/ml. Twofold dilutions of each extract were tested for reactivity with the cryptococcal latex agglutination reagent of Bloomfield et al. (N. Bloomfield, M.A. Gordon, and D.F. Elmendorf, Jr., Proc. Soc. Exp. Biol. Med. 114:64-67, 1963). All 54 C. neoformans extracts gave strong agglutinations (3+ to 4+) in dilutions of 1:4 or greater. None of the other yeasts produced any agglutination, except for 1 of 15 C. laurentii isolates, which showed a 1+ reaction that disappeared at a dilution of 1:4 and above. Subsequent testing established that a single extract made from 0.001 ml of yeast cells in 6 ml of phenolized saline contained less than 5 microgram of carbohydrate per ml, was suitable for a single rapid screening dilution, and eliminated any cross-reaction from the C. laurentii isolates. In our hands this method has provided a reliable differentiation of C. neoformans from other unknown yeast colonies in less than 20 min exclusive of a Molisch determination.

Treatment of Fungal Infections

To the Editor. —In the February issue of theArchives, an editorial by Krick and Remington (p 344) critically reviews the current status of flucytosine (Ancobon) as a therapeutic agent, mainly in cryptococcal and Candida infections. The comments of the editorialists are made in connection with our clinical experience with flucytosine published in the same issue of theArchives(p 231) and our interpretation of the results of similar studies that had appeared in the literature. Because several aspects of our study are interpreted in the editorial in a manner that is at variance with our interpretation, a clarification is needed. With regard to the combined use of amphotericin B (fungizone) and flucytosine, the editorialists refer to the demonstration of in vitro synergism between these two drugs for a small number of isolates of Cryptococcus neoformans and Candida . The possibility of synergism was the justification of our use of combined

Synergistic action of amphotericin B and 5-fluorocytosine against yeast-like organisms.

SummaryThe efficacy of amphotericin B and 5-fluorocytosine used in combination against several different isolates of Cryptococcus neoformans, Candida albicans, Candida tropicalis, and Saccharomyces cerevisiae has been investigated in vitro. In every case, the difference in killing between the drugs in combination and used alone was greater than tenfold. Against one of the six isolates the combination of drugs was fungicidal. The clinical implications of these findings regarding the synergistic activity of amphotericin B and 5-fluorocytosine are being evaluated.

Pathogenic and Potentially Pathogenic Fungi Isolated from Beach Sands and Selected Soils of Oahu, Hawaii

SUMMARYFungi capable of causing mycoses in man are known to occur in many types of soils and natural habitats, but their occurrence in Hawaii has not been studied comprehensively to date. Two known sources served as the basis for this initial Hawaiian survey: 1) sands taken from beaches of low and high swimmer-density and, 2) soils associated with avian droppings. A total of 361 sand samples representing Oahu's supra-tidal and intertidal zones was screened. More pathogenic or potentially pathogenic fungi were isolated from areas where the swimmer-density was very high and, conversely, few were isolated from low swimmer-density areas. Fifty-four colonies of these fungi were isolated. Among them were 10 genera with 13 species, including notably Candida albicans, Cladosporium werneckii, Epidermophyton floccosum, Geotrichum candidum, Microsporum canis, M. gypseum, Nocardia asteroides, Trichophyton mentagrophytes, T. rubrum, and Trichosporon cutancum.Sixty-one isolates of Cryptococcus neoformans were obtained from 170 bird droppings associated with major types of soils present on Oahu. Forty-three isolates were recovered on diphenyl agar and 33 by mouse inoculation. In the latter group 18 were not recovered on diphenyl agar and IS isolates were obtained from mice sacrificed after 6 weeks. Histoplasma capsulatum was not isolated.

Cell wall differences of patient and soil isolates of Cryptococcus neoformans.

Two isolates of Cryptococcus neoformans were studied to determine whether or not differences in chemical composition of the cell walls existed. Aerated cultures of both isolates were grown at 37°C. in a synthetic liquid medium. A purified cell wall fraction was obtained by repeated passage of cells through a French pressure cell followed by differential centrifugation. Chemical analyses of cell wall fractions of each of these strains showed quantitative differences in 3 major groups of compounds. The isolate from the patient contained a higher percentage of hexose and a lower percentage of hexosamines and lipids than did the soil isolate.

Serologic grouping of Cryptococcus neoformans.

SummaryOne hundred and six isolates of Cryptococcus neoformans were investigated by agglutination and absorption studies with anti-cryptococcal sera. In addition to the three previously reported serotypes, a fourth serotype was distinguished and designated type D. Of the 106 isolates from man and nature, 69.8% were type A, 10.4% were type B, 6.6% were type C, and 4.7% were type D, 3.8% reacted with more than one serum, and 4.7% were untypable. The majority of type B and C strains were obtained from California, suggesting the possibility of major differences in the distribution of cryptococcal serotypes.

Isolation of Pathogenic Fungus Cryptococcus Neoformans from Pigeon Droppings

Isolation of Pathogenic Fungus Cryptococcus Neoformans from Pigeon Droppings