Alejandro Ruiz

Control of Cryptococcus neoformans in nature by biotic factors

Two bacterial species (Pseudomonas aeruginosa and Bacillus subtilis) isolated from pigeon droppings, displayed anti Cryptococcus neoformans activity on 4 of 6 media and sterilized pigeon droppings. Acanthamoeba palestinensis trophozoites isolated from pigeon droppings ingested and killed 99.9% of C neoformans cells after 7 days of incubation. Mites and sow bugs (Metoponorthus pruinosus) isolated from pigeon droppings appear to be fungivorous. These findings suggest that many organisms that occur in pigeon droppings influence C. neoformans persistence, reproduction, morphology and distribution in nature.

Genotypic characterization of environmental isolates of Cryptococcus gattii from Puerto Rico.

Cryptococcus neoformans and Cryptococcus gattii are found in distinct environments with some overlap around different parts of the world. However, no systematic surveys of these two pathogens have been reported from Puerto Rico, a tropical island uniquely situated between mainland USA and countries in South America. We carried out an exhaustive environmental survey in southwestern Puerto Rico for pathogenic Cryptococcus species. Twenty-two presumptive isolates of C. gattii from cacti and tree detritus were characterized in detail by physiological and molecular methods and seventeen strains were confirmed as C. gattii. Cryptococcus gattii isolates were haploid and MAT strains. Sixteen out of seventeen C. gattii isolates belonged to VGII/AFLP6 genotype while one isolate was a VGIV/AFLP7 genotype. The results are significant as Puerto Rico strains are distinct from VGIII/AFLP5 strains reported from Southern California, but similar to C. gattii VGII/AFLP6 molecular type implicated in recent outbreaks of cryptococcosis in Pacific Northwest and British Columbia, Canada, but different in its M13 fingerprinting, and a common genotype in South America.

Distribución ambiental de Cryptococcus neoformans en el departamento de Cundinamarca-Colombia

Resumen Cryptococcus neoformans es un patogeno oportunista que afecta tanto a pacientes inmunocomprometidos como inmunocompetentes. La epidemia de sida ha senalado la importancia que tiene el estudio de la ecologia y epidemiologia del hongo. El proposito de este trabajo fue determinar si en Colombia existia relacion entre la distribucion ambiental de las diferentes variedades de C. neoformans y los pisos termicos en dos transectos ubicados en el departamento de Cundinamarca. Para el aislamiento e identificacion de la levadura se usaron metodos fenotipicos convencionales, determinando tambien la densidad poblacional (unidades formadoras de colonias por gramo de muestra) y la variedad. Se recolectaron un total de 765 muestras, provenientes de 26 municipios; de estas, 146 correspondian a excrementos de paloma ( Columba livia ), 437 a detritos de eucaliptos ( Eucalyptus camaldulensi s y especies afines) y 182 a detritos de almendros de la India ( Terminalia cattapa ). Se aislo C. neoformans del 46% de los municipios estudiados, en los dos transectos y en los pisos termicos calido, templado y frio. El 88% de los aislamientos se obtuvieron en este ultimo piso. No se encontraron muestras positivas en el piso termico de paramo. La densidad poblacional en excrementos de paloma oscilo entre 50 y 9,2 × 10 6 , en eucaliptos entre 50 y 1 × 10 7 y en almendros fue 50 UFC/g. De los 104 aislamientos 31% fueron serotipo A, 59% serotipo B y 10% serotipo C. El 96% de los aislamientos crecieron a 37 °C y todos mostraron capsula. En conclusion, C. neoformans se recupero en tres de los cuatro pisos termicos estudiados, con predileccion por el piso termico frio que presenta temperaturas entre 12 y 18 °C y alturas sobre el nivel del mar entre 2.000 y 3.000 m. Las densidades poblacionales no permitieron definir un patron estandar de ocurrencia.

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 5.1×105 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.

Virulence and antifungal susceptibility of environmental and clinical isolates of Cryptococcus neoformans from Puerto Rico.

Studies on the distribution, epidemiology and pathogenesis of Cryptococcus neoformans on the island of Puerto Rico are few. We have studied mouse virulence and in vitro antifungal susceptibility of 133 isolates of C. neoformans: 121 environmental and 12 clinical (9 from AIDS patients), that were isolated in Puerto Rico. In experimental CD-1 mice infected intravenously, the mean lethal dose 50% values (28 days) were > 5.2×106 and 1.1×105 cells/mouse for environmental and clinical isolates, respectively. Using an agar dilution assay, the minimum inhibitory concentrations of amphotericin B, ketoconazole and 5-fluorocytosine were comparable for environmental and clinical isolates in both yeast nitrogen dextrose base agar and Kimmigs agar. These data suggest a difference in lethality for mice, but no difference in antifungal susceptibility of environmental and clinical isolates of C. neoformans obtained in Puerto Rico.

Serologic grouping and sexual compatibility of airbornecryptococcus neoformans

Through the use of Anderson air samplers, 214 isolates ofCryptococcus 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 ‘a’ mating type. No isolates of ‘α’ mating type were found. The remaining 21 isolates were untypable for their mating type. These findings imply that the infectious particles ofC. neoformans in nature are relatively small, nonencapsulated yeast cells andnot basidiospores.

A one year study on the viability of Cryptococcus neoformans in nature

Using four different assay procedures we found a 50 to 86% decrease in viability of Cryptococcus neoformans over a one year period after two towers in the Oklahoma City area were modified to exclude pigeons and weather elements, especially rain. In homogeneous pigeon dropping samples a sharp decrease in viability occurred after 7 to 9 months of storage. The decrease occurred in pigeon dropping samples maintained at the natural sites and at ambient temperature in the laboratory but not in droppings stored at −4 °C. Air sampling studies done at the natural sites before and after tower modifications also showed a decrease in C. neoformans viability. The decrease in viability cannot be explained by seasonal temperature variations that occurred at the natural sites. Our findings suggest that death of C. neoformans occurred because of lack of available intracellular water exacerbated by low humidity, exclusion of precipitation and moisture from fresh pigeon droppings.In light of our experiences, at one site in south central United States, the feasibility of controlling C. neoformans at heavily contaminated sites, other than by the use of noxious or potentially dangerous chemicals, is discussed.

Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patients records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD50 at 28 days post infection ranged from 3.6–7.5×105 cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 °C and at 37 °C, but did not grow at 40 °C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.

Culture of Cryptococcus neoformans in the nonencapsulated state

Forty-one strains of Cryptococcus neoformans were examined after 3 days growth on a fresh and aged medium at pH 5 & pH 7 for comparison of capsule formation. Over one-half of the strains did not form visible capsules on aged medium at pH 5. Serotypes and source of isolation did not correlate with ability or inability to form capsules. Growth of C. neoformans in the nonencapsulated state makes it possible to culture many strains of C. neoformans in the form that more closely simulates the true infectious particles.

Erratum to: Genotypic Characterization of Environmental Isolates of Cryptococcus gattii from Puerto Rico

p. 1 Line 9: ‘‘Cryptococcus’’ should be italicized as ‘‘Cryptococcus’’. Line 14: ‘‘MAT strains’’ should be ‘‘majority of them were MATa strains’’. Figure 1, Legend, p. 3 Delete ‘‘(arrow indicates sample lesion)’’ Delete ‘‘the arrows indicate the detritus collected and the hole sampled’’ Mating Type Determination by PCR Pheromone Genes, p. 4 Left column 1st paragraph last sentence (line 9– 13): ‘‘PCR ...mating types.’’ should be ‘‘The new primer pairs of V1823 (50-CGCGATGTTGGCTGTA ATGG-30), V1824 (50-ATAAAAAGTTCGAGGTTT CCCAC-30) and V1825 (50-TAAGGAGAAAGAGC GACGTC-30), V1826 (50GGCATGTTCAATTGGG TTGC-30) were designed for PCR-Sequencing of MFa1 and MFa1 gene fragments from Cryptococcus gattii, because previous primer set V191 -192 works Yaliz Loperena-Alvarez and Ping Ren contributed equally to this work. The online version of the original article can be found under doi:10.1007/s11046-010-9296-3. Y. Loperena-Alvarez A. Ruiz C. Rios-Velazquez (&) University of Puerto Rico at Mayaguez, Mayaguez, PR, USA e-mail: crioslab@gmail.com P. Ren X. Li D. J. Schoonmaker-Bopp V. Chaturvedi New York State Department of Health, Wadsworth Center, Albany, NY, USA 123 Mycopathologia (2010) 170:287–289 DOI 10.1007/s11046-010-9317-2