Cryptococcus genetic diversity and mixed infections in Ivorian HIV patients: A follow up study.

Genetic diversity analyses were performed by sero-genotyping and multi-locus sequence typing on 252 cryptococcal isolates from 13 HIV-positive Ivorian patients followed-up for cryptococcal meningitis. Antifungal susceptibility analyses were performed according to the CLSI M27A3 method. The majority (67.8%) of the isolates belonged to the Cryptococcus neoformans (serotype A) species complex, with 93% being VNI and 7% being VNII. Cryptococcus deuterogattii VGII (serotype B) represented 16.7% of the strains, while C. neoformans/C. deneoformans VNIII (serotype AD) hybrids accounted for 15.1% of the strains. One strain (0.4%) was not identifiable. Nine different sequence types (STs 5, 6, 23, 40, 93, 207, 311, and a new ST; 555) were identified in the C. neoformans population, while the C. deuterogattii population comprised the single ST 173. The distribution of the strains showed that, while the majority of patients (9/13) harboured a single sequence type, 4 patients showed mixed infections. These patients experienced up to 4 shifts in strain content either at the species and/or ST level during their follow-up. This evolution of diversity over time led to the co-existence of up to 3 different Cryptococcus species and 4 different ST within the same individual during the course of infection. Susceptibility testing showed that all strains were susceptible to amphotericin B while 3.6% of them had a none-wild type phenotype to 5-flucytosine. Concerning fluconazole, 2.9% of C.neoformans serotype A strains and 2.4% of C. deuterogattii had also respectively a none-wild type phenotype to this molecule. All C. neoformans x C. deneoformans serotype AD hybrids had however a wild type phenotype to fluconazole. The present study showed that mixed infections exist and could be of particular importance for care outcomes. Indeed, (i) the different Cryptococcus species are known to exhibit different virulence and different susceptibility patterns to antifungal drugs and (ii) the strains genetic diversity within the samples may influence the susceptibility to antifungal treatment.

Cryptococcus laurentii diarrhea post hematopoietic stem cell transplant.

We report the recent isolation of Cryptococcus laurentii from the feces of a patient with Hodgkin's lymphoma who underwent autologous hematopoietic stem cell transplant (HSCT). The organism was identified using microscopic morphology, cultural characteristics, and biochemical tests including sugar assimilation. Minimum inhibitory concentration of various antifungals was determined by microbroth dilution method. The recovery of pure culture of C. laurentii from stool culture, and the patient's response to treatment with voriconazole support its potential etiological role. To the best of our knowledge, we report the first case of diarrhea caused by C. laurentii in an HSCT recipient.

Integrated therapy for HIV and cryptococcosis.

Cryptococcosis has been one of the most common opportunistic infections and causes of mortality among HIV-infected patients, especially in resource-limited countries. Cryptococcal meningitis is the most common form of cryptococcosis. Laboratory diagnosis of cryptococcosis includes direct microscopic examination, isolation of Cryptococcus from a clinical specimen, and detection of cryptococcal antigen. Without appropriate treatment, cryptococcosis is fatal. Early diagnosis and treatment is the key to treatment success. Treatment of cryptococcosis consists of three main aspects: antifungal therapy, intracranial pressure management for cryptococcal meningitis, and restoration of immune function with antiretroviral therapy (ART). Optimal integration of these three aspects is crucial to achieving successful treatment and reducing the mortality. Antifungal therapy consists of three phases: induction, consolidation, and maintenance. A combination of two drugs, i.e. amphotericin B plus flucytosine or fluconazole, is preferred in the induction phase. Fluconazole monotherapy is recommended during consolidation and maintenance phases. In cryptococcal meningitis, intracranial pressure rises along with CSF fungal burden and is associated with morbidity and mortality. Aggressive control of intracranial pressure should be done. Management options include therapeutic lumbar puncture, lumbar drain insertion, ventriculostomy, or ventriculoperitoneal shunt. Medical treatment such as corticosteroids, mannitol, and acetazolamide are ineffective and should not be used. ART has proven to have a great impact on survival rates among HIV-infected patients with cryptococcosis. The time to start ART in HIV-infected patients with cryptococcosis has to be deferred until 5 weeks after the start of antifungal therapy. In general, any effective ART regimen is acceptable. Potential drug interactions between antiretroviral agents and amphotericin B, flucytosine, and fluconazole are minimal. Of most potential clinical relevance is the concomitant use of fluconazole and nevirapine. Concomitant use of these two drugs should be cautious, and patients should be monitored closely for nevirapine-associated adverse events, including hepatotoxicity. Overlapping toxicities of antifungal and antiretroviral drugs and immune reconstitution inflammatory syndrome are not uncommon. Early recognition and appropriate management of these consequences can reinforce the successful integrated therapy in HIV-infected patients with cryptococcosis.

Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape.

Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO2 concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce ß-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking.

Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape

Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO₂ concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce β-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking.

Edible oil degradation by using yeast coculture of Rhodotorula pacifica ST3411 and Cryptococcus laurentii ST3412.

To develop a microbial treatment of edible oil-contaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH(4)-N]=240 mg/L) and phosphorus sources ([PO(4)-P]=90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20 degrees C was 39.8%+/-9.9% (means+/-standard deviations of eight replicates). The highest degradation rate was observed at 20 degrees C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671+/-52.0 to 143+/-96.7 ppm in 24 h, and the degradation rate was 79.4%+/-13.8% (means+/-standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells.

Evidence of symbiosis between the soil yeast Cryptococcus laurentii and a sclerophyllous medicinal shrub, Agathosma betulina (Berg.) Pillans.

The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina.

Isolation and characterization of psychrophilic yeasts producing cold-adapted pectinolytic enzymes.

AIMS: The present study was conducted to screen for psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to examine the cold-active pectinolytic enzymes produced by the isolated psychrophilic yeasts. METHODS AND RESULTS: Psychrophilic yeasts, which grow on pectin as a sole carbon source, pectinolytic-psychrophilic yeast (PPY) strains PPY-3, 4, 5 and 6, were isolated from soil from Abashiri (Hokkaido, Japan). The sequences of 28S rDNA D1/D2 of strains PPY-3 and 4 indicated a taxonomic affiliation to Cryptococcus cylindricus and Mrakia frigida, respectively, strains PPY-5 and 6 belonged to Cystofilobasidium capitatum. The isolated strains were able to grow on pectin at below 5 degrees C, and showed the activities of several cold-active pectinolytic enzymes. CONCLUSION: The findings of this study indicate the possibility that the isolated strains produce novel pectinolytic enzymes that are able to degrade pectin compounds at low temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible that the cold-active pectinolytic enzymes from the isolated strains can be applied to the food industry, e.g. the clarification of fruit juice below 5 degrees C.

[Study on the inhibitory activity, in vitro, of baicalein and baicalin against skin fungi and bacteria].

In this paper, we concentrated in examining, in vitro, the antiseptic activity of the baicalein and baicalin upon the seventeen pathogenic skin fungal and sixteen skin bacterial strains, these two flavonic compounds were known principally as the biosubstances of a traditional Chinese medicinal plant: Scutellaria baicalensis. In agar media, the baicalein possessed potent specific activity against the pathogenic yeasts with MICs of 70-100 micrograms/ml; But in the same condition, no inhibitory effect was observed upon dermatophytes and filamentous imperfect fungi for baicalein, and upon all used strains for baicalin. According to the antibacterial test of baicalein, a high efficacy was achieved against certain causative specie of axillary and foot's odour such as Micrococcus sedentarius, Staphylococcus epidermidis, S. hominis and C. xerosis with a MICs inferior to 250 micrograms/ml. The good inhibitory activity of baicalein could be linked to the group hydroxyl (-OH) in position seven of the molecule.

First report of Cryptococcus laurentii meningitis and a fatal case of Cryptococcus albidus cryptococcaemia in AIDS patients.

We report the first case of Cryptococcus laurentii meningitis and a rare case of Cryptococcus albidus cryptococcaemia in AIDS patients. Both infections were treated with amphotericin B and flucytosine. The C. laurentii meningitis was controlled after 2 weeks of treatment with no evidence of infection 20 months later. The patient with C. albidus cryptococcaemia, despite the amphotericin B/flucytosine combination therapy, died on the 14th day of treatment. The minimum inhibitory concentrations (MICs) for C. laurentii, as determined by Etest on RPMI 1640 agar, were 0.25 microg ml(-1) of amphotericin B, 1.25 microg ml(-1) flucytosine, 4 microg ml(-1) fluconazole, 0.50 microg ml(-1) itraconazole and 1.0 microg ml(-1) of ketoconazole. The MIC of amphotericin B for C. albidus was 0.5 microg ml(-1), flucytosine 1.25 microg ml(-1), fluzonazole 4 microg ml(-1), itraconazole 0.5 microg ml(-1) and ketonazole 0.25 microg ml(-1). The agreement of the amphotericin B MIC values obtained in antibiotic medium 3 by the broth microdilution method, with those obtained on casitone medium by Etest, was within a two-dilution range for both isolates. C. laurentii may cause meningitis and may also involve the lungs in AIDS patients.

[Species composition and biochemical properties of yeasts from the water of the Bratsk reservoir]. / Vidovoi sostav i biokhimicheskie svoistva drozhzhei iz vody Bratskogo vodokhranilishcha.

The specific composition of 370 yeast strains isolated from the water of the Bratsk Reservoir was studied. The strains were assigned to 7 genera and 16 species according to their morphological, cultural, and physiologo-biochemical properties. Asporogenous forms prevailed; 52 strains possessed the amylolytic activity. The proteolytic activity was found in the cultures very seldom. The capability to assimilate organic phosphorus compounds was detected in 8% of the strains, and the ability to use inorganic phosphorus compounds was registered in 38% of the strains. Up to 95% of the strains utilized oil; 88%--engine oil; 43%--phenol. The specific yeast composition varied depending on the biotype.

Further simplification of the Guizotia abyssinica seed medium for identification of Cryptococcus neoformans and Cryptococcus bacillispora.

A simplified Guizotia abyssinica seed-based medium for presumptive diagnosis of Cryptococcus neoformans and C. bacillispora (Paliwal and Randhawa 1978) was further simplified by replacing seed extract with pulverized seeds. This medium gives unambiguous results, avoids false-positive reactions with 13 other yeastlike organisms, and is simple and relatively inexpensive to prepare.

Improvement in laboratory diagnosis of pulmonary cryptococcosis.

A new differential colour medium prepared with an extract of potatoes and carrots produced ochre-brown pigmentation in Cryptococcus neoformans colonies, identical to that obtained with Guizotia abyssinica seed extract. The pigment formation was observed to be specific for this pathogenic yeast in a survey of 534 yeast-positive specimens during routine diagnostic work; among 47 C. neoformans-positive specimens were 27 sputa and bronchial aspirations, many overgrown by common yeasts.Detection of C. neoformans in the contaminated specimens was credited largely to the differential medium used. Twelve cases of cryptococcosis were investigated between 1962 and 1968, five of which presented involvement of the respiratory tract only.