Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intraand interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.

Debaryomyces hansenii is a hemiascomycetous yeast commonly found in natural substrates and in various types of cheese. Pichia guilliermondii is widely distributed in nature and is a common constituent of the normal human microflora. Both species have been described in human infections but are extremely difficult to differentiate phenotypically. Thus, frequent errors in identification occur. The 62 clinical and environmental isolates sent between 2000 and 2007 to the French National Reference Center for Mycoses and Antifungals as D. hansenii or P. guilliermondii were analyzed by using the carbon assimilation pattern, the presence of pseudohyphae, and sequencing of the ITS and D1/D2 regions of the rRNA gene. The objective of this study was to assess using nucleotide sequences whether phenotypic identification was accurate and whether phenotypic characteristics could be used to differentiate the two species when sequencing was not available. We found that 58% of the isolates were misidentified and belong to seven different species: P. guilliermondii , P. caribbica , P. jadinii , D. hansenii , Candida palmioleophila , C. haemulonii type II, and Clavispora lusitaniae . In conclusion, D. hansenii may not be as common a human pathogen as previously thought. Sequencing of either ITS or D1/D2 regions is a good tool for differentiating the species more frequently confused with D. hansenii , keeping in mind that reliable databases should be used.

Thirty-eight isolates (including 28 isolates from patients) morphologically identified as Lichtheimia corymbifera (formerly Absidia corymbifera) were studied by sequence analysis (analysis of the internal transcribed spacer [ITS] region of the ribosomal DNA, the D1-D2 region of 28S, and a portion of the elongation factor 1␣ [EF-1␣] gene). Phenotypic characteristics, including morphology, antifungal susceptibility, and carbohydrate assimilation, were also determined. Analysis of the three loci uncovered two well-delimited clades. The maximum sequence similarity values between isolates from both clades were 66, 95, and 93% for the ITS, 28S, and EF-1␣ loci, respectively, with differences in the lengths of the ITS sequences being detected (763 to 770 bp for isolates of clade 1 versus 841 to 865 bp for isolates of clade 2). Morphologically, the shapes and the sizes of the sporangiospores were significantly different among the isolates from both clades. On the basis of the molecular and morphological data, we considered isolates of clade 2 to belong to a different species named Lichtheimia ramosa because reference strains CBS 269.65 and CBS 270.65 (which initially belonged to Absidia ramosa) clustered within this clade. As neotype A. corymbifera strain CBS 429.75 belongs to clade 1, the name L. corymbifera was conserved for clade 1 isolates. Of note, the amphotericin B MICs were significantly lower for L. ramosa than for L. corymbifera (P < 0.005) but were always <0.5 g/ml for both species. Among the isolates tested, the assimilation of melezitose was positive for 67% of the L. ramosa isolates and negative for all L. corymbifera isolates. In conclusion, this study reveals that two Lichtheimia species are commonly associated with mucormycosis in humans.Mucormycosis is a life-threatening infection that occurs in immunocompromised patients, diabetic patients with ketoacidosis, and immunocompetent patients after trauma exposure to contaminated soil (7, 18). The filamentous fungi responsible for these infections belong to the Mucorales order. About 20 different species have been shown to be pathogenic for humans (4). According to a recent review (19), the species that were the most frequent encountered were Rhizopus spp., Mucor spp., and Cunninghamella spp., while Apophysomyces elegans and Absidia spp. accounted for 6% and 5% of the cases, respectively. The true frequency is, however, difficult to assess because surveys are rare and determination of the species of the Zygomycetes class by standard mycological methods remains difficult. Indeed, all the genera and species within the family Mucoraceae (the Absidia, Rhizopus, Mucor, Rhizomucor, and Apophysomyces genera) shared similar morphological characteristics (6). The precise identification to the species level often requires the specific expertise usually available only at reference laboratories. The availability of molecular tools for taxonomic and identification purposes has changed the picture. Sequencing of various DNA targets has facilitated the recognition of phylo...

Zanthoxylum chiloperone var. angustifolium was investigated. Alkaloids 1-3 from the canthin-6-one series were characterized. Derivatives 7-28 were prepared by hemisynthesis or total synthesis. All compounds were tested for in vitro antifungal activities against five pathogenic fungal strains. Analogues of canthin-6-one did not show better antifungal activities.

Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2, MTI, and ERG3, in order to obtain a rapid genotyping method for C. glabrata. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2, MTI, and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.Epidemiological surveillance of candidemia shows that although Candida albicans remains the main species, Candida glabrata is the second leading species recovered from blood cultures in Europe and in the United States (20, 28), especially in intensive care units (9). The same phenomenon is observed for recurrent vaginitis (25). Concern arises about this increase in the incidence of C. glabrata infections because of its frequent decreased sensitivity to azoles compared to C. albicans (14,22) and the high mortality rate of bloodstream infections (28,29).Investigations for C. glabrata have not been as extensive as for C. albicans. The epidemiology is thought to be similar to C. albicans (10). However, few tools are available to study the source of contamination, cross-contamination of patients at risk, and the biology of this yeast, which has, unlike C. albicans, a haploid genome (10). Studies of population structure have begun to emerge using enzymatic and randomly amplified polymorphic DNA typing (6) or sequencing of the cytochrome c oxidase subunit 2 gene (24). Recently, a technique based on the sequencing of six variable-locus genes, or multilocus sequence typing (MLST), has been proposed as a powerful typing method (7). Microsatellites represent another class of genetic markers, defined as short tandem repeats of two to six nucleotides, known to be highly polymorphic (30). Primarily developed for human genetics, they are used for fungi (15) and more specifically for pathogenic fungi such as C. albicans (3-5, 19, 23, 26) and Aspergillus fumigatus (1, 2, 16, 17).We descri...

The objective of this study was to investigate the chronobiology and infectivity of the gametocytes of Plasmodium chabaudi chabaudi. In order to increase the production of gametocytes, mice were treated with phenylhydrazine to induce a hyper-reticulocytosis. The authors observed an important stimulation of gametocytogenesis. Gametocytes were seen as soon as the second day postinoculation and were produced periodically at each schizogony, every 24 hr. The gametocytic developmental cycle lasted 60 hr and consisted of 4 successive stages: stage 0 at 36 hr, from merozoite invasion, stage I at 42 hr, stage II at 48 hr, and stage III at 54 hr. An important fraction of stage II was sequestered in small peripheral capillaries. The numbers of oocysts in the mosquitoes fed on phenylhydrazine-treated mice were larger than in controls. When mosquitoes were fed at different times of the day, circadian differences in the oocyst counts were not statistically significant. However, stage II was considered to be probably the most infective stage because, like the infective gametocyte stage of other species of murine malaria, it is sequestered in the peripheral capillaries. In contrast with Plasmodium vinckei, there is no peak of infectivity at the time of sequestration of the infective stage; this is probably due to the inhibitory effect of the schizogony occurring at this time.

No abstract

The activities of various compounds isolated from plants traditionally used against leishmaniasis in populations from the tablelands of the Guyanas have been examined. The leishmanicidal activity of plant extracts was evaluated by in uitro testing on promastigote and amastigote stages of Leishmania amazonensis and by in uiuo tests on L. amazonensis in mice. The leaves of Jacaranda copaia (Aublet) D. Don yielded two compounds: ursolic acid and jaracanone. Ursolic acid showed an interesting activity in uitro with an ED-against amastigotes of 0.02mM and no toxicity to macrophages at twice this dose. Jacaranone displayed a marked activity against promastigotes in uitro with an ED-of 0.02 mM. Both compounds have weak in uiuo antileishmanial activity. Similar synthetic compounds such as quinol and quinone acetates were prepared and showed increased activity in experimental cutaneous leishmaniasis in mice.