Several serological tests have been used successfully in the diagnosis of paracoccidioidomycosis (PCM). In contrast, data about the use of these tests in the follow-up of PCM patients have been heterogeneous. In this study, serum samples from 43 PCM patients with different clinical forms were analysed by counter-immuno-electrophoresis (CIE), complement fixation (CF) and ELISA before treatment. With CIE and ELISA, the chronic unifocal form showed significantly lower antibody levels compared with chronic multifocal and acute forms. Acute form patients had significantly higher titres than patients with multifocal disease by CIE but not by ELISA. No significant differences were observed with CF. Twenty-seven of these patients were followed-up for 2 years and showed a decline in antibody levels by all three tests, paralleling clinical improvement. However, only patients with unifocal disease cleared their antibodies after 1 year of treatment as analysed by CF and ELISA and after 2 years by CIE, suggesting that these patients may need shorter courses of therapy. Patients with the other clinical form of the disease needed > or =2 years of therapy to clear their antibodies. Sera from a further five patients who presented with a relapse were analysed. At the time of relapse all showed increases in antibody levels by CIE and ELISA, but only three showed increases by CF tests. Therefore, CIE and ELISA demonstrated a better clinical correlation than CF, probably reflecting the fungal burden of PCM patients more accurately.

The pathogen of frogs Amphibiocystidium ranae was recently described as a new genus. Due to its spherical shape, containing hundred of endospores, it was thought to be closely related to the pathogens of fish, mammals, and birds known as Dermocystidium spp., Rhinosporidium seeberi, and Sphaerothecum destruens in the Mesomycetozoea, but further studies were not conducted to confirm this relationship. To investigate its phylogenetic affinities, total genomic DNA was extracted from samples collected from infected frogs containing multiple cysts (sporangia) and endospores. The universal primers NS1 and NS8, used to amplify the 18S small-subunit rRNA by PCR, yielded Ϸ1,770-bp amplicons. Sequencing and basic local alignment search tool analyses indicated that the 18S small-subunit rRNA of A. ranae from both Rana esculenta and Rana lessonae was closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus Dermocystidium and closely related to Rhinosporidium. These data strongly supported the placement of the genus Amphibiocystidium within the mesomycetozoeans, which is in agreement with the phenotypic features that A. ranae shares with the other members of this class. Interestingly, during this study Dermocystidium percae did not group within the Dermocystidium spp. from fish; rather, it was found to be the sister group to Sphaerothecum destruens. This finding suggests that D. percae could well be a member of the genus Sphaerothecum or perhaps represents a new genus.In Italy, Rana esculenta complex water frogs constitute mixed populations of a nonhybrid taxon and hemiclonally reproducing hybrids that are directly analogous to the well-studied central European Rana lessonae/Rana esculenta systems (3,7,16,17). Since 1999, a significantly high incidence of Amphibiocystidium ranae was observed in the parental species, whose frequency has decreased (50%) relative to the hybrid Rana esculenta (12). The skin lesions were observed as small regular hemispherical elevations between 3 and 5 mm in diameter that in some cases became ulcerated. The elevations were observed as single or multiple skin lesions on the infected frogs. Histopathologically, those studies reported several ovoid, U-shape, and/or spherical cysts (sporangia in some mesomycetozoeans) of 100 to 600 m in diameter, containing 2-to 6-m-diameter endospores (2). In the vicinity of these cysts, an inflammatory infiltrated composed by lymphocytes, macrophages, and other leukocytes was always present (2, 9, 12).The phenotypic features of Dermocystidium ranae were recently determined from samples collected in a population of Rana esculenta in central Italy (12). Based on the ultrastructural characteristics of this spherical pathogen, it was found that the so-called Dermocystidium specie in frogs have some features not found in its homologous pathogens of fish, both of which were for a long time classified in the genus Dermocystidium. Thus, the epithet Amphibiocystidium ranae was introduced (12). Th...

The taxonomic relationship of Rhinosporidium seeberi with other organisms remained controversial for over a century. Recently, molecular studies have shown R. seeberi to be a protistal microbe in the newly described class Mesomycetozoea at the animal-fungal boundary. Phylogenetic analyses of R. seeberi using 18S smallsubunit (SSU) rRNA genes from several hosts suggested Rhinosporidium as a monotypic genus. To test this hypothesis, the internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 from eight humans, two swans, and a dog with rhinosporidiosis were sequenced. The ITS regions were amplified by PCR using a primer designed from a unique region of R. seeberi's 18S SSU rRNA genes in combination with the ITS4 universal primer. In addition, the universal ITS4 and ITS5 primers were also used. R. seeberi's ITS sequences showed differences in the numbers of nucleotides among strains. For instance, the eight human ITS sequences were uniformly similar with only a few mismatches and ϳ1,060 bp long. In contrast, sequences from one of the swans and the dog were 1,356 bp and ϳ1,147 bp long, respectively. Clustal analysis of all of the ITS sequences showed multiple 50-to 60-bp gaps and several mismatches among them. Parsimony analysis placed the Rhinosporidium ITS sequences in three well-supported sister groups according to the hosts' identities. This analysis strongly suggests that the genus Rhinosporidium may possess multiple host-specific strains. No correlation was found between this finding and the phenotypic features of R. seeberi in the studied samples.Rhinosporidiosis is a cutaneous and/or subcutaneous chronic disease of human and other animals caused by Rhinosporidium seeberi (2,(16)(17)(18). This granulomatous disease is characterized by the development of polyps primarily affecting the mucous membranes of the nostrils and the ocular conjunctivae of the infected hosts. Diagnosis is essentially based on the histological detection in tissues of R. seeberi's pathognomonic endosporulating sporangia in various stages of development. Rhinosporidiosis in not a life-threatening disease, and its treatment usually is limited to the surgical removal of the polyps (1, 4, 11).For the last 100 years, the taxonomic affinities of R. seeberi remained obscure, principally because this unique pathogen cannot be cultured. Thus, most of its epidemiological and taxonomical characteristics were not well understood. In 1999, a team from the United States and Sri Lanka (10) using molecular tools found that R. seeberi was phylogenetically related to a novel group of protistan microbes at the divergence between animals and fungi. This finding was soon corroborated by others (8). These microbes are presently classified in the protistal class Mesomycetozoeae. R. seeberi shares several characteristics with the other members of this class; all are aquatic microbes with spherical structures containing endospores. Most of them are unculturable and were, at one point, classified as members of the fungal or protistan kingdoms.The studies of Herr et ...

Until recently, accurate microbiological diagnosis of invasive aspergillosis (IA) was seldom established in HSCT recipients. Blood samples are rarely positive for Aspergillus species, the reliability of the cultures depends of the specimen (if taken from a normally sterile site or not) and biopsy samples require invasive procedures, rarely recommended in patients with severe thrombocytopenia. Implementing the international consensus defining the microbiological criteria for the diagnosis of Aspergillus infection, we retrospectively evaluated the role of serum galactomannan (GM) detection by EIA to diagnose IA among HSCT patients with proven invasive fungal infection (IFI) and the impact of serum storage in GM concentrations. The EIA assay allowed categorizing as "probable" 5 of the 10 cases of "possible" aspergillosis (50%). Considering a lower cut-off level for the reaction (1.0), 80% of the cases could be categorized as "probable" aspergillosis. Positive or undetermined results were detected one to 4 months before the diagnosis of IA in eight of the 11 patients (72.7%) with proven IFI. Retesting the stored samples after a second storage for four years, we could observe lower reactivity in 20% of the samples. The detection of galactomannan by the EIA test represents a major advance in the diagnosis of IA in HSCT recipients at high risk of IA. A better understanding of the kinetics of the GM in different clinical situations is necessary to maximize the benefit of the test in Aspergillus surveillance.

Lacazia loboi, the aetiological agent of lacaziosis (Jorge Lobo's disease), is an uncultivated anomalous fungal microbe closely related to Paracoccidioides brasiliensis, both restricted Latin American Pathogens. Early reports suggesting that L. loboi had been isolated in pure culture from cases of lacaziosis, only added more confusion to the already confusing aetiology of this disease. These strains were later identified as unusual contaminants and some of them as P. brasiliensis. Recent phylogenetic analysis grouped L. loboi as the sister taxon to P. brasiliensis, thus it was postulated that the original P. brasiliensis strains recovered from cases of lacaziosis, could be the aetiological agent of the disease. Using molecular methodologies, we investigated the archival P. brasiliensis isolate No. 525 from a case of lacaziosis, as well as other archival isolates, identified earlier as common contaminants, all recovered from similar cases of the disease. Phylogenetic analysis, using the 18S small subunit rDNA sequences of these isolates showed that strain No. 525 was a typical P. brasiliensis isolate and the other studied strains were indeed contaminants. This study unequivocally indicates that the aetiological agent of lacaziosis is yet to be cultured.