Raquel Vilela

Pythium insidiosum: An overview

Pythium insidiosum is an oomycete pathogenic in mammals. The infection occurs mainly in tropical and subtropical areas, particularly in horses, dogs and humans. Infection is acquired through small wounds via contact with water that contains motile zoospores or other propagules (zoospores or hyphae). The disease, though described as emerging has in fact already been described since 1884. Depending on the site of entry, infection can lead to different forms of pythiosis i.e. a cutaneous, vascular, ocular, gastrointestinal and a systemic form, which is rarely seen. The infection is not contagious; no animal-animal or animal-human transmission has been reported so far. Therapy includes radical surgery, antifungal drugs, immunotherapy or a combination of these therapies. The prevention to contract the disease in endemic areas is difficult. Avoiding stagnant waters could be of help, although the presence of P. insidiosum on grass and soil in enzootic areas renders this practice useless.

Molecular Model for Studying the Uncultivated Fungal Pathogen Lacazia loboi

ABSTRACT Lacazia loboi is an uncultivated fungal pathogen of humans and dolphins that causes cutaneous and subcutaneous infections only in the tropical areas of the Americas. It was recently found by phylogenetic analysis that this unusual pathogen is closely related to Paracoccidioides brasiliensis and to the other fungal dimorphic members of the order Onygenales. That original phylogenetic study used universal primers to amplify well-known genes. However, this approach cannot be applied to the study of other proteins. We have developed a strategy for studying the gene encoding the gp43 homologous protein of P. brasiliensis in L. loboi. The gp43 protein was selected because it has been found that this P. brasiliensis antigen strongly reacts when it is used to test sera from patients with lacaziosis. The principle behind this idea was to obtain the gp43 amino acid sequence of P. brasiliensis and other homologous fungal sequences from GenBank and design primers from their aligned conserved regions. These sets of primers were used to amplify the selected regions with genomic DNA extracted from the yeast-like cells of L. loboi from experimentally infected mice. Using this approach, we amplified 483 bp of the L. loboi gp43-like gene. These sequences had 85% identity at the nucleotide level and 75% identity with the deduced amino acid sequences of the P. brasiliensis gp43 protein. The identity of the 483-bp DNA fragment was confirmed by phylogenetic analysis. This analysis revealed that the L. loboi gp43-like deduced amino acid sequence formed a strongly supported (100%) sister group with several P. brasiliensis gp43 sequences and that this taxon in turn was linked to the other fungal sequences used in this analysis. This study shows that the use of a molecular model for investigation of the genes encoding important proteins in L. loboi is feasible.

Morphologic and Phylogenetic Characterization of Conidiobolus lamprauges Recovered from Infected Sheep

ABSTRACT Conidiobolus lamprauges, a soil and plant entomophthoralean fungus, has been reported only in a horse and, more recently, in sheep with rhinopharyngeal entomophthoramycosis. Thus, little information is available to enable proper identification of this pathogen and its differentiation from other saprotrophic and pathogenic Conidiobolus species. Using classical mycological tools and molecular methodologies, we report for the first time the taxonomic and phylogenetic description of three C. lamprauges isolates recovered from sheep with rhinopharyngeal entomophthoramycosis. The distinctive clinical and pathological features of C. lamprauges are compared with those of other Conidiobolus spp. affecting sheep, as well as with those of the stramenopilan ovine agent Pythium insidiosum. The comparative morphological attributes of Conidiobolus spp. are also diagramed; along with the sequence data generated, they should assist laboratories in the identification of these uncommon species.

The Mammalian Pathogenic Oomycetes

The oomycetes are fungal-like microbes similar to those found within some members of the kingdom Fungi. Although these two groups of microbes share morphological features, there are several contrasting differences: a) phylogenetic analysis placed the oomycetes basal to plants and green algae; b) oomycetes lack ergosterol in their cytoplasmic membrane; c) chitin is not the main compound in the cell wall of oomycetes; and d) asexual reproduction in the oomycetes occurs by the development of sporangia containing numerous biflagellate zoospores. Pythium insidiosum was considered to be the only oomycete pathogenic for mammals. However, in 1999, Grooters reported that several dogs were diagnosed with an unusual oomycete in the genus Lagenidium causing extensive cutaneous and subcutaneous infections. Thereafter, the infection has been also reported in humans and cats, and it could possibly affect other mammalian species as well. This review highlights the epidemiological, clinical and pathological features, as well as the diagnosis and management of the infections caused by this unique group of mammalian pathogenic oomycetes.

Outbreaks of rhinofacial and rhinopharyngeal zygomycosis in sheep in Paraíba, northeastern Brazil

Dois surtos de lesoes rinofaciais e dois de lesoes rinofaringeas causadas por fungos com hifas filamentosas, raramente septadas, semelhantes as dos fungos entomophthorales, sao descritos no Estado da Paraiba. Um surto da forma rinofacial afetou 5 ovinos durante a estacao chuvosa. Outro surto desta forma clinica afetou um ovino de um grupo de 40, durante a epoca seca. Os sinais clinicos da forma rinofacial foram de corrimento serosanguinolento bilateral, com aumento de volume das narinas, labio superior, e pele da face. Na necropsia, a mucosa nasal apresentava areas ulceradas de cor marrom escuro, que se estendiam desde a regiao muco-cutanea ate 10cm dentro do vestibulo nasal. A mucosa do palato duro estava, tambem, ulcerada. A superficie de corte das narinas e palato apresentava-se marrom ou avermelhada de aspecto esponjoso e friavel. Um surto de rinofaringite micotica ocorreu em uma plantacao de coco irrigado, afetando 7 ovinos de um rebanho de 60 ovinos adultos. Outro surto desta forma da enfermidade afetou um unico animal de um rebanho de 80, durante a epoca seca. Os animais apresentavam dispneia, com respiracao ruidosa devida ao bloqueio parcial das narinas, e corrimento nasal serosanguinolento. Seis dos oito animais afetados por esta forma da enfermidade apresentaram, em um olho, exoftalmia, ceratite e ulceracao da cornea. Todos os ovinos morreram ou foram sacrificados apos um curso clinico de 7-30 dias. Na necropsia exsudato amarelo consistente foi observado na regiao etmoidal, ossos turbinados, seios paranasais, palatos duro e mole, orbita, faringe, e musculos e linfonodos regionais. No estudo histologico, ambas as formas da doenca mostraram granulomas multifocais com areas necroticas eosinofilicas (material de Splendore-Hoeppli) contendo hifas com 7-30mm de diâmetro, raramente septadas, semelhantes as dos fungos entomophthorales, possivelmente Conidiobolus spp. Surtos de ambas as formas de rinite micotica sao frequentes na regiao semi-arida e em outras regioes do Brasil.

Cutaneous and meningeal sporotrichosis in a HIV patient

A male patient with HIV and past history of tuberculosis and suspected neurotoxoplasmosis was admitted to the hospital with vomiting and small nodules through all his body. Few of the nodules were found forming chains of enlarged lymphatic vessels, especially on lesions located on the limbs. Some of the nodules were ulcerated with a serosanguineous discharge. Collected samples from ulcerated and the nodular lesions showed the presence of Sporothrix schenckii in culture. Although all hemocultures were negative, a spinal fluid collected from this patient and cultures from the cutaneous lesions were both positive for S. schenckii. The patient showed improvement after treatment with Amphotericin B. Sadly, he later died of complications not related to the S. schenckii infection. This case of disseminated sporotrichosis is a remainder that in patients with immunological disorders exotic forms of this fungal clinical entity could be expected.

Molecular phylogeny of animal pathogen Lacazia loboi inferred from rDNA and DNA coding sequences

Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.

Use of Sera from Humans and Dolphins with Lacaziosis and Sera from Experimentally Infected Mice for Western Blot Analyses of Lacazia loboi Antigens

ABSTRACT Antibodies in the sera of patients with lacaziosis recognized an ∼193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.

The taxonomy and phylogenetics of the human and animal pathogen Rhinosporidium seeberi: A critical review

Rhinosporidum seeberi is the etiologic agent of rhinosporidiosis, a disease of mucous membranes and infrequent of the skin and other tissues of humans and animals. Because it resists culture, for more than 100 years true taxonomic identity of R. seeberi has been controversial. Three hypotheses in a long list of related views have been recently introduced: 1) a prokaryote cyanobacterium in the genus Microcystis is the etiologic agent of rhinosporidiosis, 2) R. seeberi is a eukaryote pathogen in the Mesomycetozoa and 3) R. seeberi is a fungus. The reviewed literature on the electron microscopic, the histopathological and more recently the data from several molecular studies strongly support the view that R. seeberi is a eukaryote pathogen, but not a fungus. The suggested morphological resemblance of R. seeberi with the genera Microcystis (bacteria), Synchytrium and Colletotrichum (fungi) by different teams is merely hypothetical and lacked the scientific rigor needed to validate the proposed systems. A fundamental aspect against the prokaryote theory is the presence of nuclei reported by numerous authors and updated in this review. Moreover, Microcystiss and Synchytriums ultra-structural and key cell cycle traits cannot be found in R. seeberi parasitic phase. The PCR amplification of a cyanobacteria 16S rDNA sequence from cases of rhinosporidiosis, while intriguing, will be viewed here as an anomaly due to contamination with environmental Microcystis or perhaps as an endosymbiotic acquisition of plastids from cyanobacteria ancestors. Thus, even if R. seeberi possesses prokaryote DNA, this does not prove that R. seeberi is a cyanobacterium. The placement of R. seeberi within the fungi is scientifically untenable. The isolation and the DNA analysis performed in a fungal strain, and the lack of appropriate controls are the main problems of this claim. Further studies are needed to validate R. seeberis acquisition of prokaryote plastids and other issues that still need careful scrutiny.

Accidental Jorge Lobo's disease in a worker dealing with Lacazia loboi infected mice: a case report

IntroductionJorge Lobos disease (Lacaziosis) is a subcutaneous infection of humans living in the Amazon region of Latin America, and in dolphins inhabiting the east coastal areas of the United States. The disease mainly affects people from rural areas living or working in close contact with vegetation and aquatic environments. Most patients refer having developed lesions after accidental trauma with plant thorns or insect bites. Inter-human transmission has never been confirmed suggesting that Lacazia loboi is acquired from environmental propagules.Case presentationWe report the case of a 41-year-old woman from São Paulo, Brazil, a non-endemic area of Jorge Lobos disease, with L. loboi skin infection most likely accidentally acquired while manipulating experimentally infected mice in the laboratory.ConclusionBecause many patients with Jorge Lobos disease do not recall accidental skin trauma before their infections, the possibility of accidentally acquired Jorge Lobos disease through unnoticed broken skin should be considered during the clinical investigation of nodular skin diseases in people who have contact with the fungus or who live in endemic areas. This is the second report of animal to human transmission of this disease.