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Early phylogenetic analysis of Pythium insidiosum, the etiologic agent of pythiosis in mammals, showed the presence of a complex comprising three monophyletic clusters. Two included isolates recovered from cases of pythiosis in the Americas (Cluster I) and Asia (Cluster II), whereas the third cluster included four diverged isolates three from humans in Thailand and the USA, and one isolate from a USA spectacled bear (Cluster III). Thereafter, several phylogenetic analyses confirmed the presence of at least three monophyletic clusters, with most isolates placed in clusters I and II. Recent phylogenetic analyses using isolates from environmental sources and from human cases in India, Spain, Thailand, and dogs in the USA, however, showed the presence of two monophyletic groups each holding two sub-clusters. These studies revealed that P. insidiosum possesses different phylogenetic patterns to that described by early investigators. In this study, phylogenetic, population genetic and protein MALDI-TOF analyses of the P. insidiosum isolates in our culture collection, as well as those available in the database, showed members in the proposed cluster III and IV are phylogenetically different from that in clusters I and II. Our analyses of the complex showed a novel group holding two sub-clusters the USA (Cluster III) and the other from different world regions (Cluster IV). The data showed the original P. insidiosum cluster III is a cryptic novel species, now identified as P. periculosum. The finding of a novel species within P. insidiosum complex has direct implications in the epidemiology, diagnosis, and management of pythiosis in mammalian hosts.

Pythiosis is frequently reported in dogs and horses inhabiting tropical, subtropical and temperate areas of the USA, but the disease is rare in domestic cats. The clinical presentation of feline pythiosis includes subcutaneous masses without ulcerated tissue and involvement of the intestinal tract. Here in we report an eight-week-old female unvaccinated stray kitten with an unusual extensive circular ulcerated lesion on her left flank. The lesion did not respond favorably to administration of systemic antibiotics. Clinical specimens submitted for culture demonstrated submerged fungal-like flat colonies later identified as Pythium insidiosum, a finding also confirmed by histopathology, serology, and DNA sequencing and thus, treated with itraconazole. Since no improvement was observed, Pythium-immunotherapy was initiated. The cat responded to the latter approach and in less than 10 days, the lesion had contracted around the edges and was crusting off to reveal healthy granulation tissue. Twenty-three days after immunotherapy was initiated the original wound had shrunken significantly to a small scabbed area. However, the cat acutely developed tachypnea, lung and intestinal complications and due to her rapid deterioration, humane euthanasia was elected. Unfortunately, necropsy was not conducted. The clinical presentation reported here suggests large ulcerative cutaneous lesions caused by P. insidiosum can also occur in domestic cats. Despite reports of unsuccessful treatment results of feline pythiosis using Pythium-immunotherapy, this report suggests this approach might be helpful in similar feline cases. Keywords: Feline, Cat, Pythiosis, Pythium insidiosum, Oomycota

OBJECTIVE To describe the geographic distribution of infections caused by Pythium insidiosum in dogs, horses, and other animal species in the US. ANIMALS For the last 20 years, we have collected data from cases of pythiosis in 1,150 horses, 467 dogs, and other species (59) from various geographic locations in the US. PROCEDURES Due to lost data (from 2006 to 2016), the selected cases include years 2000 to 2005 and 2016 to 2020. The selection of cases was based on infected host clinical features, serum samples demonstrating strong positive anti–P insidiosum IgG titers in serologic assays, and positive results on ≥ 1 of the following diagnostic modalities: microbial culture on 2% Sabouraud dextrose agar, histologic evaluation, PCR assay, and wet mount cytologic evaluation (with potassium hydroxide). RESULTS Most confirmed P insidiosum infections were found in horses and dogs in the southeastern US. Interestingly, in Texas, no cases were found west of longitude 100°W. Few pythiosis cases were diagnosed in west-coast states. Equine cases were more often diagnosed during summer and fall months, but canine cases were more often diagnosed between September and February. Cases in other species were discovered in the same geographic areas as those in dogs and horses. CLINICAL RELEVANCE To our knowledge, this is the first report providing the ecological distribution of P insidiosum infection in affected species in the US. Results of this study illustrated the importance of including P insidiosum in the differential diagnostic scheme of nonhealing skin lesions or intestinal granulomatous masses, particularly in dogs and horses inhabiting or having visited endemic areas.

Ever since the uncultivated South American fungal pathogen Lacazia loboi was first described 90 years ago, its etiology and evolutionary traits have been at the center of endless controversies. This pathogen infects the skin of humans and as long believed, dolphin skin. However, recent DNA analyses of infected dolphins placed its DNA sequences within Paracoccidioides species. This came as a surprise and suggested the human and dolphin pathogens may be different species. In this study, population genetic analyses of DNA from four infected dolphins grouped this pathogen in a monophyletic cluster sister to P. americana and to the other Paracoccidioides species. Based on the results we have emended the taxonomy of the dolphin pathogen as Paracoccidioides cetii and P. loboi the one infecting human. Our data warn that phylogenetic analysis of available taxa without the inclusion of unusual members may provide incomplete information for the accurate classification of anomalous species.

1. Diana Bordalo, MD* 2. Raquel Oliveira, MD† 3. Paula Fonseca, MD*,‡ 4. Teresa Temudo, PhD§ 1. *Department of Pediatrics, 2. ‡Adolescent Medicine Unit, Unidade de Vila Nova de Famalicao, Vila Nova de Famalicao, Portugal; 3. †Department of Pediatrics, Unidade Local de Saude do Alto Minho, Viana do Castelo, Portugal; 4. §Neuropediatric Unit, Department of Pediatrics–Centro Materno-Infantil do Norte, Centro Hospitalar do Porto, E.P.E. Porto, Portugal A 16-year-old girl presents to the pediatric emergency department after an episode of loss of consciousness, preceded by dizziness and followed by a generalized tonic-clonic seizure that lasted ∼10 minutes, with spontaneous recovery. She reports 2 previous episodes of loss of consciousness, one not witnessed, the other interpreted as a vasovagal syncope. Family history is positive for epilepsy in a great-grandmother. She has been otherwise previously healthy. Upon physical assessment, she is hemodynamically stable, nonfebrile, and has a normal neurological examination. Initial laboratory findings are normal, including urine toxicology screening, serum electrolytes, and inflammatory markers. An electrocardiography and an electroencephalography (EEG) performed 24 hours after the seizure are normal. Continuous ambulatory electrocardiography monitoring is normal. Due to the atypical seizure presentation with unwitnessed episodes, a tilt table test and a brain magnetic resonance imaging are performed, both reported as normal. During the follow-up period, the patient has recurrent seizures, and notes that these episodes follow a monthly pattern, always on the third day of the menstrual cycle. She had her menarche at ∼12 years old with irregular periods for the first 2.5 to 3 years, during which time she was seizure-free. She currently experiences some …

We studied phylogenetic and taxonomic features of isolates identified as Lagenidium giganteum recovered from six different species of mosquito larvae. The isolates grew vigorously at 25 C, moderately at 30 C, and not at all at 37 C and developed submerged, white colonies with few short, hyaline, aerial hyphae. Cultures displayed phenotypic plasticity, with broad, hyaline hyphae strongly constricted at septa that developed oval, spherical, or amorphous segments. These developed into sporangia producing one or two exit tubes, from which evanescent gelatinous vesicles containing zoospores developed. Three isolates developed oogonia consistent with features previously described for L. giganteum. Phylogenetic analysis of nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS = ITS) and cytochrome c oxidase subunit II (COXII) sequences of L. giganteum consistently grouped into eight clusters. Four of the investigated isolates grouped with sequences of an unnamed Lagenidium species infecting nematodes. Based on phenotypic and phylogenetic data, we describe the latter isolates as L. juracyae, sp. nov. In addition, we also investigated a species of Paralagenidium from a dog with lagenidiosis and describe it as new, Paralagenidium ajellopsis, sp. nov.

Ichthyophonus infection was first detected in Peruvian Oncorhynchus mykiss in 1986, but the occurrence of ichthyophonosis disease in the region is unknown. This study investigated the presence and distribution of Ichthyophonus sp. in Peruvian rainbow trout using traditional and DNA sequencing tools. Between 2007 and 2008, 205 rainbow trout from 13 hatcheries in the Mantaro river basin were examined for the presence of Ichthyophonus, and at that time only 3 farms were positive. This early study confirmed the presence of Ichthyophonus sp. in the Tranca Grande lagoon for the first time, at a prevalence of 50%. In 2012, examination of 240 trout from 24 fish farms in 2 Peruvian Departments found 9 infected farms. More recently, in 2018, Ichthyophonus sp. was found in Lake Titicaca, infecting a trout in the Ichu area (in the Department of Puno). Our molecular analysis of the infected trout showed that ichthyophonosis disease in the Peruvian trout was caused by Ichthyophonus sp. Clade C. The finding of this pathogen in Lake Titicaca should be an alert for nearby farms and entities dealing with fish of economic importance in the rivers of Peru.

Early diagnosis of SARS-CoV-2 is essential to limiting the spread of the virus and managing infected patients during hospitalization. The sensitivity of RT-qPCR is contested by the fact that it is time-consuming, executed by trained technicians in proper environment for material extraction. Here, we evaluated the first SARS-CoV-2 antigen test recommended by the World Health Organization at September, 2020 as an alternative for immediate diagnosis of symptomatic and suspected patients at a hospital in Brazil during the epidemic peak. All patients were submitted to RT-qPCR and rapid antigen test using nasopharyngeal swabs rigorously collected at the same time. Demographics, baseline comorbidities, symptoms and outcomes were considered. Prediction analysis revealed that previous stroke, chronic obstructive pulmonary disease, desaturation and tachypnea were the most relevant determinants of the death of COVID-19 patients. Comparison between the rapid antigen test and RT-qPCR revealed an overall PPV of 97%, extended to 100% when performed between 4 and 15 days of symptoms, with an accuracy of 90-91% from days 1 to 7 and a ‘Substantial’ agreement. The rapid antigen test presented no inconclusive result. Among the discordant results and RT-qPCR inconclusives, 72% presented bilateral multifocal ground-glass opacities on imaging and other exams alterations. The median time to obtain RT-qPCR results was 83.6 hours, against 15 minutes for the rapid test, precious time for deciding on patient isolation and management. Knowledge of the risk factors and a rapid diagnosis upon patient admission is critical to reduce mortality of COVID-19 patients, hospital internal costs and in-hospital transmission.

Aim: To enhance the tretinoin (TRE) safety profile through the encapsulation in nanostructured lipid carriers (NLC). Materials & methods: NLC-TRE was developed using a 23 experimental factorial design, characterized (HPLC, dynamic light scattering, differential scanning calorimetry, x-ray diffraction analysis, transmission electron microscopy, cryo-transmission electron microscopy) and evaluated by in vitro studies and in healthy volunteers. Results: The NLC-TRE presented spherical structures, average particle size of 130 nm, zeta potential of 24 mV and encapsulation efficiency of 98%. The NLC-TRE protected TRE against oxidation (p

Este estudo tem como temática a relação entre vegetarianos e a deficiência de micronutrientes por entender que, a adesão do estilo de vida vegetariano feita de forma inadequada pode resultar em uma deficiência de nutrientes essenciais. O objetivo foi realizar uma revisão integrativa sobre vegetarianos e a presença de deficiência nutricional. Para a seleção da amostra definiu-se artigos indexados de 2015 a 2020, publicados em periódicos nacionais e internacionais, disponibilizados em língua portuguesa e inglesa. A coleta de dados foi realizada na base Biblioteca Virtual de Saúde (BVS), Lilacs, Scielo, PubMed, e Google Acadêmico, utilizando os seguintes descritores: dieta vegetariana, ferro, deficiências nutricionais, deficiência de vitaminas, deficiência de minerais, micronutrientes, deficiência de vitamina B12 e deficiência de ferro. A busca resultou em 32 artigos, após utilização dos critérios de inclusão e exclusão restaram 10 artigos para análise, interpretação e discussão. Observou-se que entre vegetarianos há uma deficiência ou carência nutricional, e isso se dá por meio do consumo aumentado de fitatos e oxalatos, devido à alta ingestão de fibras. Conclui-se que os vegetarianos apresentam déficit nutricional de zinco, cálcio, ferro, vitamina B12 e vitamina D e que a ingestão adequada de todos os nutrientes, por meio de uma dieta balanceada, bem como, se há necessidade da inserção de suplementação de algum desses micronutrientes, mediante a avaliação completa do nutricionista mostra-se uma estratégia promissora na prevenção de déficit de micronutrientes em vegetarianos.

A previously healthy 15-year-old girl was sent to the Pediatric Emergency Department by the general practitioner due to suspicion of a hematological condition. The girl presented with ecchymotic lesions on the anterior region of the thorax and antero-proximal region of the upper limbs, which had apparently worsened in the last 24 hours. She denied trauma or impact sports, fever or recent infections, and blood loss or other symptoms. Laboratory evaluation showed no alterations and urine toxicology screening was negative. When confronted with normal laboratory evaluation, the girl confessed intentional self-inflicted injuries following a discussion with her mother. Parents were made aware of the situation, and a pediatric psychiatry consultation was recommended. After discharge, follow-up in the girl’s home country was assured. With this report, the author intend to raise awareness of the importance of maintaining a high index of suspicion to non-suicidal self-injury, identifying alarm signs and establishing early preventive strategies., NASCER E CRESCER - BIRTH AND GROWTH MEDICAL JOURNAL, vol. 29 n.º 1 (2020)

Pythium insidiosum is a straminopilan pathogen causing life threatening infections in mammals inhabiting temperate, tropical and subtropical areas of the world. The concept that P. insidiosum could also infect mosquitoes was mentioned earlier by investigators conducting phylogenetic analysis on available P. insidiosum isolates deposited at different culture collections. However, an official report and details on its pathological features in mosquitoes are not available. We are reporting the isolation of P. insidiosum from infected mosquito larvae during a survey conducted in central Brazil. At least three oomycotan isolates were recovered during the survey. Due to their ability to infect mosquito larvae the isolates were deposited in the U.S. Department of Agriculture, Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF; Ithaca, New York) as putative Lagenidium species. The investigated isolates developed very well at 37 °C, produced typical Pythium-like vesicles containing numerous biflagellate zoospores, hydrolyzed sucrose, and their cultured extracted proteins were recognized in serological analysis by anti-P. insidiosum antibodies. Phylogenetic analyses using ITS and partial COXII DNA sequences identified the isolates as P. insidiosum within the American Cluster I. This is the first official report of P. insidiosum recovered from infected mosquito larvae, indicating that this mammalian pathogen, in addition to plants, it could also use mosquito larvae to complete its life cycle in nature.

We studied phylogenetic and taxonomic features of isolates identified as

Background Recent molecular phylogenetic analysis of Lagenidium strains recovered from subcutaneous lesions in cats, dogs, and a human with lagenidiosis resolved into four clades; one of them was Lagenidium giganteum, but three others were novel. Aims Due to the recent increase in L. giganteum infections from mammals, we studied 21 Lagenidium strains isolated from dogs and a human available in our collection. Methods Molecular phylogenetic studies and phenotypic characteristics were used to characterize the strains. Results We report the finding of three novel species, herein designated as Lagenidium ajelloi, sp. nov., Lagenidium albertoi sp. nov, and Lagenidium vilelae sp. nov. Their morphological and growth features are also presented. Conclusions Our study revealed the presence of three novel Lagenidium species infecting mammals.

Rhinosporidium seeberi is an uncultivated Ichthyosporean infecting animals, including humans. Recent studies suggested R. seeberi undergoes synchronized nuclear division without cytokinesis. We used confocal microscopy to investigate R. seeberi nuclear division cycles in formalin-fixed tissues stained with DAPI and phalloidin. We report that R. seeberi nuclei in juvenile and intermediary sporangia synchronously divided without cytokinesis. Intermediary sporangia display numerous 3-4 μm nuclei at different mitotic stages as well as a thick inner layer with strong affinity for phalloidin. Mature sporangia showed numerous 5-12 μm cell-walled endospores, each containing a 2-4 μm in diameter nucleus. Phalloidin did not bind to the inner layers of mature sporangia or endospores. The development of a "germinative zone" in the inner layer of mature sporangia containing hundreds of nuclei was also confirmed. This study establishes that during the R. seeberi life cycle synchronous nuclear divisions without cytokinesis takes place, resulting in the formation of thousands of nuclei. Cytokinesis, on the other hand, is a 1-time event and occurs in the latest stages of intermediate sporangia, after the formation of thousands of nuclei and just before mature sporangia development.

Some species of the fungus Sporothrix cause a chronic granulomatous infection in humans and animals called sporotrichosis. In the last decades, some research into serological tests has been carried out by different groups for the rapid detection of this infection. We performed a systematic review of the literature with meta-analysis to evaluate studies using Sporothrix spp. antigens and to evaluate their accuracy for sporotrichosis diagnostic. We searched Scopus, MEDLINE, Web of Science, GALE, Technology Research Database, DOA, Elsevier, SciELO, and Google Scholar Databases. The united results of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio with their corresponding 95% confidence intervals (CI) were assessed. A total of 15 assays from 8 studies using 7 different serological methods and 8 different antigens were analyzed. The studies were performed in the USA, Brazil, and Venezuela from 1973 until 2015 and presented good quality. A high heterogeneity for sensitivity [I2 = 90.7%; 87% CI = (84–89), P

Pythiosis in Southern USA have been increasingly reported in the past ten years. The infection occurs more frequently in dogs and horses inhabiting the endemic areas. Cases of the disease are rarely diagnosed in other species including humans. Herein, we describe the first case of bovine pythiosis in a breed other than Brahman successfully treated by the used of immunotherapy.

The pathogenic entomophthoralean fungi cause infection in insects and mammalian hosts. Basidiobolus and Conidiobolus species can be found in soil and insect, reptile, and amphibian droppings in tropical and subtropical areas. The life cycles of these fungi occur in these environments where infecting sticky conidia are developed. The infection is acquired by insect bite or contact with contaminated environments through open skin. Conidiobolus coronatus typically causes chronic rhinofacial disease in immunocompetent hosts, whereas some Conidiobolus species can be found in immunocompromised patients. Basidiobolus ranarum infection is restricted to subcutaneous tissues but may be involved in intestinal and disseminated infections. Its early diagnosis remains challenging due to clinical similarities to other intestinal diseases. Infected tissues characteristically display eosinophilic granulomas with the Splendore-Höeppli phenomenon. However, in immunocompromised patients, the above-mentioned inflammatory reaction is absent. Laboratory diagnosis includes wet mount, culture serological assays, and molecular methodologies. The management of entomophthoralean fungi relies on traditional antifungal therapies, such as potassium iodide (KI), amphotericin B, itraconazole, and ketoconazole, and surgery. These species are intrinsically resistant to some antifungals, prompting physicians to experiment with combinations of therapies. Research is needed to investigate the immunology of entomophthoralean fungi in infected hosts. The absence of an animal model and lack of funding severely limit research on these fungi.

Apesar de a palavra ‘bioética’ ter sido utilizada em 1927 pelo teólogo pro­testante alemão Paul Max Fritz Jahr (1895 – 1953), o termo só se notabilizou no início da década de setenta, quando o oncologista estadunidense Van Ransselaer Potter (1911 – 2001) apresentou a bioética como uma “Ponte para o futuro”, livro publicado em 1971. Posteriormente, Potter apresentou outras perspectivas, como “Bioética global” (1988) e ainda, “Bioética pro­funda” (1998) a qual, segundo ele, visa ir à raiz dos problemas ambientais e defende os direitos de toda a comunidade biótica (...)

Infections caused by the fungal pathogen Lacazia loboi were first reported in 1931 by Jorge de Oliveira Lobo in a human with granulomatous skin lesions in Pernambuco, Brazil. Early histopathological and serological analyses found morphological similarities and cross-reactive antigens with Paracoccidioides brasiliensis. In 1971, veterinarians working with dolphins in Florida, USA, reported granulomatous skin lesions in a dolphin, similar to that in human lacaziosis. Based on histopathological findings, L. loboi was initially believed to be also the etiologic agent of cutaneous disease in dolphins. Ever since, cutaneous granulomas have been reported in different dolphin species around the coast of Asia, Europe, and North and South America. Recently, using molecular biology approaches, some investigators stated that the DNA sequences extracted from cases of cutaneous granulomas in dolphins were closely related to those of P. brasiliensis. This chapter deals with the history, taxonomy, and other features of L. loboi in humans and the unculturable P. brasiliensis var. ceti type affecting the skin of dolphins.

Severe physical facial deformities due to surgical interventions can have significant psychosocial consequences to patient’s relationships with friends and family and thus, has a considerable impact on their quality of life. We have developed a 3D prosthesis for a 56-year-old woman diagnosed with epidermoid carcinoma at the right hemiface, to improve her quality of life. The patient started radiotherapy with modulated intensity. To deal with the advance of the process, a maxilectomy of supra structure with modified radical cervical emptying on the right hemiface was performed. Reconstruction of areas surgically affected by the displacement of islands of skin and muscle (flaps) from healthy regions was initiated. Although the procedure occurred without intercurrences, the patient developed necrosis and loss of the myocutaneous flap. After the removal of the flap, the esthetic result of the treatment was evident causing exposure of subcutaneous and granulation tissues. A computational model was used to develop a 3D structure of the affected area and then used to construct the prosthesis. The prosthesis was applied over the affected area, and the patient was able see her face on the mirror for the first time in years. The patient was grateful and hopeful. We have found that the application of this new technology greatly improves the social interaction of patients with deformities due to surgical interventions.

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Cryptococcus gattii is one of the etiologic agents of cryptococcosis, a systemic mycosis that occurs in healthy and immunosuppressed humans and animals worldwide. Primary pulmonary infection caused by C. gattii is usually followed by fungal dissemination to the central nervous system, resulting in high mortality rates. In this context, animal models of cryptococcosis are useful in the study of fungal pathogenesis and host response against the pathogen, and for testing novel therapeutic options. The most frequently applied method to study fungal dissemination from the lungs to other organs is by culturing tissues, which is not accurate for the detection and quantification of fungal load at early stages of the infection. To overcome this problem, the purpose of this study was to develop a new method for the quantification of Cryptococcus dissemination. One C. gattii strain was efficiently radiolabeled with technetium-99m (99mTc), without affecting viability of the cells. Further, the 99mTc-C. gattii (111 MBq) strain was used to infect mice by intratracheal and intravenous route for biodistribution studies. 99mTc-C. gattii was successfully used in detection of the yeast in the brain of mice 6 hours postinoculation, while the detection using colony forming units was possible only 24 hours postinfection. Our results provided an alternative method that could be applied in further investigations regarding the efficacy of antifungals, fungal virulence, and host-pathogen interactions.

Pythium insidiosum immunomodulatory vaccine (PiV) has been tested in clinical and experimental pythiosis. Previous data showed that P. insidiosum immunogens have the ability to switch the Th2 immune response, normally in place during pythiosis, to a curative Th1 response. Pythiosis cannot be reproduced in experimental rodents with the exception of rabbits, and thus thorough evaluation of PiV´s immunomodulatory properties has been limited by the lack of a compatible inbred mouse model. In this study, we took advantage of the murine BALB/c Leishmania infection model, where infected mice produce a Th2 response, to evaluate the PiV Th2 to Th1 immunomodulatory potential. Twenty-one days following challenge with L. major, large cutaneous granulomas developed in control mice, consistent with the expected Th2 response. In contrast, Leishmania-induced cutaneous lesions in PiV-immunized mice were minimal or absent. Flow cytometry analysis of spleen cells from mice immunized with PiV and subsequently challenged with L. major displayed more CD4+ and CD8+ cells than the control group. Moreover, spleen cells from mice that were immunized with PiV then challenged with L. major secreted high levels of IFN-γ, with a moderate IL-2, IL-4, and IL-10 mixed cytokine profile upon in vitro re-stimulation with PiV. Anti-P. insidiosum IgG1 in immunized animals was present at low titers suggesting a minor immunological role for this Ig isotype in this model. Our preliminary data showed that BALB/c mice challenged with L. major represent an attractive model in which to study PiV´s immunomodulatory properties.

Despite numerous studies of the Rhinosporidum seeberi parasitic phase, the stages of its nuclear cycle leading to the formation of endoconidia have yet to be properly described. R. seeberi resists culture and can only be investigated on histological preparations. We have evaluated tissue sections collected from 35 host species with rhinosporidosis searching for the presence of mitotic figures during sporangia development. This study found that soon after endoconidia release, the prominent reddish vesicles typical of this stage vanished leading to the development of juvenile sporangia (JS) 12-70 µm in diameter. This stage possesses granular cytoplasm, a thick cell wall, and a central reddish nucleus with a conspicuous nucleolus. The first nuclear division takes place in the JS. It is a rarely encountered event characterized by the development of a distorted nucleus leading to the formation of two nuclei without cytokinesis. The finding of multiple nuclear divisions at prophase-, metaphase- and telophase-like stages without cytokinesis was detected in intermediate sporangia (IS). IS with multiple dividing nuclei seem to be at the same stage of nuclear partitioning, suggesting synchronized nuclear division. In these sporangia, the nuclei continue divisions without cytokinesis until the sporangia reach ≥300 µm in diameter. The last nuclear division prior to cytokinesis appears to take place in very large sporangia with thousands of nuclei. The build-up of cytoplasm around each nucleus and the formation of a thin cell wall lead to the formation of endoconidia. This study revealed the presence of several mechanisms of pathogenesis in R. seeberi that deserved further investigation.

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 inthecellwallofoomycetes;andd)asexualreproductioninthe 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 pathologicalfeatures,aswellasthediagnosisandmanagement of the infections caused by this unique group of mammalian pathogenic oomycetes.

Introducao: A assistencia ao pre-natal e fundamental para os resultados perinatais, pois quanto melhor a qualidade do amparo, mais favoraveis sao os resultados e menores sao as taxas de mortalidade materna e perinatal. Objetivos : Avaliar a qualidade do pre-natal com base no numero de consultas e na idade gestacional no inicio do pre-natal, atraves da criacao de um modelo experimental baseado na assistencia clinica e na realizacao de exames complementares. Metodos: Estudo de corte transversal, em que foram analisados 150 prontuarios de gestantes atendidas no Servico de Obstetricia da Universidade Federal de Juiz de Fora e 51 prontuarios de gestantes atendidas na Unidade Basica de Saude do bairro Santa Cecilia no municipio de Barbacena/MG. Resultados: Nao houve diferencas socioeconomicas, em relacao aos aspectos obstetricos, exames complementares, complicacoes clinicas, obstetricas, nem sorologias para toxoplasmose, sifilis e HIV. Entretanto, o numero de consultas, idade gestacional na primeira consulta, exame das mamas, sorologias para rubeola, exame macroscopico dos fluxos genitais, coleta colpocitologica, ganho de peso durante o pre-natal, anemia e a infeccao urinaria apresentaram diferenca significativa. Conclusao: O pre-natal de Barbacena foi considerado adequado em 78,4% dos casos pelo Indice de Kessner modificado por Takeda (IKMT) e adequado intermediario pelo Indice de Acesso e Assistencia ao Pre-natal (IAAPN). Ja o pre-natal de Juiz de Fora foi considerado adequado em 77,3% pelo IKMT e adequado superior pelo IAAPN. Palavras-chave: Cuidado pre-natal; gestantes; consultas

Our findings could stimulate study of public health implications of diseases caused by this fungus., Cutaneous granulomas in dolphins were believed to be caused by Lacazia loboi, which also causes a similar disease in humans. This hypothesis was recently challenged by reports that fungal DNA sequences from dolphins grouped this pathogen with Paracoccidioides brasiliensis. We conducted phylogenetic analysis of fungi from 6 bottlenose dolphins (Tursiops truncatus) with cutaneous granulomas and chains of yeast cells in infected tissues. Kex gene sequences of P. brasiliensis from dolphins showed 100% homology with sequences from cultivated P. brasiliensis, 73% with those of L. loboi, and 93% with those of P. lutzii. Parsimony analysis placed DNA sequences from dolphins within a cluster with human P. brasiliensis strains. This cluster was the sister taxon to P. lutzii and L. loboi. Our molecular data support previous findings and suggest that a novel uncultivated strain of P. brasiliensis restricted to cutaneous lesions in dolphins is probably the cause of lacaziosis/lobomycosis, herein referred to as paracoccidioidomycosis ceti.

Two camels ( Camelus dromedarius ), 3- and 4-years-old, respectively, from an eastern Tennessee wildlife farm presented with persistent weight loss and large vulvar masses. An initial biopsy of the vulvar mass of one of the camels performed by a local veterinarian showed eosinophilic dermatitis. An allergic or parasitic dermatitis was suspected. The two camels were treated with one dose of sodium iodide (66 mg/ kg, in 1.0 L of normosolR, IV) and ivermectin 1% (200 ug/kg PO). Upon presentation at the Veterinary Teaching Hospital, University of Tennessee, additional biopsies of the masses again revealed eosinophilic dermatitis. Microscopic examination of a Gomori methenamine silver (GMS)-stained section prepared from the biopsy of one of the camels revealed the presence of fungal-like hyphae of a mold which was suspected to be Pythium insidiosum . The vulvar masses were surgically debulked in both animals and sodium iodide and Pythium -immunotherapy prescribed. Pythium insidiosum was isolated in culture and hyphae elements were detected in histological sections confi rming the diagnosis of pythiosis in both animals. Despite signs of progressive healing of the vulvar surgical areas, postoperative persistent weight lost in one of the camels suggested the possibility of gastro intestinal (GI) tract pythiosis. This camel died 5 months after the fi rst onset of clinical signs and unfortunately a necropsy was not performed. The other camel responded well to the combination of surgery, iodides, and immunotherapy and has currently rejoined the other members of the herd.

In the past 100 years the microbial pathogens described in this chapter have been classified as fungal and/or parafungal protistan pathogens. Lacazia loboi, Pythium insidiosum and Rhinosporidium seeberi are the most prominent features of the pathogens covered in this chapter. Clinical samples from patients suspected of having lacaziosis submitted to the laboratory comprise deep-skin scrapings and tissue biopsy samples. The samples have to be processed and evaluated for the presence of uniform yeast like cells connected by small tubules forming short chains. In contrast with the other hydrophilic pathogens covered in the chapter, P. insidiosum can be cultured on various media. The first molecular approach for the diagnosis of P. insidiosum from clinical specimens was carried out with a patient with keratitis. The hyphal elements present in the specimen were identified by sequencing part of the 18S ribosomal DNA region using the NS1 and NS2 and internal transcribed spacer (ITS) universal primers. The identification of P. insidiosum using molecular methods are described in this chapter. The first two cases of rhinosporidiosis were reported in 1900 by Guillermo Rodolfo Seeber in his M.D. thesis in Argentina. Ashworth in 1923 stated that the genus Rhinosporidium proposed by Minchin and Fanthan should be adopted and that, based on the description of Seeber and the name Coccidioides seeberia reintroduced by Belou, the binomial R. seeberi has priority.

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.

The report of four novel mammalian pathogenic species of the genus Lagenidium prompted us to study the use of biochemical assays to differentiate the Oomycota mammalian pathogens Pythium insidiosum and Lagenidium spp. We investigated the reaction of 23 Lagenidium and eight Pythium species in various biochemical assays. Because the morphological features of the Oomycota species are similar to those of species in the Entomophthoramycota and Mucormycota, five fungal species with coenocytic hyphae were also included. We found that mammalian and plant isolates of Pythium spp. all hydrolysed sucrose, but Lagenidium species and the fungal strains did not. In addition, both Pythium spp. and Lagenidium spp. were found to be maltose-positive, whereas fungal strains did not hydrolyse this sugar. The fungal species and thermo-sensitive Lagenidium giganteum and Lagenidium humanum were urease-negative, but the mammalian Lagenidium spp. and Pythium spp. hydrolysed urea within 24 h. These findings suggest these assays can be used for the presumptive differentiation of mammalian Oomycota species in the laboratory.

In the past 100 years the microbial pathogens described in this chapter have been classified either as fungal and/or para-fungal protistan pathogens. Based on their apparent epidemiological connection with water, they were at one point also placed in a new category of hydrophilic infectious agents. However, based on taxonomic and other morphological characteristics, these three anomalous species were not well understood. This frustrating situation fueled a strong controversy that has only recently been solved with the advent of molecular methodologies. Despite the recent finding that both Pythium insidiosum and Rhinosporidium seeberi are protistan pathogens, they are still studied by medical mycologists, continuing a historical tradition. More recently, the finding of an oomycete in the genus Lagenidium affecting mammalian hosts alerted the medical community to the presence of a novel pathogen phenotypically similar to the fungi and indistinguishable from the clinical and pathological features displayed by P. insidiosum during infection. Based on rDNA phylogenetic analysis, the evolutionary location of the microbial pathogens discussed in this chapter is illustrated.

Strains pathogenic to mammals share phylogenetic and phenotypic features with strains approved for mosquito control., Infections of mammals by species in the phylum Oomycota taxonomically and molecularly similar to known Lagenidium giganteum strains have increased. During 2013–2014, we conducted a phylogenetic study of 21 mammalian Lagenidium isolates; we found that 11 cannot be differentiated from L. giganteum strains that the US Environmental Protection Agency approved for biological control of mosquitoes; these strains were later unregistered and are no longer available. L. giganteum strains pathogenic to mammals formed a strongly supported clade with the biological control isolates, and both types experimentally infected mosquito larvae. However, the strains from mammals grew well at 25°C and 37°C, whereas the biological control strains developed normally at 25°C but poorly at higher temperatures. The emergence of heat-tolerant strains of L. giganteum pathogenic to lower animals and humans is of environmental and public health concern.

In the past five years, with the use of molecular strategies the phylogenetic affinities of the two more resilient pathogens studied in medical mycology, Lacazia loboi and Rhinosporidium seeberi were finally deciphered. These studies found that L. loboi was the sister taxon to Paraccidioides brasiliensis, and R. seeberi was closely related to protistan spherical aquatic fish pathogens, located at the point were animals diverged from the fungi, in the class Mesomycetozoea. These initial studies indicated that a molecular strategy was the ideal approach to further understand these anomalous pathogens. However, the limited amount of information gathered so far from few DNA sequences, although crucial to place these organisms in the tree of life and to take a glance to their ecological preferences, did not provide answers to other important traits. In the following pages we discuss a genomic perspective for both pathogens and the benefit that such information could generate to understand more about these two uncultivated pathogens.