Canine visceral leishmaniasis: Detection of Leishmania spp. genome in peripheral blood of seropositive dogs by real-time polymerase chain reaction (rt-PCR).

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)® as a screening test and the Enzyme immunoassay test (ELISA®). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP® and ELISA® tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150-152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood.

Paracoccidioidomycosis: Detection of Paracoccidioides brasiliensis´ genome in biological samples by quantitative chain reaction polymerase (qPCR).

Paracoccidioidomycosis (PCM) is a chronic mycosis caused by the saprobic and dimorphic species Paracoccidioides brasiliensis and P. lutzii. This disease is prevalent in Latin American countries. PCM appears as a relevant concern and challenge for the mycologists, since until now there is no a methodology suitable for an efficient and safe diagnosis and species identification. Thus, the present study aimed to validate a methodology for PCM´s diagnosis, using quantitative Polymerase Chain Reaction (qPCR) through target amplification of the gene encoding the recombinant protein Pb27, a common protein to the both species Paracoccidioides brasiliensis and P. lutzii. The experiments were performed in vitro to determine the specificity, efficiency and detection limit of qPCR assay, using specific primers and probe, which sequences were subject to a patent deposited in Brazilian CTIT, under the registration number: BR1020160078830. According to the results the technique showed sensitivity of 94% and specificity of 100%, demonstrating that it will be possible to develop a new fast and safe diagnostic PCM and can be standardized in order to present a low cost, accessible to the patient served by the public health system in Brazil and Latin America.

Sporotrichosis: In silico design of new molecular markers for the Sporothrix genus.

BACKGROUND: Sporotrichosis, a cosmopolitan mycosis caused by dimorphic fungi of the Sporothrix complex, affects humans and animals. This study aimed to develop new molecular markers for Sporothrix genome detection in biological samples using PCR. METHODS: A specific region of DNA sequences from the Sporothrix genus, publicly available in GenBank, was chosen for primer design. After testing the in silico specificity of these primers, in vitro specificity was evaluated using the PCR technique. RESULTS: Three specific primers with 100% specificity for the Sporothrix genus were generated. CONCLUSIONS: PCR using the designed primers can be used to develop molecular diagnostics for sporotrichosis.

New molecular marker for phylogenetic reconstruction of black yeast-like fungi (Chaetothyriales) with hypothetical EIF2AK2 kinase gene.

In eukaryotes, phosphorylation of the α-subunit of eIF2 is a mechanism to adjust cellular gene expression profiles in response to specific signals. The eIF2α kinases are a group of serine-threonine kinases that perform important functions in response to infection, proteotoxicity, and nutrient scavenging. The conserved nature of eIF2α kinases among fungi makes them potential evolutionary markers, which may contribute to deeper understanding of taxonomy and evolution. To date, only few studies are available of eIF2α kinases in black yeasts, which are members of Chaetothyriales containing potential agents of a gamut of major human diseases, such as chromoblastomycosis, phaeohyphomycosis and mycetoma. To establish the phylogenetic validity of sequences of eIF2α kinases hypothetical genes, we compared these genes between members of different classes of fungi, including black yeasts and allies, aiming at evaluation of the phylogeny of this group using an alternative molecular marker, compared to standard ribosomal genes. Trees generated with eIF2α kinase sequences of fungi were compared with those generated by ribosomal internal transcribed spacers (ITS rDNA) sequences from the same species. Sequences used were obtained from the protein Non-redundant database of NCBI, were aligned using CLUSTALX v1.8 and alignments were analyzed with RAxML v8.2.9 on the CIPRES Science Gateway portal. The trees generated had similar topologies, demonstrating that eIF2α kinases hypothetical gene sequences present a coherent reflection of evolution among fungi, compared to trees reconstructed by the use of ribosomal sequences. Our preliminary findings with a limited dataset strongly suggest that the evolution of kinases among black yeasts follows a similar path as revealed by ribosomal data, which underlines the validity of current taxonomy of black yeasts and relatives.

Study and Development of an Anthroposophical Formula Based on Phosphorus and Formica rufa for Onychomycosis´s Treatment.

Onychomycosis is a fungal infection of the nail plate or nail bed that leads to the gradual destruction of the nail. The main difficulties in the treatment of onychomycosis refer to the duration of treatments and their side effects. Thus, it becomes relevant to look for new therapeutic alternatives in the treatment of such common diseases that are efficient without causing the undesirable side effects on the patient's body. In this way, the objective of this study was to develop an anthroposophical formula for the treatment of onychomycosis, based on Phosphorus and Formica rufa, from an extensive bibliographic survey on the functions of these components, evaluating within the principles of Anthroposophy. Considering the set of knowledge and practices on the use of these components, it was possible to arrive at a proposal therapy that can be effective for the treatment of onychomycosis. After an extensive review of several existing patents, it was observed that formulations containing Phosphorus and Formica rufa together have not been described in other studies. Subsequently, our research group published a patent of the anthroposophical formula using these two components, with the number BR1020180750755, which will be efficient to help the recovery of nails, and facilitate normal growth.

Sporotrichosis: In silico design of new molecular markers for the Sporothrix genus

ABSTRACT Background: Sporotrichosis, a cosmopolitan mycosis caused by dimorphic fungi of the Sporothrix complex, affects humans and animals. This study aimed to develop new molecular markers for Sporothrix genome detection in biological samples using PCR. Methods: A specific region of DNA sequences from the Sporothrix genus, publicly available in GenBank, was chosen for primer design. After testing the in silico specificity of these primers, in vitro specificity was evaluated using the PCR technique. Results: Three specific primers with 100% specificity for the Sporothrix genus were generated. Conclusions: PCR using the designed primers can be used to develop molecular diagnostics for sporotrichosis.

A case of disseminated sporotrichosis caused by Sporothrix brasiliensis.

This paper presents a case of disseminated sporotrichosis in a 13-year-old female, originating from a rural area in Minas Gerais state, Brazil. The patient was hospitalized in Santa Casa hospital of Belo Horizonte, with hyporexia, prostration, fever and disseminated ulcerative lesions, besides anemia, leucopenia and sepsis of probable cutaneous focus. The patient was admitted without proven immunosuppression. She was diagnosed with cutaneous-disseminated sporotrichosis. The drug therapy chosen was itraconazole during 12 months, leading to important clinical improvement and healing of cutaneous lesions.

Diagnóstico da aspergilose invasiva:Aplicação das Técnicas de Reação em Cadeia da Polimerase (PCR) e Ensaio Imunoenzimático de Detecção da Galactomanana (EIA-GM®) / Diagnosis of invasive aspergillosis: Application of Polymerase Chain Reaction Techniques (PCR) and Enzyme-Linked Immunosorbent Assay for Detectionof Galactomannan (EIA-GM®)

Introdução: a aspergilose invasiva (IA) é uma infecção fúngica grave causada por espécies do gênero Aspergillus e acomete principalmente pacientes leucêmicos,diabéticos e aqueles receptores de transplante de células-tronco, que apresentem neutropenia. Os esporos dos fungos que colonizam o epitélio pulmonar podem invadir as células endoteliais de revestimento e o acesso vascular e, assim, disseminar-se paraoutros órgãos através do sangue. A elevada mortalidade da doença está relacionada à imunossupressão grave, à rápida progressão da infecção e, principalmente, à ausência de um diagnóstico precoce e eficiente. Portanto, o diagnóstico na fase inicial da infecção é adequado, proporcionando uma terapia mais eficaz, o que pode reduzir a taxa de mortalidade da doença. Objetivo: o presente estudo teve em vista avaliar a aplicabilidadeda técnica de reação em cadeia da polimerase (PCR) no auxílio do diagnóstico de AI, em comparação com os resultados gerados pelo ensaio imunoenzimático de galactomanana (EIA-GM®), este já validado comercialmente. Métodos: foram analisadas 245 amostras de pacientes tratados no hospital Santa Casa de Belo Horizonte. Entre essas amostras, 16% (N = 39) foram positivos nos testes EIA-GM®. Em seguida, essas 39amostras positivas foram analisadas pela técnica de PCR. Resultados: de acordo com os resultados, a técnica de PCR apresentou taxa de 97,44% de sensibilidade, 97,96% de acurácia e 100% de especificidade, quando comparada ao método EIA-GM®. Conclusão:a técnica de PCR pode auxiliar no diagnóstico da AI, sempre associando os seus resultados à clinica do paciente e aos testes de imunoensaios.

Introduction: invasive aspergillosis (AI) is a serious fungal infection caused by species of the genus Aspergillus that primarily affects leukemic and diabetic patients and those recipients of stem cell transplants, which have neutropenia. The fungi spores that colonize the lung epithelium may invade the endothelial cell lining and vascular access and thus, spread to other organs through the blood. The high mortality of the disease is related tosevere immunosuppression, rapid infection progression, and especially lack of an early and efficient diagnosis. Therefore, the diagnosis in the initial infection phase is beneficial,providing a more effective therapy that can reduce the disease?s mortality rate. Objective:this study aimed at evaluating the applicability of the polymerase chain reaction (PCR) cheganin assisting the diagnosis of AI compared to the resultsgenerated by galactomannan enzyme immunoassay (EIA-GM®) that is already commercially validated. Methods: 245 samples from patients treated in the Santa Casa de Belo Horizonte hospital were analyzed. Among these samples, 16% (N = 39) were positive in EIA-GM®tests. Subsequently, these 39 positive samples were analyzedby PCR. Results: According to the results, the PCR technique showed 97.44% sensitivity, 97.96% accuracy, and 100% specificity compared to EIA-GM®. Conclusion:the PCR technique may aid in the diagnosis of AI,always associating the results to the patient's clinicaland immunoassay tests.