Development of PCR-Multiplex Assays for Identification of the Herpotrichiellaceae Family and Agents Causing Chromoblastomycosis.

The Herpotrichiellaceae family is an important group of dematiaceous filamentous fungi, associated with a variety of pathogenic fungal species causing chromoblastomycosis (CBM) and phaeohyphomycosis (PHM), both with polymorphic clinical manifestations and worldwide incidence. Currently, the identification of this family and determination of the causative agent is challenging due to the subjectivity of morphological identification methods, necessitating the use of molecular techniques to complement diagnosis. In this context, genetic sequencing of the Internal Transcribed Spacer (ITS) has become the norm due to a lack of alternative molecular tools for identifying these agents. Therefore, this study aimed to develop PCR-Multiplex methodologies to address this gap. Sequences from the ITS and Large Subunit (LSU) of ribosomal DNA were used, and after manual curation and in vitro analyses, primers were synthesized for the identification of the targets. The primers were optimized and validated in vitro, resulting in two PCR-Multiplex methodologies: one for identifying the Herpotrichiellaceae family and the bantiana clade, and another for determining the species Fonsecaea pedrosoi and Fonsecaea monophora. Ultimately, the assays developed in this study aim to complement other identification approaches for these agents, reducing the need for sequencing, improving the management of these infections, and enhancing the accuracy of epidemiological information.

Identification of Chromoblastomycosis and Phaeohyphomycosis Agents through ITS-RFLP.

Chromoblastomycosis (CBM) and phaeohyphomycosis (FEO) are infections caused by melanized filamentous fungal agents, primarily found in tropical and subtropical regions. Both infections pose significant challenges for the correct identification of the causative agent due to their morphological similarity, making conventional methods of morphological analysis highly subjective. Therefore, molecular techniques are necessary for the precise determination of these species. In this regard, this study aimed to contribute to a new methodology based on PCR-RFLP for the identification of agents causing CBM and FEO. Sequences from the Internal Transcribed Spacer (ITS) region were used to identify potential restriction enzyme sites in silico, followed by in vitro validation using the selected restriction enzymes. The obtained results were compared with species identification through morphological analyses and sequencing. The results demonstrated that the PCR-RFLP applied in this study accurately identified two major agents of chromoblastomycosis, Fonsecaea pedrosoi and Fonsecaea monophora, as well as Cladophialophora bantiana and Exophiala dermatitidis, both causative agents of phaeohyphomycosis. In this context, the proposed assay can complement current methods for identifying these species, aiding in diagnosis, and contributing to the proper management of these infections.

The marked dextro-dominance causes accentuated morphophysiological variations in the female reproductive organs of the bat Molossus molossus.

Molossus molossus is an insectivorous molossid bat that is important in the control of nocturnal insects. It is the nominal and the most representative species of the family. However, there are few studies about its reproduction. Thus, this study aimed to evaluate variations of its female reproductive organs during the different reproductive phases. Twenty adult females, divided into four sample groups (non-reproductive, early and advanced pregnancy and lactation), were submitted to morphological and morphometric analyses. Results show that the female reproductive system of M. molossus is composed of ovaries, a short bicornuate uterus, slightly convoluted uterine tubes and vagina. The system presents a distinct morphofunctional asymmetry, with a marked dextro-dominance. The right ovaries of all analyzed groups (NON, P1, P2, and LAC) showed follicles at different stages of development, a large number of interstitial glands and a small, but persistent corpus luteum. Ovulation is simple, unilateral and preferential, occurring exclusively in the right ovary. Follicular development in the left ovary usually does not pass the secondary stage. Implantation is fundic and preferential, occurring exclusively in the right uterine horn. The placenta is formed with two distinct chorioallantoic portions, one diffuse endotheliochorial, which covers the entire uterine cavity and regresses in the final stages of pregnancy, and the principal discoidal hemochorial portion, formed in the implantation site. The uterine cervix presents a pseudostratified epithelium, while the vagina has a little keratinized stratified epithelium, which does not accentually vary in the different reproductive stages, but can disrupt and shed in some cases.

Effects of surface treatment, hydration and application method on the bond strength of a silorane adhesive and resin system to dentine.

OBJECTIVES: To evaluate the effects of surface treatment, surface hydration (SH) and application method (AM) on the tensile bond strength of the Silorane Adhesive System (SAS) to dentine. METHODS: Ninety bovine teeth were used. For the control group (n=10), each dentine surface was treated according to the manufacturer's instructions of the SAS. The remaining teeth were randomly distributed into two groups (n=40), according to the type of dentine surface treatment (ST)-37% phosphoric acid or Er:YAG Laser prior to the application of the SAS. Each group was further divided into 2 subgroups (n=20), according to the SH status: dry (D) or wet (W). Each subgroup was further divided into 2 subgroups (n=10), according to the application method [AM: Active (AC) mode or Passive (PA) mode]. A coat of resin composite (Filtek P90) was applied on the surface. Artificial ageing was performed with a thermo-mechanical cycling machine. The specimens were sectioned into 1mm×1mm×10mm sticks and stressed to failure using a universal testing machine. The remaining teeth in each group were used for Scanning Electron Microscopy to examine the fractured area. Data were subjected to a three-way ANOVA, Tukey's test and Dunnet's test (α=0.05). RESULTS: The ANOVA showed significant differences for SH and AM, but not for ST. For SH, the results of Tukey's test were (in MPa): D - 14.9(±3.8)(a), W - 17.1(±4.3)(b); and for AM: PA - 14. 9(±4.2)(a), AC - 17.1(±3.9)(b). CONCLUSIONS: Acid etching, when combined with a moist dentine surface and the use of primer agitation, improves the bond strength of the SAS to dentine. CLINICAL SIGNIFICANCE: According to the results of the present in vitro study, modification of the application protocols for the silorane-based adhesive system may improve its clinical performance.