BRCA1 and BRCA2 germline mutation analysis from a cohort of 1267 patients at high risk for breast cancer in Brazil.

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country.

Leigh syndrome in a patient with a novel C12orf65 pathogenic variant: case report and literature review.

Leigh syndrome is an early onset progressive disorder caused by defects in mitochondrial oxidative phosphorylation. Pathogenic variants in nuclear and mitochondrial genes are associated with the syndrome. Homozygous pathogenic variants in the C12orf65 gene impair the mitochondrial oxidative phosphorylation system. We describe a new case of Leigh syndrome caused by a novel pathogenic variant of the C12orf65 gene resulting in the lack of the Gly-Gly-Gln (GGQ) domain in the predicted protein, and review clinical and molecular data from previously reported patients. Our study supports that the phenotype caused by C12orf65 gene variants is heterogeneous and varies from spastic paraparesis to Leigh syndrome. Loss-of-function variants are more likely to cause the disease, and variants affecting the GGQ domain tend to be associated with more severe phenotypes, reinforcing a possible genotype-phenotype correlation.

Qualitative, quantitative and genotypic evaluation of Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum isolated from individuals with different periodontal clinical conditions.

Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are strongly associated with periodontitis, and their evaluations are relevant to understand their role in the etiology and progression of periodontal diseases. In this study, the qualitative and quantitative detection of A. actinomycetemcomitans and F. nucleatum, as well as their genetic diversity, were evaluated in individuals with gingivitis, chronic periodontitis and periodontally healthy. In addition, the biotyping, serotyping, and prevalence of the ltx and cdt genes in A. actinomycetemcomitans were also determined. Subgingival biofilms obtained from gingivitis (70), periodontitis (75) and healthy (95) individuals were analyzed by cultures and PCR. Bacterial typing and presence of ltx and cdt genes in A. actinomycetemcomitans were also verified. DNA from A. actinomycetemcomitans and F. nucleatum was detected respectively, in 65.7% and 57.1% of gingivitis, 80% and 68% of periodontitis, and 57.8% and 37.8% of healthy. A. actinomycetemcomitans from gingivitis were biotypes I, II, IV, V, and X, and serotypes a, c, and e. In periodontitis, biotypes II, VI, and X, and serotypes a, b, and c were found. In healthy subjects, biotypes II and X, and serotypes b and c were found. The LTX and ltxA were observed in strains from gingivitis and periodontitis pockets. Subsequently, our data also showed no direct relationship between ltxA gene expression and leukotoxin gene 530-bp presence. On the other hand, cdt gene predominated during the inflammatory disease process. Our results strongly support a role of A. actinomycetemcomitans and F. nucleatum in advanced stage of periodontal disease.

Organochlorine pesticides levels and associated factors in a group of blood donors in São Paulo, Brazil.

The use of organochlorine pesticides (OCPs) is a worldwide public health concern given that high levels of these compounds in humans and animals can lead to serious health problems. This study aimed to evaluate the levels of 15 organochlorine pesticides in the serum of 547 blood donors in the São Paulo metropolitan region (SPMR) in 2009 and to investigate factors associated with higher levels of these compounds. The OCPs were determined by gas chromatography with micro electron capture detection. Multiple ordinal logistic regression models were employed to determine the factors associated with higher levels. Only ß-HCH and p,p'-DDE had a significant number of samples above the limit of quantification. Factors associated with higher levels of ß-HCH were age within 26 to 35 years (OR = 3.1 [1.05-9.09]), age within 36 to 45 years (OR = 18.27 [6.89-48.91]), and female gender (OR = 0.44 [0.24-0.82] for men). p,p'-DDE levels were associated with age within 26 to 35 years (OR = 2.65 [1.20-3.45]), age within 36 to 45 years (OR = 4.59 [2.64-7.92]), female gender (OR = 0.86 [0.43-0.94] for men), and previous work with pesticides (OR = 2.88 [1.22-6.84]). Lower levels of p,p'-DDE were associated with an income of 3 to 5 minimum wages (OR = 0.38 [0.19-0.75]) and with intake of foods from animal origin up to twice a week (OR = 0.15 [0.03-0.72]) and more than twice a week (OR = 0.10 [0.02-0.47]). The results of this study indicate that the study group was not exposed to high levels of OCPs.

Sialidase production and genetic diversity in Clostridium perfringens type A isolated from chicken with necrotic enteritis in Brazil.

The sialidase activity and genetic diversity of 22 Clostridium perfringens strains isolated from chickens with necrotic enteritis were determined. Sialidase activity was detected in 86.4 % of the strains. All C. perfringens showed a high value of similarity (>96 %), and they were grouped into seven clusters clearly separated from the other reference bacterial strains. From these clusters four patterns were defined in accordance with their phenotypic (sialidase production and antibiotic resistance profile) and genotypic (presence of nanI and nanJ genes) characteristics. Our results showed heterogeneity among strains, but they were genotypically similar, and it is suggested further studies are needed to better understand the pathogenesis of necrotic enteritis.

Occurrence and antimicrobial susceptibility of Porphyromonas spp. and Fusobacterium spp. in dogs with and without periodontitis.

The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis.

Intestinal inflammation and the microbiota: Beyond diversity.

The recent manuscript entitled "Relationship between clinical features and intestinal microbiota in Chinese patients with ulcerative colitis" reported a difference in the intestinal microbiota of patients with ulcerative colitis according to the severity of the colitis. The influence of the intestinal microbiota on the development and progress of gastrointestinal disorders is well established. Besides the diversity in the microbiome, the presence of virulence factors and toxins by commensal bacteria may affect an extensive variety of cellular processes, contributing to the induction of a proinflammatory environment.

Detection of Porphyromonas gulae from subgingival biofilms of dogs with and without periodontitis.

A rapid PCR approach was developed to detect Porphyromonas gulae strains from subgingival samples of dogs with and with periodontitis. The presence of P. gulae was observed in 92% and 56%, respectively, in dogs with and without periodontitis. The new primer pair was specific to detect this microorganism, and this technique could be used to evaluate a correlation between periodontitis and P. gulae in companion animals.

A novel variant in the COX15 gene causing a fatal infantile cardioencephalomyopathy: A case report with clinical and molecular review.

The cytochrome c-oxidase (COX) enzyme, also known as mitochondrial complex IV (MT-C4D), is a transmembrane protein complex found in mitochondria. COX deficiency is one of the most frequent causes of electron transport chain defects in humans. Therefore, high energy demand organs and tissues are affected in patients with mutations in the COX15 gene, with variable phenotypic expressiveness. We describe the case of a male newborn with hypertrophic cardiomyopathy and serum and cerebrospinal fluid hyperlacticaemia, whose exome sequencing revealed two variants in a compound heterozygous state: c.232G > A; p.(Gly78Arg), classified as likely pathogenic, and c.452C > G; p.(Ser151Ter), as pathogenic; the former never previously described in the literature.

An Apparently Balanced Complex Chromosome Rearrangement Involving Seven Breaks and Four Chromosomes in a Healthy Female and Segregation/Recombination in Her Affected Son.

Duplication of the distal 1q and 4p segments are both characterized by the presence of intellectual disability/neurodevelopmental delay and dysmorphisms. Here, we describe a male with a complex chromosome rearrangement (CCR) presenting with overlapping clinical findings between these 2 syndromes. In order to better characterize this CCR, classical karyotyping, FISH, and chromosomal microarray analysis were performed on material from the patient and his parents, which revealed an unbalanced karyotype with duplications at 1q41q43 and 4p15.2p14 in the proband. The rearrangements, which were derived from a maternal balanced karyotype, included an insertion of a segment from the long to the short arm of chromosome 1, a balanced translocation involving chromosomes 14 and 18, and an insertion of a segment from the short arm of chromosome 4 into the derived chromosome 14. This study aimed to better define the clinical history and prognosis of a patient with this rare category of chromosomal aberration. Our results suggest that the frequency of CCR in the general population may be underestimated; when balanced, they may not have a phenotypic effect. Moreover, they emphasize the need for cytogenetic techniques complementary to chromosomal microarray for proper genetic counseling.

Presence of periodontopathic bacteria in coronary arteries from patients with chronic periodontitis.

In this study the presence of periodontopathic pathogens in atheromatous plaques removed from coronary arteries of patients with chronic periodontitis and periodontally healthy subjects by PCR was detected. Our results indicate a significant association between the presence of Porphyromonas gingivalis and atheromas, and the periodontal bacteria in oral biofilm may find a way to reach arteries.

Congenital afibrinogenemia in a patient with vascular abnormalities and a novel variant: clinical-molecular description and literature review.

: The objective is to report a patient with congenital afibrinogenemia and vascular abnormalities and also review the clinical and molecular issues. The female proband, diagnosed with congenital afibrinogenemia, was admitted at a hospital due to a hemorrhagic shock. Angiotomography revealed ectasias from ascending branch to the abdominal aorta, with multiple calcifications and atheroma. Clinical exome identified a homozygous novel pathogenic variant in FGG gene. In our review the main symptom, at diagnosis, was umbilical cord bleeding and the degree of clinical involvement varied from asymptomatic to severe. The FGA gene was the most affected and possible hot spots were observed. Variants considered as loss of function were the most frequent. The association of vascular abnormalities in a patient with congenital afibrinogenemia alerts for a closer follow-up of vascular issues in these patients.

Prevalence of Enteropathogens and Virulence Traits in Brazilian Children With and Without Diarrhea.

The use of molecular diagnostics for pathogen detection in epidemiological studies have allowed us to get a wider view of the pathogens associated with diarrhea, but the presence of enteropathogens in asymptomatic individuals has raised several challenges in understanding the etiology of diarrhea, and the use of these platforms in clinical diagnosis as well. To characterize the presence of the most relevant bacterial enteropathogens in diarrheal episodes, we evaluated here the prevalence of diarrheagenic E. coli pathotypes, Salmonella spp., and Yersinia enterocolitica in stool samples of children with and without diarrhea using real-time quantitative PCR (qPCR). We found that the presence of genetic markers associated with bacterial pathogens was significantly higher in stool samples from the diarrhea group compared to the control (P < 0.001). Bacterial loads in samples positive for eae and aggR markers were also determined. Compared to samples from asymptomatic children, a significantly higher number of copies of the eae gene were found in diarrhea samples. Also, the presence of genetic markers associated with STEC strains with clinical significance was evaluated in eae-positive samples by high-throughput real-time PCR. The data presented herein demonstrated that asymptomatic children of an urban area in Brazil might be enteropathogen reservoirs, especially for STEC.

Occurrence of yeasts, enterococci and other enteric bacteria in subgingival biofilm of HIV-positive patients with chronic gingivitis and necrotizing periodontitis.

The purpose of this study was to determine the prevalence of enteric bacteria and yeasts in biofilm of 80 HIV-positive patients with plaque-associated gingivitis or necrotizing periodontitis. Patients were subjected to extra, intra oral and radiographic examinations. The oral hygiene, bleeding on probing, gingival conditions, and attachment loss were evaluated. Clinical specimens were collected from gingival crevices or periodontal pockets, transferred to VMGA III, diluted and transferred to Sabouraud Dextrose agar with 100 µg/ml of chloramphenicol, peptone water, EVA broth, EMB agar, SS agar, Bile esculin agar and Brilliant green agar. Isolation of yeasts was carried out at room temperature, for 3-7 days; and for the isolation of enteric microorganisms plates were incubated at 37°C, for 24-48 h. The yeasts identification was performed according to the carbon and nitrogen assimilation, fermentation of carbohydrates and germ tube formation. Bacteria were identified according to their colonial and cellular morphologies and biochemical tests. Yeasts were identified as Candida albicans and its occurrence was more common in patients with CD4+ below 200/mm(3) and was affected by the extension of periodontal involvement (P = 0.0345). Enteric bacteria recovered from clinical specimens were identified as Enterobacter sakazakii, Enterobacter cloacae, Serratia liquefaciens, Klebsiella oxytoca and Enterococcus sp. Enterobacteriaceae and enterococci were detected in 32.5% of clinical samples from patients with necrotizing periodontitis. In conclusion, non-oral pathogenic bacteria and C. albicans were more prevalent in periodontal sites of HIV-positive patients with necrotizing periodontitis and chronic gingivitis.

Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis.

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases.

A Novel MGP Gene Mutation Causing Keutel Syndrome in a Brazilian Patient.

Keutel syndrome is caused by mutations in the matrix gamma-carboxyglutamic acid (MGP) gene (OMIM 154870) and is inherited in an autosomal recessive fashion. It is characterized by brachydactyly, pulmonary artery stenosis, a distinctive facial phenotype, and cartilage calcification. To date, only 36 cases have been reported worldwide. We describe clinical and molecular findings of the first Brazilian patient with Keutel syndrome. Keutel syndrome was suspected based on clinical and morphological evaluation, so we sequenced the MGP gene using the TruSight One Sequencing Panel (Illumina). The obtained MGP gene sequence was then validated by Sanger sequencing. We identified a novel pathogenic homozygous variant of the MGP gene (c.2T>C; p.Met1Thr) confirming Keutel syndrome. Proper diagnosis of this syndrome is important for clinical management and is an indication for genetic counseling. Keutel syndrome should be suspected in patients with cartilage calcifications and brachydactyly when associated with a distinctive facial phenotype and pulmonary artery stenosis.

Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility.

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.

Pathogenicity and genetic profile of oral Porphyromonas species from canine periodontitis.

OBJECTIVE: In this study, the presence of the prtC and fimA genes involved in the pathogenicity of oral Porphyromonas spp. isolated from dogs with periodontitis and healthy, as well as their genetic diversity was investigated. DESIGN: Thirty-two Beagle dogs, 24 with periodontitis and 8 healthy were evaluated. Subgingival samples from only one gingival site of both groups were collected. Bacteria grown in anaerobiosis were identified by RAPID ID 32A kits. From each strain the respective DNA was obtained and used to genotyping by conventional PCR and AP-PCR. RESULTS: Dogs with periodontitis harbored 28 P. gulae, 2 P. creviocaricanis, 1 P. cangingivalis and 7 P. macacae; and from healthy dogs, 11 P. gulae and 5 P. circumdentaria. In P. gulae isolated from periodontal dogs the gene prtC was observed in 19 (67.85%) and in 7 (63.63%) from healthy dogs. P. gulae strains from periodontal dogs harbored either the gene fimA I or fimA II; while strains from healthy dogs harbored the gene fimA I, fimA II, fimA III or fimA IV, as well as 1 P. circumdentaria the gene fimA II. By AP-PCR strains were grouped in different clusters suggesting heterogeneity of these microorganisms. CONCLUSIONS: The results presented herein inform that Porphyromonas spp. isolated from dogs with and without periodontitis harbored the prtC and fimA genes and it could be a role in the establishment of the infectious process.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells.

Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Alterations of Intestinal Microbiome by Antibiotic Therapy in Hospitalized Children.

The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.