Hereditary breast cancer next-generation sequencing (NGS) panel evaluation in the south region of Brazil: a novel BRCA2 candidate pathogenic variant is reported.

In this article, we delineate a loosely selected cohort comprising patients with a history of early-onset breast cancer and/or a familial occurrence of cancer. The aim of this study was to gain insights into the presence of breast cancer-related gene variants in a population from a micro-region in southern Brazil, specifically the Metropolitan Region of Curitiba. This area exhibits a highly genetically mixed population, mirroring the general characteristics of the Brazilian people. Comprehensive next-generation sequencing (NGS) multigene panel testing was conducted, involving the evaluation of twelve patients. Two pathogenic variants and one candidate pathogenic variant were identified: BRCA2:c.8878C>T, p.Gln2960Ter; CHEK2:c.1100delAG>A, p.Thr367Metfs*15 and BRCA2:c.3482dupG>GA, p.Asp1161Glufs*3, a novel variant, previously unpublished, is reported.

Digital PCR detection of EGFR somatic mutations in non-small-cell lung cancer formalin fixed paraffin embedded samples.

BACKGROUND: Digital PCR (dPCR) is proposed to replace real time PCR and Sanger sequencing for detection and quantification of rare mutations, frequently unnoticed in the mass of tumoral cells. Screening of endothelial growth factor receptor (EGFR) mutations is mandatory before treatment with EGFR-targeted therapy with small-molecule tyrosine kinase inhibitors, which has been approved for the treatment of advanced non-small-cell lung cancer (NSCLC). OBJECTIVE: In order to establish a cost-effective method for detection of mutations, we optimized dPCR identification of EGFR mutations in exons 18-21, and determined dPCR sensitivity, limits of detection (LoD) and quantification (LoQ). METHODS: For clinical validation, we compared the performance of dPCR and castPCR in 57 NSCL formalin fixed paraffin embedded samples and 10 lung cancer-free formalin fixed paraffin embedded samples. RESULTS: EGFR mutations DEL19, p.L858R, p.G719X, p.L861Q and p.T790 M were detected by dPCR in 27 samples versus 11 detected by castPCR (p = 0.014). LoD was determined as 100 molecules of DNA/uL and LoQ as 1%. Most of the samples (87%) identified by competitive Allele-Specific TaqMan (castPCR) as wild-type and by dPCR as mutated, presented less than 10% mutated DNA molecules (mean 4.57%). Accuracy of dPCR was 94.44%, as measured with the assay recommended by the College of American Pathologists. CONCLUSION: These results indicated higher sensibility and specificity of dPCR for screening EGFR mutations in NSCLC biopsies, compared to castPCR.

An outbreak of tuberculosis by Mycobacterium bovis in coatis (Nasua nasua).

Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, infrequently causes severe or lethal disease in captive wildlife populations. A dead coati from a wildlife triage center showing pulmonary lesions compatible with tuberculosis had raised suspicion of a potential disease caused by mycobacteria species and was further investigated. Four native coatis (Nasua nasua) with suspected mycobacterial infection were sedated, and bronchoalveolar lavages and tuberculin skin tests (TSTs) were performed. All animals tested positive upon TST. Mycobacterial culturing, Ziehl-Neelsen staining, and genetic testing were performed on postmortem samples and the etiologic agent was identified as M. bovis. Molecular genetic identification using a polymerase chain reaction panel was crucial to achieving a definitive diagnosis.

Detection of RD(Rio) strain of Mycobacterium tuberculosis in tapirs (Tapirus terrestris) from a zoo in Brazil.

Tuberculosis is a chronic infection caused by strains of the Mycobacterium tuberculosis complex and occurs in both animal and human populations. The death of a tapir showing purulent material and a hard mass in the lungs at necropsy raised suspicion of a potential disease caused by mycobacteria species in a Brazilian zoo. Later, two other tapirs with similar signs died and were further investigated. Polymerase chain reaction (PCR) from bronco-alveolar lavages was performed, and both animals tested positive for the RD(Rio) strain of M. tuberculosis, which is a recently discovered Latin American-Mediterranean sublineage and the main cause of human tuberculosis in Rio de Janeiro, Brazil. To investigate the possibility of human infection and the source of transmission, all 50 zoo employees underwent tuberculin skin testing; four were reactive, but radiographic exams and direct sample staining did not suggest tuberculosis. Thus, direct human to animal transmission was not proven. However, the presence of RD(Rio) M. tuberculosis in tapirs highlights the lack of attention to diseases that human beings may transmit to wildlife.

Molecular identification and typing of Mycobacterium massiliense isolated from postsurgical infections in Brazil

OBJECTIVE: One hundred thirty-one cases of postsurgical infections were reported in Southern Region of Brazil between August 2007 and January 2008. Thirty-nine (29.8 percent) cases were studied; this report describes epidemiological findings, species identification, antimicrobial susceptibility and clonal diversity of rapidly growing mycobacteria isolated in this outbreak. METHODS: All 39 isolates were analyzed by Ziehl-Nielsen stained smear, bacterial culture and submitted to rpoB partial gene sequencing for identification. The isolates were also evaluated for their susceptibility to amikacin, cefoxitin, clarithromycin, ciprofloxacin, doxycycline, tobramycin and sulfamethoxazole. RESULTS: Thirty-six isolates out of the confirmed cases were identified as Mycobacterium massilienseand the remaining three were identified as Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum. All M. massiliense isolates were susceptible to amikacin (MIC90 = 8 µg/mL) and clarithromycin (MIC90 = 0.25 µg/mL) but resistant to cefoxitin, ciprofloxacin, doxycycline, tobramycin and sulfamethoxazole. Molecular analysis by pulsed-field gel electrophoresis clustered all 36 M. massiliense isolates and showed the same pattern (BRA 100) observed in three other outbreaks previously reported in Brazil. CONCLUSIONS: These findings suggest a common source of infection for all patients and reinforce the hypotheses of spread of M. massiliense BRA100 in Brazilian hospital surgical environment in recent years.

Molecular identification and typing of Mycobacterium massiliense isolated from postsurgical infections in Brazil.

OBJECTIVE: One hundred thirty-one cases of postsurgical infections were reported in Southern Region of Brazil between August 2007 and January 2008. Thirty-nine (29.8%) cases were studied; this report describes epidemiological findings, species identification, antimicrobial susceptibility and clonal diversity of rapidly growing mycobacteria isolated in this outbreak. METHODS: All 39 isolates were analyzed by Ziehl-Nielsen stained smear, bacterial culture and submitted to rpoB partial gene sequencing for identification. The isolates were also evaluated for their susceptibility to amikacin, cefoxitin, clarithromycin, ciprofloxacin, doxycycline, tobramycin and sulfamethoxazole. RESULTS: Thirty-six isolates out of the confirmed cases were identified as Mycobacterium massiliense and the remaining three were identified as Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum. All M. massiliense isolates were susceptible to amikacin (MIC90 = 8 µg/mL) and clarithromycin (MIC90 = 0.25 µg/mL) but resistant to cefoxitin, ciprofloxacin, doxycycline, tobramycin and sulfamethoxazole. Molecular analysis by pulsed-field gel electrophoresis clustered all 36 M. massiliense isolates and showed the same pattern (BRA 100) observed in three other outbreaks previously reported in Brazil. CONCLUSIONS: These findings suggest a common source of infection for all patients and reinforce the hypotheses of spread of M. massiliense BRA100 in Brazilian hospital surgical environment in recent years.

Tuberculose bovina: saúde animal e saúde pública / Bovine tuberculosis: animal and human health / Tuberculosis bovina: salud animal y salud pública

A tuberculose é uma doença infecto-contagiosa, de abrangência mundial, que afeta seres humanos e outras diversas espécies animais. A principal espécie infectante para os bovinos é o Mycobacterium bovis, o qual é endêmico no Brasil e causa prejuízos econômicos, por redução da produtividade do rebanho e perdas de carcaças no frigorífico. Por se tratar de uma zoonose, nos seres humanos pode causar uma doença clinica e patologicamente indistinguível da infecção por Mycobacterium tuberculosis. Apesar da carência de informações sobre a relevância da tuberculose bovina para os seres humanos, o aumento do número de pacientes imunossuprimidos no país, principalmente pelo vírus da imunodeficiência humana, causa preocupação em relação a doenças causadas por espécies atípicas. A espécie M. bovis é transmitida principalmente através da via aerógena para bovinos e através do consumo de leite e derivados crus para pessoas. A imunidade do hospedeiro contra os bacilos da tuberculose envolve uma variedade de eventos celulares, podendo resultar em apresentações clínicas e patologia diferenciada, variando desde infecções autolimitantes até doença sistêmica grave. O diagnóstico em bovinos é realizado principalmente por tuberculinização. Entretanto, é importante realizar a identificação laboratorial da espécie causadora da doença. Embora programas de controle e erradicação tenham sido implantados apenas na última década no país, o controle da tuberculose é fundamental para saúde pública, saúde animal e produção animal.

Tuberculosis is a worldwide infectious disease that affects human beings and several other animal species. The main specie infecting bovine is Mycobacterium bovis, which is endemic in Brazil and causes economic losses due to productivity reduction and losses at the slaughterhouse. Since tuberculosis is a zoonosis, M. bovis may infect human beings, causing a clinically or pathologically disease indistinguishable from M. tuberculosis infection. Despite the lack of information about bovine tuberculosis relevance to human beings, immunosuppressed pacients increasing in the country, mainly due to human immunodeficiency virus, causes concern about diseases caused by atypical species. Mycobacterium bovis specie is mainly transmitted by respiratory route to cattle and by raw milk and other products to human beings. The host immunity against tuberculosis bacillus includes a variety of cellular events, and may result in differentiated clinical and pathology results, ranging from self-limiting infection to severe systemic disease. Bovine diagnosis is mainly by tuberculin skin test, however, differentiate the infecting specie is important. Although control and eradication programs have been implanted only in the last decade in the country, tuberculosis control is essential for both public and animal health.

La tuberculosis es una enfermedad infecto-contagiosa, de distribución mundial, que afecta seres humanos y otras diversas especies animales. La principal especie infecciosa para los bovinos es el Mycobacterium bovis, la cual es endémica en Brasil y causa pérdidas económicas, por reducción de la productividad del rebaño y pérdidas de carcasas en el frigorífico. Además, por ser una zoonosis, en los seres humanos puede causar una enfermedad clínica y patológicamente indistinguible de la infección por M. tuberculosis. A pesar de la falta de información sobre la importancia de la tuberculosis bovina para los seres humanos, el aumento del número de pacientes inmune suprimidos en el país, principalmente por el virus de la inmunodeficiencia humana, causa preocupación en relación a enfermedades causadas por especies atípicas. La especie M. bovis es transmitida principalmente a través de la vía respiratoria para bovinos y a través del consumo de leche y derivados crudos para personas. La inmunidad del hospedador contra los bacilos de la tuberculosis incluye una variedad de eventos celulares, los cuales pueden resultar en presentaciones clínicas y patología diferenciada, variando desde infecciones autolimitantes hasta enfermedad sistémica grave. El diagnóstico en bovinos se realiza principalmente a través de prueba de la tuberculina. Sin embargo, es importante realizar la identificación en laboratorio de la especie causante de la enfermedad. Aunque programas de control y erradicación hayan sido implementados en la última década en el país, el control de tuberculosis es fundamental para la salud pública, salud animal y producción animal.

Genotipagem do vírus da hepatite C por PCR em tempo real com base na análise da região NS5B / Genotyping of hepatitis C vírus by real time PCR based in analysis of NS5B region

A genotipagem do vírus da hepatite C (VHC) é a principal ferramenta para prognóstico e tempo de tratamento. Dependendo do genótipo infectado existem diferentes esquemas e tempo de tratamento. O objetivo deste trabalho foi desenvolver padronizar e validar um método de genotipagem por PCR em tempo real com base na análise da região NS5B. Esta região apresenta um grau de polimorfismo que permite identificar de modo mais acurado tanto os tipos como os subtipos do VHC. Para isto foram desenhados dois conjuntos de primers e sondas. Neste trabalho desenvolvemos um método one-step modificado em uma reação triplex em que ocorre a identificação dos genótipos (1a, 1b, 3a) e em outro set a identificação dos genótipos (2a, 2b, 2c). Os resultados obtidos pelo método de genotipagem em tempo real concordaram em 100% com os resultados de seqüênciamento da região NS5B quando excluímos amostras que foram identificados como mistura de genótipos no método desenvolvido e classificados somente como um único genótipo no seqüênciamento. Houve uma boa concordância entre o método desenvolvido e o seqüênciamento da região NS5B pelo coeficiente de Kappa (k= 0,6222; p=0,0020). O método de desenvolvido conseguiu detectar 97,93% (190/194) do genótipo 1, 86,11% (31/36) do genótipo 2 e 100% (80/80) do genótipo 3. A média da sensibilidade foi de 97%. Quando comparamos a genotipagem por PCR em tempo real e LiPA nas 310 amostras analisadas não houve resultados discordantes em relação ao genótipo. Entretanto, 26,24% (79/301) das amostras analisadas apresentaram resultados discordantes em relação ao subtipo quando comparados os dois métodos. Foi determinada a sensibilidade analítica do método através de um ponto do painel da OptiQuant que foi diluído de modo seriado e a sensibilidade relativa foi realizada através de amostras de plasma de pacientes com carga viral determinada pelo Cobas Amplicor...

Hepatitis C virus (HCV) genotyping is the most significant predictor of response to antiviral therapy. Depending on the infecting HCV genotyping different antiviral regimens have been proposed as well as the length of different treatment. The aim of this study was to develop and evaluate a new real time PCR of HCV genotyping based in NS5B region. This region has sequencing heterogeneity and can accurately identify both type and subtype of HCV. Furthermore, we compared the real time PCR with LiPA and sequencing of NS5B region. We developed a new one-step modified method in triplex reaction where we identified in two sets genotypes (1a, 1b, 3a) and (2a, 2b, 2c). Results obtained by real time PCR agreed 100% with those obtained by NS5B sequencing when excluded samples with mixed of HCV genotypes identified by real time PCR genotyping and in NS5B sequencing all samples were classified only as only one genotype. We found a good concordance for the analysis of genotype concordance between genotyping by real time and sequencing of NS5B region through the coefficient kappa (k= 0,6222; p=0,0020). The method developed detected 97,93% (190/194) of genotype 1, 86,11% (31/36) of genotype 2 and 100% (80/80) of genotype 3, with the overall sensitivity of this new method being 97%. Among 310 samples only two samples had discordant results at type level when comparing real time PCR and LiPA. However, 26,24% (79/301) had discordant results at subtype level when comparing LiPA and real time PCR genotyping of HCV. In order to measure the analytical sensitivity of the real time assay, one member of the panel OptiQuant HCV RNA was diluted. The relative sensitivity was determined by analysis the clinical specimens based upon the initial HCV RNA concentration determined by Cobas Amplicor...