ABSTRACT
CONTEXT AND OBJECTIVE: Mycobacterium avium subsp. paratuberculosis (MAP) has attracted the interest of researchers because of similarities between paratuberculosis and Crohn's disease (CD). The aim of this study was to evaluate the frequency of MAP through cultures, histology and polymerase chain reaction (PCR) on intestinal biopsies from Brazilian CD patients. Quantitative real time PCR (qRT-PCR) was performed on positive samples. DESIGN AND SETTING: Analytical cross-sectional study with control group at two federal universities. METHODS: Fresh samples were collected from 25 patients; five with CD, eight with ulcerative colitis (UC) and 12 controls with non-inflammatory bowel disease (nIBD). Formalin-fixed paraffin-embedded (FFPE) samples from 143 patients were also collected: 44 CD, 49 UC and 56 nIBD. RESULTS: None of the fresh samples was positive for MAP. Five FFPE samples (one CD, two UC and two nIBD) and three fresh samples (one in each group) were positive through IS900-PCR. qRT-PCR was performed on these eight samples. Among the FFPE samples, there were 192.12 copies/μl in the CD group, 72.28 copies/μl in UC and 81.43 copies/μl in nIBD. Among the fresh samples, there were 432.99 copies/μl, 167.92 copies/μl and 249.73 copies/μl in the CD, UC and nIBD groups, respectively. The highest bacterial load was in the CD group. CONCLUSION: This study does not provide evidence for a role of MAP in the etiology of CD, although MAP DNA was detected in all three patient groups. This is the first report of MAP presence in human intestinal biopsies in Brazil.
CONTEXTO E OBJETIVO: Mycobaterium avium subsp. paratuberculosis (MAP) tem atraído o interesse de pesquisadores devido às semelhanças entre a paratuberculose e a doença de Crohn (CD). Este estudo objetivou avaliar a frequência de MAP por meio de cultura, histologia e reação da polimerase em cadeia (PCR), em biópsias intestinais de pacientes brasileiros com CD. PCR quantitativa em tempo real (qRT-PCR) foi realizada nas amostras positivas. TIPO DE ESTUDO E LOCAL: Estudo transversal analítico com grupo controle realizado em duas universidades federais. MÉTODOS: Amostras frescas foram coletadas de 25 pacientes; cinco com CD, oito com colite ulcerativa (UC) e 12 controles sem doença inflamatória intestinal (nIBD). Também foram coletadas 149 amostras fixadas em parafina (FFPE): 44 CD, 49 UC e 56 nIBD. RESULTADOS: Nenhuma das amostras frescas foi positiva para MAP. Cinco amostras FFPE (uma CD, duas UC e duas nIBD) e três amostras frescas (uma de cada grupo) foram positivas por IS900-PCR. qRT-PCR foi realizada nessas oito amostras. Nas amostras FFPE, havia 192,12 cópias/μl no grupo CD, 72,28 cópias/μl no UC e 81,43 cópias/μl no nIBD. Nas amostras frescas, havia 432,99 cópias/μl, 167,92 cópias/μl e 249,73 cópias/μl nos grupos CD, UC e nIBD, respectivamente. A maior carga bacteriana foi encontrada no grupo CD. CONCLUSÃO: Este estudo não fornece evidências do papel de MAP na etiologia da CD, embora DNA de MAP tenha sido detectado em pacientes dos três grupos. Este é o primeiro relato da presença de MAP em biópsias intestinais humanas no Brasil.
Subject(s)
Humans , Male , Female , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Inflammatory Bowel Diseases/microbiology , Colitis, Ulcerative/microbiology , Crohn Disease/microbiology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Case-Control Studies , Polymerase Chain Reaction/methods , Cross-Sectional Studies , Mycobacterium avium subsp. paratuberculosis/pathogenicity , Sequence Analysis, DNA/methodsABSTRACT
CONTEXT AND OBJECTIVE: Mycobacterium avium subsp. paratuberculosis (MAP) has attracted the interest of researchers because of similarities between paratuberculosis and Crohn's disease (CD). The aim of this study was to evaluate the frequency of MAP through cultures, histology and polymerase chain reaction (PCR) on intestinal biopsies from Brazilian CD patients. Quantitative real time PCR (qRT-PCR) was performed on positive samples. DESIGN AND SETTING: Analytical cross-sectional study with control group at two federal universities. METHODS: Fresh samples were collected from 25 patients; five with CD, eight with ulcerative colitis (UC) and 12 controls with non-inflammatory bowel disease (nIBD). Formalin-fixed paraffin-embedded (FFPE) samples from 143 patients were also collected: 44 CD, 49 UC and 56 nIBD. RESULTS: None of the fresh samples was positive for MAP. Five FFPE samples (one CD, two UC and two nIBD) and three fresh samples (one in each group) were positive through IS900-PCR. qRT-PCR was performed on these eight samples. Among the FFPE samples, there were 192.12 copies/µl in the CD group, 72.28 copies/µl in UC and 81.43 copies/µl in nIBD. Among the fresh samples, there were 432.99 copies/µl, 167.92 copies/µl and 249.73 copies/µl in the CD, UC and nIBD groups, respectively. The highest bacterial load was in the CD group. CONCLUSION: This study does not provide evidence for a role of MAP in the etiology of CD, although MAP DNA was detected in all three patient groups. This is the first report of MAP presence in human intestinal biopsies in Brazil.
Subject(s)
Colitis, Ulcerative/microbiology , Crohn Disease/microbiology , Inflammatory Bowel Diseases/microbiology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Mycobacterium avium subsp. paratuberculosis/pathogenicity , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Young AdultABSTRACT
A contagem de células somáticas (CCS) determina a quantidade de leucócitos e células epiteliais presentes no leite. Em caso de inflamação (mastite subclínica ou clínica), há um aumento considerável na CCS, principalmente de neutrófilos. É importante quantificar e qualificar os tipos celulares presentes para conhecer o grau de inflamação e caracterizar como aguda ou crônica. O objetivo deste trabalho foi testar quatro métodos de coloração de células somáticas; broadhurst-Paley, hematoxilina-eosina, corante Panótico® e May Grunwald-Giemsa por meio da microscopia óptica e determinar a mais adequada para nossas condições de laboratório. Diante dos dados obtidos, elegeu-se a coloração de associação de eosina e azul de metileno (May Grunwald-Giemsa) com a qual se obteve resultados variando entre 60.000 e 58.690.000 células/mL em diferentes amostras de leite bovino. Não foi possível quantificar as células pelos outros três métodos de coloração devido às alterações morfológicas exacerbadas das mesmas.
The somatic cell count (SCC) determines the amount of leukocytes and epithelial cells present in milk. In case of inflammation (subclinical or clinical), there is a considerable increase in SCC, especially neutrophils. It is important to quantify and qualify the cell types present to know the degree of inflammation and characterized as acute or chronic. The objective was to test four staining methods of somatic cells; Broadhurst-Paley, hematoxylin-eosin stain Panoptic and May Grunwald-Giemsa by light microscopy and determine the most suitable for our laboratory conditions. Considering the available data, was elected the association of eosin staining and methylene blue (May-Grunwald Giemsa) with which they obtained results ranging from 60,000 to 58,690,000 cells / ml in different samples of bovine milk. It was not possible to quantify the cells by the other three staining methods due to morphological changes exacerbated them.