RESUMO
Campylobacter jejuni (CJ) is the most commonly isolated stool pathogen in the United States. Biopsy findings are typically those of focal active colitis (FAC), a nonspecific pattern usually indicating infection or adverse drug effect that is characterized by focal cryptitis and preservation of crypt architecture. We developed a molecular test for CJ that can be performed on routinely processed gastrointestinal biopsy specimens, and assessed what percentage of patients with biopsy findings of FAC have molecular evidence of CJ infection. One hundred and ten colon biopsies diagnosed as FAC were retrieved from three institutions. Polymerase chain reaction (PCR) was performed following DNA extraction; primers were designed to target a 286-bp fragment of the mapA gene that is specific to CJ. Pure genomic DNA derived from cultures served as the positive control; reagent blanks and 50 normal colon specimens served as negative controls. Nineteen percent (21/110) of the FAC biopsies were positive for CJ DNA by PCR analysis. Fourteen CJ-positive patients presented with diarrhea, 3 presented with gastrointestinal bleeding, and 3 had incidental FAC found on screening colonoscopy. Ten patients had abnormal colonoscopic findings, including erythema (4), ulcers (4), colitis (1), and hemorrhage (1). As CJ is an enteric pathogen that is not present in the gut as a commensal organism, the presence of CJ DNA suggests current or recent previous infection in these patients. CJ infection should be considered in patients with diarrhea and colon biopsies showing FAC. Furthermore, PCR analysis performed on fixed, routinely processed colon biopsies is an excellent diagnostic method for detection of this organism.
Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/isolamento & purificação , Colite/diagnóstico , Colite/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Campylobacter/complicações , Colite/complicações , Diagnóstico Diferencial , Diarreia/etiologia , Feminino , Gastroenteropatias/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Yersinia enterocolitica (YE) is associated with several inflammatory gastrointestinal disorders. Pathogenic YE organisms are classified as biogroup 1B (high-virulence [HV] serovars) or biogroups 2 through 5 (low virulence [LV]). We developed the first molecular assay designed to distinguish between these groups and correlated the molecular results with histologic patterns of inflammation. Eleven known pathogenic YE culture isolates (6 biogroup 1B and 5 biogroups 2-5) and 6 YE-positive archival cases were subjected to polymerase chain reaction analysis using primer pairs targeting a strain-dependent variable region, allowing discrimination between biogroups with a single assay. All 11 known culture isolates were confirmed. Of the 6 archival cases, 4 were LV, and 2 were HV. Histologic correlation revealed granulomatous inflammation in the LV cases and suppurative inflammation in the HV cases. This novel assay is useful for diagnosis using culture samples and archival tissues. It also could yield important information correlating YE epidemiology, pathogenesis, and morphology because these preliminary data suggest that LV strains may be associated with chronic granulomatous processes and HV strains with suppurative inflammation.
Assuntos
DNA Bacteriano/análise , Reação em Cadeia da Polimerase/métodos , RNA Bacteriano/análise , Yersiniose/diagnóstico , Yersinia enterocolitica , Fixadores , Formaldeído , Humanos , Inclusão em Parafina , RNA Ribossômico 23S/análise , Estudos Retrospectivos , Yersiniose/microbiologia , Yersinia enterocolitica/classificação , Yersinia enterocolitica/genética , Yersinia enterocolitica/patogenicidadeRESUMO
Previously, we detected pathogenic (invasive) DNA in the appendices of two patients who later developed Crohn's disease (CD). This subsequent investigation is the first to evaluate a series of specimens from CD patients for the presence of pathogenic DNA. A total of 54 intestinal resection specimens from 52 patients with confirmed CD were evaluated. Lesional tissue was tested by polymerase chain reaction analysis for the presence of genes occurring only in pathogenic Primer pairs are specific for each species, with no known cross reactions with other bacteria. Forty normal bowel specimens, 30 cases of acute appendicitis, and 50 cases of various active colitides served as disease controls. Medical records were reviewed following polymerase chain reaction and histologic evaluation. A total of 17 of 54 resections (31%) contained DNA by polymerase chain reaction. Mesenteric lymph nodes were also positive in eight of these cases. All controls were negative. -positive patients had carried the diagnosis of CD for a median of 10 years before resection (range 1 month to 40 years). We report the first documentation of DNA in a series of CD cases. Further studies are needed, including serial study, over time, of -positive CD patients, as well as prospective studies of newly diagnosed CD patients for evidence of infection. Like previous studies associating infectious organisms with CD, much work remains to elucidate whether the presence of DNA is an epiphenomenon or actually a factor in the pathogenesis of CD.
Assuntos
Apendicite/microbiologia , Doença de Crohn/microbiologia , DNA Bacteriano/análise , Yersiniose/microbiologia , Yersinia enterocolitica/isolamento & purificação , Adulto , Idoso , Apendicite/patologia , Doença de Crohn/patologia , Feminino , Humanos , Íleo/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Yersiniose/patologia , Yersinia enterocolitica/genética , Yersinia enterocolitica/patogenicidadeRESUMO
Cat-scratch disease (CSD) is largely due to infection with Bartonella henselae. Microbiologic detection is difficult, and molecular testing is not readily available. A monoclonal antibody (mAB) to B henselae has become commercially available. We evaluated the usefulness of immunohistochemical analysis (IHC) for diagnosing CSD on surgical specimens and compared these results with polymerase chain reaction (PCR) detection and serologic testing for B henselae. We studied 24 formalin-fixed, paraffin-embedded (FFPE) cases of lymphadenitis with histologic and/or clinical suspicion of CSD. Control cases included 14 cases of lymphadenopathy other than CSD. FFPE tissue sections were evaluated with an mAB to B henselae, Steiner silver stain (SSS), and PCR that targeted B henselae and Bartonella quintana. Positive cases were as follows: SSS, 11 (46%); PCR, 9 (38%); and IHC, 6 (25%). Only 2 cases (8%) were positive for all 3 studies. All control cases were negative for IHC and PCR. The diagnostic sensitivity of these 3 tests is low for CSD. SSS seems to be the most sensitive test but is the least specific. PCR is more sensitive than IHC and may, therefore, serve as a helpful second-line test on all IHC- cases.
Assuntos
Bartonella henselae/isolamento & purificação , Doença da Arranhadura de Gato/diagnóstico , Imuno-Histoquímica/métodos , Adolescente , Adulto , Antígenos de Bactérias/análise , Bartonella henselae/genética , Bartonella henselae/imunologia , Doença da Arranhadura de Gato/sangue , Doença da Arranhadura de Gato/microbiologia , Criança , Pré-Escolar , DNA Bacteriano/análise , Feminino , Humanos , Lactente , Linfadenite/diagnóstico , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Testes Sorológicos , Coloração pela Prata/métodos , Adulto JovemRESUMO
Activation of PPARgamma in MOSER cells inhibits anchorage-dependent and anchorage-independent growth and invasion through Matrigel-coated transwell membranes. We carried out a longitudinal two-class microarray analysis in which mRNA abundance was measured as a function of time in cells treated with a thiazolidinedione PPARgamma agonist or vehicle. A statistical machine learning algorithm that employs an empirical Bayesian implementation of the multivariate HotellingT2 score was used to identify differentially regulated genes. HotellingT2 scores, MB statistics, and maximum median differences were used as figures of merit to interrogate genomic ontology of these targets. Three major cohorts of genes were regulated: those involved in metabolism, DNA replication, and migration/motility, reflecting the cellular phenotype that attends activation of PPARgamma. The bioinformatic analysis also inferred that PPARgamma regulates calcium signaling. This response was unanticipated, because calcium signaling has not previously been associated with PPARgamma activation. Ingenuity pathway analysis inferred that the nodal point in this cross-talk was Down syndrome critical region 1 (DSCR1). DSCR1 is an endogenous calcineurin inhibitor that blocks dephosphorylation and activation of members of the cytoplasmic component of nuclear factor of activated T cells transcription factors. Lentiviral short hairpin RNA-mediated knockdown of DSCR1 blocks PPARgamma inhibition of proliferation and invasion, indicating that DSCR1 is required for suppression of transformed properties of early stage colorectal cancer cells by PPARgamma. These data reveal a novel, heretofore unappreciated link between PPARgamma and calcium signaling and indicate that DSCR1, which has previously been thought to function by suppression of the angiogenic response in endothelial cells, may also play a direct role in transformation of epithelial cells.
Assuntos
Sinalização do Cálcio , Neoplasias Colorretais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , PPAR gama/genética , PPAR gama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Biologia Computacional/métodos , Citoplasma/metabolismo , Proteínas de Ligação a DNA , Genômica/métodos , Humanos , Lentivirus/metabolismo , Modelos Biológicos , Fosforilação , Transdução de SinaisRESUMO
The role of enteric bacteria in the pathogenesis of acute appendicitis is a controversial subject. Campylobacter jejuni has been previously demonstrated in a minority of cases of acute appendicitis using microbiological or immunohistochemical methods, notably in cases where inflammation was limited to the mucosa/submucosa. Our goal was to evaluate cases of acute appendicitis for C. jejuni DNA using molecular methods, and to compare our findings to the histologic features. In total, 50 archival cases of acute appendicitis were selected, and PCR was performed using primers targeting a 286-bp fragment of the mapA gene specific to C. jejuni. Twenty histologically unremarkable appendectomy specimens served as negative controls. Cases were reviewed with attention to particular histological features including mucosal ulceration, cryptitis, depth of inflammatory infiltrate, and the presence of mural necrosis. Of acute appendicitis cases, 22% (11/50) were positive for C. jejuni DNA by PCR analysis. Control cases were negative for C. jejuni DNA. All patients presented with signs and symptoms typical of acute appendicitis. Of the C. jejuni positive cases, only 27% contained acute inflammation limited to the mucosa/submucosa, whereas the remainder had mural or transmural inflammation; therefore, the histological features of C. jejuni-positive acute appendicitis cases were indistinguishable from C. jejuni-negative cases. In summary, C. jejuni DNA was detected in a significant percentage (22%) of acute appendicitis cases, a much higher percentage than previous studies using other methodologies. As C. jejuni is an enteric pathogen that does not exist as a commensal or nonpathogenic organism, the presence of C. jejuni DNA implies current or recent infection. Further study is needed to determine whether the presence of C. jejuni DNA in acute appendicitis indicates appendiceal involvement by C. jejuni enteritis, or if there is a true causative role for C. jejuni in acute appendicitis.
Assuntos
Apendicite/microbiologia , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , DNA Bacteriano/análise , Adulto , Apêndice/microbiologia , Apêndice/patologia , Campylobacter jejuni/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Francisella tularensis (FT), a zoonotic bacterium that causes tularemia, has received attention as a possible bioterrorism threat. We developed a PCR assay for use in fixed, processed tissues, which are safer to handle and allow archival testing. PCR analysis for a 211-bp fragment of the FT lipoprotein gene was performed on tissues from 16 cases of tularemia. In all, 14/15 cases with intact DNA (93%) were positive for FT by PCR. Frequent histologic findings in PCR-positive tissues included irregular microabscesses and granulomas in liver, spleen, kidney, and lymph nodes, and necrotizing pneumonia. Unusual cases featuring suppurative leptomeningitis and gastrointestinal ulcers were also seen. As this disease is endemic in North America, and has been identified as a potential bioterroristic threat, awareness of the clinicopathologic spectrum of disease and available detection methods is increasingly important. This PCR assay, the first designed for use in processed tissues, is an excellent method for diagnosis of tularemia.