Detection of respiratory viruses in primary cholesteatoma tissues.

Cholesteatomas are frequent middle ear benign tumors of unknown etiology. Infectious agents have been considered as possible contributing factors in the pathogenesis of cholesteatomas. Aiming to investigate the presence of respiratory viruses in primary cholesteatoma tissues, 26 formalin-fixed paraffin-embedded primary cholesteatoma tissues obtained from patients seen at the of the Clinical Hospital of the University of São Paulo School of Medicine, in Ribeirão Preto, Brazil were tested by real-time polymerase chain reaction (PCR). Considering the PCR results, 35% of the tissues were positive for human rhinovirus (HRV), 15.3% for human enterovirus (EV), 3.8% for human metapneumovirus (HMPV), and 3.8% for human bocavirus (HBoV). Serial immunohistochemistry for virus antigens and cell surface markers evidenced that the viruses were associated with fibroblasts, dendritic cells, macrophages, B lymphocytes, CD4+ , and CD8+ T lymphocytes. These findings indicate for the first time the presence of active respiratory virus infection in primary cholesteatoma tissues, suggesting that persisting virus infection in the middle could play a role in the pathogenesis and evolution of cholesteatomas.

Real-time multiplex PCR assay and melting curve analysis for identifying diarrheagenic Escherichia coli.

A real-time multiplex PCR assay was designed to amplify the virulence genes eae, pEAF, aatA, daaC, elt, est, ipaH, stx(1), and stx(2) for the detection of all diarrheagenic Escherichia coli pathotypes. This assay proved to be more sensitive and rapid than a conventional multiplex PCR for diarrheagenic E. coli isolates from children with diarrhea.

Genotypic and phenotypic analysis of diarrheagenic Escherichia coli strains isolated from Brazilian children living in low socioeconomic level communities.

BACKGROUND: Childhood diarrheal diseases remain highly endemic in developing areas of Brazil. The importance of Escherichia coli among children with diarrhea in these areas was unknown. This study determined the prevalence of different E. coli categories in symptomatic and asymptomatic children from low socioeconomic level rural communities in southeastern Brazil. METHODS: A total of 560 stool samples were collected from 141 children with diarrhea (< 10 years) and 419 apparently healthy controls who resided in 23 communities. E. coli isolates (n = 1943) were subjected to two multiplex PCRs developed for the detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and Shiga toxin-producing E. coli (STEC). Strains were also examined for the presence of EPEC, EAEC, and DAEC by assays of adhesion to HEp-2 cells and by hybridization with specific DNA probes. RESULTS: Diarrheagenic E. coli strains were isolated from 253 (45.2%) children, and were associated with diarrhea in children aged < 5 years (p < 0.001). EAEC (20.9%), DAEC (11.6%), EPEC (9.3%) were the most frequent pathotypes, followed by ETEC (2.7%), EIEC (0.5%), and STEC (0.2%). Depending of the assay, EPEC, EAEC, and DAEC (collectively termed enteroadherent E. coli) strains were isolated in 45% to 56% of diarrhea cases, a significantly higher incidence than in controls (P < 0.05). Individually, only DAEC showed significant association with diarrhea (p < 0.05), particularly in children aged 2-5 years. CONCLUSION: This study indicates that enteroadherent E. coli is an important cause of diarrhea in children living in low socioeconomic level communities in southeastern Brazil. Our results reveal that the PCR1 assay is an excellent tool for the identification of EAEC and DAEC.

An outbreak due to a norovirus GII.Pe-GII.4 Sydney_2012 recombinant in neonatal and pediatric intensive care units.

BACKGROUND: Noroviruses are the major cause of gastroenteritis outbreaks worldwide in all age groups. The several genotypes, mutation, and recombination events guarantee the broad diffusion and maintenance of these viruses in the general human population. We described an outbreak caused by a norovirus recombinant strain screened in samples obtained from children and hospital staff affected in an outbreak of diarrhea at neonatal and pediatric intensive care units in Southeastern Brazil. METHODS: Noroviruses were investigated by PCR and the genotype was determined by sequencing both partial genes regions of RNA-dependent RNA-polymerase (ORF1) and capsid protein (ORF2); the recombination event was performed using SimPlot. Rotaviruses were investigated by real-time PCR targeting the NSP3 gene. RESULTS: The GII.Pe-GII.4 Sidney_2012 norovirus recombinant was detected in four from among six children with diarrhea and in four symptomatic contacts; the rotavirus was negative in all samples. The virus was introduced by a hospital staff member affecting other staff members and patients of the pediatric intensive care unit. Five days lapsed between the first and last diarrhea case caused by norovirus and the outbreak was restrained through strict hand hygiene, environment disinfection and limiting the contact of infected persons. CONCLUSIONS: The outbreak was due to the GII.Pe-GII.4 Sydney_2012 variant that occurred in the same year of its first description in the world.

Age-specific prevalence of diffusely adherent Escherichia coli in Brazilian children with acute diarrhoea.

In a prospective study between February 2003 and June 2004, stool specimens of children less than 2 years of age with diarrhoea (n=218) and without diarrhoea (n=86), living in Vitória, Espírito Santo, Brazil, were examined for the presence of diarrhoeagenic Escherichia coli. E. coli isolates were tested by colony blot hybridization with specific DNA probes designed to detect EPEC, ETEC, EIEC, EAEC, DAEC and EHEC/STEC. Diarrhoeagenic E. coli strains were detected as the sole pathogen in stools of 92 (30.3 %) children, including 72 (33.0 %) with diarrhoea and 20 (23.2 %) without diarrhoea. DAEC was the most frequent pathotype and was found significantly more often from patients (18.3 %) than from controls (8.1 %) (P<0.05), particularly among children more than 1 year of age (P=0.01). Atypical EPEC and EAEC isolates were isolated from both patients (5.5 % and 4.6 %, respectively) and controls (6.9 % and 6.9 %, respectively). ETEC was more frequently isolated from patients (3.2 %) than controls (1.2 %). Typical EPEC (0.9 %) and EIEC (0.4 %) isolates were detected only in children with diarrhoea. In conclusion, our data suggest that DAEC should be considered potential pathogens in the region of Brazil studied.

High-grade cervical lesions among women attending a reference clinic in Brazil: associated factors and comparison among screening methods.

BACKGROUND: Although screening for cervical cancer is recommended for women in most countries, the incidence of cervical cancer is greater in developing countries. Our goal was to determine the prevalence and factors associated with high-grade lesions/cervical cancer among women attending a reference clinic in Brazil and evaluate the correlation of histology with cytology, colposcopy and the high-risk HPV (HR-HPV) tests. METHODS: A cross-sectional study of women attending a colposcopy clinic was carried out. The patients were interviewed to collect demographic, epidemiological and clinical data. Specimens were collected for cervical cytology, Chlamydia trachomatis and HPV testing using the Hybrid Capture (HC) and PCR tests. Colposcopy was performed for all patients and biopsy for histology when cell abnormalities or cervical intraepithelial neoplasia (CIN) were present. RESULTS: A total of 291 women participated in the study. The median age was 38 years (DIQ: 30-48 years). The prevalence of histologically confirmed high-grade lesions/cervical cancer was 18.2% (95%, CI: 13.8%-22.6%), with 48 (16.5%) cases of CIN-2/CIN-3 and 5 (1.7%) cases of invasive carcinoma. In the final logistic regression model, for ages between 30 and 49 years old [OR = 4.4 (95%: 1.01-19.04), history of smoking [OR = 2.4 (95%, CI: 1.14-5.18)], practice of anal intercourse [OR = 2.4 (95%, CI: 1.10-5.03)] and having positive HC test for HR-HPV [OR = 11.23 (95%, CI: 4 0.79-26, 36)] remained independently associated with high-grade lesions/cervical cancer. A total of 64.7% of the cases CIN-3\Ca in situ were related to HPV-16. Non-oncogenic HPV were only found in CIN-1 biopsy results. Compared to histology, the sensitivity of cytology was 31.8%, the specificity 95.5%; the sensitivity of colposcopy for high-grade lesions/cervical cancer was 51.0%, specificity was 91.4% and the concordance with HPV testing was high. CONCLUSIONS: The results confirm an association of HR-HPV with precursor lesions for cervical cancer. These data emphasize that cytological screening to detect precursor lesions is still important in some regions and that HR-HPV should be included for screening.

Adeno-associated virus and human papillomavirus types in cervical samples of pregnant and non-pregnant women.

OBJECTIVE: To investigate the prevalence of AAV and HPV DNA and their types in cervical secretion from pregnant and non-pregnant women. STUDY DESIGN: The samples were obtained from 40 pregnant and 62 non-pregnant women who were attended at the outpatient clinic of the Federal University Hospital of Espírito Santo, Southeastern Brazil. AAV and HPV were investigated by PCR and typed by PCR and/or RFLP. RESULTS: The occurrence of AAV in all samples was 25.5% (26/102): 81% (21/26) and 19% (5/26) for AAV2/3 and AAV5 species, respectively. AAV were observed in 35% (14/40) and 19% (12/62) of pregnant and non-pregnant women, respectively. HPV occurred in 22% of all samples; 25% (10/40) in pregnant and 20% (12/60) in non-pregnant women. HPV types were determined for 72.7% of the strains, most of which classified as high-risk. AAV-HPV co-infection was observed in 15.4% (4/26), mostly in pregnant women. CONCLUSIONS: There was a greater prevalence of AAV and HPV in pregnant than in non-pregnant women, which suggests that the gestational state may play a role in reactivating the viruses.