Inês Aparecida Tozetti

Adenovirus, calicivirus and astrovirus detection in fecal samples of hospitalized children with acute gastroenteritis from Campo Grande, MS, Brazil

We analyzed fecal samples from hospitalized children up to three years of age with acute gastroenteritis at Campo Grande, Mato Grosso do Sul, Brazil, from May 2000-January 2004. Astrovirus and calicivirus were detected by Reverse Transcription-Polymerase Chain Reaction and adenovirus was detected using the Rotavirus and Adenovirus combined immunoenzyme assay. Astrovirus, adenovirus and calicivirus were detected at rates of 3.1%, 3.6% and 7.6%, respectively. These results re-emphasize the need for the establishment of regional vigilance systems to evaluate the impact of enteric viruses on viral gastroenteritis.

Local immunosuppression induced by high viral load of human papillomavirus: characterization of cellular phenotypes producing interleukin‐10 in cervical neoplastic lesions

A specific immune response to human papillomavirus (HPV) in the cervical microenvironment plays a key role in eradicating infection and eliminating mutated cells. However, high‐risk HPVs modulate immune cells to create an immunosuppressive microenvironment, and induce these immune cells to produce interleukin 10 (IL‐10). This production of IL‐10, in conjunction with HPV infection, contributes to the appearance of cervical neoplastic lesions. We sought to characterize the IL‐10‐producing cellular phenotype, and investigate the influence of host and HPV factors upon the induction of an immunosuppressive microenvironment. Immunohistochemical analysis demonstrated an increase in IL‐10 production by keratinocytes, macrophages and Langerhans cells in high‐grade cervical lesions and cervical cancer. This increase was more pronounced in patients older than 30 years, and was also correlated with high viral load, and infection with a single HPV type, particularly high‐risk HPVs. Our results indicate the existence of a highly immunosuppressive microenvironment composed of different IL‐10‐producing cellular phenotypes in cervical cancer samples, and samples classified as high‐grade cervical lesions (cervical intraepithelial neoplasia stages II and III). The immunosuppressive microenvironment that developed for these different cellular phenotypes favours viral persistence and neoplastic progression.

Rotavírus A em crianças de até três anos de idade, hospitalizadas com gastroenterite aguda em Campo Grande, Estado do Mato Grosso do Sul

Polyacrylamide gel electrophoresis and combined immunoenzyme assay for rotavirus and adenovirus were used to analyze 380 fecal samples from children up to three years of age who were hospitalized with acute diarrhea in Campo Grande, State of Mato Grosso do Sul, between May 2000 and January 2004. Among all the samples, 88 (23.2%) were positive for Rotavirus A. Out of these, 81 (92%) had a defined electrophoretic pattern: 77 (87.5%) with a long pattern and four (4.5%) with a short pattern. Genotype G and P characterization was done by nested RT-PCR for 85 samples, of which 56 (65.9%) were genotyped as type G. Among these, 49 (87.5%) were G1, five (8.9%) were G4, one (1.8%) was G3 and one (1.8%) was G9. The genotype was found to be type P in 37 samples (43.5%) and all of these were P[8]. The G and P association most observed was G1P[8], with 33 samples (89.2%), followed by G4P[8], two samples (5.4%); G3P[8], one sample (2.7%); and G9P[8], one sample (2.7%).

Multiple types of human papillomavirus in cervical samples in women in Campo Grande, MS, Brazil.

We analyzed 87 cervical samples from Campo Grande, Mato Grosso do Sul, with a PGMY/GP+ nested PCR system. Positive samples were typed using E7 type-specific primer pairs for HPV 6/11, 16, 18, 45 and 66. Eighteen samples (22%) were infected with HPV6/11, 18 samples (22%) with HPV66, 13 samples (15.9%) with HPV45, 8 samples (9.8%) with HPV18 and 7 samples (8.5%) with HPV16. Seventeen samples (20.7%) were infected by two HPV types, and five samples (6.1%) by three HPV types. We conclude that infection with multiple types is present at a high frequency in our population and that there is a relation between some types and cytological finds.

Glucocorticoid-induced tumor necrosis factor receptor expression in patients with cervical human papillomavirus infection

INTRODUCTION The progression of human papillomavirus (HPV) infection in the anogenital tract has been associated with the involvement of cells with regulatory properties. Evidence has shown that glucocorticoid-induced tumor necrosis factor receptor (GITR) is an important surface molecule for the characterization of these cells and proposes that GITR ligand may constitute a rational treatment for many cancer types. We aimed to detect the presence of GITR and CD25 in cervical stroma cells with and without pathological changes or HPV infection to better understand the immune response in the infected tissue microenvironment. METHODS We subjected 49 paraffin-embedded cervical tissue samples to HPV DNA detection and histopathological analysis, and subsequently immunohistochemistry to detect GITR and CD25 in lymphocytes. RESULTS We observed that 76.9% of all samples with high GITR expression were HPV-positive regardless of histopathological findings. High GITR expression (77.8%) was predominant in samples with ≥ 1,000 RLU/PCB. Of the HPV-positive samples negative for intraepithelial lesion and malignancy, 62.5% had high GITR expression. High GITR expression was observed in both carcinoma and high-grade squamous intraepithelial lesion (HSIL) samples (p = 0.16). CD25 was present in great quantities in all samples. CONCLUSIONS The predominance of high GITR expression in samples with high viral load that were classified as HSIL and carcinoma suggests that GITR+ cells can exhibit regulatory properties and may contribute to the progression of HPV-induced cervical neoplasia, emphasizing the importance of GITR as a potential target for immune therapy of cervical cancer and as a disease evolution biomarker.

Determination of HPV DNA viral load by hybrid capture assay and its association with cytological findings

OBJECTIVE: To compare the relation between HPV viral load by hybrid capture II test (HCII) and cytological findings. METHODS: Three hundred sixty-two reagent samples to HPV DNA by HCII had their viral loads classified in four categories and correlated to cytological results. RESULTS: Twenty-two samples (6.1%) were reagent only to low-risk oncogenic types (group A) and 340 (93.9%) were reagent to high-risk oncogenic types (group B). The correlation between viral load for the reagent samples to group A and cytological results showed low-grade squamous intraepithelial lesion (LSIL) predominance (50%). Most of this group samples had viral load between 1 to <10RLU/PCA. Of the patients that were reagent to group B 52.1% had LSIL cytology and 38.2% were negative to intraepithelial lesion and malignancy (NILM) cytology. The patients with LSIL had viral load well distributed with a slight predominance of 100 to < 1,000RLU/PCB category. The samples had viral load between 1 to <10RLU/PCB showed NILM cytology predominance (48.1%). High-grade squamous intraepithelial lesions (3.4%) were present on the samples with viral load between 100 to <1,000RLU/PCB (p = 0.023). There was a correlation between the median for group B viral load and LSIL/HSIL results. CONCLUSIONS: The quantification of viral load, mainly of high-risk HPV types, may be a useful tool for dealing with patients who have suspicious lesions.

Good agreements between self and clinician-collected specimens for the detection of human papillomavirus in Brazilian patients

Women infected with human papillomavirus (HPV) are at a higher risk of developing cervical lesions. In the current study, self and clinician-collected vaginal and cervical samples from women were processed to detect HPV DNA using polymerase chain reaction (PCR) with PGMY09/11 primers. HPV genotypes were determined using type-specific PCR. HPV DNA detection showed good concordance between self and clinician-collected samples (84.6%; kappa = 0.72). HPV infection was found in 30% women and genotyping was more concordant among high-risk HPV (HR-HPV) than low-risk HPV (HR-HPV). HPV16 was the most frequently detected among the HR-HPV types. LR-HPV was detected at a higher frequency in self-collected; however, HR-HPV types were more frequently identified in clinician-collected samples than in self-collected samples. HPV infections of multiple types were detected in 20.5% of clinician-collected samples and 15.5% of self-collected samples. In this study, we demonstrated that the HPV DNA detection rate in self-collected samples has good agreement with that of clinician-collected samples. Self-collected sampling, as a primary prevention strategy in countries with few resources, could be effective for identifying cases of HR-HPV, being more acceptable. The use of this method would enhance the coverage of screening programs for cervical cancer.

Presence of highly oncogenic human papillomavirus in the oral mucosa of asymptomatic men

OBJECTIVES The aim of this study was to identify highly oncogenic forms of human papillomavirus in the oral mucosa of asymptomatic men. METHODS In this study, we analyzed samples of exfoliated cells from the oral cavity of 559 asymptomatic men. DNA-human papillomavirus was detected using the consensus primers PGMY09/11; viral genotyping was performed using type-specific PCR and restriction fragment length polymorphism. RESULTS DNA-human papillomavirus was detected in 1.3% of the study participants and of those 42.8% were infected by more than one type of virus. Viral types included HPV6, 11, 89 (low oncogenic risk), and HPV52, 53 (high oncogenic risk). Increased vulnerability to human papillomavirus infection was observed in individuals aged over 26 years, among those who reported oral sex practices, and in those who have had more than 16 sexual partners since first engaging in sexual intercourse. CONCLUSIONS There was a low prevalence of human papillomavirus detection in the oral mucosa of asymptomatic men. Highly oncogenic human papillomavirus types and infection by more than one viral type was observed. Oral sex practices and a large number of sexual partners may increase the risk of acquiring human papillomavirus infection.

Predominant overexpression of CD25/FOXP3, IFN-γ, and suppressive cytokines in high-grade lesion samples infected with human papillomavirus

Introduction: Human papillomavirus (HPV) persistent infection is the leading cause of cervical cancer and its precursor lesions, and the inappropriate immune response is among the factors that contribute to viral persistence. This may be influenced by regulatory T (Treg) cells and the production of immunosuppressive cytokines, such as transforming growth factor beta (TGF-β) and interleukin-10 (IL-10). Objective: We established the profile of the predominant response, Th1 or immunosuppressive response, in the tissue microenvironment, by detecting interferon-gamma (IFN-γ), TGF-β, and IL-10, as well as the co-expression of IL-2 receptor alpha (CD25) and forkhead box P3 (FOXP3). Methods: Seventy-four samples from uterine cervix biopsies that underwent HPV deoxyribonucleic acid (DNA) detection and histopathology analysis were immunostained to detect CD25/FOXP3, IFN-γ and suppressive cytokines in lymphocytes. Results: The microenvironment of high-grade squamous intraepithelial lesion (HSIL) samples with high numbers of viral particles (≥ 10,000 copies/ml) contained high numbers of CD25/FOXP3+, TGF-β+, IL-10+, and IFN-γ+ cells. Conclusion: The co-expression of CD25/FOXP3 and the expression of TGF-β, and IL-10 in HSIL samples suggest the existence of Treg cells in these locations, although IFN-γ expression was observed in several cells in these samples. Our data suggest that this cytokine could be related to immunosuppressed microenvironment maintenance, favoring the persistent HPV infection and the progression to carcinoma.

Molecular epidemiology of the human papillomavirus infection in self-collected samples from young women

The prevalence of human papillomavirus (HPV) infection is the highest in young, sexually active women less than 35 years of age. Direct diagnosis of infection by enabling genotyping methods is important considering that the viral types are divided into high (HR‐HPV) and low (LR‐HPV) oncogenic risk. This study aimed to evaluate the epidemiological and molecular characteristics of HPV infection in self‐collected samples from young women. A cross‐sectional study of 245 sexually active students (18 to 35 years of age) was undertaken with self‐collected samples. Extracted DNA was analyzed by polymerase chain reaction (PCR) with the PGMY 09/11 and PC04/GH20 primers for the detection of HPV DNA and the β‐globin gene, respectively. Viral genotyping was performed by type‐specific PCR (TS‐PCR) and restriction fragment length polymorphism (RFLP). Of the 236 valid samples, 68 (28.9%) were positive for HPV DNA, as genotyped by TS‐PCR and RFLP. The HR‐HPV were most prevalent, especially HPV‐16, ‐31, ‐33, and ‐45, and the most prevalent LR‐HPV were HPV‐6 and ‐83. Multi‐type HPV infections were detected in 17 (25%) samples. HPV infection was statistically more prevalent among younger women with lower educational levels and who had more partners in the past 2 years. A high prevalence of HPV infection was found in the age group examined, especially HR‐HPV types, as well as the presence of risk behaviors associated with HPV infection were observed. Considering these results, vaccinating females before the onset of sexual activity in Brazil should be emphasized. J. Med. Virol. 86:266–271, 2014.