Detecção do HPV e da Chlamydia trachomatis em amostras de cérvice uterina de mulheres da cidade de São Miguel do Oeste

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2014.O Papilomavirus Humano (HPV) e Chlamydia trachomatis (CT) são responsáveis pelas Doenças Sexualmente transmissíveis, de origem viral e bacteriana, mais prevalentes na população sexualmente ativa. O HPV desempenha papel central na etiologia de praticamente todos os casos de câncer cervical, entretanto, admite-se que outros fatores possam, conjuntamente com o vírus, modular o risco de transição da infecção cervical para a malignidade. Além do HPV, diversos estudos estão considerando a CT como um cofator no desenvolvimento de neoplasias intraepiteliais cervicais e outras alterações celulares significativas em mulheres com histórico de infecção por HPV. Diante disto, o objetivo deste estudo foi estimar a prevalência de HPV, CT e a coinfecção HPV/CT por métodos moleculares (PCR-multiplex, microarray, RFLP e sequenciamento) em amostras cervicais de mulheres atendidas no Sistema Único de Saúde da cidade de São Miguel do Oeste/SC. Trata-se de um estudo transversal, cujo desfecho é a positividade ao HPV e à CT. Durante o período de novembro de 2011 a março de 2013, foram coletadas amostras cervicais de 325 mulheres sexualmente ativas e que procuraram o serviço de saúde para rastreio rotineiro do câncer cervical. As amostras foram submetidas à PCR-multiplex padronizada neste estudo, utilizando os conjuntos de iniciadores PGMY0911, CTP1/CTP2 e PCO3/PCO4. As amostras positivas para HPV foram submetidas à técnica de PCR-RFLP (Polimorfismo dos Fragmentos de Restrição), microarray e sequenciamento. A prevalência do DNA de HPV foi de 24,6% na amostra estudada. Já em relação à CT a prevalência foi de 11,1% e 4,6% da amostragem total, apresentando coinfecção para HPV/CT. Observou-se associação do HPV com as seguintes variáveis: citologia (pAbstract : The Human Papillomavirus (HPV) and Chlamydia trachomatis (CT) are responsible for the most prevalent sexually transmitted disease caused by virus and bacteria. HPV has a central role in the etiology of virtually all cervical cancers; however it is believed that other factors may also modulate the risk of a cervical infection progress to malignancy. Many studies consider CT as a cofactor in cervical intraepithelial neoplasia development as well other significant cell abnormalities in women with history of HPV infection. The aim of this study was to estimate the prevalence of HPV, CT and HPV/CT coinfection by molecular methods in cervical samples from women attending public health services at São Miguel do Oeste/SC. This is a cross-sectional study and the outcomes are positive results to HPV, CT or HPV/CT coinfection. A total of 325 cervical samples were collect from sexually active women seeking cervical cancer screening from November 2011 to March 2013. HPV, CT and coinfections were detected by PCR-multiplex using the consensus primers, PGMY0911, CTP1/CTP2 e PCO3/PCO4. HPV-positive samples were typed by PCR-RFLP, microarray and sequencing. The HPV, CT and HPV/CT prevalence were 24.6%, 11.1% and 4.6% respectively. Statistics associations were observed for HPV with the variables cytology (p<0,001), age (p=0,001), first sexual intercourse (p= 0,008) and oral contraceptive use (p=0,048). There was a significant association between CT and number of partners along the life and age (p = 0.006), and oral contraceptive use (p = 0.011) with HPV/CT coinfection. A total of 32 HPV different genotypes were identified by RFLP, microarray and sequencing and the most prevalent types were 16, 39, 53, 68 and 56. The multiplex-PCR proved to be useful to simultaneously detect HPV and CT. The RFLP showed good performance to identify up to two viral types in the same sample. The sequencing methodology proved to be an excellent tool for the identification of a single viral type, while the Papillocheck® was the best method for samples infected for more than two viral types. The PCR-RFLP has a high predictive negative value and can be used as a screening method. To samples with more than two viral types a complementary methodology with high discriminatory power can be used. Alternatives protocols like combination of a cheap in house RFLP and an expensive commercial methodology with high discriminatory power can be used, specially in places with few financial resources to ensure best quality to screening

Caracterização molecular e determinação do perfil de resistência de isolados clínicos de Neisseria gonorrhoeae circulantes na Grande Florianópolis: série histórica 2008-2016

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2018.Neisseria gonorrhoeae tem apresentado uma rápida e extraordinária capacidade de desenvolver resistência a todos os antimicrobianos introduzidos para o tratamento durante os últimos 70-80 anos. O presente estudo se constitui em uma análise retrospectiva da sensibilidade antimicrobiana, dos determinantes moleculares da resistência antimicrobiana e da genotipagem por NG-MAST de 153 isolados clínicos de N. gonorrhoeae circulantes na grande Florianópolis no período de 2008-2016. A Concentração Inibitória Mínima (MIC) foi determinada pelo método de ágar diluição para os antimicrobianos: penicilina, tetraciclina, ciprofloxacino, azitromicina, ceftriaxona e cefixima. Detecção da enzima beta-lactamase foi realizada pelo método de nitrocefina em disco. Foram detectadas altas taxas de resistência à penicilina (26,1%), tetraciclina (41,2%) e ao ciprofloxacino (52,3%). Resistência à azitromicina foi detectada em 5,2% dos isolados. Todos os isolados de N. gonorrhoeae foram sensíveis às ESCs (cefixima e ceftriaxona). No entanto, 8,5% (n=13) apresentaram MIC=0,125µg/mL para cefixima, um log abaixo do ponto de corte de resistência estabelecido pelo EUCAST. Produção da enzima ß-lactamase foi detectada em 12,4% dos isolados e um deles apresentou o plasmídeo Toronto/Rio carreando o alelo blaRTEM-135R. Resistência plasmidial à tetraciclina foi observada em 5,2% dos isolados e ambos os genes tetM Americano e Holandês foram identificados. Nos anos de 2013-2016 a resistência à penicilina foi quase 2,5 vezes maior que no período de 2008-2012. A resistência à tetraciclina foi alta em todo o período estudado. A resistência ao ciprofloxacino foi elevada desde os primeiros anos do estudo e aumentou significativamente ao longo dos nove anos (p < 0,001). Mutações na região QRDR de gyrA e parC foram observadas em 87,5% dos isolados resistentes ao ciprofloxacino. Sessenta e quatro diferentes STs foram determinados, 15 clusters foram identificados e 19 STs foram descritos pela primeira vez. Os STs mais prevalentes foram: ST225 (n=11), ST2992 (n=11) ST1582 (n=10), ST338 (n=7), ST1407 (n=4), ST2202 (n=4) e ST6827 (n=4). Trinta e três isolados permaneceram com STs desconhecidos. Dois genogrupos G225 e G1407 foram formados. O genogrupo G225 foi significativamente associado à resistência ao ciprofloxacino (p<0,001) à penicilina (p=0,016) e à tetraciclina (p< 0,001) enquanto o genogrupo G1407 foi estatisticamente associado com MIC=0,125µg/mL para cefixima (p=0,001), resistência ao ciprofloxacino (p < 0,001), à penicilina (p=0,016) e à tetraciclina (p< 0,001). Isolados que compartilharam o mesmo alelo tbpB 29 foram significativamente associados com resistência à azitromicina (p= 0,008) e à penicilina (p= 0,035). O ST338 foi associado com resistência plasmidial à penicilina (gene blaRTEM-1R) (p<0,001) e à tetraciclina (tetM) (p= 0,006), enquanto o cluster que compartilhava o mesmo alelo tbpB 137, foi associado à resistência plasmidial à tetraciclina (gene tetM). Os resultados do presente estudo demostraram altas taxas de resistência à penicilina, à tetraciclina e ao ciprofloxacino associadas à persistência e disseminação de linhagens gonocócicas com resistência ao ciprofloxacino e resistência cromossômica e plasmidial à penicilina e à tetraciclina. Além disso, ressalta-se a importância do estabelecimento da vigilância para monitorar possíveis mudanças no perfil de sensibilidade da ceftriaxona e azitromicina, atualmente utilizadas no tratamento da gonorreia no Brasil.Abstract : Neisseria gonorrhoeae has shown a rapid and extraordinary ability to develop resistance to all antimicrobials introduced for the treatment during the last 70-80 years. The present study was based on a retrospective analysis of antimicrobial sensitivity, molecular determinants of antimicrobial resistance and genotyping by NG-MAST of 153 clinical isolates of N. gonorrhoeae circulating in the Greater Florianopolis in the period of 2008-2016. Minimal Inhibitory Concentration (MIC) was determined by agar dilution method for penicillin, tetracycline, ciprofloxacin, azithromycin, ceftriaxone and cefixime antimicrobials. Beta-lactamase enzyme production was determined by nitrocefin disks method. High rates of resistance were detected to penicillin (26.1%), tetracycline (41.2%) and ciprofloxacin (52.3%). Resistance to azithromycin was detected in 5.2% of the isolates. All isolates of N. gonorrhoeae were sensitive to ESCs (cefixime and ceftriaxone). However, 8.5% (n=13) had MIC=0.125µg/ml for cefixime, one log below the resistance cut-off point established by EUCAST. ß-lactamase enzyme production was detected in 12.4% of the isolates and one of them presented the Toronto/Rio plasmid carrying the blaRTEM-135 Rallele. Plasmid resistance to tetracycline was observed in 5.2% of the isolates and both American and Dutch tetM genes were identified. In the years of 2013-2016 resistance to penicillin was almost 2.5 times higher than the 2008-2012 period. Tetracycline resistance was high throughout the study period. Resistance to ciprofloxacin was elevated from the early years of the study and increased significantly over the nine years (p<0.001). Mutations in the QRDR region of gyrA and parC were observed in 87.5% of isolates resistant to ciprofloxacin. Sixty-four different STs were determined, 15 clusters were identified and 19 STs were first described. The most prevalent STs were: ST225 (n=11), ST2992 (n=11), ST1582 (n=10), ST338 (n= 7), ST1407 (n=4), ST2202 (n= 4) and ST6827 (n= 4). Thirty three isolates belonged to unknown STs. Two genogroups were formed: G225 and G1407. G225 genogroup was significantly associated with resistance to ciprofloxacin (p<0.001), penicillin (p=0.016) and tetracycline (p<0.001) whereas G1407 genogroup was statistically associated with MIC=0.125 µg/ml for cefixime (p=0.001), resistance to ciprofloxacin, penicillin (p=0.016) and tetracycline (p<0.001). Isolates that shared the same tbpB 29 allele were significantly associated with resistance to azithromycin (p=0.008) and penicillin (p=0.035). ST338 was associated with plasmid resistance to penicillin (blaRTEM-1 Rgene) (p<0.001) and tetracycline (tetM) (p=0.006), whereas the cluster that shared the same tbpB 137 allele was associated with plasmid resistance to tetracycline (tetM gene). The results of the study demonstrated high rates of resistance to penicillin, tetracycline and ciprofloxacin associated with persistence and dissemination of gonococcal strains resistant to ciprofloxacin and plasmid resistance to penicillin and tetracycline. In addition, it is important to establish surveillance to monitor possible changes in the sensitivity profile of ceftriaxone and azithromycin, currently used in the treatment of gonorrhea in Brazil

LOWER BIFIDOBACTERIA COUNTS IN ADULT PATIENTS WITH CELIAC DISEASE ON A GLUTEN-FREE DIET

Context The ingestion of gluten is responsible for the symptoms of Celiac disease, but other environmental factors can also influence. Strains of the Bifidobacterium genus have been shown to afford protection against the inflammatory response and mucosal damage caused by gliadin peptides in vitro. Objectives This study was designed to compare the concentration of fecal bifidobacteria and pH of patients with celiac disease on gluten-free diet and control subjects in order to identify if the imbalance on fecal microbiota still remain during the treatment of celiac disease and identify the necessity of dietary supplementation with pre- or probiotics. Methods It was analyzed the feces of 42 healthy subjects and 14 celiac patients. The bifidobacteria count in feces was done in selective medium BIM-25. Microscopic analysis of the colonies was performed by Gram stain. The identification of the genus Bifidobacterium was performed by determination of fructose-6-phosphate phosphoketolase. Fecal pH was measured using a pH meter. Results The concentration of bifidobacteria per gram of feces was significantly higher in healthy subjects (controls) (1.5 ± 0.63 x108 CFU/g) when compared to celiac patients (2.5 ± 1.5 x107 CFU/g). The fecal pH was not different between celiac patients (7.19 ± 0.521) and controls (7.18 ± 0.522). Conclusions These results suggest that with lower levels of bifidobacteria, celiac patients have an imbalance in the intestinal microbiota, regardless of pH, even while on a gluten-free diet. This fact could favor the pathological process of the disorder

External quality assurance with dried tube specimens (DTS) for point-of-care syphilis and HIV tests: experience in an indigenous populations screening programme in the Brazilian Amazon.

OBJECTIVES: The availability of point-of-care (POC) tests for infectious diseases has revolutionised the provision of healthcare for remote rural populations without access to laboratories. However, quality assurance for POC tests has been largely overlooked. We have evaluated the use and stability of dry tube specimens (DTS) for External Quality Assurance (EQA) for HIV and syphilis screening in remote indigenous populations in the Amazon region of Brazil. METHODS: All healthcare workers (HCWs) participating in the community-screening were trained. We used HIV and syphilis DTS panels developed by the reference laboratory, containing samples with negative and positive results at different antibody concentrations, for both infections. DTS panels were distributed to HCWs in the communities for reconstitution and testing using POC HIV and syphilis tests. The results of testing were sent to the reference laboratory for marking and remedial action taken where necessary. RESULTS: In total 268 HCWs tested 1607 samples for syphilis and 1608 samples for HIV. Results from HCWs showed a concordance rate of 90% for syphilis and 93% for HIV (κ coefficients of 0.74 and 0.78, respectively) with reference laboratories. Most false negatives were in samples of very low antibody concentration. DTS syphilis specimens produced the expected results after storage at 2-8°C or at 18-24°C for up to 3 weeks. CONCLUSIONS: The results show that POC tests for syphilis and HIV give valid results in environments where traditional tests do not work, but errors in the interpretation of POC test results were identified by the EQA programme using DTS. EQA using DTS can help to improve the quality of screening programmes using POC tests in remote regions