Detec����o de DNA de HPV no plasma para potencial identifica����o precoce de recidiva de c��ncer do colo de ��tero

O c��ncer do colo do ��tero constitui a terceira neoplasia maligna mais comum na popula����o feminina, com aproximadamente 520 mil novos casos e 260 mil ��bitos por ano e origina-se a partir da infec����o genital persistente pelo Papiloma V��rus Humano (HPV) oncog��nico. Os principais HPVs considerados de alto risco oncog��nico s��o os tipos HPV-16 e 18, respons��veis por cerca de 70% de todos os casos de c��nceres cervicais (CC) no mundo. Pacientes com CC apresentam taxa de recidiva variando de 8% a 49%. Dentro de dois anos de seguimento, 62% a 89% das recidivas s��o detectadas. Atualmente, os testes usados para detec����o de recidiva s��o a citopatologia da c��pula vaginal e exames de imagem, por��m ainda n��o est��o dispon��veis testes espec��ficos. O DNA livre-circulante (cf-DNA) representa um biomarcador n��o-invasivo facilmente obtido no plasma e soro. V��rios estudos mostram ser poss��vel detectar e quantificar ��cidos nucl��icos no plasma de pacientes com c��ncer e que as altera����es no cfDNA potencialmente refletem mudan��as que ocorrem durante a tumorig��nese. Essa ferramenta diagn��stica n��o-invasiva pode ser ��til no rastreio, progn��stico e monitoramento da resposta ao tratamento do c��ncer. Portanto, o desenvolvimento e a padroniza����o de testes laboratoriais n��o invasivos capazes de identificar marcadores tumorais e diagnosticar precocemente a recidiva da doen��a aumentam a chance de cura atrav��s da utiliza����o dos tratamentos preconizados. Sendo assim, este estudo tem o objetivo de detectar o DNA de HPV no plasma de pacientes com CC para avaliar sua potencial utilidade como marcador precoce de recidiva. Um fragmento de tumor e sangue de pacientes com CC, atendidas no ICESP e HC de Barretos, foram coletados antes do tratamento. Entraram no estudo 137 pacientes nas quais o tumor foi positivo para HPV-16 ou 18, sendo 120 amostras positivas para HPV-16 (87,6%), 12 positivas para HPV-18 (8,8%) e cinco positivas para HPV-16 e 18 (3,6%). A m��dia de idade das pacientes deste estudo foi de 52,5 anos. Plasma de 131 pacientes com CC da data do diagn��stico e de 110 pacientes do seguimento foram submetidas ao PCR em Tempo Real HPV tipo espec��fico. A presen��a do DNA de HPV no plasma pr��-tratamento foi observada em 58,8% (77/131) com carga viral variando de 204 c��pias/mL a 2.500.000 c��pias/mL. A positividade de DNA no plasma pr��-tratamento aumentou com o estadio cl��nico do tumor: I - 45,2%, II - 52,5%, III - 80,0% e IV - 76,9%, (p=0,0189). A presen��a do DNA de HPV no plasma p��s-tratamento foi observada em 27,3% (30/110). A m��dia de tempo das recidivas foi de 3,1 anos (2,7 - 3,5 anos). O DNA de HPV foi positivo at�� 460 dias antes do diagn��stico cl��nico da recidiva. As pacientes com DNA de HPV no plasma apresentaram pior progn��stico, tanto sobrevida como o tempo livre de doen��a, em rela����o ��s que foram negativas. Nas pacientes com CC a presen��a de HPV no plasma de seguimento pode ser um marcador precoce ��til para o monitoramento da resposta terap��utica e detec����o de pacientes com risco aumentado de recidiva e progress��o da doen��a.

An��lise de express��o g��nica de Xylella fastidiosa cultivada em diferentes concentra����es de glicose pela t��cnica de microarrays de DNA

Xylella fastidiosa (Xf) �� o agente etiol��gico causador de doen��as em uma grande variedade de cultivos de grande import��ncia econ��mica, causando a c1orose variegada dos citros, uma das doen��as mais danosas �� ind��stria de citros no Brasil. Os genomas de algumas cepas deste fitopat��geno foram completamente seq��enciados promovendo assim estudos funcionais do genoma em larga escala. Neste trabalho n��s nos propusemos a investigar o perfil de transcri����o de Xf atrav��s da t��cnica microarranjos (no t��tulo da disserta����o microarrays, mas a partir de agora usaremos microaarranjos) de DNA usando todo o genoma do fitopat��geno e cultivando-a sob meio definido variando a concentra����o de glicose. O objetivo inicial deste trabalho era observar se Xf comportava-se da mesma forma que Xac, que tem a express��o de goma aumentada devido ao aumento da concentra����o de glicose do meio. Nossas an��lises revelaram que enquanto os transcritos relacionados �� goma n��o se mostraram afetados com a concentra����o de glicose, genes que codificam para an��logos a Colicina-V e precursores de fimbria foram induzidos em alta concentra����o de glicose. Baseados nestes resultados, n��s propusemos um modelo de mecanismo de produ����o e defesa contra a Colicina em Xf.

Les��es em DNA promovidas por produtos de oxida����o do β-caroteno: poss��veis implica����es biol��gicas

Apesar de diversos estudos in vitro e em popula����es indicarem um efeito protetor do β-caroteno em sistemas biol��gicos, estudos epidemiol��gicos como o \"The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study\" (ATBC) e o \"The Beta-Carotene and Retinol Efficacy Trial\" (CARET) mostraram um aumento na incid��ncia de c��ncer pulmonar em indiv��duos fumantes suplementados com β-caroteno. Essa a����o contradit��ria tem sido chamada na literatura de \"Paradoxo do β-Caroteno\". Sabe-se que este caroten��ide sob altas press��es de oxig��nio ou na presen��a de per��xidos pode sofrer oxida����o e levar a forma����o de compostos como alde��dos, ep��xidos, etc, que s��o capazes de se adicionarem covalentemente ao DNA. Estudos, in vitro e in vivo t��m demonstrado a possibilidade de os metab��litos do β-caroteno agirem como agentes pr��-carcinog��nicos. Estes agentes quando ativados quimicamente podem levar �� forma����o de adutos de DNA. J�� se sabe que alguns desses adutos encontramse em n��veis aumentados em diversas situa����es de risco de c��ncer. Diversos grupos, incluindo o nosso, t��m demonstrado a forma����o de les��es em DNA a partir de alde��dos e ep��xidos ex��genos ou gerados endogenamente. O presente trabalho mostra que a rea����o do β-caroteno e dois de seus produtos de oxida����o, retinal e β-apo-8\'-carotenal, com 2\'-desoxiguanosina e DNA leva �� forma����o de adutos. Dentre os adutos formados, foi caracterizado o aduto 1,N2eteno-2\'-desoxiguanosina (1 ,N2-εdGuo). Os n��veis de outro aduto de DNA, a 8-oxo-7,8-dihidro-2\'-deoxiguanosina (8-oxodGuo), tamb��m foram monitoradas para estudo comparativo. A forma����o dos adutos tamb��m foi verificada em fibroblastos normais de pulm��o humano (linhagem IMR-90) expostos ao β-caroteno e aos seus produtos de oxida����o. Experimentos com ratos suplementados com β-caroteno e expostos �� fuma��a de cigarro em per��odos de 7, 30 e 180 dias, mostraram n��veis aumentados de 1,N2-εdGuo nos animais suplementados com o caroten��ide comparado ao grupo ve��culo. Aumento no n��vel de 8-oxodGuo tamb��m foi verificado nos tratamentos de 7 e 180 dias. Um aumento significativo no n��vel do eteno aduto tamb��m foi verificado nos animais suplementados com β-caroteno e expostos �� fuma��a de cigarro, comparado ao grupo apenas exposto �� fuma��a ap��s 7 e 180 dias de exposi����o. Nestes mesmos grupos, o aumento do 8-oxodGuo s�� foi observado no tratamento por 180 dias. Sabendo que estas les��es s��o comprovadamente mutag��nicas, nossos estudos podem contribuir para o esclarecimento dos mecanismos envolvidos na forma����o de c��ncer em fumantes suplementados ou n��o com β-caroteno.

O fator DNA ��� ferramentas a favor da constru����o de marcas diferenciadas

As organiza����es est��o se adaptando a um novo mercado, onde as pessoas t��m um papel mais ativo na forma como devem agir no meio em que est��o inseridas. De entre estas mudan��as, o design surge como uma ferramenta de diferencia����o entre seus concorrentes, tornando-se um dos itens de maior enfoque perante as empresas. Segundo Martins e Merino, o design transcende a cria����o de simples pe��as gr��ficas, pois contribui adicionando valores como uma imagem adequada, com responsabilidades perante a sociedade. Tais valores e outros conceitos, como a vis��o, miss��o e cultura da organiza����o fazem parte da ess��ncia da sua marca, identificados por meio do seu ���DNA���, que �� constru��do, de acordo com Olhats et al., dentro de um processo cocriativo, envolvendo a empresa e seus stakeholders, compartilhando as experi��ncias que ambos possuem a respeito da marca. O surgimento de metodologias para a constru����o de uma marca e seus valores vem fortalecer os estudos da ��rea de branding, como em Govindarajan e Trimble, que defendem a constru����o do ���DNA corporativo��� como uma forma inovadora de aplica����o de conceitos e valores que auxiliam a empresa a ter uma melhor compreens��o do seu funcionamento e de todos os elementos que a cercam, para que possa se diferenciar das outras empresas. Neste contexto, surge o seguinte questionamento de pesquisa: �� poss��vel existir uma ferramenta f��sica ou digital que apoie uma metodologia de gest��o de marca apoiada no design? Para autores como Gob��, programas de identidade empresarial precisam ser vision��rios e integrados, refletindo o compromisso das empresas em compartilhar seus valores com os consumidores. Ainda para o autor, as identidades corporativas devem funcionar e se comunicar com maior afinco com as pessoas, considerando-se os valores gastos no desenvolvimento de um programa de identidade empresarial. Metodologias cocriativas como a do Brand DNA, de acordo com Cardoso, s��o vistas como elementos motivadores que oferecem uma experi��ncia gratificante e marcante aos participantes do processo, refor��ando a identifica����o entre os colaboradores e a marca da organiza����o. A presente pesquisa tem como objetivo desenvolver, para a metodologia do Brand DNA Process, um conjunto de ferramentas (toolkit) que abarque toda a teoria cient��fica e facilite sua aplica����o. Dentre os conceitos a serem abordados, encontram-se os de Gest��o do Design, Branding, DNA da Marca e Toolkits. Para o desenvolvimento e cumprimento dos objetivos, prop��e-se como metodologia a base proposta por Gil (2002), que separa a classifica����o da pesquisa em dois grupos: quanto aos objetivos e quanto os procedimentos t��cnicos utilizados; iniciando-se como uma pesquisa explorat��ria visando maior profundidade no tema desenvolvido, definindo seus objetivos e, em seguida, partindo para uma pesquisa bibliogr��fica, adquirindo material e refer��ncias te��ricas sobre o que est�� sendo feito atualmente dentro da ��rea a ser explorada. Nortear��o esta pesquisa artigos, teses e livros da ��rea de gest��o de design, branding e inova����o.

Dna Extraction From Filter-paper Spots Of Vaginal Samples Collected After Sexual Violence.

To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human ��-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. ��-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

Analysis Of The Dna Fourier Transform-infrared Microspectroscopic Signature Using An All-reflecting Objective.

The Fourier transform-infrared (FT-IR) signature of dry samples of DNA and DNA-polypeptide complexes, as studied by IR microspectroscopy using a diamond attenuated total reflection (ATR) objective, has revealed important discriminatory characteristics relative to the PO2(-) vibrational stretchings. However, DNA IR marks that provide information on the sample's richness in hydrogen bonds have not been resolved in the spectral profiles obtained with this objective. Here we investigated the performance of an all reflecting objective (ARO) for analysis of the FT-IR signal of hydrogen bonds in DNA samples differing in base richness types (salmon testis vs calf thymus). The results obtained using the ARO indicate prominent band peaks at the spectral region representative of the vibration of nitrogenous base hydrogen bonds and of NH and NH2 groups. The band areas at this spectral region differ in agreement with the DNA base richness type when using the ARO. A peak assigned to adenine was more evident in the AT-rich salmon DNA using either the ARO or the ATR objective. It is concluded that, for the discrimination of DNA IR hydrogen bond vibrations associated with varying base type proportions, the use of an ARO is recommended.

Prognostic Value Of Dna And Mrna E6/e7 Of Human Papillomavirus In The Evolution Of Cervical Intraepithelial Neoplasia Grade 2.

This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL). This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months. Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant. The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution.

Phylogenetics, Ancestral State Reconstruction, And A New Infrafamilial Classification Of The Pantropical Ochnaceae (medusagynaceae, Ochnaceae S.str., Quiinaceae) Based On Five Dna Regions.

Ochnaceae s.str. (Malpighiales) are a pantropical family of about 500 species and 27 genera of almost exclusively woody plants. Infrafamilial classification and relationships have been controversial partially due to the lack of a robust phylogenetic framework. Including all genera except Indosinia and Perissocarpa and DNA sequence data for five DNA regions (ITS, matK, ndhF, rbcL, trnL-F), we provide for the first time a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogenetic backbone of the family. Based on this, we present a new classification of Ochnaceae s.l., with Medusagynoideae and Quiinoideae included as subfamilies and the former subfamilies Ochnoideae and Sauvagesioideae recognized at the rank of tribe. Our data support a monophyletic Ochneae, but Sauvagesieae in the traditional circumscription is paraphyletic because Testulea emerges as sister to the rest of Ochnoideae, and the next clade shows Luxemburgia+Philacra as sister group to the remaining Ochnoideae. To avoid paraphyly, we classify Luxemburgieae and Testuleeae as new tribes. The African genus Lophira, which has switched between subfamilies (here tribes) in past classifications, emerges as sister to all other Ochneae. Thus, endosperm-free seeds and ovules with partly to completely united integuments (resulting in an apparently single integument) are characters that unite all members of that tribe. The relationships within its largest clade, Ochnineae (former Ochneae), are poorly resolved, but former Ochninae (Brackenridgea, Ochna) are polyphyletic. Within Sauvagesieae, the genus Sauvagesia in its broad circumscription is polyphyletic as Sauvagesia serrata is sister to a clade of Adenarake, Sauvagesia spp., and three other genera. Within Quiinoideae, in contrast to former phylogenetic hypotheses, Lacunaria and Touroulia form a clade that is sister to Quiina. Bayesian ancestral state reconstructions showed that zygomorphic flowers with adaptations to buzz-pollination (poricidal anthers), a syncarpous gynoecium (a near-apocarpous gynoecium evolved independently in Quiinoideae and Ochninae), numerous ovules, septicidal capsules, and winged seeds with endosperm are the ancestral condition in Ochnoideae. Although in some lineages poricidal anthers were lost secondarily, the evolution of poricidal superstructures secured the maintenance of buzz-pollination in some of these genera, indicating a strong selective pressure on keeping that specialized pollination system.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.

Changes In Liver Cell Dna Methylation Status In Diabetic Mice Affect Its Ft-ir Characteristics.

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including ��as -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

A Modified Acidic Approach For Dna Extraction From Plant Species Containing High Levels Of Secondary Metabolites.

Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

Human Herpesvirus-6 And Cytomegalovirus Dna In Liver Donor Biopsies And Their Correlation With Hla Matches And Acute Cellular Rejection.

Herpesvirus reactivation is common after liver transplantation. Analyze the presence of cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) DNA in liver donor biopsies, seeking to better understand issues involving human donor leukocyte antigens (HLA)-A, B and DR, as well as correlations with acute cellular rejection. Fifty-nine liver transplantation patients were investigated for the presence of HCMV and HHV-6 DNA in liver donor biopsies, using the Nested-PCR technique. The clinical donor information and HLA matches were obtained from the S��o Paulo State Transplant System. The recipients' records regarding acute cellular rejection were studied. Seven (11.8%) biopsies were positive for HCMV DNA and 29 (49%) were positive for HHV-6 DNA. In 14 donors with HLA-DR 15 nine had HHV-6 DNA positive liver biopsy with a tendency for significant association (p=0.09), 22 recipients developed acute cellular rejection and 9/22 were positive for HLA-DR 15 (p=0.03; ��(2)=4.51), which was statistically significant in univariate analysis and showed a tendency after multivariate analysis (p=0.08). HHV-6 DNA was prevalent in liver donors studied as well as HLA-DR 15. These findings suggest that patients with HLA-DR 15 in liver donor biopsies develop more rejection after liver transplantation.

On The Consistency Of Monte Carlo Track Structure Dna Damage Simulations.

Monte Carlo track structures (MCTS) simulations have been recognized as useful tools for radiobiological modeling. However, the authors noticed several issues regarding the consistency of reported data. Therefore, in this work, they analyze the impact of various user defined parameters on simulated direct DNA damage yields. In addition, they draw attention to discrepancies in published literature in DNA strand break (SB) yields and selected methodologies. The MCTS code Geant4-DNA was used to compare radial dose profiles in a nanometer-scale region of interest (ROI) for photon sources of varying sizes and energies. Then, electron tracks of 0.28 keV-220 keV were superimposed on a geometric DNA model composed of 2.7 �� 10(6) nucleosomes, and SBs were simulated according to four definitions based on energy deposits or energy transfers in DNA strand targets compared to a threshold energy ETH. The SB frequencies and complexities in nucleosomes as a function of incident electron energies were obtained. SBs were classified into higher order clusters such as single and double strand breaks (SSBs and DSBs) based on inter-SB distances and on the number of affected strands. Comparisons of different nonuniform dose distributions lacking charged particle equilibrium may lead to erroneous conclusions regarding the effect of energy on relative biological effectiveness. The energy transfer-based SB definitions give similar SB yields as the one based on energy deposit when ETH ��� 10.79 eV, but deviate significantly for higher ETH values. Between 30 and 40 nucleosomes/Gy show at least one SB in the ROI. The number of nucleosomes that present a complex damage pattern of more than 2 SBs and the degree of complexity of the damage in these nucleosomes diminish as the incident electron energy increases. DNA damage classification into SSB and DSB is highly dependent on the definitions of these higher order structures and their implementations. The authors' show that, for the four studied models, different yields are expected by up to 54% for SSBs and by up to 32% for DSBs, as a function of the incident electrons energy and of the models being compared. MCTS simulations allow to compare direct DNA damage types and complexities induced by ionizing radiation. However, simulation results depend to a large degree on user-defined parameters, definitions, and algorithms such as: DNA model, dose distribution, SB definition, and the DNA damage clustering algorithm. These interdependencies should be well controlled during the simulations and explicitly reported when comparing results to experiments or calculations.

Post Hoc Analysis Of The Patricia Randomized Trial Of The Efficacy Of Human Papillomavirus Type 16 (hpv-16)/hpv-18 As04-adjuvanted Vaccine Against Incident And Persistent Infection With Nonvaccine Oncogenic Hpv Types Using An Alternative Multiplex Type-specific Pcr Assay For Hpv Dna.

The efficacy of the human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against cervical infections with HPV in the Papilloma Trial against Cancer in Young Adults (PATRICIA) was evaluated using a combination of the broad-spectrum L1-based SPF10 PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA25) system with type-specific PCRs for HPV-16 and -18. Broad-spectrum PCR assays may underestimate the presence of HPV genotypes present at relatively low concentrations in multiple infections, due to competition between genotypes. Therefore, samples were retrospectively reanalyzed using a testing algorithm incorporating the SPF10 PCR-DEIA/LiPA25 plus a novel E6-based multiplex type-specific PCR and reverse hybridization assay (MPTS12 RHA), which permits detection of a panel of nine oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 45, 52, 58, and 59). For the vaccine against HPV types 16 and 18, there was no major impact on estimates of vaccine efficacy (VE) for incident or 6-month or 12-month persistent infections when the MPTS12 RHA was included in the testing algorithm versus estimates with the protocol-specified algorithm. However, the alternative testing algorithm showed greater sensitivity than the protocol-specified algorithm for detection of some nonvaccine oncogenic HPV types. More cases were gained in the control group than in the vaccine group, leading to higher point estimates of VE for 6-month and 12-month persistent infections for the nonvaccine oncogenic types included in the MPTS12 RHA assay (types 31, 33, 35, 45, 52, 58, and 59). This post hoc analysis indicates that the per-protocol testing algorithm used in PATRICIA underestimated the VE against some nonvaccine oncogenic HPV types and that the choice of the HPV DNA testing methodology is important for the evaluation of VE in clinical trials. (This study has been registered at ClinicalTrials.gov under registration no. NCT00122681.).

Human Herpesvirus Infections Of The Central Nervous System: Laboratory Diagnosis Based On Dna Detection By Nested Pcr In Plasma And Cerebrospinal Fluid Samples.

Infections of the central nervous systems (CNS) present a diagnostic problem for which an accurate laboratory diagnosis is essential. Invasive practices, such as cerebral biopsy, have been replaced by obtaining a polymerase chain reaction (PCR) diagnosis using cerebral spinal fluid (CSF) as a reference method. Tests on DNA extracted from plasma are noninvasive, thus avoiding all of the collateral effects and patient risks associated with CSF collection. This study aimed to determine whether plasma can replace CSF in nested PCR analysis for the detection of CNS human herpesvirus (HHV) diseases by analysing the proportion of patients whose CSF nested PCR results were positive for CNS HHV who also had the same organism identified by plasma nested PCR. In this study, CSF DNA was used as the gold standard, and nested PCR was performed on both types of samples. Fifty-two patients with symptoms of nervous system infection were submitted to CSF and blood collection. For the eight HHV, one positive DNA result-in plasma and/or CSF nested PCR-was considered an active HHV infection, whereas the occurrence of two or more HHVs in the same sample was considered a coinfection. HHV infections were positively detected in 27/52 (51.9%) of the CSF and in 32/52 (61.5%) of the plasma, difference not significant, thus nested PCR can be performed on plasma instead of CSF. In conclusion, this findings suggest that plasma as a useful material for the diagnosis of cases where there is any difficulty to perform a CSF puncture.