Successful and failed mini-implants: microbiological evaluation and quantification of bacterial endotoxin

Abstract Objectives Using two groups of mini-implants (successful and failed) the objectives of this in vivo study were: to evaluate the microbial contamination by the checkerboard DNA-DNA hybridization technique and to quantify the bacterial endotoxin by the limulus amebocyte lysate assay. Material and Methods The 15 successful and 10 failed mini-implants (1.6 mm diameter × 7.0 or 9.0 mm long), placed in the maxilla and/or mandible, were obtained from 15 patients undergoing orthodontic treatment. Data were analyzed statistically by the Wilcoxon rank-sum test using the SAS software (a=0.05). Results All 40 microbial species were detected in both groups of mini-implants, with different frequencies. No differences were observed between the groups with respect to microbial complexes (blue, purple, yellow, green, orange, red and other species) and endotoxin quantification (p>0.05). Conclusion Neither microbial contamination nor endotoxin quantification was determinant for the early loss of stability of the mini-implants.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Diagnóstico molecular do papilomavírus humano por captura híbrida e reação em cadeia da polimerase / Molecular diagnosis of human papillomavirus in hybrid capture and polymerase chain reaction

A infecção pelo papilomavírus humano (HPV) é extremamente comum e está associada a várias condições clínicas, que variam de infecções assintomáticas a doenças benignas e malignas da mucosa genital, como as verrugas genitais, a neoplasia intraepitelial cervical e o câncer do colo do útero. O objetivo deste estudo foi apresentar as técnicas de biologia molecular por captura híbrida (CH) e reação em cadeia da polimerase (PCR), utilizadas no diagnóstico do HPV e suas aplicações. Métodos mais precisos, quando aplicados em situações especiais, principalmente no caso de divergência entre outros métodos diagnósticos, podem ser aplicados às políticas de saúde pública, visando diminuir a mortalidade causada pelo câncer do colo do útero em consequência do HPV.(AU)

HPV infection is extremely common and is associated with various clinical conditions, ranging from asymptomatic infections to benign and malignant diseases of the genital mucosa, such as genital warts, cervical intraepithelial neoplasia and cervical cancer. The aim of this study was to present the techniques of molecular biology by hybrid capture and PCR, used in the diagnosis of HPV and its applications. More accurate methods when applied in special situations, especially in the case of divergence between other diagnostic methods can be applied to public health policies in order to reduce mortality caused by cervical cancer because of HPV.(AU)

Isolation and characterization of drought-responsive genes from peanut roots by suppression subtractive hybridization

Background Peanut (Arachis hypogaea L.) is an important economic and oilseed crop. Long-term rainless conditions and seasonal droughts can limit peanut yields and were conducive to preharvest aflatoxin contamination. To elucidate the molecular mechanisms by which peanut responds and adapts to water limited conditions, we isolated and characterized several drought-induced genes from peanut roots using a suppression subtractive hybridization (SSH) technique. Results RNA was extracted from peanut roots subjected to a water stress treatment (45% field capacity) and from control plants (75% field capacity), and used to generate an SSH cDNA library. A total of 111 non-redundant sequences were obtained, with 80 unique transcripts showing homology to known genes and 31 clones with no similarity to either hypothetical or known proteins. GO and KEGG analyses of these differentially expressed ESTs indicated that drought-related responses in peanut could mainly be attributed to genes involved in cellular structure and metabolism. In addition, we examined the expression patterns of seven differentially expressed candidate genes using real-time reverse transcription-PCR (qRT-PCR) and confirmed that all were up-regulated in roots in response to drought stress, but to differing extents. Conclusions We successfully constructed an SSH cDNA library in peanut roots and identified several drought-related genes. Our results serve as a foundation for future studies into the elucidation of the drought stress response mechanisms of peanut.

O uso do diagnóstico genético pré-implantacional em pacientes com aborto de repetição: revisão do uso da técnicade array-CGH / The use of preimplantatian genetic diagnasis in patients with recurrent abortion: a review of the technique of array-CGH

Tendo em vista a grande frequência de alterações cromossômicas, seja nos casais com quadros de abortamento de repetição ou nos fetos abortados, uma possibilidade para o tratamento para esses pacientes seria o uso de tratamentos de reprodução assistida, associados ao diagnóstico genético pré-implantacional (PGD) com a técnica de hibridização genética comparativa por array (array-CGH), para a transferência apenas de embriões geneticamente normais. O objetivo desta revisão é avaliar se é possível melhorar o prognóstico gestacional, com redução do número de perdas e o do tempo para conseguir uma gestação saudável, desses casais com aborto de repetição ao usarem o PDG por array-CGH. Foram executadas duas revisões bibliográficas dos últimos 10 anos, a primeira relacionando o uso do PGD nos casos de aborto de repetição e a outra com o uso do array-CGH e PGD. A literatura, apesar de discordante quanto à real eficácia do PGD nos casos de aborto de repetição, tende a se mostrar favorável ao uso dessa técnica, da mesma forma que o método de fluorescence in situ hybridization (Fish) é inferior a array-CGH para o PGD. Dessa forma, apesar de ser uma técnica promissora para casais com AR, o PGD com array-CGH necessita de mais estudos que comprovem sua real eficácia.

Given the high frequency of chromosomal abnormalities, either in couples with recurrent miscarriage or in aborted fetuses, a possibility for treatment for these patients is the use of assisted reproduction treatment, associated with preimplantation genetic diagnosis (PGD) with technique by array comparative genomic hybridization (array-CGH), to transfer only genetically normal embryos. The aim of this review is to assess the feasibility of improving the prognosis ofpregnancy, reducing the number of losses and the time to achieve a healthy pregnancy, for couples with recurrent abortion when using PDC with array-CGH. Two literature reviews were performed for the last 10 years, the first relating the use of PGD and recurrent miscarriage, and the other using the array-CGH and PGD.The literature, although discordant about the real efficacy of PGDin cases of recurrent abortion, tends to show favorableto use this technique, just as the method of array-GGHshows to be better than Fish (fluorescence in-situ hybridization) for PGD. Thus, despire of being a promising technique for couples with RA,the use of PDG with array-GGH needs more study to prove its actual effectiveness.

Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L. )

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

The ability of the BANA test to detect different levels of P. gingivalis, T. denticola and T. forsythia

The aim of this study was to evaluate the ability of the BANA Test to detect different levels of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia or their combinations in subgingival samples at the initial diagnosis and after periodontal therapy. Periodontal sites with probing depths between 5-7 mm and clinical attachment level between 5-10 mm, from 53 subjects with chronic periodontitis, were sampled in four periods: initial diagnosis (T0), immediately (T1), 45 (T2) and 60 days (T3) after scaling and root planing. BANA Test and Checkerboard DNA-DNA hybridization identified red complex species in the subgingival biofilm. In all experimental periods, the highest frequencies of score 2 (Checkerboard DNA-DNA hybridization) for P. gingivalis, T. denticola and T. forsythia were observed when strong enzymatic activity (BANA) was present (p < 0.01). The best agreement was observed at initial diagnosis. The BANA Test sensitivity was 95.54 percent (T0), 65.18 percent (T1), 65.22 percent (T2) and 50.26 percent (T3). The specificity values were 12.24 percent (T0), 57.38 percent (T1), 46.27 percent (T2) and 53.48 percent (T3). The BANA Test is more effective for the detection of red complex pathogens when the bacterial levels are high, i.e. in the initial diagnosis of chronic periodontitis.

A acurácia dos testes de Biologia molecular para HPV no diagnóstico de doença residual após tratamento das lesões intraepiteliais escamosas / HPV molecular test accuracy in the diagnosis of residual diseaseafter intraepithelial lesions treatment

As pacientes submetidas ao tratamento de lesões intra-epiteliais de alto grau (LIE-AG) necessitam realizar rigoroso controle, pois apresentam maior risco de lesão residual ou persistente. O presente estudo tem por objetivo avaliar a acurácia da captura híbrida do tipo II, comparada à citologia oncológica no seguimento de pacientes submetidas a tratamento de LIE-AG. Foi realizada revisão sistemática, incluindo-se todos os artigos encontrados até agosto de 2009 e, combinando-se os descritores para cada base de dados específica. Os 260 estudos encontrados foram submetidos a rigorosa avaliação e selecionados a partir de sua qualidade metodológica. Conclui-se que o teste de captura híbrida do tipo II é um método de maior sensibilidade e menor especificidade quando comparado à citologia oncológica, e sua utilização é adequada no seguimento de pacientes com maior risco de lesões intraepiteliais.

Patients submitted to treatment for high squamous intraepithelial lesions (HSIL) need stricter follow-up exams due to a higher risk of persistent or residual lesion. This study aims to evaluate hybrid capture accuracy (type II), compared with Pap smear accuracy,in HSIL patients follow-up. A systematic review was made including all the papers published until August 2009, and combining descriptors for each specific database. The 260 studies were submitted to rigorous evaluation and selected based on their methodological quality. It was concluded that the hybrid capture test type II is a more sensitive method with lower specificity when compared with Pap smear and its use is recomended during the follow-up of patients with increased risk of intraepithelial lesions.

Comparação das técnicas de captura de híbridos e PCR para a detecção de HPV em amostras clínicas / Comparison of hybrid capture and PCR for HPV detection in clinical samples

INTRODUÇÃO E OBJETIVOS: São conhecidos mais de 100 tipos de papilomavírus humano (HPV), dos quais 30 têm sido reportados em infecções anogenitais. A infecção tem importância clínica, pois alguns tipos virais estão associados a lesões que podem progredir para o câncer cervical. Sabe-se que os métodos moleculares são muito importantes para o diagnóstico dessa infecção. O objetivo do estudo é comparar a detecção de HPV de alto risco pelo método de captura híbrida 2 (CH2) com a detecção do vírus pela reação em cadeia da polimerase convencional (PCRc) e em tempo real (PCR-TR). METODOLOGIA: Foram analisadas 56 amostras ectocervicais por CH2 e, após, por PCRc e PCR-TR. RESULTADOS: Ambas, PCRc e PCR-TR, apresentaram alta concordância entre si (95,1 por cento), enquanto a comparação entre as PCRs e a CH2 mostrou concordância razoável entre os resultados (PCRc = 90,2 por cento e PCR-TR = 87,8 por cento). DISCUSSÃO E CONCLUSÃO: A CH é aceita para a detecção do HPV, entretanto pode ser menos sensível em comparação com as técnicas de PCR. A PCR-TR tem a vantagem sobre a PCRc em termos de velocidade, sendo também um pouco mais sensível. Devido à alta sensibilidade e à rapidez, os métodos de PCR poderiam ser usados para a triagem de HPV em amostras ectocervicais.

INTRODUCTION AND OBJECTIVE: More than 100 types of human papillomaviruses (HPV) are known, of which 30 have been reported in anogenital infections. The infection has clinical importance, inasmuch as some viral types are associated with lesions that can progress to cervical cancer. Molecular methods are considered an important tool for the diagnosis of this infection. The objective of this study was to compare the detection of high risk HPV using hybrid capture 2 with HPV detection by conventional and real time PCR. METHODOLOGY: 56 ectocervical samples were analyzed by hybrid capture and after that by conventional and real time PCR. RESULTS: Both PCR and RT-PCR showed a high degree of correlation (95.1 percent), whereas the comparison between PCR and HC2 showed a fair correlation (90.2 percent and 87.8 percent for PCR and RT-PCR, respectively). DISCUSSION AND CONCLUSIONS: HC is widely accepted for the detection of HPV, however, it may lack sensitivity in comparison with PCR techniques. RT-PCR has further advantages over the conventional PCR in terms of speed as well as it is slightly more sensitive. Due to their high sensitivity and fast response, PCR methods could be used as a screening method for HPV detection in ectocervical samples.

Periimplantitis: checkerboard DNA-DNA hybridization for bacterial pathogen identification

A recent innovation in medical field is the use of DNA probes in microbiological diagnosis of the oral cavity. Thus, this study has the objective to present the mainly characteristics of Checkerboard DNA-DNA Hybridization method for bacterial pathogens identification related to periimplantitis, commonly disease found in the oral cavity, as wells as, to show the uses and applications of this technique.

Una innovación reciente en medicina es la utilización de sondas de DNA para diagnóstico microbiológico. Este trabajo tiene como objetivo, presentar las principales características del método Checkerboard DNA-DNA Hybridization para la identificación de bacterias patógenas associadas a periimplantite en la cavidad oral, mostrando las diferentes utilizaciones y aplicaciones de esta técnica.

Identificação e análise de expressão de RNAs intrônicos não codificadores humanos / Identification and expression analysis of human intronic noncoding RNAs

Nesse trabalho, nós mostramos estudos em larga-escala de RNAs não codificadores antisenso que são transcritos em regiões intrônicas de genes humanos. Alguns destes transcritos intrônicos possuem níveis de expressão correlacionados ao grau de diferenciação tumoral de câncer de próstata, apontando para uma relevância biológica destas mensagens em doenças complexas como o câncer. Nós também avaliamos a existência de um mecanismo comum de regulação de transcrição, compartilhado por mRNAs codificadores de proteína e RNAs intrônicos, através de análises de perfís de expressão de uma linhagem tumoral de próstata estimulada por andrógeno. A análise de ESTs e mRNAs depositados em bancos públicos de seqüência revelou mais de 55 mil RNAs Totalmente Intrônicos Não-codificadores (TIN), transcritos dos íntrons de 74% de todos os genes RefSeq únicos. Guiados por esta informação, nós desenhamos uma plataforma de oligonucleotídeos contendo sondas senso e antisenso para cada um de 7.520 transcritos TIN selecionados aleatoriamente, além de sondas para os genes codificadores de proteína correspondentes. Nos identificamos assinaturas intrônicas e exônicas de expressão tecido-específicas em fígado, próstata e rim.Os RNAs TIN antisenso mais altamente expressos eram transcritos de íntrons de genes codificadores de proteína enriquecidos na categoria “Regulação da transcrição...

Screening of differently expressed genes in human prostate cancer cell lines with different metastasis potentials / 华中科技大学学报（医学）（英德文版）

In order to screen the genes differentially expressed in two human prostate cancer cells with different metastasis potentials, suppression subtractive hybridization (SSH) was done twice on human prostate cancer cell line with high potential of metastasis PC3M-1E8 and its synogenetic cell line PC3M-2B4 with low metastasis potential. In the first subtraction PC3M-2B4 was used as tester and PC3M-1E8 as driver and the forward subtractive library was constructed. In the second on the tester and driver were interchanged and the reverse subtractive library was constructed. The screened clones of both libraries were sequenced and Gene Bank homology search was performed. Some clones were confirmed by quantitative real-time PCR. The results showed that two subtractive libraries containing 238 positive clones were constructed. Analysis of 16 sequenced clones randomly picked from two libraries showed that 4 differentially expressed gene fragments were identified as new EST with unknown functions. It was concluded that two subtractive libraries of human prostate cancer cell lines with different metastasis potentials were constructed successfully.

Comparison of VERSANT Hepatitis B Virus DNA 3.0 Assay with Digene Hybrid Capture II Hepatitis B Virus DNA Test in Relation to Clinical Status of Hepatitis B Virus / 대한진단검사의학회지

BACKGROUND: Some differences exist among various Hepatitis B virus (HBV) DNA quantification assays due to lack of standardization and besides clinical usefulness has not been firmly elucidated in Korean HBV patients. METHODS: We compared Bayer VERSANT HBV DNA 3.0 Assay (VERSANT 3.0) with Digene Hybrid Capture II HBV DNA Test (HC-II) according to HBeAg status and ALT levels in 232 HBV-infected Korean patients. One hundred and seventeen sera with undetectable DNA levels by HC-II were further analyzed by Real-Q HBV quantification assay (BioSewoom). RESULTS: Although VERSANT 3.0 and HC-II showed an excellent correlation (r=0.9739), the results (copies/mL) by VERSANT 3.0 were 0.45 log10 higher than those by HC-II. HBV DNA levels were higher in HBeAg-positive group than in HBeAg-negative group (P=0.002), and in abnormal ALT group than in normal ALT group (P<0.0001). The detection rate of HBV DNA by VERSANT 3.0 was lower in HBeAg-negative and normal ALT group (n=68) than in HBeAg-positive or abnormal ALT group (n=164) (35.3% vs 89.6%, P<0.0001). Fifty two sera out of 61 sera with undetectable DNA by VERSANT 3.0 were measurable by Real-Q with mean value of 3.26 log10 copies/mL. CONCLUSIONS: VERSANT 3.0 and HC-II showed an excellent correlation, but a little difference (0.45 log10) existed. VERSANT 3.0 effectively measured clinically relevant HBV DNA levels in most HBVinfected patients in Korea. However, more sensitive assays are needed for patients with negative HBeAg and normal ALT to see the low copies of HBV DNA levels.

Human Papilloma Virus Genotyping Assay using Restriction Fragment Mass Polymorphism Analysis, and Its Comparison with Sequencing and Hybrid Capture Assays / 대한진단검사의학회지

BACKGROUND: Infection with human papilloma virus (HPV) is the main cause of cervical cancer, and HPV genotyping is of increasing importance for determining clinical course and management of the disease based on the HPV genotypes. Here, we established a novel matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) assay, termed restriction fragment mass polymorphism (RFMP) that is suitable for genotyping multiple HPV in an accurate and high-throughput manner. We evaluated the performance of the RFMP assay in HPV genotyping by comparing the results with those of direct or clonal sequencing and hybrid capture (HC) assays. METHODS: The study population consisted of 50 patients with histologically confirmed cervical lesions and a positive test for HPV DNA. HPV genotyping was performed with RFMP, sequencing, and HC assays. The assigned genotypes and risk groups were compared among the methods. RESULTS: Concordance rates in the genotype level between RFMP vs sequencing, sequencing vs HC, and HC vs RFMP were 98% (49/50), 88% (44/50), and 88% (44/50), respectivley. Especially, RFMP and sequencing were 100% concordant when assigned high-risk group was considered identical in 1 case of mixed genotypes identified only in RFMP. The observed discrepancy between HC and the other two methods is due to the assignment of six cases of low, intermediate, or unassigned risk genotypes as high-risk group in HC method. CONCLUSIONS: RFMP, sequencing, and HC assays were highly concordant with each other in HPV genotyping. Compared to sequencing assay, RFMP assay is found to be advantageous in detecting mixed genotype infections. The accuracy and amenability to high-throughput analysis should make the RFMP assay suitable for reliable screening of HPV genotypes in clinical laboratories.

Determinação da carga viral de DNA de HPV pelo ensaio de captura híbrida e sua associação com achados citológicos / 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 percent) were reagent only to low-risk oncogenic types (group A) and 340 (93.9 percent) 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 percent). Most of this group samples had viral load between 1 to <10RLU/PCA. Of the patients that were reagent to group B 52.1 percent had LSIL cytology and 38.2 percent 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 percent). High-grade squamous intraepithelial lesions (3.4 percent) 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.

OBJETIVO: Comparar a relação entre a carga viral do HPV por captura híbrida II (HCII) e os achados citológicos. MÉTODOS: Trezentas e sessenta e duas amostras reagentes para DNA de HPV por HCII tiveram suas cargas virais classificadas em quatro categorias e correlacionadas aos resultados citológicos. RESULTADOS: Vinte e duas amostras (6,1 por cento) foram reagentes somente para os tipos de baixo risco oncogênico (grupo A) e 340 (93,9 por cento) foram reagentes para os tipos de alto risco oncogênico (grupo B). A correlação entre carga viral das amostras reagentes para o grupo A e resultados citológicos mostrou predominância (50 por cento) de lesão escamosa intraepitelial de baixo grau (LSIL). A maioria das amostras desse grupo teve carga viral entre 1 e < 10RLU/PCA. Nos pacientes reagentes para o grupo B observamos que 52,1 por cento tiveram citologia LSIL e 38,2 por cento tiveram citologia negativa para lesão intraepitelial e malignidade (NILM). Os pacientes com LSIL tiveram a carga viral bem distribuída, com ligeira predominância da categoria de 100 a < 1.000RLU/PCB. As amostras com carga viral entre 1 e < 10RLU/PCB mostraram predominância de citologia NILM (48.1 por cento). Lesões escamosas de alto grau (3,4 por cento) foram presentes nas amostras com carga viral entre 100 e < 1.000RLU/PCB (p = 0,023). Houve correlação entre a mediana da carga viral para o grupo B e os resultados LSIL/HSIL. CONCLUSÕES: A quantificação da carga viral, principalmente para os tipos de HPV de alto risco oncogênico, pode ser uma ferramenta útil para o acompanhamento de pacientes com lesões suspeitas.

Prevalence of human papillomavirus infection in the genital tract determined by hybrid capture assay

Human Papillomavirus (HPV) infection is the most prevalent sexually-transmitted virus worldwide. It is known to be the etiological agent of cervical cancer and cervical intraepithelial neoplasia (CIN). Consequently, there is strong motivation to evaluate HPV testing in cervical cancer screening. Recently developed, the second generation of the hybrid capture test (HCA II) is a non-radioactive, relatively rapid, hybridization assay, designed to detect 18 HPV types divided into high and low-risk groups. We evaluated 7,314 patients (5,833 women and 1,481 men) for HPV infection by HCA II. Among them, 3,008 (41.1 percent) presented HPV infection: 430 (14.2 percent) had HPV DNA of low risk for cancer, 1,631 (54.2 percent) had high risk HPV types and 947 (31.5 percent) had both types. The prevalence in females was 44.9 percent. The prevalence of HPV DNA in the group for which cytological results were available was slightly higher: 55.3 percent (1007/1824). Significant differences were detected in the frequency of HPV infection of the cervix between normal cases and those with high-grade squamous-intraepithelial lesions (HSIL)(P<0.0001). Among males, the prevalence was 26.2 percent, composed of 9.1 percent in Group A, 9.7 percent in Group B and 7.4 percent with multiple infections. We observed that male prevalence was lower and that low-risk types were more frequent than in females. HPV viral load was significantly greater in SILs than in normal or inflammatory cases (P<0.0001), suggesting an association between high viral load values and risk of SIL. Because of high costs, the HCA II test cannot be recommended for routine mass screening for cervical infection in poor countries. Nevertheless, it was found to be a useful tool, when combined with cytology, discovering high-risk infections in apparently normal tissues and revealing silent infections that may be responsible for the maintenance of HPV in the general population. These findings point...

Genetic Alterations in Primary Gastric Carcinomas Correlated with Clinicopathological Variables by Array Comparative Genomic Hybridization

Genetic alterations have been recognized as an important event in the carcinogenesis of gastric cancer (GC). We conducted high resolution bacterial artificial chromosome array-comparative genomic hybridization, to elucidate in more detail the genomic alterations, and to establish a pattern of DNA copy number changes with distinct clinical variables in GC. Our results showed some correlations between novel amplified or deleted regions and clinical status. Copy-number gains were frequently detected at 1p, 5p, 7q, 8q, 11p, 16p, 20p and 20q, and losses at 1p, 2q, 4q, 5q, 7q, 9p, 14q, and 18q. Losses at 4q23, 9p23, 14q31.1, or 18q21.1 as well as a gain at 20q12 were correlated with tumor-node-metastasis tumor stage. Losses at 9p23 or 14q31.1 were associated with lymph node status. Metastasis was determined to be related to losses at 4q23 or 4q28.2, as well as losses at 4q15.2, 4q21.21, 4q 28.2, or 14q31.1, with differentiation. One of the notable aspects of this study was that the losses at 4q or 14q could be employed in the evaluation of the metastatic status of GC. Our results should provide a potential resource for the molecular cytogenetic events in GC, and should also provide clues in the hunt for genes associated with GC.

Genetic Alterations in Intrahepatic Cholangiocarcinoma as revealed by Degenerate Oligonucleotide Primed PCR-Comparative Genomic Hybridization

Intrahepatic cholangiocarcinoma (ICC), a malignant neoplasm of the biliary epithelium, is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy. The genetic mechanisms involved in the development of ICC are not well understood and only a few cytogenetic studies of ICC have been published. Recently, technique of degenerate oligonucleotide primed (DOP)-PCR comparative genomic hybridization (CGH) permits genetic imbalances screening of the entire genome using only small amounts of tumor DNA. In this study chromosomal aberrations in 33 Korean ICC were investigated by DOP-PCR CGH. The common sites of copy number increases were 20q (67%), 17 (61%), 11q11-q13 (42%), 8p12-qter (39%), 18p (39%), 15q22-qter (36%), 16p (36%), 6p21 (30%), 3q25-qter (27%), 1q41-qter (24%), and 5p14-q11.2 (24%). DNA amplification was identified in 16 carcinomas (48%). The frequent sites of amplification were 20q, 17p, 17q23-qter, and 7p. The most frequent sites of copy number decreases were 1p32-pter (21%) and 4q (21%). The recurrent chromosomal aberrations identified in this study provide candidate regions involved in the tumorigenesis and progression of ICC.