In-house standardization and validation of a multiplex RT-PCR assay for the detection of 13 respiratory viruses / Estandarización interna y validación de un ensayo RT-PRC múltiple para la detección de 13 virus respiratorios

Background. Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, such as culture and antigen detection. Objective. Standardization and validation of a multiplex real-time PCR assay for the detection of 13 respiratory viruses. Methods. The assay was validated using RNA control targets and comparing results to single-target PCR's. Results. Using RNA controls the multiplex format was found to be as sensitive and specific as the single-target PCRs, and no competition was observed between targets. The efficiencies for most of the reactions were approximately 90%, but a lower efficiency was found for Parainfluenza 2 with a rate of amplification in each cycle of 86.63%. On the other hand, a higher efficiency was observed in respiratory syncytial virus A and respiratory syncytial virus B ((93.07% each). Conclusion: This multiplex RT-PCR format shows an adequate efficiency, demonstrating an excellent sensitivity, specificity and repeatability for all the studied respiratory viruses.

Antecedentes. PRC múltiple en tiempo real es usada cada vez más para el diagnóstico de virus respiratorios y ha mostrado ser superior a metodos tradicionales, como cultivo y detección de antígeno. Objetivo. Estandarizar y validar una PRC múltiple en tiempo real para la detección de 13 virus respiratorios. Metodos. El ensayo fue realizado usando blanco de RNA control y comparando los resultados a blancos únicos de PCR. Resultados. Usando el RNA control, el formato de multiplex era tan sensible y específico como la PCR. Las eficiencias para la mayoría de las reacciones de aproximadamente el 90%, pero una eficiencia baja fue encontrada para influenza 2 con una tasa de amplificación en cada ciclo de 86.63%. Por otra parte, una mayor eficiencia fue observada en virus sincitial respitario A y B (93, 67% cada uno). Conclusión. Este formato RT-PCR múltiple muestra una adecuada eficiencia, demostrando un excelente especificidad y reproducibilidad para todos los virus respiratorios estudiados.

Type-Specific Identification of Genital Human Papillomavirus Infection in Women with Cytological Abnormality.

OBJECTIVES: To estimate the frequency of human papillomavirus (HPV) infection and the genotype distribution of HPV among women with a Pap smear showing atypical squamous cells of undetermined significance (ASC-US) attending the Program for the Detection and Control of Cervical Cancer in Bogotá, Colombia. STUDY DESIGN: Cervical samples from 200 women with an ASC-US Pap smear were analyzed for the presence of HPV DNA and genotype distribution using a commercial molecular technique (Linear Array®; Roche Molecular Systems, USA). RESULTS: HPV infection was found in 140 women (70%). High-risk HPV types were present in 46.4% of the samples; 16.4% showed a low-risk HPV type, and 37.1% showed both. Of the positive samples, 42.9% were infected with a single viral genotype, whereas 57.1% exhibited multiple HPV infections. The most common HPV genotypes were HPV 16, 53, and 52 with a prevalence of 26.4, 16.4, and 13.6%, respectively. CONCLUSION: The epidemiological characterization of HPV infections described in this study might guide actions for epidemiological surveillance to strengthen the program in Bogotá and to develop appropriate HPV vaccination programs.

XRCC2 R188H (rs3218536), XRCC3 T241M (rs861539) and R243H (rs77381814) single nucleotide polymorphisms in cervical cancer risk.

Human Papillomavirus (HPV) is the main cause of cervical cancer and its precursor lesions. Transformation may be induced by several mechanisms, including oncogene activation and genome instability. Individual differences in DNA damage recognition and repair have been hypothesized to influence cervical cancer risk. The aim of this study was to evaluate whether the double strand break gene polymorphisms XRCC2 R188H G>A (rs3218536), XRCC3 T241M C>T (rs861539) and R243H G>A (rs77381814) are associated to cervical cancer in Argentine women. A case control study consisting of 322 samples (205 cases and 117 controls) was carried out. HPV DNA detection was performed by PCR and genotyping of positive samples by EIA (enzyme immunoassay). XRCC2 and 3 polymorphisms were determined by pyrosequencing. The HPV-adjusted odds ratio (OR) of XRCC2 188 GG/AG genotypes was OR = 2.4 (CI = 1.1-4.9, p = 0.02) for cervical cancer. In contrast, there was no increased risk for cervical cancer with XRCC3 241 TT/CC genotypes (OR = 0.48; CI = 0.2-1; p = 0.1) or XRCC3 241 CT/CC (OR = 0.87; CI = 0.52-1.4; p = 0.6). Regarding XRCC3 R243H, the G allele was almost fixed in the population studied. In conclusion, although the sample size was modest, the present data indicate a statistical association between cervical cancer and XRCC2 R188H polymorphism. Future studies are needed to confirm these findings.

TNF-α and IL-10 promoter polymorphisms, HPV infection, and cervical cancer risk.

Although the implication of genetic factors in cervical cancer development remains to be elucidated, accumulative epidemiological evidence suggests that polymorphisms of cytokine genes may be involved in the etiology of cervical carcinoma. Tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) are two multifunctional cytokines implicated in inflammation, immunity, and cellular organization, and were proposed to play important roles in cancer biology. In order to determine whether IL-10 -1082 (G/A) and TNF-α -238 (G/A) and -308 (G/A) polymorphisms are associated with susceptibility to cervical cancer, a case-control study of 122 cancer patients and 176 healthy controls was conducted. Cervical samples were genotyped for both TNF-α polymorphisms by PCR-RFLP assay. SNP-1082 from IL-10 gene was genotyped using pyrosequencing technology. The association between cervical cancer risk and the studied SNPs was evaluated by logistic regression. Under univariate analysis, none of these polymorphisms appeared associated with susceptibility of cervical cancer development or HPV infection. However, individuals carrying heterozygous genotype for TNF-α -238 polymorphism seem to be at lower risk for cervical cancer development, with borderline significance (OR = 0.42, P = 0.069), as well as those carrying heterozygous genotypes for IL-10 and TNF-α -238 (OR = 0.40, P = 0.08). In conclusion, these results suggest a potential effect of TNF-α -238 G/A in the reduction of cervical cancer risk in Argentine women, but not TNF-α -308 or IL-10. Larger studies are needed to fully understand the genetic predisposition for the development of cervical cancer.

The effect of TP53 codon 72 and RNASEL codon 462 polymorphisms on the development of cervical cancer in Argentine women.

Epidemiological evidence suggests that genetic factors, such as variants in cancer suppressor genes, may play an important role in the etiology of cervical carcinoma. TP53 is an outstanding cell cycle regulator, mutated in most human cancers, and RNASEL is thought to be involved in antiviral and apoptotic responses. To determine whether TP53 Arg72Pro and RNASEL Arg462Gln polymorphisms are associated with susceptibility to cervical cancer, a case-control study of 98 cancer patients and 123 healthy controls was conducted. Cervical samples were genotyped for both polymorphisms by pyrosequencing technology. The association between cervical cancer risk and the studied SNPs was evaluated by logistic regression, and potential gene-gene interactions were studied by Multifactor Dimensionality Reduction analysis. In the single-locus analysis, only the heterozygous TP53 Arg72Pro genotype was significantly associated with the risk of developing a cervical carcinoma, while the RNASEL polymorphism showed no association after age adjustment. In addition, the combination of both polymorphisms gives near-null information gain. Consequently, the effect provided by each single nucleotide polymorphism individually is considered higher than the effect resulting from the interaction between these two genes in cervical cancer risk. These results suggest that a heterozygous TP53 Arg72Pro genotype may contribute to cervical cancer susceptibility.

Human papillomavirus DNA and oncogene alterations in colorectal tumors.

The aim of the present study is to determine the presence and molecular integrity of high-risk HPV types in colorectal adenocarcinomas and to assess whether viral DNA is related to common proto-oncogene alterations, such as k-ras mutations and c-myc gene amplification, in colorectal cancer. Seventy-five colorectal adenocarcinomas were screened for HPV infection using nested-PCR (MY09/11-GP5+/6+). HPV typing was performed by type-specific PCR for HPV 16 and HPV 18 DNA. Unidentified samples were subsequently sequenced to determine the viral genotype. The physical status of HPV was determined by a nested PCR approach for type-specific E2 sequences. C-myc amplification was assessed by co-amplification with ß-globin as control locus, and mutation in k-ras codons 12 and 13 by ARMS-PCR. Overall, HPV was detected in thirty-three colorectal specimens (44%). HPV 16 was the prevalent type (16/75), followed by HPV 18 (15/75), HPV 31 (1/75) and HPV 66 (1/75). E2 disruption was detected in 56.3% of HPV 16 and in 40% of HPV 18 positive tumors. C-myc amplification was detected in 29.4% of cases, while k-ras mutations in 30.7%. There was no significant trend for HPV infection in tumors harboring either k-ras or c-myc alterations. This study demonstrates HPV DNA and viral integration in colorectal tumors, suggesting a potential role of this virus in colorectal carcinogenesis. There was no concurrence, however, of k-ras and c-myc activation with viral infection.

Genotoxic effects of benzo[a]pyrene and dibenzo[a,l]pyrene in a human lung cell line.

Several studies have shown that polycyclic aromatic hydrocarbons (PAHs) produce genotoxic effects in assays performed in vivo and in vitro. This study was undertaken to investigate the ability of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to induce DNA damage in a human lung fibroblast cell line (MRC-5), using sister-chromatid exchanges test (SCEs), the comet assay, and evaluating point mutations in codon 12 of the K-ras protooncogene by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCPs) and restriction fragment length polymorphisms (RFLP)-enriched PCR methods. Sister-chromatid exchanges frequencies were significantly increased in cells exposed to benzo[a]pyrene and dibenzo[a,l]pyrene in relation to controls (p < .001). Using the standard alkaline comet assay, significant differences between groups were found for the variable comet moment (CM) when cells were exposed to BP (p < .001) and DBP (p < .001). Nevertheless, PCR-SSCP and RFLP-enriched PCR methods did not show any association between treatments with BP and DBP and K-ras point mutations. The data presented in this study indicated that BP and DBP induced both DNA strand breaks and sister-chromatid exchanges but not significant point mutations at codon 12 of K-ras gene in the MRC-5 cell line.

Evaluation of p53 codon 72 polymorphism in adenocarcinomas of the colon and rectum in La Plata, Argentina.

AIM: To evaluate the potential association between p53 codon 72 polymorphism and sporadic colorectal adenocarcinoma development,and human papillomavirus (HPV) infection. METHODS: One-hundred and nine controls and 53 patients with colon cancer from the city of La Plata, Argentina were analyzed. p53 codon 72 genotypes and HPV infection were identified using allele-specific polymerase chain reaction and nested polymerase chain reaction, respectively. RESULTS: The differences in the distribution of p53 codon 72 polymorphism between the cases and controls were statistically significant. The arginine allele had a prevalence of 0.65 in controls and 0.77 in cases. The corresponding odds ratio for the homozygous arginine genotype was 2.08 (95% CI, 1.06-4.05; P<0.05). Lack of association was found between p53 polymorphism and HPV infection in the set of adenocarcinomas. CONCLUSION: The findings of the present study indicate that p53 codon 72 arginine homozygous genotype may represent a genetic predisposing factor for colon cancer development. However,further studies are needed in order to elucidate the role of p53 codon 72 polymorphism in colorectal cancer.

Induction of DNA strand breaks, DNA-protein crosslinks and sister chromatid exchanges by arsenite in a human lung cell line.

Based on in vitro studies, several modes of action for arsenic have been suggested, although the mechanisms responsible for arsenic carcinogenesis have not been well established. In our previous study a dose-dependent increment in DNA migration was detected at low doses of sodium arsenite, but at higher dose levels a reduction in the migration was observed, suggesting the induction of DNA adducts. In order to confirm this hypothesis we performed the experiments considering other parameters and modifications of the standard alkaline comet assay. Additionally, the induction of sister chromatid exchanges was evaluated. The present study showed the induction by sodium arsenite of single strand breaks and DNA-protein adducts assessed by comet assay as well as of sister chromatid exchanges in the human lung fibroblast cell line MRC-5. The standard alkaline comet assay also revealed, at the highest arsenic concentration tested, a reduction in all the considered parameters in relation to untreated cells and the other doses. On the other hand, the incubation with proteinase K induced a dose-dependent increment in DNA migration as a consequence of the release of proteins joined to the DNA. Thus, sodium arsenite was able to induce both DNA-strand breaks and protein-DNA adducts in arsenic exposed MRC-5 cells, depending on the concentrations of arsenic salts tested.

Herpes simplex virus and human papillomavirus infection in cervical disease in Argentine women.

The aim of the present study was to determine that prevalence of herpes simplex virus (HSV) type 1 and 2 in cervical samples from Argentine women and to assess the role of HSV-2 in cervical cancer. A sample of 79 normal and 200 neoplastic cervical tissues (35 invasive cervical carcinomas, 75 high-grade squamous intraepithelial lesions, 79 low-grade squamous intraepithelial lesions and 11 abnormal squamous cells of undermined significance) was analyzed for herpes simplex and human papillomavirus DNA using the polymerase chain reaction method. Viral genotyping was performed by single strand conformation polymorphisms and restriction fragment length polymorphisms. The overall prevalence of HSV was 21.5% in controls and 29% in cases. Among women with normal cytology, herpes simplex prevalence in HPV positive (20.8%) women was approximately the same as in negative (21.8%) women. HPV- and age- adjusted ORs of high-grade squamous intraepithelial lesions and invasive cervical carcinomas for HSV-2 were 1.4 (p = 0.6) and 1.6 (p = 0.5), respectively. The obtained results indicated that herpes simplex virus may not be involved in cervical cancer development. Future investigations are needed to provided conclusive evidence on the role of this pathogen in cervical cancer.

DNA-protein cross-links and sister chromatid exchanges induced by dimethylarsinic acid in human fibroblasts cells.

Biotransformation of inorganic arsenic to form both methylarsinic acid (MA) and dimethylarsinic acid (DMA) has traditionally been considered as a mechanism to facilitate the detoxification and excretion of arsenic. However, the methylation of inorganic arsenic as a detoxification mechanism has been questioned due to recent studies revealing an important role of organic arsenic in the induction of genetic damage. In a previous report a reduction of DNA migration after treatment of cells with DMA was described. In order to further evaluate the possible induction of protein-DNA adducts, an experiment was performed taking into account other parameters and modifications of the standard alkaline comet assay. In addition, the results obtained with the comet assay were compared with those obtained by analyzing the induction of sister chromatid exchanges (SCEs). SCE frequencies were significantly increased in treated cells in relation to controls (p<0.001). Furthermore, in the standard alkaline comet assay, as well as in the control assay for proteinase K treatment, a significant dose-dependent reduction in tail moment was observed. Nevertheless, the post-treatment with proteinase K induced the release of proteins joined to the DNA and consequently, a dose-dependent increment in DNA migration was observed (p<0.001). These results suggest that DNA-protein cross-links may be an important genotoxic effect induced by dimethylarsinic acid in human MRC-5 cells.

Human papillomavirus DNA detection and typing in male urine samples from a high-risk population from Argentina.

The aim of the present study was to evaluate the first void urine (FVU) as a non-invasive sampling method for HPV detection and genotyping in a high-risk population. Men presenting with HPV associated penile lesions or HPV positive partners attending a urological department in La Plata, Argentina were enrolled for HPV detection and genotyping. DNA from 185 first-void urine samples was evaluated for the presence of HPV by nested polymerase chain reaction using MY09/11 and GP05/06 primers. The viral genotype was analyzed by means of the single-stranded conformation polymorphisms (SSCP) method. Seventy-three percent (135/185) of the FVU specimens were positive for HPV-DNA. The viral prevalence in patients with HPV-DNA positive partners was 68.8% (77/112), and 79.5% (58/73) of patients with penile lesions were found to be HPV positive. The most frequent viral type was HPV-11 (26.7%), followed by HPV-6 (23%), HPV-16 (21.5%), HPV-18 (6%), and HPV-31 (4.4%). In this study, 11.1% (15/135) of the HPV positive specimens were double infections. These results indicate that high-risk HPVs can be found in clinical lesions in a high percentage (43.8%), as simple or double infections. In this sense, the male population represents an important reservoir for the virus and may play a role in the transmission and perpetuation of the infection in the general population. The method described below provides a tool for detection and typing of HPV-DNA using samples obtained by non-invasive techniques and thus easy to obtain.

Chlamydia trachomatis and Human papillomavirus infections in cervical disease in Argentine women.

OBJECTIVES: To examine the prevalence of Human papillomavirus and Chlamydia trachomatis DNA in cervical samples among women with normal and abnormal cervical cytology from La Plata, Argentina. METHODS: Two hundred and seventy-nine women (200 with cervical neoplasia or ICC and 79 women with normal cytology) provided cervical samples for the detection of HPV and C. trachomatis DNA by PCR-based assays. RESULTS: HPV DNA increased with the cervical lesion severity, ranging from 30% among women with normal cytology to 99-100% among women with HSIL or ICC. C. trachomatis DNA prevalence increased from low levels in women with normal cytology (11%) to 47% in those with HSIL, but was uncommon among ICC patients (20%). Among women with normal cytology, C. trachomatis prevalence was higher in HPV DNA positive (12.5%) than HPV DNA negative women (10.9%), but this difference was not significant. CONCLUSIONS: HPV prevalence in the general population is slightly higher than those reported for other developing countries. C. trachomatis DNA positivity was associated with a higher risk of both LSIL and HSIL lesions, but not with ICC.

A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells.

Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl(2)) and cadmium sulphate (CdSO(4)) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p<0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p<0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p<0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene.

Herpes simplex virus genotyping: multiple optional PCR-based RFLP systems and a non-isotopic single-strand conformation polymorphism method.

Polymerase chain reaction (PCR)-based methods for herpes simplex virus (HSV) types I/II genotyping are described. These methods are based on the single-stranded conformation properties of DNA molecules obtained by PCR (PCR-SSCP) and restriction fragment analysis of PCR products (PCR-RFLP). With the aim to analyse these two genotyping techniques, genomic DNA from the standard viral strains KOS and G, and also 79 normal cervical samples were studied for HSV I/II. Sequence analysis showed 19 different RFLP possible systems for HSV typing. Five systems were used in this study and all led to the expected fragments obtained by sequence analysis. PCR-SSCP showed distinct patterns for both viral types and had 100% of concordance with PCR-RFLP results. The prevalence of herpes simplex virus in the normal cervical samples were 21.5% (17 out of 79), the most frequent viral type was HSV-2, with a prevalence of 71% (12 out of 17). Both techniques appeared suitable for HSV I/II genotyping and are easy to perform in most clinical laboratories.

The c-myc activation in cervical carcinomas and HPV 16 infections.

Despite the prominent role for Human Papillomavirus (HPV) infection in the development of genital cancer, other genetic or environmental co-factors have also been involved. Studies of c-myc activation in cervical carcinomas have reported that gene over-expression (mainly gene amplification) are common in cervical squamous cell carcinomas and may correlate with the biologic behavior of the neoplasm. Using PCR based technology, DNAs from 79 normal cervical samples and 225 abnormal cervical tissue scrapes were analyzed for HPV detection and typing and for c-myc gene amplification. Significant differences were found between the different cyto/histology groups (P<0.0001) and also with HPV high-risk infected samples (P<0.0002). In this sense, we showed that the average c-myc copy number increased according to the histological grade of the lesion (OR=6.3, CI=2.1-18.8). Also, the results showed that the infection with HPV 16 was tightly associated with c-myc amplification (OR=10.6, CI=3.1-36). These results could indicate that oncogene amplification take place in pre-invasive stages of cervical disease and could cooperate not only in tumor progression but also in cell transformation. Moreover, the results strongly associate the c-myc gene amplification to the infection with the oncogenic HPV 16, showing that the pattern of virus infection and oncogene activation could be specific for different viral genotypes.

The p53 codon 72 genotypes in HPV infection and cervical disease.

OBJECTIVE: [corrected] An experimental study has indicated that individuals homozygous for the Arg-encoding allele of p53 gene may have an increased susceptibility to HPV-related cervical cancer but many epidemiological studies have failed to repeat this result. Many epidemiological studies have failed in the attempt to repeat this results. The aim of the present work was to investigate whether the p53 arginine allele confers a risk factor for cervical carcinogenesis. STUDY DESIGN: Using PCR based technology, DNAs from 90 normal cervical samples and 205 abnormal cervical tissue scrapes were analyzed for the type of HPV present and p53 codon 72 polymorphism. RESULTS: Non statistically significant differences were found for the frequencies of p53 genotypes in the different cytological/histological groups (chi2=1.4; P=0.97) nor for the risk for HPV infection (chi2=1; P=0.9). CONCLUSION: This study showed that polymorphism at codon 72 of TP53 gene is not associated with an increased susceptibility to cervical disease and/or HPV infection in the Argentine women population.

Amplificación del proto-oncogén HER-2/neu asociada a la infección por VPH de bajo riesgo en citología exfoliativa cervical / HER-2/neu prot-oncogen amplification related to low risk VPH infection in cervical exfoliative cytology

Se analizaron 69 muestras positivas para el virus papiloma humano (VPH) tipos -6, -16 y -18, y 24 muestras con citología cervical normal negativas para el VPH como grupo control. El análisis de la amplificación génica del proto-oncogén HER-2/neu se realizó utilizando la técnica de coamplificación con locus de referencia. Se encontró una asociación entre la amplificación del gen HER-2/neu y el grupo viral de "bajo riesgo" (VPH-6) (p < 0.005). Dentro de este grupo, se observó una asociación entre la amplificación del proto-oncogén y el status citopatológico CIN I (p < 0.01). Debido a que la mayoría de las muestras CIN I analizadas presentaron un patrón coilocítico, la amplificación de HER-2/neu parecería estar relacionada con este tipo de alteración celular. Por otra parte, sería importante estudiar la amplificación génica y la expresión de HER-2/neu en los diferentes estadios de las neoplasias intraepiteliales cervicales a fin de poder evaluar su papel en la progresión del cáncer cervical.