Classification and identification of human papillomavirus based on its prevalence and development of cervical lesion among Iranian women.

Introduction: Cervical cancer is the most common female cancer in large areas of the developing world, and almost half of these cases (54%) arises in Asia, where cervical cancer is still threatening women's health and survival, which makes it a considerable public problem. Human papillomavirus (HPV) is one of the most powerful human carcinogens. Today, it has been proven that all cervical cancers and primary precancerous lesions are caused by carcinogenic types of HPV infections. HPV genotyping can therefore evaluate the screening programs. Methods: Five hundred fifty women referring to the gynecological centers were subjected to Pap smear cell samples. The cytopathological diagnosis of obtained cervical samples was based on the Bethesda system. HPV genotyping was carried out using the INNO-LiPA HPV Genotyping Extra II Amp assay. Results: In a total of 244 HPV positive cases, single­type HPV infec-tion was observed in 49.6%, while multi­type HPV infections (including ≥ 2 types) were found in 45.5% of cases. Among the 110 cases with abnormal cytology results, going-over analyses led to the identification of atypical squamous cell of unknown significance (ASCUS) in 73 cases, low­grade squamous intraepithelial lesions (LSIL) in 24 cases, and high­grade squamous intraepithelial lesion (HSIL) in 12 cases. In these groups, the infection rate of high-risk HPV (HR-HPV) was 89%, 82%, and 100%, respectively. Conclusion: In this study, the total population of women suffering from different cervical lesions and malignancy was found to be infected with various HPV genotypes. High prevalence of HPV- 53 and HPV- 16 detected among participants with normal cytology can be considered as a tip-off development of cervical cancer among Iranian women.

Assessment of Cervical Cancer Molecular-Based Screening Tools; HPV-DNA Detection versus E6/E7 mRNA Testing; First Report of a Prospective Cohort Study among Iranian Women.

BACKGROUND: Human papillomavirus (HPV) has been found as the most considerable causes of cervical cancer. Recently, several molecular methods have been introduced to increase the accuracy of the screening programs and decrease the mortality rate. Among these methods, mRNA-based methods have more advantages as they assess the expression level of HPV E6 and E7 oncogenic mRNAs. This study aimed to evaluate the results of HPV RNA- and DNA-based methods among Iranian women population with normal cytology results. METHODS: Overall, 4640 women were enrolled referred to the Gynecology Oncology Ward of Vali-e-Asr Hospital, private and academic clinics, Tehran, Iran from Jan 2016 to Apr 2018. To assess the HPV-DNA infection INNO-LiPA® HPV Genotyping Extra-II kit was used. For HPV-RNA assessment, Aptima HPV Assay and in house HPV-RNA genotyping methods were applied. RESULTS: The positivity rates of HPV infection according to DNA- and RNA-based methods were 18.0% and 11.2%, respectively (P<0.001). The positive predictive value, negative predictive value, specificity and sensitivity of DNA-based method in contrast with RNA-based method were 59.2% (56.6-61.6), 99.4% (99.0-99.6), 91.7% (90.8-92.6) and 95.2% (93.0-96.9) respectively. CONCLUSION: At the present study for prognosis of cervical cancer, RNA-based method seemed to be more specific in contrast to DNA-based method. Patient follow up and further studies will be conducted in order to clarify the clinical sensitivity and specificity of the two methods.

A facile PCR-RFLP method for genotyping of ITPA rs1127354 and rs7270101 polymorphisms.

BACKGROUND: Inosine triphosphate pyrophosphatase (ITPA) gene single nucleotide polymorphisms (SNPs), rs1127354 and rs7270101, may cause a functional impairment in ITPase enzyme, resulting anemia protection in patients with chronic hepatitis C virus (HCV) infection undergoing ribavirin (RBV)-dependent regimens. The main purpose of this study was to provide and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for genotyping of ITPA rs1127354 and rs7270101 polymorphisms in chronic HCV-infected patients. METHODS: In the current study, 100 Iranian patients with chronic hepatitis C were examined and genotyped for ITPA rs1127354 and rs7270101 gene polymorphisms. To genotype rs1127354 and rs7270101 polymorphisms, PCR-RFLP technique and sequencing technique were performed on these samples. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results. RESULTS: The rs1127354 and rs7270101 polymorphisms of ITPA gene were genotyped by PCR-RFLP technique and sequencing simultaneously, and the results of both techniques were 100% concordant in all 100 patients. Both PCR-RFLP and sequencing techniques indicated that the genotypic frequency of rs7270101 was 80% AA, 19% AC and 1% CC, and for rs1127354 was 79% CC, 20% CA and 1% AA, respectively. CONCLUSION: We developed and validated a rapid and inexpensive PCR-RFLP technique for the detection of ITPA rs1127354 and rs7270101 gene polymorphisms.

A Simple PCR-RFLP Method for Genotyping of IFNL4 rs368234815 Polymorphism in Patients With Chronic Hepatitis C.

BACKGROUND: The IFNL4 rs368234815 polymorphism plays a prominent role in spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infection. This study aimed to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for assessment of rs368234815 polymorphism. METHODS: We genotyped the rs368234815 polymorphism in 87 patients with chronic HCV by PCR sequencing and PCR-RFLP methods, simultaneously. RESULTS: Genotyping of IFNL4 rs368234815 via PCR-RFLP was concordant with PCR sequencing in all 87 individuals (100%). The analytical sensitivity and specificity of the developed PCR-RFLP method for genotyping of rs368234815 polymorphism were each 100%. Among these patients with chronic HCV, the frequency of rs368234815 TT/TT, TT/ΔG, and ΔG/ΔG were 44.8%, 37.9%, and 17.3%, respectively. CONCLUSIONS: The PCR-RFLP method that we developed is accurate, rapid, inexpensive, and easy to perform for genotyping of the IFNL4 rs368234815 polymorphism. This method can be used for clinical and research work.

Impact of IFNL4 rs12979860 and rs8099917 polymorphisms on response to Peg-Interferon-α and Ribavirin in patients with congenital bleeding disorder and chronic hepatitis C.

BACKGROUND: The aim of this study was to determine whether two polymorphisms of the human interferon lambda 4 (IFNL4) gene (rs12979860 and rs8099917) can predict sustained virologic response (SVR) following antiviral therapy in patients with inherited bleeding disorder and chronic hepatitis C (CHC). METHODS: This retrospective study was conducted on 294 patients with congenital bleeding disorder and CHC who were treated with Peg-Interferon-α (PegIFN) and Ribavirin (RBV). Baseline patient and viral parameters were measured and analyzed statistically to assess their combined and individual contributions to SVR prediction. RESULTS: The most prevalent variants of rs12979860 and rs8099917 identified among the study patients were CT (45.9%) and TT (57.6%), respectively. Overall, SVR was achieved in 69% of the study patients. The rate of SVR was lower in patients with HCV genotype-1 than in those with HCV genotype-3 (62% vs 88%; P<.001; OR=0.23). Multivariate analysis of SVR predictors in patients with HCV genotype-1 infection included age (<24 years), BMI (<25), absence of cirrhosis, HCV RNA level (<400 000 IU/mL), rs8099917 TT and rs12979860 CC, all of which were associated with a higher SVR rate. In HCV genotype-3 infection, only rs12979860 CC was significantly associated with SVR. CONCLUSION: These results demonstrate that polymorphisms of the IFNL4 gene are highly associated with SVR to PegIFN and RBV combination therapy in patients with a congenital bleeding disorder and CHC. Assessment of rs12979860 and rs8099917 genotypes can guide physicians in choosing an optimal treatment regimen, including less expensive therapies that may only be available in many geographic locales.

The interferon lambda 4 rs368234815 predicts treatment response to pegylated-interferon alpha and ribavirin in hemophilic patients with chronic hepatitis C.

BACKGROUND: A dinucleotide variant rs368234815 in interferon lambda 4 (IFNL4) gene was recently found to be associated with the hepatitis C virus (HCV) treatment response. This study aimed to assess the impact of IFNL4 rs368234815 polymorphism on treatment response to pegylated-IFN alpha (Peg-IFN-α) and ribavirin (RBV) in hemophilic patients with chronic hepatitis C (CHC). MATERIALS AND METHODS: In this retrospective study, 92 hemophilic patients with CHC who were treated with Peg-IFN-α/RBV were investigated. Single-nucleotide polymorphisms (SNPs) in IFNL genomic region including rs368234815, rs12979860, and rs8099917 were analyzed by DNA sequencing. RESULTS: Of the 92 patients, 63 (68.5%) achieved sustained virological response (SVR). Of the 43 patients with rs368234815 TT/TT genotype, 36 (83.7%) achieved SVR, while in 49 patients with non-TT/TT genotypes, 27 (55.1%) achieved SVR. Other pretreatment parameters predicted SVR were patients' body mass index, HCV genotype, rs12979860, and rs8099917 SNPs. In multivariate analysis, all above-mentioned parameters except rs8099917 remained as predictors of SVR. IFNL4 rs368234815 was a strong predictor of SVR; however, the prediction power of this SNP was the same as that of rs12979860 SNP in the patients of the current study. CONCLUSION: IFNL4 rs368234815 SNP can be considered for decision-making in the treatment of HCV-infected patients.

The Prevalence of Hepatitis C Virus Core Amino Acid 70 Substitution and Genotypes of Polymorphisms Near the IFNL3 Gene in Iranian Patients With Chronic Hepatitis C.

BACKGROUND: Molecular studies have demonstrated that the hepatitis C virus (HCV) genotype and host genetics play predictive roles in the management of patients infected with HCV. OBJECTIVES: This study aimed to investigate the HCV genotype, core amino acid (aa) 70 substitution, and polymorphisms near the IFNL3 gene (including rs12979860 and rs8099917) among Iranian patients with chronic hepatitis C (CHC). PATIENTS AND METHODS: In this cross-sectional study, the molecular profiles of the HCV genotype, core aa 70 substitution, and rs12979860 and rs8099917 polymorphisms and plasma HCV RNA levels were determined in 429 CHC patients including 141 hemophilic, 84 thalassemic, and 204 non-hemophilic, non-thalassemic patients. RESULTS: The hepatitis C virus subtype 1a was the most common subtype in the study population. Core aa substitution Arg70Gln was strongly associated with cirrhosis (OR = 2.49; 95% CI = 1.13 - 5.50; P = 0.020). Core aa 70 substitutions were more frequently observed in patients with the HCV subtype 1b than in patients with any other HCV subtypes (P < 0.001). Core aa 70 substitutions were also more common in patients with the rs12979860 TT genotype than in patients with non-TT genotypes (17.3% vs. 8.5%, P = 0.022) and also in rs8099917 non-TT genotypes than in the TT genotype (14.0% vs. 7.0%, P = 0.026). The HCV genotypes and rs8099917 polymorphisms were correlated in which HCV subtype 1b was in favor of rs8099917 GG and HCV subtype 3a favored rs8099917 TT (P = 0.021). Furthermore, the rs12979860 TT and rs8099917 GG genotypes showed significantly lower HCV RNA levels than the other genotypes (P < 0.001). CONCLUSIONS: There is an as yet unexplained association between HCV and host parameters with unknown mechanisms in patients with chronic HCV infection. The assessments of core aa 70 substitution and polymorphisms near the IFNL3 gene could offer promising steps to improve the management of patients with HCV.

The ITPA and C20orf194 Polymorphisms and Hematological Changes During Treatment With Pegylated-Interferon Plus Ribavirin in Patients With Chronic Hepatitis C.

BACKGROUND: It has been found that ITPase deficiency is caused by ITPA gene polymorphisms. It was observed that ITPA polymorphisms have impact on hematological changes, including hemoglobin (Hb)-decline during treatment of chronic hepatitis C (CHC) patients with pegylated-interferon (PEG-IFN) plus ribavirin (RBV). OBJECTIVES: This study aimed to assess the effect of ITPA and C20orf194 polymorphisms on hematological changes at week 4 of treatment with PEG-IFN plus RBV in patients with CHC. PATIENTS AND METHODS: In this retrospective study, 168 patients with CHC (56% HCV genotype-1 and 44% HCV genotype-3) under the treatment of PEG-IFN plus RBV were genotyped for rs1127354, rs7270101 and rs6051702 polymorphisms by the polymerase chain reaction-restriction fragment length polymorphism. Hematological changes including Hb-, platelet (Plt)- and white blood cell-decline at week 4 of the treatment were assessed. RESULTS: In univariate analysis, rs1127354 and HCV genotypes were found to influence the Hb-decline at week 4 of the treatment. In multivariate analysis, rs1127354 CA + AA and HCV genotype-3 were found to have a great role on prevention of Hb-decline. Furthermore, rs1127354 and HCV RNA levels were found to influence the Plt-decline at week 4 of the treatment in the univariate analysis. In multivariate analysis, rs1127354 CA + AA and HCV RNA levels less than 600,000 IU/mL were found to be associated with a higher level of Plt-decline. CONCLUSIONS: In patients with CHC, who were treated with PEG-IFN plus RBV, Hb-decline was affected by rs1127354 and HCV genotypes. However, Plt-decline may be altered by rs1127354 and baseline HCV RNA levels.

The Role of Polymorphisms Near the IL28B Gene on Response to Peg-Interferon and Ribavirin in Thalassemic Patients With Hepatitis C.

BACKGROUND: Hepatitis C Virus (HCV) is the major cause of liver failure in thalassemic patients. In these patients, iron overload and their comorbidities make difficulties during Pegylated-Interferon (PEG-IFN) and Ribavirin (RBV) therapy. OBJECTIVES: We aimed to assess the impact of polymorphisms near the IL28B gene on virological response in HCV - infected thalassemic patients, who were treated with PEG-IFN and RBV. PATIENTS AND METHODS: This cross - sectional study was conducted on 143 thalassemic patients with chronic hepatitis C, who were treated with a combination of PEG-IFN and RBV regimen. The rs12979860 and rs8099917 polymorphisms were assessed as the most common polymorphisms near the IL28B gene by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The rate of sustained virological response (SVR) was significantly lower in thalassemic patients with HCV genotype-1 infection compared to patients with HCV genotype-3 infection. Among baseline predictors, rs12979860 and rs8099917 polymorphisms were found to be the only parameters associated with achievement of SVR in thalassemic patients with HCV genotype-1 infection however, there was no association between these polymorphisms and the rate of SVR in thalassemic patients with HCV genotype-3 infection. CONCLUSIONS: In HCV genotype-1- infected thalassemic patients with rs12979860 CC genotype and without severe comorbidities, PEG-IFN and RBV combination therapy can be tried yet in those with rs12979860 CT/TT it may be reasonable to treat cases with new direct-acting antivirals.

Interferon-λ polymorphisms and response to pegylated interferon in Iranian hepatitis C patients.

AIM: To evaluate the efficacy of pegylated interferon in Iranian chronic hepatitis C patients in relation to interferon-λ (IFNL) polymorphisms. METHODS: This study enrolled patients with chronic hepatitis C referred to the Tehran Blood Transfusion Hepatitis Clinic in 2011. Patients were included in the study if they had no concomitant hepatic illness, were negative for human immunodeficiency virus antibodies, and had no prior history of treatment with any type of pegylated interferon. Patients were treated with 180 µg pegylated interferon alpha-2a (Pegaferon(®)) weekly and 800-1200 mg ribavirin daily for 24 or 48 wk depending on weight and hepatitis C virus (HCV) genotype. Blood samples were collected from patients to obtain DNA for determination of IFNL rs12979860 and rs8099917 polymorphisms. The virologic response in patients was then evaluated and compared between the different IFNL genotypes. RESULTS: A total of 152 patients with a mean age of 41.9 ± 10.0 years were included in the study, of which 141/152 were men (92.8%). The most frequent HCV genotype was type-1, infecting 93/152 (61.2%) patients. Sustained virologic response (SVR) was achieved in 81.9% of patients with HCV genotype-1 and 91.1% of patients with HCV genotype-3. Treatment success was achieved in 91.2% (52/57) of patients with the IFNL rs12979860 CC genotype and 82.1% (78/95) in those with other genotypes. Similar treatment response rates were also observed in patients with rs8099917 TT (39/45; 86.7%) and non-TT (61/68; 89.7%) genotypes. Univariate analyses identified the following factors which influenced treatment response for inclusion in a multivariate analysis: age, HCV RNA level, stage of liver fibrosis, rs12979860 CC genotype, and aspartate transaminase level. A logistic regression analysis revealed that only the rs12979860 CC genotype was significantly associated with achievement of SVR (OR = 6.2; 95%CI: 1.2-31.9; P = 0.03). CONCLUSION: The rs12979860 CC genotype was associated with SVR in patients receiving pegylated interferon plus ribavirin, however, the SVR rate in other rs12979860 genotypes was also relatively high.

The Impact of IFNL4 rs12979860 Polymorphism on Spontaneous Clearance of Hepatitis C; A Case-Control Study.

BACKGROUND: About 30% of individuals with hepatitis C virus (HCV) infection are able to clear HCV spontaneously. Differences in host genetics affect the outcome of HCV infection. Single nucleotide polymorphisms (SNPs) of the Interferon lambda (IFNL) genes were associated with spontaneous and treatment-induced clearance of HCV infection. OBJECTIVES: The aim of this study was to evaluate the association between the IFNL4 rs12979860 SNP and spontaneous clearance of HCV infection in Iranian population. MATERIALS AND METHODS: A case-control study was designed on 91 cases with spontaneous HCV infection clearance and 259 patients with persistent HCV infection as the control group. The rs12979860 SNP was assessed as the most common IFNL polymorphism by PCR-RFLP method. RESULTS: Distribution of rs12979860 CC genotype in the spontaneous clearance group was around two folds of its distribution in chronic hepatitis C group (P < 0.001, OR = 4.09, 95% CI = 2.44-6.86). CONCLUSIONS: The rs12979860 SNP was observed as a strong host genetic factor associated with spontaneous clearance of hepatitis C infection.

Development and Validation of a Simple, Rapid and Inexpensive PCR-RFLP Method for Genotyping of Common IL28B Polymorphisms: A Useful Pharmacogenetic Tool for Prediction of Hepatitis C Treatment Response.

BACKGROUND: In 2009, 3 genome-wide association studies implicated IL28B single-nucleotide polymorphisms (SNPs) as the strongest genetic pretreatment predictor of sustained virological response (SVR) in hepatitis C infection. Recently, the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) included IL28B testing in their guidelines. OBJECTIVES: The main aim of this study was to develop and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for genotyping of common IL28B polymorphisms (rs12979860 and rs8099917). PATIENTS AND METHODS: Two methods were developed to genotype common IL28B polymorphisms: 1) PCR-sequencing as a reference method and 2) PCR-RFLP as a rapid and inexpensive method. Both polymorphisms were genotyped in 104 Iranian hepatitis C patients by both methods simultaneously. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results. RESULTS: Genotyping of rs12979860 and rs8099917 by PCR-RFLP was concordant with PCR-sequencing in 104 (100%) individuals. The analytical sensitivity and specificity of the PCR-RFLP method for genotyping of both SNPs are 100%. Among these 104 patients with chronic hepatitis C, the frequency of the rs12979860 CC, CT and TT genotypes were 40.4%, 47.1% and 12.5% and the frequency of the rs8099917 TT, GT and GG genotypes were 59.6%, 35.6% and 4.8%, respectively. Also, three IL28B haplotypes (rs12979860-rs8099917) were found among our patients including C-T, T-G and T-T with 63.9%, 22.6% and 13.5% frequency, respectively. C-G haplotype was absent in all of our patients. CONCLUSIONS: We have developed a validated, fast, and simple PCR-RFLP method for genotyping of common IL28B SNPs that is more cost-effective than sequencing.

Distribution of IL28B Genotypes in Iranian Patients with Chronic Hepatitis C and Healthy Individuals.

BACKGROUND: IL28B polymorphism is recognized as one of the most prominent predictors of hepatitis C spontaneous and treatment-induced clearance. Interestingly, the favorable genotypes of IL28B are found to be more frequent in Asian ethnicity than Caucasian and African populations, respectively. A few studies reported that there is a mysterious association between the IL28B polymorphism and the hepatitis C virus (HCV) genotype in patients with chronic hepatitis C but they did not give any reason for this phenomenon. OBJECTIVES: The foremost purpose of this study was to compare the distribution of IL28B genotypes between Iranian healthy individuals and patients with chronic hepatitis C. PATIENTS AND METHODS: In this study, 921 patients with chronic hepatitis C and 142 healthy individuals were included. The IL28B rs12979860 and rs8099917 polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The frequency of IL28B rs12979860 CC, CT, and TT genotypes in chronic hepatitis C patients was 38%, 48.8%, and 13.2% and in healthy individuals was 43.7%, 48.6%, and 7.7%. Also, the frequency of IL28B rs8099917 TT, GT, and GG genotypes in chronic hepatitis C patients was 58.3%, 37.1%, and 4.6% and in healthy individuals was 64.1%, 32.4% and 3.5%. The differences in the distribution of IL28B rs12979860 and rs8099917 genotypes between patients with chronic hepatitis C and healthy individuals were not statistically significant. When we compared the distribution of IL28B genotypes between the healthy group and the HCV infected patients by HCV genotype, we found 9.8% higher frequency of rs12979860 CC genotype in the healthy individuals than HCV genotype 1 infected patients (P = 0.03) however there was no significant difference in the distribution of rs12979860 genotypes between the healthy and HCV genotype 3 infected groups (P = 0.46). CONCLUSIONS: It seems that the impact of IL28B polymorphism on the spontaneous clearance of HCV genotype 1 is more prominent than HCV genotype 3 which results in the observation of higher rs12979860 C allele frequency in chronic hepatitis C patients with HCV genotype 3 than HCV genotype 1.