Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real-time PCR assay for the detection of high-risk human papillomaviruses.

The detection of high-risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high-level hospitals or the limited resources in primary-level hospitals hinder widespread testing. To address this issue, we explored a sample-to-answer genotyping system and assessed its performance by comparing it with the traditional real-time polymerase chain reaction (PCR) method conducted manually. Samples randomly selected from those undergoing routine real-time PCR detection were re-analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray-based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real-time PCR results, 76 were high-risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high-risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high-risk HPV 51 were revealed to be false positive in real-time PCR by other method. When counting by samples without discriminating the high-risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having an HPV type not covered by real-time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real-time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab-on-chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

Partial Lipectomy of the Epididymal Fat Alters Expression of the Steroidogenic Enzymes in the Mouse Testis at Different Postnatal Ages.

The epididymal fat is a type of gonadal adipose tissue, which is localized closely to the testis. Even though it has been suggested that the epididymal fat is necessary for maintenance of spermatogenesis in the testis, the influence of epididymal fat on expression of testicular steroidogenic enzymes has not been examined. In the present research, expressional changes of steroidogenic enzymes in the mouse testis after 2 weeks of the surgical partial lipectomy of epididymal fat at different postnatal ages were determined by real-time polymerase chain reaction analysis. The transcript levels of all molecules at 2 months of postnatal age were significantly increased by the lipectomy of epididymal fat. However, the lipectomy at 5 months of postnatal age resulted in decreases of expression levels of all molecules examined in the testis. Except a reduced transcript level of hydroxysteroid 17-beta dehydrogenase 3, there were no significant changes of expression levels of other steroidogenic enzymes by the lipectomy at 8 months of postnatal age. At 12 months of postnatal age, the lipectomy caused a significant increase of transcript level of steroidogenic acute regulatory protein and a significant decrease of transcript level of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1, without any expressional change of cytochrome P450 side chain cleavage, hydroxysteroid 17-beta dehydrogenase 3, and hydroxysteroid 17-beta dehydrogenase 3 in the testis. These findings suggest that the substances derived from epididymal fat could differentially influence on expression of steroidogenic enzymes in the testis during postnatal period.

Dual Oxidase 1, 2 Gene Expression in Women With Polycystic Ovary Syndrome (PCOS).

Objective: Dual oxidases (DUOX1, DUOX2) belong to the NADPH oxidase (NOX) family, which produce H2O2 necessary for thyroid hormone biosynthesis. This study aims to evaluate gene expression for DUOX1, DUOX2 in PCOS patients and its relation with thyroid hormone and magnesium levels. Materials and methods: Totally 88 cases were studied including 24 people with PCOS and hypothyroidism, 44 people with PCOS and normal thyroid function, and 20 hypothyroid patients without PCOS. In comparison 40 healthy controls in the age group of 16-35 years matched for age group and BMI were evaluated. Using Vegaro syringe 5 cc of blood was sampled from all 128 people and after RNA extraction and cDNA synthesis using Real-Time PCR technique, the expression level of DUOX1 and DUOX2 genes was investigated. Results: The results of hormonal tests showed that there is a significant difference between the level of T4, T3, and TSH hormones in hypothyroid patients with or without PCOS in comparison to the control group. Regarding the level of Mg, the results showed that there is a significant difference between the levels of Mg in PCOS group with or without hypothyroidism in comparison to the control group. Gene expression results showed that the relative changes of DUOX1 gene expression in different groups compared to the control group were significantly reduced P<0.05. In the polycystic group with hypothyroidism, the gene expression level showed a decrease compared to the normo-thyroid polycystic group and the hypothyroid non-PCO group, which was statistically significant P<0.05. Conclusion: According to the results of the present study and the previous studies that have been published in the field of Duox1, it can be assumed that the reduction of Duox1 expression can interfere with the oxidative stress system. Further studies with other molecular techniques may help to understand the exact action mechanism of these genes.

Comparison of COBAS AmpliPrep/COBAS TaqMan HCV Qualitative Test v2.0 with COBAS AMPLICOR Hepatitis C Virus Test v2.0 for the Qualitative Detection of Hepatitis C Virus RNA in Korean Clinical Samples / 임상검사와정도관리

BACKGROUND: We comparatively evaluated the performance of the conventional COBAS Amplicor HCV test v2.0 (CAM; Roche Molecular Systems, USA) and the newly developed COBAS AmpliPrep/COBAS TaqMan HCV test v2.0 (CAP/CTM; Roche Molecular Systems) for qualitative detection of hepatitis C virus (HCV) RNA in clinical samples. METHODS: Six hundred serum samples (100 HCV-positive, 500 HCV-negative, as determined by CAM) were selected and analysed using the new qualitative HCV RNA test, CAP/CTM qualitative test. Results were compared by confirmatory CAP/CTM quantitative test, which is a quantitative HCV RNA real-time polymerase chain reaction by Roche Molecular Systems, and anti-HCV test (Roche Diagnostics GmbH, Germany). Twenty-two additional serum samples, which gave a gray zone result by CAM, were selected for comparison. RESULTS: The two qualitative HCV RNA assays yielded concordant results for 586 of 600 tested samples (concordance rate, 97.7%; kappa coefficient, 0.92; 95% confidence interval [CI], 0.87 to 0.96; P<0.001). Upon re-testing by CAM, we found that the concordance rate increased to 98.2% (kappa coefficient, 0.93; 95% CI, 0.89 to 0.97; P<0.001). The additional 22 samples showing gray zone results for CAM were retested and were also tested by CAP/CTM. The results for 13 of these samples changed to negative and were now concordant with the CAP/CTM and confirmatory CAP/CTM quantitative results. For the remaining samples, the results were variable. For all the 22 samples, the results of the new CAP/CTM were in agreement with those obtained by confirmatory CAP/CTM quantitative test. CONCLUSIONS: The results of the two assays were in good agreement, with 97.7% concordance rate. However, CAP/CTM is more sensitive than CAM and showed no gray zone results. Therefore, it can be a more efficient and useful test for the qualitative detection of HCV RNA in clinical samples.

Relationship Between Hepatitis B Virus DNA Level in Saliva and Periodontal Status in Chronic Hepatitis B Patients / 中华医院感染学杂志

OBJECTIVE To quantify the HBV DNA in saliva of chronic hepatitis B(CHB) patients and to study its relation to the periodontal status of the patients. METHODS HBV DNA were isolated from 60 patients with CHB by Trizol-chloroform method.HBV DNA levels were tested by real-time PCR technique and their relationship with plaque index,gingival index and probing depth was analyzed. RESULTS The positive rate of HBV DNA was (56.7%,) and the level of HBV DNA was 4.16+0.57(LogE,copies/ml) in saliva.The detectability of HBV DNA in saliva had no significant correlation with the values of oral clinical parameters. CONCLUSIONS The detectability and level of HBV DNA in saliva have no correlation with the oral hygienic parameters.Other sources of the HBV DNA may exist besides the serum in saliva.