Evaluation of clinical usefulness of HPV-16 and HPV-18 genotyping for cervical cancer screening.

OBJECTIVE: High-risk human papillomavirus (HR-HPV) infection is a leading cause of cervical cancer, of which human papillomavirus (HPV)-16 and HPV-18 account for about 70% of cases. Since HPV infection is common, it is important to focus on the HPV genotypes that pose the highest risk for effective cervical cancer screening. In this study, we evaluated the clinical usefulness of HPV-16/HPV-18 genotyping for cervical cancer screening. METHODS: A total of 86,022 women aged 25 years or older was analyzed in this study. Sensitivity, specificity, positive predictive value, and negative predictive value of HPV genotyping and cytology were analyzed. In addition, we subdivided participants into two groups according to cytology results, negative for intraepithelial lesion of malignancy (NILM) and atypical squamous cells of undetermined significance (ASC-US), and analyzed absolute risk (AR) and relative risk (RR) of cervical intraepithelial neoplasia (CIN) 3 or worse according to HPV genotype. RESULTS: The AR of CIN 3 or worse was 77.0 times higher in HR-HPV-positive compared to HR-HPV-negative. Compared to 12 other HR-HPV-positive, the AR of CIN 3 or worse was 4.2 times higher in HPV-16 and/or HPV-18 positive. This finding was more evident in women with NILM than in women with ASC-US. The RR of CIN 3 or worse was 7.0 in women with NILM and 4.5 in women with ASC-US. CONCLUSION: Regardless of the cytology results, the risk of CIN 3 or worse was higher in HPV-16/HPV-18 than in other HR-HPV. HPV-16/HPV-18 genotyping is recommended to screen women with a high risk of cervical cancer.

Prevalence and molecular basis of null blood group phenotypes in the Korean population: Analysis using a public database.

BACKGROUND: Null phenotypes are characterized by complete absence of all antigens within a blood group system and caused by null variants (e.g., nonsense, frameshift, initiation codon, and canonical splice site variants) in the genes encoding the antigens. Knowing the prevalence and molecular basis of null phenotypes is essential to establish a rare donor program, and the aim of this study was to reveal the prevalence and molecular basis of null phenotypes using the Korean Reference Genome Database (KRGDB) containing whole-genome sequences of 1722 Korean individuals. STUDY DESIGN AND METHODS: Population allele frequencies of null alleles in 39 blood group systems except ABO, MNS, Rh, Lewis, and FORS were obtained from the KRGDB. The prevalence of null phenotypes was calculated using Hardy-Weinberg equation. RESULTS: The prevalence of null phenotypes were estimated to be less than 0.001% in all blood group systems except JR and SID. The prevalence of the Jr(a-) and Sd(a-) phenotypes were estimated to be 0.0453% and 0.2323%, respectively. The most frequent null allele of the JR system was ABCG2*01N.01, accounting for approximately 85% of null alleles. DISCUSSION: Our approach using a public database allowed us to investigate the prevalence and molecular basis of null phenotypes in the Korean population, which will serve as a guide for establishing a rare donor program in Korea. Considering the clinical significance, Jr(a-) is an important null phenotype that should be typed in the Korean population, and molecular assays targeting the most frequent allele ABCG2*01N.01 may be useful in detecting this phenotype.

Evaluation of molecular methods for plasma detection of EGFR mutations in non-small cell lung cancer.

AIM: Epidermal growth factor receptor (EGFR) mutations are detected in non-small cell lung cancer (NSCLC) and associated with responses to therapy with tyrosine kinase inhibitors (TKIs). We compared the analytical performances of two real-time PCRs and droplet digital PCR (ddPCR) to detect EGFR mutations using plasma. METHODS: Plasma EGFR tests were performed using 86 plasma samples from 75 prospectively enrolled NSCLC patients with early and advanced stages. Analytical performances of plasma-using two real-time PCR, Cobas EGFR mutation v2 and PANAMutyper, EGFR kit, and ddPCR were evaluated based on the tissue EGFR test results. The frequencies of EGFR mutations and acquired T790M mutation after TKI therapy were also assessed. RESULTS: The incidence of all EGFR mutations was 52.3% (23/44) in tissue and was up to 43.2% (19/44) in plasma. The Cobas detection rates of three EGFR mutations (exon 19 deletions, L858R, and T790M) in plasma were similar to those in tissue. The Cobas showed a higher detection rate (76.7%) than that by the PANAMutyper (60.5%). Sensitivity for T790M mutation was lower than the sensitivity for the exon 19 deletions or L858R in both tests. Mutant allele frequency measured by ddPCR was significantly correlated with the semi-quantitative values of the Cobas. CONCLUSIONS: Plasma EGFR tests showed similar detection rates for common EGFR mutations compared to the tissue EGFR tests. Cobas showed higher sensitivity in detection of EGFR mutations in body fluids than the PANAMutyper. Real-time PCR using plasma or body fluids could be a suitable first test for the detection of EGFR mutations.

Evaluation of Cobas 8000 Analyzer Series Module e801 Analytical Performance.

BACKGROUND: Immunoassays are important tests that provide essential information for patient care. The e801, a module of the recently released Cobas 8000 series (Roche Diagnostics, Mannheim, Germany), is an automated immunoassay system based on streptavidin-biotin interactions. In this study, we evaluated the analytical performance of the e801. METHODS: We evaluated the precision, linearity, assay comparison, and reference range validation of 16 analytes (AFP, CA19-9, CA125, CEA, CYFRA, ferritin, NSE, PSA, Vitamin D, E2, fT4, TSH, FSH, insulin, NT-proBNP, and T) according to the guidelines of the Clinical Laboratory Standards Institute. RESULTS: In precision evaluations, the coefficients of variation were less than each allowable total error for all analytes. Linearity was observed for all analytes over the entire tested analytical range (r2≥0.99). Performance comparisons revealed that the two systems are comparable, with correlation coefficients (r)>0.975 for all analytes. The reference range validation was also within the allowable criteria. CONCLUSIONS: In this study, the Cobas 8000 e801 analyzer demonstrated acceptable performance with respect to precision, linearity, reference range validation, and correlation. Therefore, the e801 analyzer is expected to be useful for routine immunoassays in clinical laboratories, although education and awareness about biotin interference is necessary for successful implementation in clinical practice.

Evaluation of the Elecsys Syphilis electrochemiluminescence immunoassay as a first-line screening test in the reverse algorithms for syphilis serodiagnosis.

OBJECTIVES: With the development of the automated treponemal test, new syphilis serodiagnosis algorithms, reverse algorithm, and European Centre for Disease Prevention and Control (ECDC) algorithm have been recommended recently. We investigated the efficacy of an electrochemiluminescence immunoassay (ECLIA) as an initial screening test in the reverse and ECDC algorithms. METHODS: Samples from 4,771 subjects were included in this study. We performed rapid plasma reagin (RPR), ECLIA, and Treponema pallidum particle agglutination (TPPA) according to these three algorithms. The fluorescent treponemal antibody absorbed (FTA-ABS) test was additionally applied for discordant cases between the RPR and ECLIA results. The FTA-ABS results and the consensus of three algorithms were considered a gold standard. RESULTS: A total of 208 subjects were diagnosed with syphilis. The traditional algorithm had a sensitivity of 25.96%, specificity of 100%, and accuracy of 96.77%. Both the reverse and ECDC algorithms showed the same diagnostic performance, sensitivity of 95.19%, specificity of 99.96%, and accuracy of 99.75%. The agreements between the traditional algorithm and the other algorithms were 96.9% with a kappa value of 0.415. CONCLUSIONS: The diagnostic accuracy of the reverse and ECDC algorithms using the ECLIA as a first-line screening test was superior to that of the traditional algorithm.

Urine Cotinine level with smoking history predicts a risk of coronary artery calcification.

We investigated whether urine cotinine level, alone or combined with smoking status and cumulative smoking amount, could predict coronary calcium (CAC) score increase over time. The study population included 10,980 subjects. We analysed an association between CAC score increase over time and single or combined smoking-related factors. Urine cotinine level of ≥100 ng/mL, current or ex-smokers, and cumulative smoking amount of ≥1 pack-years (PY) showed significantly higher odds ratios (ORs) for CAC score increase over time. A combination of current smokers with >10 PY and urine cotinine level of ≥100 ng/mL showed the highest OR. Irrespective of smoking status and cumulative smoking amount, all combinations with urine cotinine of ≥100 ng/mL showed higher ORs than other combinations with urine cotinine level of <100 ng/mL. Urine cotinine levels can be useful to predict coronary artery calcification and encourage smokers to quit smoking.

Comparison of AdvanSure HPV GenoBlot and Hybrid Capture 2 assays for detection of HPV infection.

BACKGROUND: We sought to compare the performance of the AdvanSure assay to the Hybrid Capture (HC) 2 for the detection of high-risk human papillomavirus (HR HPV). METHODS: A total of 855 cervical swab specimens were obtained. We submitted all specimens for HR HPV detection with HC2 and the AdvanSure assay. We subsequently analyzed discordant results and specimens that were positive on both assays using restriction fragment mass polymorphism (RFMP) genotyping analysis. RESULTS: HC2 yielded positive results in 12.0% of specimens, while the AdvanSure assay detected one of 13 HR HPV types in 11.5% of specimens. The overall agreement rate between the assays was 98.5% with a kappa coefficient of 0.928. Discordant results between these two assays were observed in 12 cases, seven were positive only on HC2 and five were positive only on AdvanSure. RFMP analysis of the 12 discordant cases revealed three false-positive results using HC2, and one false-positive and five false-negative results using AdvanSure. CONCLUSIONS: Considering the high agreement rate with HC2 and the ability to differentiate 35 HPV genotypes including HPV 16/18, the AdvanSure assay could be used as a laboratory testing method for HPV infection screening.

Comparison of GeneFinder human papillomavirus (HPV) Liquid Beads Microarray PCR Kit and Hybrid Capture 2 Assay for Detection of HPV Infection.

BACKGROUND: Along with advances in methodological technologies, various assays for detecting high-risk human papillomavirus (HR HPV) have been introduced. The GeneFinder HPV liquid beads microarray PCR kit is one of the recently developed. Our aim was to compare the performance of GeneFinder to Hybrid Capture 2 for detection of HR HPV. METHODS: A total of 900 cervical swab specimens were obtained. All specimens were submitted for HR HPV detection with Hybrid Capture 2 (HC2) and GeneFinder and then additionally analyzed the discordant or both positive results using restriction fragment mass polymorphism (RFMP) genotyping analysis. RESULTS: Hybrid Capture 2 detected 12.8% cases and GeneFinder detected 15.8% cases with 13 HR HPV types. Also, GeneFinder detected 27.4% cases for 32 detectable HPV types. The overall agreement rate was 93.2% with 0.724 kappa coefficient. Discordant results between these two assays were observed in 56 cases. HC2 showed sensitivity of 83.5% and specificity of 95.9%, while GeneFinder showed sensitivity of 85.4% and specificity of 91.9%. For HPV 16 or HPV 18 detection, GeneFinder showed 95.0% or 66.7% of sensitivity and 99.2% or 100%, respectively. Overall coinfection rate was 15.4% (38/247) in GeneFinder analysis. CONCLUSIONS: Considering the high agreement rate with HC2, high sensitivity and the ability to differentiate 32 HPV genotypes including HPV 16/18, GeneFinder could be used as a laboratory testing method for the screening of HPV infections. The use of GeneFinder may also contribute to future research associated with the significance of various HPV types and multiple coinfections.

Comparison of Abbott RealTime High-Risk HPV and Hybrid Capture 2 Assays for Detection of HPV Infection.

BACKGROUND: Various assays for detecting high-risk human papillomavirus (HR HPV) have been introduced recently, including the Abbott RealTime High-Risk HPV assay. We sought to compare the performance of Abbott PCR to Hybrid Capture 2 for the detection of HR HPV. METHODS: A total of 941 cervical swab specimens were obtained. We submitted all specimens for HR HPV detection with HC2 and Abbott PCR, and then additionally analyzed discordant and concordant positive results using restriction fragment mass polymorphism (RFMP) genotyping analysis. RESULTS: HC2 detected one of 13 HR HPV types in 12.3% (116/941) of cases, while Abbott PCR detected one of 14 detectable HR HPV types in 12.9% (121/941) of cases. The overall agreement rate was 97.3% with a kappa coefficient of 0.879. Discordant results between these two assays were observed in 25 cases. HC2 showed a sensitivity of 90.0% and specificity of 95.9%, while Abbott PCR showed a sensitivity of 98.0% and specificity of 96.8% when using RFMP results as the gold standard. For HPV 16/18 detection, Abbott PCR showed 95.8%/88.9% sensitivity and 99.2%/99.8% specificity, respectively. The overall coinfection rate between HPV 16, 18 and non-16/18 was 9.9% (12/121) in Abbott PCR analysis. CONCLUSIONS: Considering its high agreement rate with HC2, higher sensitivity/specificity compared to HC2, and ability to differentiate HPV 16/18 from other HPV types, Abbott PCR could be a reliable laboratory testing method for the screening of HPV infections.

Evaluation of Serum Cotinine Cut-Off to Distinguish Smokers From Nonsmokers in the Korean Population.

BACKGROUND: Cotinine has been widely used as an objective marker to identify current smokers. We conducted this study to address the absence of Korean studies investigating the efficacy of immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the detection of serum cotinine and to determine the optimal serum cotinine cut-off level for differentiating current smokers from nonsmokers. METHODS: Serum specimens were obtained from 120 subjects. They were randomly chosen to represent a broad distribution of urine cotinine levels based on a retrospective review of questionnaires and results of urine cotinine levels. We determined serum cotinine levels using the IMMULITE 2000 XPi Immunoassay System (Siemens Healthcare Diagnostics Inc., USA) and LC-MS/MS (API-4000, Applied Biosystems, USA). Correlation was analyzed between IMMULITE serum cotinine, urine cotinine, and LC-MS/MS serum cotinine levels. ROC curve was analyzed to identify the optimal IMMULITE serum cotinine cut-off level for differentiating current smokers from nonsmokers. RESULTS: IMMULITE serum cotinine levels correlated with both urine cotinine and LC-MS/MS serum cotinine levels, with correlation coefficients of 0.958 and 0.986, respectively. The optimal serum cotinine cut-off level for distinguishing current smokers from nonsmokers was 13.2 ng/mL (95.7% sensitivity, 94.1% specificity) using IMMULITE. CONCLUSIONS: This is the first study to investigate the use of LC-MS/MS for the measurement of serum cotinine and to determine the optimal serum cotinine cut-off level for the IMMULITE immunoassay. Our results could provide guidelines for differentiating current smokers from nonsmokers in the Korean population.

Diagnostic performance and establishment of reference limits of HE4 in Korean healthy women.

OBJECTIVE: We aimed to establish distribution and reference limits of HE4 and risk of ovarian malignancy algorithm (ROMA) in healthy Korean women and investigated the factors influencing HE4 levels. We also investigated the diagnostic performances of HE4 and ROMA score, compared with CA125. METHODS: We collected specimens from 1809 healthy Korean women, 140 specimens from patients with ovarian cancers (OCs) and 123 specimens from patients with benign ovarian tumor. Serum HE4 and CA125 concentrations were measured using an electrochemiluminescence immunoassay. The receiver operator characteristic (ROC) curve analysis was done for ROMA, HE4, CA125 and combining of HE4 and CA125. RESULTS: HE4 level was influenced by age, not by menopausal status. The 97.5th percentile upper reference limit of HE4 of subjects <50years and ≥50year-old was 63.87pmol/L and 88.28pmol/L, respectively. The 97.5th percentile upper reference limits of ROMA score were 13.66 in premenopausal and 19.30 in postmenopausal women. The serum HE4 level was even lower in the patients with benign tumor compared to those in healthy controls. HE4 had significantly higher concentrations in OCs than benign ovarian tumor (P<0.001). ROMA and HE4 combined with CA125 or not performed better diagnostically than CA125 alone for distinguishing OCs, with AUCs of 0.844 for ROMA, 0.827 for combining of HE4 and CA125, 0.825 for HE4, and 0.795 for CA125. CONCLUSIONS: The reference limit of HE4 was different from those reported by other studies, suggesting racial or regional difference. HE4 and ROMA were better than CA125 for differentiation normal and benign ovarian tumor from OCs. (Word count: 253).

Performance Evaluation of Three URiSCAN Devices for Routine Urinalysis.

BACKGROUND: This study compares the diagnostic performance (in routine urinalysis) of three URiSCAN devices and three Roche analyzers to manual microscopy and quantitative assays. METHODS: We analyzed eight dipstick tests using three URiSCAN devices. The results were compared to those of the tests performed using three Roche analyzers. The results of leukocyte and erythrocyte screens were compared to those obtained using manual microscopy. Protein, glucose, pH, and specific gravity (SG) assays performed on the URiSCAN devices were compared with the results of corresponding quantitative assays. RESULTS: The rates of agreement within one grade difference were found to be more than 94.3%. When compared with manual microscopy, the Optima provided better diagnostic performance for the detection of leukocytes compared with the Urisys 1100. Compared to the Urisys 2400, the Super plus provided better diagnostic performance with regard to both leukocytes and erythrocytes. There was good correlation between the three URiSCAN devices and each quantitative assay, except for SG detection. CONCLUSION: There were well correlated results between those of the three URiSCAN devices and those obtained using the corresponding Roche analyzers, quantitative assays, and manual microscopy. URiSCAN series devices are therefore suitable for routine urinalysis in clinical laboratories.

Comparison of clinical performances among Roche Cobas HPV, RFMP HPV PapilloTyper and Hybrid Capture 2 assays for detection of high-risk types of human papillomavirus.

The cervical cancer screening guidelines suggest that early detection of HPV16 and HPV18 is helpful for identifying women with cervical intraepithelial neoplasia (CIN) grade two or higher. We comparatively evaluated three HPV DNA assays, Roche Cobas HPV, RFMP HPV PapilloTyper, and Hybrid Capture 2 (HC2). A total of 861 cervical swab samples from women over 30 years of age were classified into two groups, that is, high grade squamous intraepithelial lesion (HSIL) and non-HSIL, according to cervical cytology results and analyzed by three assays. The results of direct sequencing or Linear array HPV genotyping test were considered true when the three assays presented discrepancies. The concordance rates between Roche Cobas HPV versus RFMP HPV PapilloTyper, RFMP HPV PapilloTyper versus HC2, and Roche Cobas versus HC2 were 94.5%, 94.3%, and 95.9%, respectively. For detection of HPV16 and HPV18, Roche Cobas HPV showed the concordance rates of 98.3% (κ = 0.73) and 99.4% (κ = 0.40) with the confirmation tests, respectively; and RFMP HPV PapilloTyper showed the concordance rates of 99.5% (κ = 0.92) and 100.0% (κ = 1.00), respectively. In conclusion, Roche Cobas HPV, RFMP HPV PapilloTyper, and HC2 showed high agreement rates. Roche Cobas HPV and RFMP HPV PapilloTyper are particularly useful, since both provide HPV specific genotypes, HPV16 and HPV18.

Investigation of Transition Types of HPV DNA Test Results over Time in Korean Women.

BACKGROUND: Understanding the history of human papilloma virus (HPV) infection is important for interpretation of a positive HPV DNA screening test, future work-up and treatment. We investigated the transition of HPV DNA test results in Korean women, and analyzed the association of cytology result with transition type. MATERIALS AND METHODS: We retrospectively reviewed annual HPV DNA test results for 5,274 subjects between January 2005 and December 2012. Each subject had a minimum of five annual tests over the eight-year period. Based on the pattern of results, the transition type for each subject was assigned to one of the following: negative, persistent, latent, transient, and unclassifiable. Associations of cytology results with the HPV DNA transition types, number of positive results, and the durations of positive results were also analyzed. RESULTS: The proportion of abnormal cytology findings decreased in the following order of transition patterns: persistent, latent, transient, and negative. Among transient patterns, a duration of three years or more significantly correlated with cytology results of non-high grade squamous intraepithelial lesion (HSIL; p<0.001). In the persistent group, duration of five years or more correlated with both non-HSIL and HSIL (p<0.001). Latent group showed no correlation with duration. Irrespective of patterns, having five or more positive results was significantly associated with HSIL (p<0.001). CONCLUSIONS: Our findings may contribute to better understanding of HPV infection, interpretation of HPV DNA screening results, and prediction of prognosis according to transition type.

Associations of single nucleotide polymorphisms related to insulin resistance with polycystic ovary syndrome.

BACKGROUND: We investigated and validated most of the hitherto-suggested polymorphisms for PCOS. MATERIALS AND METHODS: DNA samples from 359 pre-menopausal Korean women with PCOS (n=196) and without (n=166) were analyzed by primer extension and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using iPLEX Gold technology from Sequenom (Sequenom Inc, San Diego, CA, USA) to determine the genotypic frequency of 80 single nucleotide polymorphisms (SNPs) from the 49 genes. RESULTS: Four SNPs (rs10060745, rs1719889, rs680, rs705379) showed moderate association with a risk of PCOS. Three (rs1719889, rs680, and rs705379) out of four SNPs were related to insulin resistance. The rs705379 showed significant association with increased fasting glucose (p=0.045) and serum 2-h C-peptide level (p=0.026). The rs1719889 showed significant association with fasting glucose level (p=0.03). CONCLUSION: Our results provide evidence for the relationship between high prevalence of insulin resistance and PCOS.