Mortality and alcohol-related morbidity in patients with delirium tremens, alcohol withdrawal state or alcohol dependence in Norway: A register-based prospective cohort study.

BACKGROUND AND AIMS: Little is known about long-term consequences of delirium tremens (DT). This study aimed to compare all-cause and cause-specific mortality and alcohol-related morbidity between patients with: (i) DT, (ii) alcohol withdrawal state (AWS) and (iii) alcohol dependence (AD). DESIGN: A national longitudinal health registry study with linked data from the Norwegian Patient Registry and the Norwegian Cause of Death Registry. SETTING: Norway. PARTICIPANTS: All patients registered in the Norwegian Patient Registry between 2009 and 2015 with a diagnosis of AD (ICD-10 code F10.2), AWS (F10.3) or DT (F10.4) and aged 20-79 years were included (n = 36 287). MEASUREMENTS: Patients were categorized into three mutually exclusive groups; those with DT diagnosis were categorized as DT patients regardless of whether or not they had received another alcohol use disorder diagnosis during the observation period or not. Outcome measures were: annual mortality rate, standardized mortality ratios (SMR) for all-cause and cause-specific mortality and proportion of alcohol-related morbidities which were registered in the period from 2 years before to 1 year after the index diagnosis. FINDINGS: DT patients had higher annual mortality rate (8.0%) than AWS (5.0%) and AD (3.6%) patients, respectively. DT patients had higher mortality [SMR = 9.8, 95% confidence interval (CI) = 8.9-10.7] than AD patients (SMR = 7.0, 95% CI = 6.8-7.2) and AWS patients (SMR = 7.8, 95% CI = 7.2-8.4). SMR was particularly elevated for unnatural causes of death, and more so for DT patients (SMR = 26.9, 95% CI = 21.7-33.4) than for AD patients (SMR = 15.2, 95% CI = 14.2-16.3) or AWS patients (SMR = 20.1, 95% CI = 16.9-23.9). For all comorbidities, we observed a higher proportion among DT patients than among AWS or AD patients (P < 0.001). CONCLUSIONS: People treated for delirium tremens appear to have higher rates of mortality and comorbidity than people with other alcohol use disorders.

Transition From Substance-Induced Psychosis to Schizophrenia Spectrum Disorder or Bipolar Disorder.

Objective: The authors investigated transitions to schizophrenia spectrum or bipolar disorder following different types of substance-induced psychosis and the impact of gender, age, number of emergency admissions related to substance-induced psychosis, and type of substance-induced psychosis on such transitions. Methods: All patients in the Norwegian Patient Registry with a diagnosis of substance-induced psychosis from 2010 to 2015 were included (N=3,187). The Kaplan-Meier method was used to estimate cumulative transition rates from substance-induced psychosis to either schizophrenia spectrum disorder or bipolar disorder. Cox proportional hazard regression was used to estimate hazard ratios for transitions to schizophrenia spectrum or bipolar disorders associated with gender, age, number of emergency admissions, and type of substance-induced psychosis. Results: The 6-year cumulative transition rate from substance-induced psychosis to schizophrenia spectrum disorder was 27.6% (95% CI=25.6­29.7). For men, the risk of transition was higher among younger individuals and those with either cannabis-induced psychosis or psychosis induced by multiple substances; for both genders, the risk of transition was higher among those with repeated emergency admissions related to substance-induced psychosis. The cumulative transition rate from substance-induced psychosis to bipolar disorder was 4.5% (95% CI=3.6­5.5), and the risk of this transition was higher for women than for men. Conclusions: Transition rates from substance-induced psychosis to schizophrenia spectrum disorder were six times higher than transition rates to bipolar disorder. Gender, age, number of emergency admissions, and type of substance-induced psychosis affected the risk of transition.

Annual incidence of substance-induced psychoses in Scandinavia from 2000 to 2016.

BACKGROUND: Substance-induced psychosis (SIP) is a serious condition and may predispose for schizophrenia. We know too little about SIP incidence over time and across countries, including substance-specific SIPs. We estimated annual incidence rate of SIP in Denmark, Norway, and Sweden according to substance, age, gender, and socioeconomic background. METHODS: Data were drawn from registries covering the whole adult population in the countries. Annual incidence rate per 100 000 persons of SIPs was estimated for Denmark and Sweden from 2000 to 2016 and for Norway from 2010 to 2015. RESULTS: The annual incidence rate of any SIP fluctuated between 9.3 and 14.1. The most commonly occurring SIPs were those induced by alcohol, cannabis, amphetamines, and multiple substances. There was a steady decrease in the incidence rate of alcohol-induced psychosis from the first to the last year of the observation period in Denmark (from 4.9 to 1.5) and Sweden (from 4.5 to 2.2). The incidence rate of cannabis-induced psychosis increased in all countries, from 2.6 to 5.6 in Denmark, from 0.8 to 2.7 in Sweden, and from 1.8 to 3.0 in Norway. Median age of any SIP decreased in Denmark (from 36 to 29 years) and Sweden (from 41 to 31 years). Incidence rates were higher in men and in individuals on disability pension, and increased more among those with high parental education. CONCLUSIONS: We found similar and stable incidence rates of any SIP in all Scandinavian countries through the observation period. The incidence of alcohol-induced psychosis decreased. The incidence of cannabis-induced psychosis increased.

Disparities in Screening and Treatment of Cardiovascular Diseases in Patients With Mental Disorders Across the World: Systematic Review and Meta-Analysis of 47 Observational Studies.

OBJECTIVE: This study used meta-analysis to assess disparities in cardiovascular disease (CVD) screening and treatment in people with mental disorders, a group that has elevated CVD incidence and mortality. METHODS: The authors searched PubMed and PsycInfo through July 31, 2020, and conducted a random-effect meta-analysis of observational studies comparing CVD screening and treatment in people with and without mental disorders. The primary outcome was odds ratios for CVD screening and treatment. Sensitivity analyses on screening and treatment separately and on specific procedures, subgroup analyses by country, and by controlling for confounding by indication, as well as meta-regressions, were also run, and publication bias and quality were assessed. RESULTS: Forty-seven studies (N=24,400,452 patients, of whom 1,283,602 had mental disorders) from North America (k=26), Europe (k=16), Asia (k=4), and Australia (k=1) were meta-analyzed. Lower rates of screening or treatment in patients with mental disorders emerged for any CVD (k=47, odds ratio=0.773, 95% CI=0.742, 0.804), coronary artery disease (k=34, odds ratio=0.734, 95% CI=0.690, 0.781), cerebrovascular disease (k=8, odds ratio=0.810, 95% CI=0.779, 0.842), and other mixed CVDs (k=11, odds ratio=0.839, 95% CI=0.761, 0.924). Significant disparities emerged for any screening, any intervention, catheterization or revascularization in coronary artery disease, intravenous thrombolysis for stroke, and treatment with any and with specific medications for CVD across all mental disorders (except for CVD medications in mood disorders). Disparities were largest for schizophrenia, and they differed across countries. Median study quality was high (Newcastle-Ottawa Scale score, 8); higher-quality studies found larger disparities, and publication bias did not affect results. CONCLUSIONS: People with mental disorders, and those with schizophrenia in particular, receive less screening and lower-quality treatment for CVD. It is of paramount importance to address underprescribing of CVD medications and underutilization of diagnostic and therapeutic procedures across all mental disorders.

Cohort profile: Copenhagen Hospital Biobank - Cardiovascular Disease Cohort (CHB-CVDC): Construction of a large-scale genetic cohort to facilitate a better understanding of heart diseases.

PURPOSE: The aim of Copenhagen Hospital Biobank-Cardiovascular Disease Cohort (CHB-CVDC) is to establish a cohort that can accelerate our understanding of CVD initiation and progression by jointly studying genetics, diagnoses, treatments and risk factors. PARTICIPANTS: The CHB-CVDC is a large genomic cohort of patients with CVD. CHB-CVDC currently includes 96 308 patients. The cohort is part of CHB initiated in 2009 in the Capital Region of Denmark. CHB is continuously growing with ~40 000 samples/year. Patients in CHB were included in CHB-CVDC if they were above 18 years of age and assigned at least one cardiovascular diagnosis. Additionally, up-to 110 000 blood donors can be analysed jointly with CHB-CVDC. Linkage with the Danish National Health Registries, Electronic Patient Records, and Clinical Quality Databases allow up-to 41 years of medical history. All individuals are genotyped using the Infinium Global Screening Array from Illumina and imputed using a reference panel consisting of whole-genome sequence data from 8429 Danes along with 7146 samples from North-Western Europe. Currently, 39 539 of the patients are deceased. FINDINGS TO DATE: Here, we demonstrate the utility of the cohort by showing concordant effects between known variants and selected CVDs, that is, >93% concordance for coronary artery disease, atrial fibrillation, heart failure and cholesterol measurements and 85% concordance for hypertension. Furthermore, we evaluated multiple study designs and the validity of using Danish blood donors as part of CHB-CVDC. Lastly, CHB-CVDC has already made major contributions to studies of sick sinus syndrome and the role of phytosterols in development of atherosclerosis. FUTURE PLANS: In addition to genetics, electronic patient records, national socioeconomic and health registries extensively characterise each patient in CHB-CVDC and provides a promising framework for improved understanding of risk and protective variants. We aim to include other measurable biomarkers for example, proteins in CHB-CVDC making it a platform for multiomics cardiovascular studies.

Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus).

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Total and cause-specific standardized mortality ratios in patients with schizophrenia and/or substance use disorder.

Individuals with schizophrenia or substance use disorder have a substantially increased mortality compared to the general population. Despite a high and probably increasing prevalence of comorbid substance use disorder in people with schizophrenia, the mortality in the comorbid group has been less studied and with contrasting results. We performed a nationwide open cohort study from 2009 to 2015, including all Norwegians aged 20-79 with schizophrenia and/or substance use disorder registered in any specialized health care setting in Norway, a total of 125,744 individuals. There were 12,318 deaths in the cohort, and total, sex-, age- and cause-specific standardized mortality ratios (SMRs) were calculated, comparing the number of deaths in patients with schizophrenia, schizophrenia only, substance use disorder only or a co-occurring diagnosis of schizophrenia and substance use disorder to the number expected if the patients had the age-, sex- and calendar-year specific death rates of the general population. The SMRs were 4.9 (95% CI 4.7-5.1) for all schizophrenia patients, 4.4 (95% CI 4.2-4.6) in patients with schizophrenia without substance use disorder, 6.6 (95% CI 6.5-6.8) in patients with substance use disorder only, and 7.4 (95% CI 7.0-8.2) in patients with both schizophrenia and substance use disorder. The SMRs were elevated in both genders, in all age groups and for all considered causes of death, and most so in the youngest. Approximately 27% of the excess mortality in all patients with schizophrenia was due to the raised mortality in the subgroup with comorbid SUD. The increased mortality in patients with schizophrenia and/or substance use disorder corresponded to more than 10,000 premature deaths, which constituted 84% of all deaths in the cohort. The persistent mortality gap highlights the importance of securing systematic screening and proper access to somatic health care, and a more effective prevention of premature death from external causes in this group.

Validation of automatic joint space width measurements in hand radiographs in rheumatoid arthritis.

Computerized methods promise quick, objective, and sensitive tools to quantify progression of radiological damage in rheumatoid arthritis (RA). Measurement of joint space width (JSW) in finger and wrist joints with these systems performed comparable to the Sharp-van der Heijde score (SHS). A next step toward clinical use, validation of precision and accuracy in hand joints with minimal damage, is described with a close scrutiny of sources of error. A recently developed system to measure metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints was validated in consecutive hand images of RA patients. To assess the impact of image acquisition, measurements on radiographs from a multicenter trial and from a recent prospective cohort in a single hospital were compared. Precision of the system was tested by comparing the joint space in mm in pairs of subsequent images with a short interval without progression of SHS. In case of incorrect measurements, the source of error was analyzed with a review by human experts. Accuracy was assessed by comparison with reported measurements with other systems. In the two series of radiographs, the system could automatically locate and measure 1003/1088 (92.2%) and 1143/1200 (95.3%) individual joints, respectively. In joints with a normal SHS, the average (SD) size of MCP joints was [Formula: see text] and [Formula: see text] in the two series of radiographs, and of PIP joints [Formula: see text] and [Formula: see text]. The difference in JSW between two serial radiographs with an interval of 6 to 12 months and unchanged SHS was [Formula: see text], indicating very good precision. Errors occurred more often in radiographs from the multicenter cohort than in a more recent series from a single hospital. Detailed analysis of the 55/1125 (4.9%) measurements that had a discrepant paired measurement revealed that variation in the process of image acquisition (exposure in 15% and repositioning in 57%) was a more frequent source of error than incorrect delineation by the software (25%). Various steps in the validation of an automated measurement system for JSW of MCP and PIP joints are described. The use of serial radiographs from different sources, with a short interval and limited damage, is helpful to detect sources of error. Image acquisition, in particular repositioning, is a dominant source of error.

Calcinosis cutis presenting in the context of long-term therapy for chronic myeloid leukemia: a case report and review of the literature.

Calcinosis cutis is a poorly understood process in which calcium salts deposit in the skin and subcutaneous tissues. Due to its multifactorial pathogenesis, several subtypes and potential etiologies have been described. Presented here is a case of bilateral pretibial calcinosis cutis in a patient on long-term tyrosine kinase inhibitor therapy for chronic myeloid leukemia. The patient initially presented with a right tibial ulceration treated with multiple surgical debridements, antibiotics, and negative pressure wound therapy. The wound was ultimately closed with a split-thickness skin graft. Relevant literature is examined and several possible mechanisms are discussed.

Contribution of assessing forefoot joints in early rheumatoid arthritis patients: insights from item response theory.

OBJECTIVE: To evaluate the contribution of assessing forefoot joints to the measurement range and measurement precision of joint counts in early rheumatoid arthritis (RA) using item response theory. METHODS: Baseline measures of tender and swollen joint counts were analyzed in 459 early RA patients from the Dutch Rheumatoid Arthritis Monitoring remission induction cohort. The contribution of forefoot joints was studied by evaluating their effect on the measurement range and measurement precision of measures based on 28-joint counts. In addition, the alignment between the patient and joint distributions was investigated to determine whether the forefoot joints were informative for measuring joint tenderness or swelling of an early RA patient. RESULTS: In total, 233 patients (50.76%) experienced tenderness and 200 patients (43.57%) experienced swelling in ≥1 forefoot joint. Forefoot joints were more informative for measuring joint tenderness than joint swelling, but did not significantly improve the measurement range and measurement precision of the 28-joint counts. Furthermore, including forefoot joints did not remove the existing discrepancy between the joint and patient distributions in both joint counts. CONCLUSION: Forefoot joints were frequently affected on an individual level, but did not significantly improve the measurement range or precision of 28-joint counts in patients with early RA. From a measurement perspective, reduced joint counts are appropriate for use on a population level. The contribution of assessing forefoot joints on an individual level requires further investigation. Additionally, the results should be cross-validated in patients with longer disease durations to determine whether the pattern of joint involvement is similar in later stages of RA.

Changes in type-specific human papillomavirus load predict progression to cervical cancer.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with the development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). However, HPV infection is common and usually transient. Viral load measured at a single time-point is a poor predictor of the natural history of HPV infection. The profile of viral load evolution over time could distinguish HPV infections with carcinogenic potential from infections that regress. A case-cohort natural history study was set-up using a Belgian laboratory database processing more than 100,000 liquid cytology specimens annually. All cytology leftovers were submitted to real-time PCR testing identifying E6/E7 genes of 17 HPV types, with viral load expressed as HPV copies/cell. Samples from untreated women who developed CIN3+ (n = 138) and women with transient HPV infection (n = 601) who contributed at least three viral load measurements were studied. Only single-type HPV infections were selected. The changes in viral load over time were assessed by the linear regression slope for the productive and/or clearing phase of infection in women developing CIN3+ and women with transient infection respectively. Transient HPV infections generated similar increasing (0.21 copies/cell/day) and decreasing (-0.28 copies/cell/day) viral load slopes. In HPV infections leading to CIN3+, the viral load increased almost linearly with a slope of 0.0028 copies/cell/day. Difference in slopes between transient infections and infections leading to CIN3+ was highly significant (P < .0001). Serial type-specific viral load measurements predict the natural history of HPV infections and could be used to triage women in HPV-based cervical cancer screening.

Productivity loss due to absenteeism and presenteeism by different instruments in patients with RA and subjects without RA.

OBJECTIVES: To explore the impact of at-work productivity loss on the total productivity cost by different instruments in patients recently diagnosed with RA and controls without RA. METHODS: Cross-sectional data were collected from outpatients with RA between December 2007 and February 2008. The control group was formed by subjects without RA matched on age and gender. Absenteeism and presenteeism were estimated by the Quantity and Quality (QQ) Questionnaire, Work Productivity and Activity Impairment Questionnaire General Health V2.0 (WPAI-GH) and Health and Labor Questionnaire (HLQ) questionnaires. Differences between groups were tested by Mann-Whitney U-test. Costs were valued by the human capital approach. RESULTS: Data were available from 62 patients with a paid job and 61 controls. QQ- and WPAI-GH scores of presenteeism were moderately correlated (r = 0.61) while the HLQ presenteeism score correlated poorly with the other instruments (r = 0.34). The contribution of presenteeism on total productivity costs was estimated at ∼70% in the RA group. The mean costs per person per week due to presenteeism varied between €79 and €318 per week in the RA group, dependent on the instrument used. The costs due to presenteeism were about two to four times higher in the RA group compared with the control group. CONCLUSION: This study indicates that the impact of presenteeism on the total productivity costs in patients with RA is high. However, work productivity in individuals without RA was not optimal either, which implies a risk of overestimation of cost when a normal score is not taken into account. Finally, different presenteeism instruments lead to different results.

Validating the 28-tender joint count using item response theory.

OBJECTIVE: To examine the construct validity of the 28-tender joint count (TJC-28) using item response theory (IRT)-based methods. METHODS: A total of 457 patients with early stage rheumatoid arthritis (RA) were included. Internal construct validity of the TJC-28 was evaluated by determining whether the TJC-28 fit a 2-measure logistic IRT model. As well, we tested whether the discrimination and difficulty parameters of the joints properly reflected the known left-right symmetry of joint involvement. External validity was evaluated by correlations with other established measures of disease activity, including pain, disability, general health, erythrocyte sedimentation rate (ESR), and the 28-swollen joint count. RESULTS: The TJC-28 showed a good fit with the 2-parameter logistic model, with no relevant differential item functioning across sex, age, and time and with excellent reliability. The 28 joints covered a reasonable range of disease activity, even though they were mainly targeted at patients with moderate or high disease activity levels. The joint parameters reflected the left-right symmetry of joint involvement for all pairs of joints except one. All disease activity measures, except ESR, were significantly correlated with the TJC-28. Most correlations were of the expected magnitude. CONCLUSION: The TJC-28 showed good internal and acceptable external construct validity for patients with early-stage RA. The IRT analyses did point to some potential limitations of the instrument, a major problem being its limited measurement range. Future research should examine whether instrument modifications might lead to a more robust assessment of disease activity in patients with RA.

Prior knowledge of HPV status improves detection of CIN2+ by cytology screening.

OBJECTIVE: The objective of the study was to investigate whether knowledge of human papillomavirus (HPV) deoxyribonucleic acid test results increases sensitivity of guided cytology screening for the detection of cervical intraepithelial neoplasia (CIN)-2 or higher-grade cervical lesions. STUDY DESIGN: This was a prospective colposcopy-controlled study of 2905 BD SurePath samples to identify cases with CIN2+ within a 24 month follow-up period. Sensitivity and specificity to detect CIN2+ was evaluated, comparing guided cytology screening with and without prior knowledge of HPV status. RESULTS: Prior knowledge of HPV status resulted in significantly higher detection rate of CIN2+ compared with screening blinded to HPV status (P = .005) with limited loss of specificity (P = .026). Gain in sensitivity is higher in older women (43.8%, P = .008) vs in younger women (10.2%, P = .317), whereas loss of specificity is more pronounced in younger women (P < .001) vs older women (P = .729). CONCLUSION: Guided cytological screening performed with prior knowledge of HPV status results in an improved detection of CIN2 or higher-grade lesions.

Does outdoor work during the winter season protect against depression and mood difficulties?

OBJECTIVE: At temperate latitudes, 1-5% of the population suffer from winter depression; during winter, mood difficulties tend to increase but may be alleviated by bright light therapy. Unlike indoor workers, outdoor workers are exposed to therapeutic levels of sunlight during winter. We hypothesized that outdoor work may protect against mood difficulties and depression. METHOD: We studied this hypothesis among 2910 civil servants from Århus, Denmark, who participated in a survey in January-February 2009. Mental symptoms (N=422) defined a common case category that we broke down into two parts: depression (N=66) and mood difficulties but no depression (N=356). A total of 222 controls were also sampled from the study population. All 644 participants reported the extent of outdoor work. RESULTS: The confounder-adjusted odds ratio (OR) of mood difficulties showed a decreasing trend by increasing hours of outdoor work of borderline statistical significance. The OR was 0.63 [95% confidence interval (95% CI) 0.34-1.18)] for those working outdoors for >2 hours a day. No such effect was suggested for depression. CONCLUSION: Our study is limited by its cross-sectional design and low statistical power but nevertheless suggests that outdoor work during winter may protect against mood difficulties. If this finding holds true it may have significant impact on workers' health as well as public health in general. Therefore, further studies are recommended.

BD-ProExC as adjunct molecular marker for improved detection of CIN2+ after HPV primary screening.

BACKGROUND AND METHODS: We investigated the efficacy of 8 cervical cancer screening strategies relative to cytology with emphasis on immunocytochemical detection of high-risk human papillomavirus (hrHPV)-induced cell transformation (BD-ProExC) as a tool of triage following primary cytology or hrHPV testing. 3,126 women were tested with BD-SurePath liquid-based cytology, hrHPV PCR genotyping and BD-ProExC immunostaining, and colposcopy verification to calculate sensitivity and positive predictive value (PPV) in detecting cervical intraepithelial neoplasia (CIN2(+)). RESULTS: Compared to cytology screening, double testing with cytology and hrHPV resulted in the same sensitivity with a significant increase in the PPV (relative PPV: 1.83). However, twice as many tests were needed. Cytology with atypical squamous cells of undetermined significance (ASC-US) triage and hrHPV testing showed comparative results to double testing requiring only a small increase in number of tests. Screening for hrHPV subtypes 16/18, and ASC-US triage with hrHPV16/18 resulted in significant reductions in sensitivity (ratio: 0.74 and 0.96, respectively). Primary hrHPV/BD-ProExC screening was significantly more sensitive (ratio: 1.63/1.33), but had a significantly lower PPV (ratio: 0.64/0.88). ASC-US triage by BD-ProExC increased the PPV (ratio: 1.90) but decreased the sensitivity (ratio: 0.96). Primary hrHPV screening followed by BD-ProExC triage, led to significant increases in sensitivity (ratio: 1.30) and PPV (ratio: 2.89), and resulted in 55% fewer referrals for colposcopy. CONCLUSIONS: From the investigated screening strategies, primary hrHPV DNA-based screening followed by BD-ProExC triage was determined to be the best screening strategy. IMPACT: Immunocytological triage could be used to perfect hrHPV primary screening.

High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR.

BACKGROUND: The establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies. METHODS: The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and ß-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67). RESULTS: An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of <6.4% was observed. CONCLUSIONS: The type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.

Type-specific HPV geno-typing improves detection of recurrent high-grade cervical neoplasia after conisation.

The aim of this case-control study was to examine if type-specific human papillomavirus (HPV) DNA geno-typing before and after treatment of high-grade cervical intra-epithelial neoplasia (CIN) improves prediction of recurring or persisting CIN 2 or 3 compared with follow-up cytology or high-risk (hr)HPV testing. Women with biopsy-proven recurrence of CIN 2 or 3 (cases) in a follow-up period of at least 24 months after treatment of high-grade CIN were compared with women without recurrence (controls). These cohorts were identified by a database search of the Riatol Laboratoria (Antwerp, Belgium). In a cohort of 823 women treated with conisation for high-grade CIN between January 2001 and December 2007, 21 patients with a histologically proven recurrence of CIN2+ were identified. A group of women (n=42) from the same cohort without recurrence was randomly chosen. We found that hrHPV testing at 6 months post-treatment is significantly more sensitive compared with follow-up cytology (ratio: 1.31, 95% confidence interval (CI): 1.10-1.54), but less specific (ratio: 0.85, 95% CI: 0.81-0.90) to predict failure of treatment. When compared with hrHPV testing, HPV geno-typing is more efficient (equal sensitivity, but higher specificity, ratio: 1.43, 95% CI: 1.280-1.62). When compared with follow-up cytology, HPV geno-typing is more sensitive (ratio: 1.31, 95% CI: 1.10-1.54) and more specific (ratio: 1.22, 95% CI: 1.14-1.36). All women who developed a recurrence tested positive for hrHPV. The negative predictive value in the absence of hrHPV DNA was 100%. Six months after treatment HPV geno-typing is the most sensitive and specific method to predict recurrent or persistent CIN 2-3 in the next 24 months.

Nucleic acid sequence-based amplification assay for human papillomavirus mRNA detection and typing: evidence for DNA amplification.

Human papillomavirus (HPV) E6/E7 mRNA has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA. This study evaluated the RNA specificity of nucleic acid sequence-based amplification (NASBA)-based HPV detection using HPV DNA plasmids (HPV type 16 [HPV16], HPV18, HPV31, HPV33, and HPV45) and nucleic acid extracts of several cell lines, which were systematically subjected to enzymatic treatments with DNase and RNase. HPV plasmid dilutions (10(6) to 10(0) copies/microl) and nucleic acid extracts (total DNA, RNA-free DNA, total RNA, and DNA-free RNA) of unfixed and fixed (PreServCyt and SurePath) HaCaT, HeLa, and CaSki cells were tested with the NucliSENS EasyQ HPV test. The RNA-free DNA extracts of HeLa and CaSki cells could be amplified by HPV18 and -16 NASBA, respectively. Fixation of the cells did not influence NASBA. All HPV plasmids could be detected with NASBA. Based on the plasmid dilution series, a lower detection limit of 5 x 10(3) HPV DNA copies could be determined. Our study identified viral double-stranded DNA as a possible target for NASBA-based HPV detection. The differences in diagnostic accuracy between the NASBA-based tests and conventional HPV DNA detection assays seem to be attributable not to the more specific amplification of viral mRNA but to the limited type range and the lower analytical sensitivity for HPV DNA.