Comparison of the analytical and clinical performance of five tests for the detection of human papillomavirus genital infection.

HPV-based screening provides greater protection against cervical cancer (CC) than cytology-based strategies. Currently, several molecular diagnostic assays for the detection of human papillomavirus (HPV) are available. In this study, we analyzed 5 different HPV testing and genotyping techniques (Hybrid Capture 2 [HC2; Qiagen, Hilden, Germany], AnyplexTMII HPV28 [Anyplex; Seegene, Seoul, Korea], Linear Array [Roche, Branchburg, NJ, USA], GP5+/6+ PCR-EIA-RH [Labo Bio-medical Products, Rijswijk, The Netherlands] and CLART2 [Genomica, Madrid, Spain]) in 295 women referred to the hospital Colposcopy Clinic from 2007 to 2008 due to positive HPV test results or an abnormal Pap test. DNA extraction for HPV genotyping was performed in cervical sample specimens after Pap test and HPV detection by HC2. The inclusion criteria were: (1) adequate cervical sampling with sufficient material for the Pap test and HPV detection and genotyping, and (2) colposcopically-directed biopsy and/or endocervical curettage. HC2 showed the highest sensitivity for high-grade squamous intraepithelial lesion and CC (HSIL+) detection (96.1%), but all the HPV genotyping tests showed a higher specificity. (Anyplex 86.8%; Linear Array 86.0%; GP5+/6+ 78.8%; CLART2 76.5%). The agreement between HC2 results and the other techniques was similar: 82.4%, kappa=0.650 for Anyplex; 83.4%, kappa=0.670 for Linear Array, 79.93%, kappa=0.609 for GP5+/6+ and 82.4%, kappa=0.654 for CLART2. HPV 16 and/or 18 infection was a risk factor for underlying HSIL+ in the univariate analysis. Anyplex showed the highest risk of underlying HSIL+ after positive HPV 16 and/or 18 tests (OR 31.1; 95% CI 12.1-80.0).

Clinical and analytical performance of the PapilloCheck HPV-Screening assay using the VALGENT framework.

BACKGROUND: The benefit of HPV testing for cervical cancer screening and disease management has been shown in many recent studies and is part of several new evidence-based guidelines. Assessment of emerging HPV tests in this context is essential, using well-annotated samples, such as those generated via the Validation of Genotyping Tests-HPV (VALGENT) framework. OBJECTIVE: Our aim was to assess the PapilloCheck HPV assay in terms of absolute and relative accuracy for primary cervical cancer screening, using a standard comparator test (GP5+/6+EIA)already validated in randomised trials. STUDY DESIGN: Type-specific HPV prevalence was stratified by age and cytology grade and compared with the luminex typing assay incorporating a GP5+6+ PCR (GP5+/6+ LMNX Assay). Clinical outcomes were compared with GP5+/6+EIA. RESULTS: Prevalence of hrHPV types (high-risk HPV) increased with severity of cytology. The concordance between PapilloCheck and the GP5+/6+ LMNX Assay was excellent when assessed at the qualitative hrHPV presence/absence level also at the type-specific level in the whole population and in women over 30 years of age. Absolute clinical sensitivity and specificity of the PapilloCheck was high and ranged between 95.5% and 98.2% for sensitivity and between 82.7% and 91.6% for specificity, depending on the outcome and population. CONCLUSION: The sensitivity and specificity of this assay for the outcomes of CIN2+ were similar to those of the standard comparator assay, GP5+/6+ EIA.

Clinical and Analytical Performance of the Onclarity HPV Assay Using the VALGENT Framework.

As the demand for human papillomavirus (HPV)-related cervical screening increases, emerging HPV tests must be evaluated robustly using well-annotated samples, such as those generated in the Validation of HPV Genotyping Tests (VALGENT) framework. Through VALGENT, we assessed the performance of the BD Onclarity HPV assay, which detects 14 high-risk (HR) types and resolves six individual types and three groups of types. Consecutive samples from a screening population (n = 1,000), enriched with cytologically abnormal samples (n = 300), that had been tested previously with the GP5+/6+ PCR enzyme immunoassay (EIA) and the GP5+/6+ PCR LMNX assay (Diassay) were tested with the Onclarity assay. Type-specific HPV prevalences were analyzed according to age and cytological result. The accuracy of the Onclarity assay for the detection of cervical intraepithelial neoplasia grade 2+ (CIN2+) and CIN3+ was assessed relative to the GP5+/6+ EIA results by using noninferiority criteria. Overall agreement and type-specific agreement between the Onclarity assay and the GP5+/6+ LMNX assay were assessed. The prevalence of HPV types 16, 18, 31, and 45 increased with the severity of cytological results (P for trend, <0.05). For the detection of CIN2+, the Onclarity assay had a relative sensitivity of 1.02 (95% confidence interval [CI], 0.99 to 1.05; P < 0.001 for noninferiority) and a relative specificity of 0.99 (95% CI, 0.97 to 1.00; P = 0.186 for noninferiority). The kappa for agreement between the Onclarity assay and the GP5+/6+ LMNX assay for HR-HPV was 0.92 (95% CI, 0.89 to 0.94), and values for the six individual types ranged from 0.78 (95% CI, 0.68 to 0.87) for HPV-52 to 0.96 (95% CI, 0.93 to 0.99) for HPV-16. These data suggest that the Onclarity assay offers applications for clinical workstreams while providing genotyping information that may be useful for risk stratification beyond types 16 and 18.

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC.

BACKGROUND: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the ß-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the ß-catenin paradox. METHODS: We analysed the expression patterns of SMAD4, p53 and ß-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity. RESULTS: Eighty-four percent of CRCs with high nuclear ß-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner. CONCLUSIONS: The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.

Clinical evaluation of a GP5+/6+-based luminex assay having full high-risk human papillomavirus genotyping capability and an internal control.

The LMNX genotyping kit HPV GP (LMNX) is based on the clinically validated GP5+/6+ PCR, with a genotyping readout as an alternative for the more established enzyme immunoassay (EIA) detection of 14 targeted high-risk human papillomavirus (HPV) types. LMNX is additionally provided with an internal control probe. Here, we present an analysis of the clinical performance of the LMNX using a sample panel and infrastructure provided by the international VALGENT (Validation of Genotyping Tests) project. This panel consisted of cervical specimens from approximately 1,000 women attending routine screening, "enriched" with 300 women with abnormal cytology. Cases were defined as women classified with cervical intraepithelial neoplasia (CIN) grade 2+ (CIN2+) (n = 102) or CIN3+ (n = 55) within the previous 18 months. Controls were women who had normal cytology results over two subsequent screening rounds at a 3-year interval (n = 746). The GP5+/6+-PCR EIA (EIA) was used as a comparator assay and showed sensitivities of 94.1% and 98.2% for CIN2+ and CIN3+, respectively, with a clinical specificity of 92.4% among women aged ≥ 30 years. The LMNX demonstrated clinical sensitivities of 96.1% for CIN2+ and of 98.2% for CIN3+ and a clinical specificity of 92.6% for women aged ≥ 30 years. The LMNX and EIA were in high agreement (Cohen's kappa = 0.969) for the detection of 14 hrHPVs in aggregate, and no significant difference was observed (McNemar's P = 0.629). The LMNX internal control detected 0.6% inadequate specimens. Based on our study results, we consider the LMNX, similarly to the EIA, useful for HPV-based cervical cancer screening.

Large contribution of human papillomavirus in vaginal neoplastic lesions: a worldwide study in 597 samples.

AIM: This work describes the human papillomavirus (HPV) prevalence and the HPV type distribution in a large series of vaginal intraepithelial neoplasia (VAIN) grades 2/3 and vaginal cancer worldwide. METHODS: We analysed 189 VAIN 2/3 and 408 invasive vaginal cancer cases collected from 31 countries from 1986 to 2011. After histopathological evaluation of sectioned formalin-fixed paraffin-embedded samples, HPV DNA detection and typing was performed using the SPF-10/DNA enzyme immunoassay (DEIA)/LiPA25 system (version 1). A subset of 146 vaginal cancers was tested for p16(INK4a) expression, a cellular surrogate marker for HPV transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance. RESULTS: HPV DNA was detected in 74% (95% confidence interval (CI): 70-78%) of invasive cancers and in 96% (95% CI: 92-98%) of VAIN 2/3. Among cancers, the highest detection rates were observed in warty-basaloid subtype of squamous cell carcinomas, and in younger ages. Concerning the type-specific distribution, HPV16 was the most frequently type detected in both precancerous and cancerous lesions (59%). p16(INK4a) overexpression was found in 87% of HPV DNA positive vaginal cancer cases. CONCLUSIONS: HPV was identified in a large proportion of invasive vaginal cancers and in almost all VAIN 2/3. HPV16 was the most common type detected. A large impact in the reduction of the burden of vaginal neoplastic lesions is expected among vaccinated cohorts.

Clinical evaluation of high-risk HPV detection on self-samples using the indicating FTA-elute solid-carrier cartridge.

BACKGROUND: High-risk human papillomavirus (hrHPV) testing in cervical screening is usually performed on physician-taken cervical smears in liquid-based medium. However, solid-state specimen carriers allow easy, non-hazardous storage and transportation and might be suitable for self-collection by non-responders in screening and in low-resource settings. OBJECTIVES: We evaluated the adequacy of self-collected cervicovaginal (c/v) samples using a Viba-brush stored on an Indicating FTA-elute cartridge (FTA-based self-sampling) for hrHPV testing in women referred to a gynecology clinic due to an abnormal smear. STUDY DESIGN: 182 women accepted to self-collect a c/v sample. After self-sampling, a physician obtained a conventional liquid-based cervical smear. Finally, women were examined by colposcopy and a biopsy was taken when clinically indicated. Self-samples required only simple DNA elution, and DNA was extracted from physician-obtained samples. Both samples were tested for 14 hrHPVs by GP5+/6+-EIA-LQ Test and SPF(10)-DEIA-LiPA(25). RESULTS: Both assays detected significantly more hrHPV in physician-collected specimens than in self-collected samples (75.3% and 67.6% by SPF(10); 63.3% and 53.3% by GP5+/6+, respectively). The combination of physician-collected specimen and GP5+/6+ testing demonstrated the optimal balance in sensitivity (98.0%) and specificity (48.1%) for CIN2+ detection in this referral population. A test system of FTA-based self-collection and SPF(10) hrHPV detection approached this sensitivity (95.9%) and specificity (42.9%). CONCLUSIONS: These results show that the clinical performance of hrHPV detection is determined by both the sample collection system and the test method. FTA-based self-collection with SPF(10) testing might be valuable when a liquid-based medium cannot be used, but requires further investigation in screening populations.

Universal human papillomavirus genotyping by the digene HPV Genotyping RH and LQ Tests.

BACKGROUND: High-risk (hr)HPV testing plays an important role in primary cervical cancer screening. Subsequent hrHPV genotyping might contribute to better risk stratification. The majority of hrHPV tests do not include identification of individual hrHPV genotypes. OBJECTIVES: The digene HPV Genotyping RH Test (strip-based) and LQ Test (xMAP-based) allow genotyping of GP5+/6+ amplimers, but their probes target a region in the L1 ORF, which is also amplified by other broad-spectrum hrHPV assays, e.g., the Roche Amplicor HPV Test (Amplicor) and the Roche Linear Array. The goal was to test whether the RH Test and LQ Test can be used as an universal hrHPV genotyping test. STUDY DESIGN: Self-collected cervico-vaginal specimens (n=416) from an epidemiologic study were analyzed with Amplicor. The amplimers obtained were also tested with the RH Test and LQ Test for identification of 18 HPV types, including the 13 hrHPVs targeted by Amplicor. RESULTS: 197 specimens were positive by Amplicor, in which the RH Test and LQ Test identified one of the 13 hrHPVs in 94.4% and 98.0%, respectively. In 219 specimens remaining negative by Amplicor, the RH Test and LQ Test, performed on the Amplicor amplification products, still detected one of the 13 hrHPVs in 3.7% and 5.5%, respectively, and include identification of HPV53, 66, and 82. Overall, the RH and LQ Tests demonstrated high concordance with Amplicor for hrHPV detection (κ=0.908 and κ=0.923, respectively). CONCLUSIONS: The digene HPV Genotyping RH and LQ Tests can be directly used for amplimers generated by the Amplicor HPV Test.

High genotyping concordance between the digene HPV Genotyping RH Test and the Reverse Line Blot genotyping assay on GP5+/6+-PCR products.

BACKGROUND: Based on epidemiologic studies, 18 mucosal human papillomavirus (HPV) types have been classified as (probably) high-risk (HR) (i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82). Recognition of HR HPV at the individual type level may be valuable in clinical management of HR HPV-positive women. OBJECTIVES: The goal of this study was to evaluate the performance of the novel digene HPV Genotyping RH Test (digene RH Test), which uses type-specific probes for the 18 HR HPV genotypes, in comparison to the established in-house Reverse Line Blot (RLB) genotyping assay on PCR products generated with the clinically validated GP5+/6+-PCR method. STUDY DESIGN: GP5+/6+ amplimers, generated from 493 digene High Risk HPV HC2 DNA Test (HC2)-positive and 95 HC2-negative cervical smears, were genotyped by both the digene RH Test and the RLB assay. RESULTS: Both genotyping assays demonstrated high concordance for overall HR HPV detection (ú = 0.886) and type-specific identification of the 18 HR types (overall ú = 0.951, individual ú range 0.777 to 1.000) in 493 HC2-positive samples. The digene RH Test revealed positivity for one or more HR HPV type(s) in 86.6% of the HC2-positive women, and negativity was confirmed in 97.9% of the HC2-negative women. CONCLUSIONS: The digene HPV Genotyping RH Test revealed a high genotyping agreement with the established RLB assay on GP5+/6+ amplimers. Accordingly, this assay following GP5+/6+-PCR could serve as a follow-up test in a clinical setting for women who are HC2-positive to identify the respective HR HPV genotype(s).

High-throughput genotyping of high-risk HPV by the digene HPV Genotyping LQ Test using GP5+/6+-PCR and xMAP technology.

BACKGROUND: Epidemiologic studies have classified 18 genotypes of the human papillomavirus (HPV) as (probably) high-risk (HR) based on their association with cervical cancer, i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. Given the fact that certain HR HPV types confer an increased risk of cervical (pre)cancer, type-specific identification might aid clinical management of women tested positive for HR HPV. Therefore, the development of robust, high-throughput genotyping assays is important. OBJECTIVES: An analytical comparison of the digene HPV Genotyping LQ Test (digene LQ Test), capable of identifying 18 HR types using bead-based xMAP suspension array technology, with the established Reverse Line Blot (RLB) genotyping assay was carried out on amplimers generated with the clinically validated GP5+/6+-PCR method. STUDY DESIGN: GP5+/6+ amplimers, generated from 434 digene High Risk HPV HC2 DNA Test (HC2)-positive and 95 HC2-negative cervical smears, were genotyped by both the digene LQ Test and the RLB genotyping assay. RESULTS: The genotyping assays revealed high agreement for overall HR HPV detection (ú = 0.884) and type-specific identification of the 18 HR HPV types (overall ú = 0.958, individual ú range 0.795 to 1.000). The digene LQ Test demonstrated a very good inter-laboratory reproducibility (ú = 0.987). Among the HC2-positive women, the digene LQ Test revealed positivity for one or more HR HPV type(s) in 85.9%, and negativity was observed in 97.9% of the HC2-negative women. CONCLUSIONS: The digene LQ Test demonstrated a high genotyping agreement with the established RLB genotyping assay on GP5+/6+ amplimers. This novel assay allows for high-throughput genotyping following HR HPV testing by HC2.

Reduced quality of life in veterans at risk for drug-related problems.

The relationships between drug therapy and health-related quality of life in 1054 patients who received care from Department of Veterans Affairs medical centers (VAMCs) were assessed. Patients at high risk for drug-related problems were enrolled into a pharmaceutical care study at nine VAMCs. On enrollment, the short form (SF)-36 was completed and medical records were examined for evidence of coexisting illness. Drug therapy in the year before enrollment was analyzed in relation to SF-36 scores. Mean +/- SD SF-36 scores ranged from 37.99+/-41.70 for role physical to 70.78+/-18.97 for mental health domains, with all domain scores significantly below age-adjusted national norms (p<0.05). Patients taking a drug that required therapeutic monitoring had significantly lower SF-36 scores (p=0.0001 to p=0.0033) across all domains except for bodily pain and mental health, compared with patients not taking these agents.

Torsades de pointes associated with chlorpromazine: case report and review of associated ventricular arrhythmias.

PURPOSE: To present a case of chlorpromazine-associated torsades de pointes, review established cases of ventricular arrhythmias associated with chlorpromazine, and describe the proarrhythmic characteristics of this drug. DATA SOURCES: Articles identified through a search of MEDLINE and IDIS from January 1966-November 2000 and thorough review of the article bibliographies. Patient cases also were identified from a search of the Food and Drug Administration's Adverse Event Reporting System database (November 1997-March 2001). Cases involving intentional overdoses of chlorpromazine were excluded. RESULTS: In addition to the case reported herein, 12 cases of documented, chlorpromazine-associated ventricular arrhythmias were identified; five had characteristic features of torsades de pointes. Chlorpromazine delayed repolarization and produced electrocardiographic abnormalities; although, whether chlorpromazine induced torsades de pointes through a mechanism of early afterdepolarizations is unclear. Similar to other instances of drug-induced torsades de pointes, concurrent factors such as electrolyte deficiencies may place the patient at increased risk for arrhythmia. CONCLUSIONS: Chlorpromazine can delay repolarization and produce electrocardiographic abnormalities. These can result infrequently in ventricular arrhythmias and torsades de pointes, particularly in patients with confounding factors.

Interpreting the findings of the IMPROVE study.

Various findings of the Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers (IMPROVE) study are reviewed. Suggestions for future methodologies that will enhance this study are discussed. The IMPROVE study is one of the largest pharmaceutical care studies conducted. Although it was an intervention study that examined global outcomes following management by pharmacists, it was designed as an effectiveness study. Several new practice and research methods were developed, including a method to identify patients at high risk for drug-related problems utilizing pharmacy databases, a method to identify chronic diseases using pharmacy databases, a method to evaluate the structure and process for delivering pharmaceutical care in Veterans Affairs medical centers (VAMCs), and guidelines for providing care to patients in the IMPROVE study. Nine VAMCs participated in the study, and 1054 patients were randomized to either an intervention group (n = 523) or a control group (n = 531). Pharmacists documented a total of 1855 contacts with the intervention group patients and made 3048 therapy-specific interventions over the 12-month study period. There was no meaningful difference in patient satisfaction or quality of life in the two groups. Selected disease-specific indicators found an improved rate of measurement of hemoglobin A1c tests and better control of total and low-density-lipoprotein (LDL) cholesterol levels in the intervention group compared with the control group. Total health care costs increased in both groups over the 12-month period. The mean increase in costs in the intervention group was $1020, which was lower than the control group's value of $1313. The lessons learned from the IMPROVE study suggest to future investigators how to study and measure the effects of clinical pharmacy services on patient outcome.

Can clinical pharmacists affect SF-36 scores in veterans at high risk for medication-related problems?

BACKGROUND: An objective of pharmaceutical care is for pharmacists to improve patients' health-related quality of life (HRQOL) by optimizing medication therapy. OBJECTIVES: The objective of this study was to determine whether ambulatory care clinical pharmacists could affect HRQOL in veterans who were likely to experience a drug-related problem. RESEARCH DESIGN: This was a 9-site, randomized, controlled trial involving Veterans Affairs Medical Centers (VAMCs). Patients were eligible if they met > or = 3 criteria for being at high risk for drug-related problems. Enrolled patients were randomized to either usual medical care or usual medical care plus clinical pharmacist interventions. HRQOL was measured with the SF-36 questionnaire administered at baseline and at 6 and 12 months. RESULTS: In total, 1,054 patients were enrolled; 523 were randomized to intervention, and 531 to control. After patient age, site, and chronic disease score were controlled for, the only domain that was significantly different between groups over time was the bodily pain scale, which converged to similar values at the end of the study. Patients' rating of the change in health status in the past 12 months was statistically different between groups, intervention patients declining less (-2.4 units) than control subjects (-6.3 units) (P < 0.004). This difference was not considered clinically meaningful. However, a dose-response relationship was observed for general health perceptions (P = 0.004), vitality (P = 0.006), and change in health over the past year (P = 0.007). CONCLUSIONS: These results suggest that clinical pharmacists had no significant impact on HRQOL as measured by the SF-36 for veterans at high risk for medication-related problems.

Clinical and economic impact of ambulatory care clinical pharmacists in management of dyslipidemia in older adults: the IMPROVE study. Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers.

We examined the impact of ambulatory care clinical pharmacist interventions on clinical and economic outcomes of 208 patients with dyslipidemia and 229 controls treated at nine Veterans Affairs medical centers. This was a randomized, controlled trial involving patients at high risk of drug-related problems. Only those with dyslipidemia are reported here. In addition to usual medical care, clinical pharmacists were responsible for providing pharmaceutical care for patients in the intervention group. The control group did not receive pharmaceutical care. Seventy-two percent of the intervention group and 70% of controls required secondary prevention according to the National Cholesterol Education Program guidelines. Significantly more patients in the intervention group had a fasting lipid profile compared with controls (p=0.021). The absolute change in total cholesterol (17.7 vs 7.4 mg/dl, p=0.028) and low-density lipoprotein (23.4 vs 12.8 mg/dl, p=0.042) was greater in the intervention than in the control group. There were no differences in patients achieving goal lipid values or in overall costs despite increased visits to pharmacists. Ambulatory care clinical pharmacists can significantly improve dyslipidemia in a practice setting designed to manage many medical and drug-related problems.

An economic analysis of a randomized, controlled, multicenter study of clinical pharmacist interventions for high-risk veterans: the IMPROVE study. Impact of Managed Pharmaceutical Care Resource Utilization and Outcomes in Veterans Affairs Medical Centers.

STUDY OBJECTIVE: To determine if clinical pharmacists could affect economic resource use and humanistic outcomes in an ambulatory, high-risk population. DESIGN: Prospective, randomized, controlled study. SETTING: Nine Veterans Affairs medical centers. PATIENTS: Patients who were at high risk for medication-related problems. INTERVENTION: Patients were randomized to usual medical care with input from a clinical pharmacist (intervention group) or just usual medical care (control group). MEASUREMENTS AND MAIN RESULTS: Of 1,054 patients enrolled, 523 were randomized to the intervention group and 531 to the control group. The number of clinic visits increased in the intervention group (p=0.003), but there was no difference in clinic costs. Mean increases in total health care costs were $1,020 for the intervention group and $1,313 for the control group (p=0.06). CONCLUSION: Including the cost of pharmacist interventions, overall health care expenditures were similar for patients randomized to see a clinical pharmacist versus usual medical care.

Types of interventions made by clinical pharmacists in the IMPROVE study. Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers.

The purpose of this study was to describe and evaluate the activities and interventions provided by ambulatory care clinical pharmacists during the IMPROVE (Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers) study. A total of 523 patients were randomized into the intervention arm at nine Veterans Affairs medical centers if they were considered to be at high risk for drug-related problems. Patients randomized to the control group had no interventions and they are not reported. Using a standard form, pharmacists were asked to document the length of visit, method of contact, medical conditions addressed, and drug-related problems addressed and resolved during each contact. Seventy-eight ambulatory care clinical pharmacists documented 1855 contacts over 12 months, an average of 3.54 +/- 2.31/patient. The length of visits was 15 minutes or more for 73% of contacts. In-person contacts accounted for 1421 visits (76.6%), with the remainder being telephone contacts. During each contact the average number of drug-related problems addressed and resolved were 1.64 +/- 1.16 and 1.14 +/- 0.98, respectively. More drug-related problems were addressed and resolved when visits were 15 minutes or longer (p=0.001) and when the contact was in person (p=0.001). These data may provide information to clinical pharmacists developing pharmacy-managed clinics for patients at high risk for drug-related problems. The information may be a benchmark for types of interventions that can be made, as well as the time commitments required to make them.

High-resolution HLA typing for the DRB3/4/5 genes by sequence-based typing.

The high degree of polymorphism of the HLA genes at the nucleotide sequence level has proven sequence-based typing a major typing strategy. For DRB1 the allelic variability is predominantly present in the second exon and by DNA sequencing of exon 2 all hitherto known DRB1 alleles can be detected. For the associated genes DRB3, DRB4 and DRB5 the situation is slightly different. Allelic differences are not limited to exon 2 and the sequence of exon 3 and sometimes exon 4 is needed for complete subtyping. Oligonucleotides to amplify the exons needed for subtyping of DRB3, DRB4 and DRB5 were designed. Gene-specific products were generated to make simultaneous detection of alleles in heterozygous combinations possible. In this way 238 individuals were fully typed for their DRB3, 4 and 5 subtypes. Additional samples were typed for only one of the genes. All samples had been previously typed by PCR-SSP. Concordant typing results were obtained for all individuals tested. The DRB3 alleles typed for included *0101, *0201, *0202 and *0301, for DRB4 they were *01011, *0102 and *0103 and for DRB5 *0101, *0102, *0103, *0105, *0201, *0202 and *0203. All alleles were easily detected by the protocol described except for DRB5*0201. Sequencing of exon 3 and 4 of the DRB5*0201 allele showed this allele to be a sequencing error and the sequences obtained were identical to the exon 2, 3 and 4 sequences of DRB5*0202. Two new alleles were identified in the samples studied, DRB4*0105 and DRB3*0207. Sequence based typing has been recognized as a valuable tool for HLA typing of DRB1, DQB1 and DPB1 since several years. It is shown to be a superior typing method as well in the detection of the different DRB3, 4 and 5 subtypes.

Comparison of DRB sequence-based typing using different strategies.

Sequence-based typing (SBT) has become an important tool in the identification of HLA alleles. In this study a comparison was made between SBT of DRB1/3/4/5 alleles performed in two laboratories each using a different strategy for SBT. The laboratories in Utrecht and in Maastricht performed direct sequencing of PCR amplified genomic DNA from 30 selected samples. Primers and conditions for PCR amplification were different. Sequencing was either performed with T7 polymerase, using internal sequencing primers, or with cycle sequencing using an M13 tailed system. Two different automated DNA sequencers were used; the ALFexpress from Pharmacia and Applied Biosystems 373A. We concluded that nor the method of sequencing nor the sequencing machine influences typing results. However the PCR reaction used for generating template DNA is the most critical step. Different primers and different conditions can lead to false negative reactions. The fact that these false negative reactions can occur with different alleles in different combinations but not in all, implicates that extensive quality control is needed to assure correct typing results.