Determining the frequency of prescription errors in an Irish hospital.

BACKGROUND: Faults in writing prescriptions are a common source of medical error. Iatrogenic injury due to medication error increases patient morbidity and hospital stay, thereby encouraging litigation. AIM: To assess the accuracy and legibility of the prescriptions in patients' medication charts. METHODS: A cross-sectional observational study examined prescribing records of inpatients randomly selected in two surgical wards. Medication charts were assessed by a committee consisting of a nurse, a pharmacist and a doctor for omission and legibility of prescribing information. RESULTS: Important patient information and medication administration details were frequently omitted from charts. Overall, 27% of individual prescriptions had potential to cause prescription error because of illegibility or omission of medication administration details. CONCLUSIONS: The results of this study demonstrate that prescription error frequently occurs in the clinical workplace and may contribute to medical error. Improving legibility of handwriting and use of novel prescribing devices may reduce prescription error.

Randomized controlled trial of oral vs intravenous therapy for the clinically diagnosed acute uncomplicated diverticulitis.

OBJECTIVE: Despite the high prevalence of hospitalization for left iliac fossa tenderness, there is a striking lack of randomized data available to guide therapy. The authors hypothesize that an oral antibiotic and fluids are not inferior to intravenous (IV) antibiotics and 'bowel rest' in clinically diagnosed acute uncomplicated diverticulitis. METHOD: A randomized controlled trial was constructed in two District General Hospitals. All clinically diagnosed patients presenting with acute uncomplicated diverticulitis were eligible for the study. Oral and IV regimens utilizing ciprofloxacin and metronidazole were compared. The primary outcomes studied were surrogates for resolution of symptoms (including tenderness on day 3 and length of stay) and failure of oral therapy. Secondary endpoints studied were serial constitutional and biomarker trends. RESULTS: There were 41 patients in the oral arm and 38 in the IV arm (n = 79). No patient had to be converted to IV antibiotics from the oral group. There was a complete resolution of symptoms in both groups. Tenderness was equivalent in both groups on day 3. Among secondary endpoints, a serial decrease in C reactive protein was the best serological predictor of resolution for both groups. CONCLUSION: Oral antibiotics are not inferior to intravenous antibiotics in achieving resolution of clinically diagnosed diverticulitis.

Legibility of doctor's signatures: novel approaches to improving an age-old problem.

UNLABELLED: In today's medico-legal environment, the importance of identification of the authors of notes in patient medical charts cannot be overemphasized. We evaluated three different techniques of signing patient notes, over a one month period, in order to determine which technique was the most effective in identifying the author of the note. Surgical NCHDs in our hospital were divided into three groups. Group 1 was asked to sign the notes as they normally would. Group 2 was asked to print their name in block capitals after their signature and Group 3 was given pens with a personal self inking stamp to be used in addition to signing the notes. The number of signatures in all the charts, compliance with the assigned technique and the legibility of signatures were calculated. RESULTS: in Group 1, all NCHDs signed their name when writing notes (100% compliance), however the NCHD's signature was identified only 37% of the time. In Groups 2 (who signed in block capitals) and Group 3 (who used the pen with personalised stamp) the author was identifiable 100% of the time when the respective signing method was used. Using the pen with personalised self inking stamp was significantly more popular (77% compliance) compared to signing in block capitals (46% compliance). In conclusion the pen, with personalised self inking stamp, provides a fast and effective means to clarify signatures of NCHD's documentation, which is not only important in a day to day patient management, but is essential from a medico-legal stand point.

Low molecular weight heparin prophylaxis in day case surgery.

BACKGROUND: The role of Low molecular weight heparins (LMWH) in day case/short-stay surgery is unknown. AIM: To characterise the current national use of LMWH prophylaxis in specific day and short stay surgeries. METHODS: A standardised anonymous postal questionnaire was sent to all consultant general surgeons in Ireland. The operations selected were herniorraphy, anorectal, varicose vein and laparoscopic cholecystectomy. RESULTS: Questionnaires were sent to 82 surgeons in 2003. There was a response rate of 68.3% (56). Fifty-four per cent of respondents said there was a protocol in place for administration of LMWH in day case surgery. Of these 41% were not confident that their protocols were being adhered. Fifty-nine per cent of all respondents said they stratified patients according to individual risk. Thirteen per cent reported occurrence of VTE post day case surgery CONCLUSION: This study demonstrates a heterogeneous pattern of administration of LMWH. In the absence of published validated protocols, the authors suggest a consensus day case protocol.

Strategic targeting of essential host-pathogen interactions in chlamydial disease.

The chlamydiae are obligate intracellular gram-negative bacteria that are exquisitely adapted for exploitation of their hosts and contribute to a wide range of acute and chronic human diseases. Acute infections treated with non-cidal antibiotics can lead to the development of persistent, non-replicating bacteria with the corollary that these persistent (yet viable) chlamydiae can resist eradication by further antimicrobial treatment and cause chronic disease. These findings highlight an urgent need for therapeutics that are effective against persistent infections and call for creative approaches to identify potential drug targets. The C. pneumoniae and C. trachomatis genome projects have greatly expanded our knowledge of chlamydial pathogenesis and have provided an enormous potential for the identification and characterization of unknown genes and potential virulence factors in these bacteria. As intracellular pathogens, chlamydiae rely on host cells for all aspects of their survival, from the initial attachment with host cell membranes, to cellular invasion, acquisition of host cell metabolites and intracellular replication. As such, the molecules participating in interactions with the host could be attractive targets for therapeutic intervention. This review describes recent advances in chlamydial genomics, proteomics and cell biology that have cast light on host-pathogen relations that are essential for chlamydial survival. Using this knowledge, we discuss how strategically interfering with essential interactions between chlamydiae and the host cell could be exploited to develop an innovative, and potentially more relevant arsenal of therapeutic compounds.

Randomised, blinded, placebo controlled trial of doxycycline for chronic seronegative arthritis.

OBJECTIVE: To determine whether long term doxycycline improves symptoms in patients with chronic seronegative or reactive arthritis. METHODS: A randomised, triple blind, controlled clinical trial of three months' treatment with doxycycline or placebo of patients with chronic reactive or seronegative arthritis was conducted. The primary study end points were three month pain and functional status measured by a self administered Arthritis Impact Measurement Scales version 2 (AIMS2) quality of life questionnaire. Secondary end points were pain and functional status at 6-12 months, three month rheumatologist assessed joint count, pain, and arthritis activity, and treatment efficacy in those with previous exposure to chlamydia. RESULTS: Of 60 patients randomly allocated to receive doxycycline or placebo, results from 37 were evaluable at three months. Groups were well balanced for major prognostic variables. Doxycycline had no detectable effect at three months on pain change scores (mean difference 1.5, 95% CI -1.2 to 4.2, p=0.25) or composite functional change scores (mean difference 0.8, 95% CI -5.6 to 7.1, p=0.81). Furthermore, there were no differences in secondary study end points, and no apparent treatment effect in patients with previous chlamydia infection. CONCLUSION: Three months' treatment with doxycycline did not improve pain or functional status in patients with chronic reactive or seronegative arthritis.

Dendritic cell discoveries provide new insight into the cellular immunobiology of DNA vaccines.

The evolution of increasingly virulent human pathogens, together with the rapid onset of antimicrobial resistance has created a need for new vaccination strategies. Nucleic acid vaccines, based on recombinant DNA technology are a promising new vaccine formulation capable of eliciting both humoral and cellular immune responses. This technology has been experimentally validated in animal models of pathogen challenge and tumor protection following administration of a DNA vaccine and has led to extensive research into the mechanisms of protective immunity. We focus here on the cellular and molecular mechanisms leading to cell-mediated immune responses to DNA vaccines and discuss these mechanisms in light of recent advances in the field of dendritic cell immunobiology. In particular, the potential involvement of: (i) the CpG pattern-recognition receptor, toll-like receptor-9; (ii) the dendritic cell-specific surface adhesion molecule, DC-SIGN; and (iii) the molecular interactions between CD40 and CD154 in the evolution of protective cell-mediated immunity to DNA vaccines are discussed. An improved understanding of the precise mechanisms leading to protective cellular immunity following DNA vaccination may help in the design of novel DNA constructs containing immunostimulatory features that target one or more of these mechanisms, with the aim of increasing the immunogenic potential and protective efficacy of DNA vaccines.

Replicate PCR testing and probit analysis for detection and quantitation of Chlamydia pneumoniae in clinical specimens.

Nucleic acid amplification of clinical specimens with low target concentration has variable sensitivity. We examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of Chlamydia pneumoniae detection by PCR. Nested and non-nested C. pneumoniae PCR assays were compared using 10 replicates of 16 serial dilutions of C. pneumoniae ATCC VR-1310. The proportion positive versus the C. pneumoniae concentration was modeled by probit regression analysis. To validate the model, 10 replicates of 26 previously positive patient specimens of peripheral blood mononuclear cells (PBMC), sputum, or nasopharyngeal swabs (NPS) were tested. The proportion of replicates that were positive varied with the concentration of C. pneumoniae in the sample. At concentrations above 5 infection-forming units (IFU)/ml, both nested and non-nested PCR assay sensitivities were 90% or greater. The nested PCR was more sensitive (median detection, 0.35 versus 0.61 IFU/ml; relative median detection, 0.58; 95% confidence interval, 0.31 to 0.99; P = 0.04). In clinical specimens, replicate PCR detected 15 of 26 (nested) versus 1 of 26 (non-nested, P < 0.001). For PBMC specimens, testing 1, 3, or 5 replicates detected 3, 5, or 9 of 10 positive specimens, respectively. Median C. pneumoniae concentrations were estimated at 0.07 IFU/ml for PBMC and at <0.03 IFU/ml for NPS specimens. We conclude that performing 5 or 10 replicates considerably increased the sensitivity and reproducibility of C. pneumoniae PCR and enabled quantitation for clinical specimens. Due to sampling variability, PCR tests done without replication may miss a large proportion of positive specimens, particularly for specimens with small amounts of target C. pneumoniae DNA present.

Chlamydia pneumoniae and atherosclerosis: does the evidence support a causal or contributory role?

The intracellular bacterial pathogen Chlamydia pneumoniae causes respiratory tract infection and has been associated with atherosclerosis and coronary artery disease. Since atherosclerosis is a progressive disease and is considered to be a chronic inflammation of the artery vessel wall, the interaction of C. pneumoniae with cells of the vasculature that can result in a local inflammatory response is of paramount importance. In this essay we review the pathophysiology of atherosclerosis in the context of C. pneumoniae infection and present an integrated model that includes the involvement of C. pneumoniae in all stages of atherogenesis including initiation, inflammation, fibrous plaque formation, plaque rupture and thrombosis. We hypothesize that acute and persistent infection of professional immune cells (T-cells, monocytes and macrophages) and non-immune cells (endothelial cells and smooth muscle cells) contributes to a sustained inflammatory response mediated by extensive cellular 'crosstalk' and numerous cytokines/chemokines. This cascade of inflammatory mediators may contribute to cellular dysfunction and tissue remodelling of the arterial intima. An improved understanding of the precise mechanism(s) of C. pneumoniae involvement in atherogenesis may help resolve the question of causality however, at the present time, we interpret the data as favoring a contributory rather than a causal role. Future research directed at the discovery of chlamydial virulence factors necessary for intracellular survival and subsequent alterations in host cell gene expression including signalling pathways may be important for the design of future clinical trials.

Evaluation of the NucliSens Basic Kit for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genital tract specimens using nucleic acid sequence-based amplification of 16S rRNA.

We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of C. trachomatis and N. gonorrhoeae and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of C. trachomatis, 1 CFU of N. gonorrhoeae, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for N. gonorrhoeae by culture and NASBA and a total of 96 specimens for C. trachomatis by PCR and NASBA. The Basic Kit detected 139 of 142 N. gonorrhoeae culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. For C. trachomatis, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both N. gonorrhoeae and C. trachomatis, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.

Previous exposure to Chlamydia pneumoniae, Helicobacter pylori and other infections in Canadian patients with ischemic heart disease.

OBJECTIVE: Previous exposures to Chlamydia pneumoniae (CP), Helicobacter pylori (HP) or cytomegalovirus (CMV) have been associated with atherosclerotic heart disease. These associations were studied in Canadian patients, and the exposure to five infections measured. DESIGN: Case-control study. SETTING: In the coronary care units (Hamilton General site, Henderson General site, McMaster University Medical Centre site of the Hamilton Health Sciences Corporation and St Joseph's Hospital) and from the regional angiography suite (Hamilton General site), Hamilton, Ontario. PATIENTS AND METHODS: One hundred seven consecutive patients presenting with myocardial infarction or unstable angina (coronary care unit patients), or with previous angina or myocardial infarction (angiography suite patients), were compared with 107 family practice or outpatient clinic control subjects. INTERVENTIONS: Cardiovascular risk factors were measured, as was serology for CP, HP, CMV, adenovirus and hepatitis A virus. Statistical analysis was by logistic regression, adjusted for age and sex. RESULTS: Exposure to CP was more frequent in patients than in control subjects (85.4% versus 70.3%, adjusted odds ratio [OR] 2.3, 95% CI 1.1 to 5.1, P=0.03). Dividing CP immunoglobulin G absorbance into quarters, with the lowest quarter as the reference group, the adjusted ORs were 2.8, 3.0 and 4.3, respectively, for the second, third and fourth quarters (P=0.001 for trend). The seroprevalences of HP (61.7%), CMV (64.0%), adenovirus (75.6%) and hepatitis A virus (59.2%) were high, with no association with disease. CONCLUSIONS: An association was found between heart disease and previous exposure to CP, with a stepwise increase in ORs at higher antibody levels, whereas no association was found with HP, CMV or other infections. A prospective validation of this association is needed.

cDNA array analysis of altered gene expression in human endothelial cells in response to Chlamydia pneumoniae infection.

Strong epidemiological and pathological evidence supports a role for Chlamydia pneumoniae infection in atherosclerosis and human coronary heart disease. Animal models have shown that C. pneumoniae disseminates hematogenously in infected monocytes and macrophages, while in vitro data suggest that infected macrophages can transmit C. pneumoniae infection directly to endothelial cells. Endothelial cells may be key in vivo targets for C. pneumoniae infection; given that these cells are important in regulating the dynamics of the vessel wall, we used cDNA microarrays to study the transcriptional response of endothelial cells to infection with C. pneumoniae. cDNA arrays were used to characterize the mRNA expression profiles for 268 human genes following infection with C. pneumoniae, which were compared to mRNA profiles of uninfected cells. Selected genes of interest were further investigated by reverse transcription-PCR throughout a 24-h period of infection. C. pneumoniae infection upregulated mRNA expression for approximately 20 (8%) of the genes studied. Genes coding for cytokines (interleukin-1), chemokines (monocyte chemotactic protein 1 and interleukin-8), and cellular growth factors (heparin-binding epidermal-like growth factor, basic fibroblast growth factor, and platelet-derived growth factor B chain) were the most prominently upregulated. In addition to these families of genes, increases in mRNA levels for intracellular kinases and cell surface receptors with signal transduction activities were observed. Time course experiments showed that mRNA levels were upregulated within 2 h following infection. These results expand our knowledge of the response of endothelial cells to C. pneumoniae by further defining the repertoire of C. pneumoniae-inducible genes and provide new insight into potential mechanisms of atherogenesis. In addition, the use of cDNA microarrays may prove useful for the study of host cell responses to C. pneumoniae infection during latent and replicative stages of infection and related pathology.

Circulating nucleic acids of Chlamydia pneumoniae and cytomegalovirus in patients undergoing coronary angiography.

Peripheral blood mononuclear cells from 208 consecutive patients undergoing elective coronary angiography or angioplasty were collected before, immediately after, and 4 h after the procedure. Nucleic acids of Chlamydia pneumoniae and of cytomegalovirus (CMV) were detected by PCR and confirmed by hybridization. Circulating C. pneumoniae DNA was identified in 24 patients (11.5%) and was associated with current smoking (odds ratio [OR] = 4.5, 95% confidence interval [CI] = 1.6 to 12.2, P = 0.004) but not with arterial narrowing on coronary angiogram or with serological results positive for C. pneumoniae. Circulating CMV DNA was identified in 36 patients (17.3%) and was associated with anti-CMV immunoglobulin G (OR = 2.7, 95% CI = 1.2 to 6.3, P = 0.02) but not with angiographic arterial narrowing or with the need for revascularization. Neither C. pneumoniae nor CMV DNA detection increased after angioplasty, a procedure in which endothelium is disrupted. Larger prospective studies are needed to determine the prognostic significance of DNA detection.

Effect of routine use of a multiplex PCR for detection of vanA- and vanB- mediated enterococcal resistance on accuracy, costs and earlier reporting.

A multiplex PCR (MPCR) for detection of vanA-and vanB-mediated resistance to vancomycin was optimized and adapted for use in the routine microbiology laboratory. Consecutive specimens (1196) submitted for vancomycin resistant Enterococci (VRE) surveillance were processed by clinical technologists on Bile Esculin Azide Agar containing 6 mg/L vancomycin (BEAA/Vanco6) plates and 466 showing black colony growth were processed by conventional biochemical testing (CBT) and by MPCR. CBT identified 208 VRE positives. MPCR detected 205 of the CBT- positives plus an additional 10. Analysis of the discordant specimens determined that 5 CBT- negative/MPCR-positive specimens also contained Enterococci with vanC resistance, 3 CBT-positive/MPCR-negative specimens were true positives, and 5 CBT-negative/MPCR-positive specimens occurred due to technical error. The sensitivity and specificity of MPCR were 98.4% and 96.1%. MPCR identifications of VRE were achieved approximately 48 h earlier than CBT and at 60% of the costs.

Comparison of human papillomavirus DNA testing and repeat Papanicolaou test in women with low-grade cervical cytologic abnormalities: a randomized trial. HPV Effectiveness in Lowgrade Paps (HELP) Study No. 1 Group.

BACKGROUND: Results of cervical cytology screening showing atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesions (LSIL) indicate risk for high-grade cervical intraepithelial neoplasia (CIN 2 or 3). In a community-based randomized trial we compared the test performance of human papillomavirus (HPV) DNA testing with that of 6-month repeat Papanicolaou (Pap) test in detecting histologically confirmed CIN 2 or 3. METHODS: We randomly assigned 212 women aged 16-50 years with ASCUS or LSIL on cervical cytology screening to undergo either immediate HPV DNA testing or a repeat Pap test in 6 months. Cervical swabs for the HPV DNA testing and the Pap smears were obtained by their family physicians. We tested the swabs for oncogenic HPV using the Hybrid Capture II assay (Digene Corp., Beltsville, Md.). Community-based pathologists examined the Pap smears. All women were referred for colposcopy by their family physicians. Two gynecological pathologists assessed the histology findings. We calculated test performance in women who completed the trial using CIN 2 or 3 as the reference standard. RESULTS: A total of 159 women completed the study. Compared with HPV DNA testing, which detected 87.5% (7/8) of the cases of CIN 2 or 3, repeat Pap smear showing high-grade intraepithelial neoplasia (HSIL) detected 11.1% (1/9) of cases (p = 0.004), and repeat Pap smear showing ASCUS, LSIL or HSIL detected 55.6% (5/9) (p = 0.16). Corresponding specificities were 50.6%, 95.2% (p = 0.002) and 55.6% (p = 0.61). Loss to follow-up was 17.1% in the HPV test group and 32.7% in the repeat Pap group (p = 0.009). Given the 7 cases of CIN 2 or 3 detected by HPV testing and the 5 cases detected by the repeat Pap smear, the incremental cost of HPV testing was calculated to be $3003 per additional case of CIN identified. INTERPRETATION: HPV DNA testing was more costly but was associated with significantly less loss to follow-up. It may detect more cases of CIN 2 or 3 in women with low-grade cytologic abnormalities.

Quantitation of Chlamydia trachomatis 16S rRNA using NASBA amplification and a bioluminescent microtiter plate assay.

We developed a nucleic acid sequence based amplification (NASBA) assay which employs the recombinant photoprotein Aequorin in a microtiter plate format for detection and quantitation of C. trachomatis that may be useful in large scale epidemiological studies aimed at improving our understanding of factors affecting transmission of this sexually transmitted pathogen. The conditions for NASBA amplification of the16S rRNA target were optimized (90 mM KCl, 12 mM MgCl(2), 0.2 microM P1 and P2 primers), amplified RNA was captured by a biotin-labelled capture probe immobilized onto streptavidin coated microtiter plates and detected with an FITC-labelled oligonucleotide probe and Aequorin-anti-FITC antibody conjugate. The analytical sensitivity of NASBA was 1,000 in vitro generated RNA transcripts and 1.6 IFU of C. trachomatis. The sensitivity of NASBA using the bioluminescent assay was 10 fold higher than Northern blotting. Time course amplification experiments performed with 10 fold serial dilutions of target established that amplification was linear at 75 min and extended over a range of five log units of input RNA copy number. Linear regression analysis confirmed a linear fit for the data with r(2) = 0.959 (p < 0.004). A double log plot of RLU signal versus copy number was linear; analysis of residuals from a series of runs tests confirmed a fit with a linear model (number of runs = 3, p = 0.5 where p < 0.05 indicates statistical deviation from a linear model). NASBA amplification coupled with bioluminescent detection in a microtiter plate format should provide a useful tool for quantitation of C. trachomatis in clinical specimens for use in epidemiological studies.

Nucleic acid sequence based amplification (NASBA) of Chlamydia pneumoniae major outer membrane protein (ompA) mRNA with bioluminescent detection.

Chlamydia pneumoniae has been associated with chronic conditions such as atherosclerosis and coronary heart disease but the precise role of this intracellular bacteria in the pathogenesis of these diseases is not well defined. Several techniques have been developed for detection of C. pneumoniae in atheromatous lesions, however it remains unclear whether persistent forms of the organism and/or actively replicating bacteria contribute to associated pathology. The aim of this study was to utilize nucleic acid sequence based amplification (NASBA) technology together with a highly sensitive aequorin bioluminescent hybridization assay for the detection of C. pneumoniae ompA mRNA transcripts. A NASBA targeting the ompA gene of C. pneumoniae was developed, and the sensitivity was evaluated using both C. pneumoniae ompA RNA generated in vitro, and purified C. pneumoniae inclusion forming units (IFU). C. pneumoniae NASBA was capable of detecting between 100 and 1000 ompA RNA molecules and could detect 0.2 IFU of C. pneumoniae using the aequorin bioluminescent assay. The sensitivity of the bioluminescent assay was at least 10-fold higher than Northern blot detection. The linearity of NASBA amplification was assessed in time-course amplification experiments with different input numbers of RNA molecules. When NASBA products were analyzed during the linear phase of amplification, the dynamic range of bioluminescent detection extended over 8-log units of input RNA copy number. NASBA amplification coupled with bioluminescent detection may prove to be a useful molecular tool for the detection, quantitation and analysis of differentially expressed chlamydial genes during various stages of infection and disease pathology or for other mRNAs of interest in different disease processes.

Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group.

BACKGROUND: Human papillomavirus (HPV) is thought to be the primary cause of cervical intraepithelial neoplasia and cervical cancer. We determined the age-specific prevalence of HPV infection and its risk factors in Ontario women. METHODS: We obtained 2 cervical specimens from randomly selected women (in 5-year age categories, from 15 to 49 years) who were being seen in 32 family practices for cytologic screening. The specimens were tested for carcinogenic HPV by the hybrid capture II assay (Digene Corp., Silver Spring, Md.) and by polymerase chain reaction (PCR) and genotyping. RESULTS: Of 1004 women eligible to participate, samples were obtained from 955 (95.1%). The prevalence of HPV (as determined by the hybrid capture II method) was highest, at 24.0% (95% confidence interval [CI] 16.5% to 31.5%), among women 20 to 24 years of age and was progressively lower in older age groups, reaching 3.4% (95% CI 0.1% to 6.7%) in women 45 to 49 years old. The prevalence of HPV (any type) as determined by PCR showed a similar pattern but was significantly higher (p = 0.01) among women 45 to 49 years old than among those 40 to 44 years old (13.0% [95% CI 6.4% to 19.6%] v. 3.3% [95% CI 0.1% to 6.5%]). Risk factors for positivity with the hybrid capture II method were never-married status, divorced or separated status, more than 3 lifetime partners, more than 1 partner in the preceding year, cigarette smoking and current use of oral contraceptives. The presence of squamous intraepithelial lesions on cytologic examination was strongly associated with positivity with the hybrid capture II assay (odds ratio 96.0, 95% CI 22.3 to 413.4; p < 0.01). INTERPRETATION: The highest prevalence of HPV was 24.0%, in women 20 to 24 years old. Risk factors supported a sexual mode of transmission, and there was a strong association between HPV and abnormal cervical cytologic results.

Comparison of self-collected vaginal, vulvar and urine samples with physician-collected cervical samples for human papillomavirus testing to detect high-grade squamous intraepithelial lesions.

BACKGROUND: Certain types of human papillomavirus (HPV) in cervical samples are strongly associated with squamous intraepithelial lesions (SIL) and invasive cervical carcinoma. We determined and compared the test characteristics of testing for HPV with samples obtained by patients and with samples obtained by their physicians. METHODS: In a consecutive series of women referred to a colposcopy clinic at a teaching hospital because of abnormalities on cervical cytologic screening, 200 agreed to collect vulvar, vaginal and urine samples for HPV testing. The physician then collected cervical samples for HPV testing, and colposcopy, with biopsy as indicated, was performed. Presence of HPV was evaluated using the hybrid capture II assay (Digene Corp., Silver Spring, Md.) with a probe cocktail for 13 carcinogenic types. Cervical specimens were also tested for HPV by polymerase chain reaction and hybridization with type-specific probes. Cervical smears for cytologic examination were obtained from all women. RESULTS: High-grade lesions (high-grade squamous intraepithelial lesions [HSIL], equivalent to cervical intraepithelial neoplasia [CIN] grade 2 or 3, and adenocarcinoma) were found in 58 (29.0%) of the 200 women. Carcinogenic types of HPV were detected in the self-collected vaginal samples of 50 (86.2%) of these 58 women, in the self-collected vulvar samples of 36 (62.1%) and in the self-collected urine samples of 26 (44.8%). Carcinogenic types of HPV were detected in the cervical samples collected by physicians for 57 (98.3%) of these 58 women. The remaining 142 women (71.0%) had normal findings or low-grade squamous intraepithelial lesions (LSIL, CIN grade 1). Test results were negative or noncarcinogenic types of HPV were detected in the self-collected vaginal samples of 76 (53.5%) of these 142 women, in the self-collected vulvar samples of 89 (62.7%) and in the self-collected urine samples of 99 (69.7%). The sensitivity for self-collected samples ranged from 44.8% to 86.2%, and the specificity from 53.5% to 69.7%. For the samples collected by physicians, the sensitivity was 98.3% and the specificity 52.1%. The self-sampling methods were generally acceptable to the women: 98.4% of respondents (126/128) deemed urine sampling acceptable, 92.9% (118/127) found vulvar sampling acceptable, and 88.2% (112/127) found vaginal sampling acceptable. INTERPRETATION: Self-collection of samples for HPV testing was acceptable to women attending a colposcopy clinic for investigation of suspected cervical lesions and shows sufficient sensitivity to warrant further evaluation as a screening test for cervical cancer prevention programs.

Analytical sensitivity, reproducibility of results, and clinical performance of five PCR assays for detecting Chlamydia pneumoniae DNA in peripheral blood mononuclear cells.

Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P<0.001), with the PstI fragment (P<0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P<0.001), and the other 3 assays detected no positive specimens (P<0.001, compared with the ompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detect C. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.