A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Fitness effects of mutation: testing genetic redundancy in Arabidopsis thaliana.

Screens of organisms with disruptive mutations in a single gene often fail to detect phenotypic consequences for the majority of mutants. One explanation for this phenomenon is that the presence of paralogous loci provides genetic redundancy. However, it is also possible that the assayed traits are affected by few loci, that effects could be subtle or that phenotypic effects are restricted to certain environments. We assayed a set of T-DNA insertion mutant lines of Arabidopsis thaliana to determine the frequency with which mutation affected fitness-related phenotypes. We found that between 8% and 42% of the assayed lines had altered fitness from the wild type. Furthermore, many of these lines exhibited fitness greater than the wild type. In a second experiment, we grew a subset of the lines in multiple environments and found whether a T-DNA insert increased or decreased fitness traits depended on the assay environment. Overall, our evidence contradicts the hypothesis that genetic redundancy is a common phenomenon in A. thaliana for fitness traits. Evidence for redundancy from prior screens of knockout mutants may often be an artefact of the design of the phenotypic assays which have focused on less complex phenotypes than fitness and have used single environments. Finally, our study adds to evidence that beneficial mutations may represent a significant component of the mutational spectrum of A. thaliana.

Risk of environmental transmission of norovirus infection from prior room occupants.

BACKGROUND: Environmental contamination of norovirus (NoV) is believed to be a significant source for further transmission in hospitals. AIM: To investigate the risk of acquiring NoV in a cleaned room previously occupied by a patient with NoV infection. The risk of having a roommate with recent NoV infection was also assessed. METHODS: In a retrospective cohort, comprising 33,788 room stays at five infectious Disease wards in southern Sweden from 2013 to 2018, the risk of acquiring NoV infection after admission to an exposed or non-exposed room was analysed with uni- and multivariable statistical analysis, controlling for age, colonization pressure and any roommate. RNA sequencing of the NoV strains involved in suspected room transmission was also performed. RESULTS: Five of the 1106 patients exposed to a room with a prior occupant with NoV infection and 49 in the non-exposed group acquired NoV infection. An association between NoV acquisition was found in the univariable analysis (odds ratio (OR) 3.3, P=0.01), but not when adjusting for potential confounders (OR 1.9, P=0.2). Sequencing of the NoV samples showed that only two of the five exposed patients with acquired NoV infection were infected by identical strains to the prior room occupant, inferring a room transmission risk of 0.2% (95% confidence interval 0.05-0.78%). None of the 52 patients who shared room with a roommate with NoV symptoms resolved for ≥48 h acquired NoV infection. CONCLUSIONS: In absolute terms, the risk of room transmission of NoV is low. Discontinuation of isolation ≥48 h after resolution of symptoms seems adequate.

Self-sampling of the vaginal fluid at home combined with high-risk HPV testing.

BACKGROUND: Around 65% of women with cervical carcinoma in Sweden have not attended an organised screening. We therefore investigated the value of using self-sampling at home in combination with a test for high-risk human papilloma virus (HPV) to increase participation. METHODS: A total of 2829 women 30-58 years old, who had not attended the organised screening for > or = 6 years, were recruited. They were offered self-sampling at home (Qvintip) and recommended to send the collected vaginal fluid to a laboratory for analysis of the presence of high-risk HPV (Hybrid Capture 2 method). RESULTS: A total of 39.1% of the women accepted home sampling. These women disclosed a relatively high prevalence of high-risk HPV, which decreased with age, from 11.1% in women 30-39 years old to 2.9% in women > or =50 years . Follow-up disclosed histological cervical intraepithelial neoplasm (CIN) 2-3 lesions in 43.2% of the women with a persistent HPV infection, corresponding to 2.0% of the total number of participating women. The sensitivity of a single smear to detect the histological CIN 2-3 lesions were only 52.6%, even if all abnormal smears (atypical squamous cells of unknown significance (ASCUS)-CIN 3)) were included. CONCLUSION: The use of self-sampling at home in combination with testing for high-risk HPV increases the participation rate of the organised screening and detects almost twice as many women with pre-malignant cell alterations (CIN 2-3) in comparison those with a single cytological smear.

Tissue Doppler imaging describes diastolic function in men prone to develop hypertension over twenty years.

AIMS: Hypertension is one of several risk factors of cardiovascular disease and is associated with left ventricular (LV) systolic and diastolic dysfunction. A method for reliably detecting the onset of LV dysfunction before transition to irreversible damage of the myocardium would be of crucial importance in subjects with essential hypertension. METHODS AND RESULTS: Subjects with clear differences in BP level, development and duration of the hypertensive disease were examined at the age of 60 yrs: normotensives (n = 17), new hypertensives who developed hypertension over a 20 year period (n = 15) and hypertensives (n = 19). Relationships between conventional echocardiographic and tissue velocities imaging (TVI) parameters compared to LV parameters, and TVI as an estimate of LV function were explored. E'(Lat) (TVI peak early diastolic velocity) (P = 0.006) and E/E'(Lat) (P = 0.002) demonstrated differences in diastolic function between the groups. There were no significant differences regarding systolic myocardial velocities. E'(Lat) correlated to S'(Lat) (TDI peak systolic velocity) (r = 0.32, P = 0.026) and was independently predicted by S'(Lat) (R(2) = 0.24, P = 0.025) in multivariate analysis. E'(Lat) correlated negatively to LV mass index (r = -0.34, P = 0.012), also in multivariate regression analysis (R(2) = 0.12, P = 0.032). CONCLUSIONS: Myocardial diastolic velocities and mitral flow to annulus velocity ratio differentiated LV function between the hypertensive and normotensive groups. The parameters probably reflect changes in relaxation, recoil and contraction and parallel changes in LV mass index.

Risk factors for hospital norovirus outbreaks: impact of vomiting, genotype, and multi-occupancy rooms.

BACKGROUND: Norovirus is frequently introduced to the hospital and is a frequent cause of hospital outbreaks. Recognition of the factors that facilitate or impede norovirus transmission is an important step to effectively prevent hospital outbreaks. AIM: To investigate risk factors for norovirus outbreaks in hospital settings. METHODS: Clinical data, ward setting, and norovirus genotype were collected from all 65 norovirus-positive index cases in outbreaks and all 186 sporadic norovirus cases at 192 wards in southern Sweden during 2010-2012 in a nested case-control study. Uni- and multivariate statistical analyses were conducted. FINDINGS: Outbreak was independently associated with the number of patients sharing a room with the norovirus case (odds ratio (OR): 1.9 per additional patient in the room; P < 0.01), vomiting (OR: 2.6; P = 0.04), age >80 years (OR: 3.2; P < 0.01), comorbidity (OR: 2.3; P = 0.05), and onset of symptoms after admission to the ward (OR: 3.5; P < 0.01) in the multivariate analysis. Infection with genotype GII.4 was found to be strongly associated with outbreak in the univariate analysis (OR: 5.7; P < 0.01). Moreover, associations between GII.4 and vomiting (OR: 2.5; P = 0.01) and old age (OR: 4.3: P < 0.01) were found. CONCLUSION: This is the first study to investigate clinical, ward and genotype risk factors for norovirus hospital outbreaks. Recognition of these factors may help direct and prioritize infection control actions based on the outbreak risk. The results also suggest that the outbreak association with GII.4 partly may be explained by an enhanced ability to induce vomiting.

Access to and use of research by rural nurses.

INTRODUCTION: The use of relevant research findings to inform clinical practice is important for nurses, regardless of setting. Although there have been studies addressing the use of research among various practitioners, little is known about how nurses in rural areas access health information (specifically research findings), nor how such findings are incorporated into daily practice. The purpose of this study was to explore rural nurses' access, use and perceived usefulness of research for rural practice. METHODS: The study was conducted in a sparsely populated state located in the western part of the USA. An ethnographic method was chosen to answer the research questions for this descriptive study. Semi-structured interviews were conducted with 29 rural nurses from nine communities by graduate nursing students enrolled in a rural nursing course following in-class instruction and practice. Field notes taken by the students supplemented the interview data. The students' notes included a windshield survey or description of the context and location within which the participants lived and/or practiced as well as the interviewers' observations, thoughts and impressions about the research project. Interviews were audiotaped and transcribed verbatim. Once transcribed, the interview narratives, windshield data and field notes were analyzed by the students for common themes; the students then wrote and submitted papers to the faculty addressing the themes that emerged from their interviews. The analysis conducted by the faculty members included four sources of data: transcriptions of interviews; field notes; windshield data; and students' papers. The process of identifying themes was facilitated by using the software program NUD*IST (QSR International; Melbourne, VIC, Australia). Demographic information was entered into the Statistical Package for Social Scientists (SPSS Inc; Chicago, IL, USA) to compile descriptive information about the sample. FINDINGS: Twenty-seven female and two male nurses participated in the study. The nurses' ages ranged from 31-72 years and their experience in nursing spanned 3-50 years with a range of 1 to 35 years in rural nursing. The interviews revealed that most of the nurses used the term 'research' to mean 'gathering information'. When asked how often they used 'research' the responses ranged from 2-3 times per day to 2-3 times per month. The preferred means of obtaining information was asking a colleague. Additional resources included work-place journals, books, in-services, conferences and the internet. Twenty-three of the nurses reported having internet access at work; 25 had internet access at home. Supportive supervisors and articles in general nursing journals were identified as helpful. Barriers to using research included: lack of knowledge of research methods; lack of time at work or at home to look up information; and the lack of computers and internet access on the nursing units. When computers were available, the nurses reported that poor computer literacy decreased their ability to quickly find and evaluate information. Additional barriers included diminishing financial support from employers and the long travel distances required to attend conferences. The nurses reported finding little clinical research specifically related to rural practice. CONCLUSIONS: Education and mentorship is needed about how to evaluate the types and strength of evidence, access research using the internet, interpret findings, and incorporate evidence in clinical practice. Interventions that foster the appreciation and use of research by staff nurses and managers are needed in order to build an evidence based culture. Research is needed, specifically as related to rural clinical practice.

Epidermal exfoliation of over 95% after a burn in an 18-month-old boy: Case report and review of the literature.

This report concerns an 18-month-old boy who presented with a 6% total body surface area scald. The subject of this report is unique in that he developed the largest exfoliation described in literature. After 3 days an epidermal exfoliation with the appearance of a deliberately inflicted scald developed. As the exfoliation progressed to over 95% total body surface area the suspicion of child abuse or neglect could be abandoned. The diagnosis Staphylococcal scalded skin syndrome was set, due to the finding of Staphylococcus aureus on swabs, the lack of mucosal engagement, and the patient's age. The boy's skin healed within 3 weeks. The few reports published are all case reports and most frequently described visually infected burns with smaller epidermal exfoliations, and clinically based exfoliation diagnosis. S. aureus often cause burn wound infections that can lead to complications caused by cross-infection. It is important for burn surgeons and intensive care specialists to be aware of the increased possibility of Staphylococcal scalded skin syndrome occurring in patients who have a reduced barrier to infection such as burn patients and also, that the diagnosis can be difficult to make.

Glutamate stimulates the formation of N-acylphosphatidylethanolamine and N-acylethanolamine in cortical neurons in culture.

The formation of anandamide (N-arachidonoylethanolamine), N-acylethanolamine, and N-acylphosphatidylethanolamine was studied in primary cultures of rat cortical neurons. The cells were incubated for 22 h with [14C]ethanolamine, [U-14C]arachidonic acid, [3H]arachidonic acid, [32P]phosphate, [14C]stearic acid, or [3H]myristic acid. The lipids from the cells and media were separated by thin layer chromatography. [14C]Ethanolamine labelling revealed two compounds (I and II), which on different thin layer chromatography systems migrated as N-acylethanolamine (0.06-0.55% of total radioactivity) and N-acylphosphatidylethanolamine (0.66-6.49% of total radioactivity), respectively. Compound II was also labelled with [32P]phosphate, and radioactive fatty acids. Treatment of compound II with phospholipase D (Streptomyces chromofuscus) resulted in two compounds, one comigrating as phosphatidic acid and the other as N-acylethanolamine. Compound I could be labelled with [14C]stearic acid and [3H]myristic acid, but not with [3H]- or [14C]arachidonic acid. Exogenous [3H]anandamide was metabolised with a t1/2 of 2.6 h. The labelling of the two compounds identified as N-acylethanolamine and N-acylphosphatidylethanolamine were more pronounced the older the culture. The neurotoxic amino acid, glutamate, stimulated within 2 h dose-dependently (ED50 = 40 microM) the formation of both compounds. It is suggested that N-acylethanolamine and N-acylphosphatidylethanolamine are formed in relation to the cytotoxicity induced by glutamate, and that these compounds may be markers of neurotoxicity. We could not detect any formation of anandamide using radioactive arachidonic acid.

Changes in the DNase I sensitivity of DNA sequences within the yeast 2 micron plasmid nucleoprotein complex effected by plasmid-encoded products.

We examined the effect of plasmid-encoded gene products on two DNase-I-sensitive regions of DNA in the yeast 2 micron plasmid nucleoprotein complex. For these studies, each sensitive region was cloned into an appropriate vector, and the chimeric plasmids were transformed into yeast. Nucleoprotein complexes of the chimeric plasmids were partially purified and tested for sensitivity to DNase I digestion. One sensitive region is between the 3' end of the 2 micron plasmid coding region D and the plasmid REP3 locus. This region was more sensitive and exhibited a different cleavage pattern when purified from a yeast strain containing endogenous 2 micron plasmid copies than when purified from a yeast strain lacking plasmid copies. Examination of the effect of individual gene products and combinations of the various gene products revealed that the plasmid's REP1, REP2 and D loci were all necessary to restore the pattern to that found in the preparation containing endogenous 2 micron plasmid copies. The other sensitive region studied brackets the binding site of the plasmid-encoded FLP protein, which catalyzes site-specific recombination between the 2 micron plasmid's inverted repeated sequences. In contrast to the first sensitive region examined, the sensitive region in the inverted repeat was less sensitive in chimeric plasmids isolated from a yeast strain containing endogenous 2 micron plasmid copies than from one lacking endogenous copies. Presumably, this protection results from the binding of the FLP protein.

Cold Hardening of Spring and Winter Wheat and Rape Results in Differential Effects on Growth, Carbon Metabolism, and Carbohydrate Content.

The effect of long-term (months) exposure to low temperature (5[deg]C) on growth, photosynthesis, and carbon metabolism was studied in spring and winter cultivars of wheat (Triticum aestivum) and rape (Brassica napus). Cold-grown winter rape and winter wheat maintained higher net assimilation rates and higher in situ CO2 exchange rates than the respective cold-grown spring cultivars. In particular, the relative growth rate of spring rape declined over time at low temperature, and this was associated with a 92% loss in in situ CO2 exchange rates. Associated with the high photosynthetic rates of cold-grown winter cultivars was a 2-fold increase per unit of protein in both stromal and cytosolic fructose-1,6-bisphosphatase activity and a 1.5- to 2-fold increase in sucrose-phosphate synthase activity. Neither spring cultivar increased enzyme activity on a per unit of protein basis. We suggest that the recovery of photosynthetic capacity at low temperature and the regulation of enzymatic activity represent acclimation in winter cultivars. This allow these overwintering herbaceous annuals to maximize the production of sugars with possible cryoprotective function and to accumulate sufficient carbohydrate storage reserve to support basal metabolism and regrowth in the spring.

Bacterial vaginosis. Transmission, role in genital tract infection and pregnancy outcome: an enigma.

Whether bacterial vaginosis (BV) is acquired from an endogenous or an exogenous source is subject to controversy. Despite findings of an association between sexual behaviour and BV, some data indicate that BV is not a sexually transmitted infection in the traditional sense, while other data indicate that BV is an exogenous infection. A third aspect of BV is its tendency to go unnoticed by affected women. All of this will have a strong impact on how physicians view the risks of asymptomatic BV. This review focuses on whether or not BV should be regarded as a sexually transmitted infection (STI), its role in postoperative infections and pelvic inflammatory disease (PID), and on whether or not treatment of BV during pregnancy to reduce preterm delivery should be recommended. The reviewed studies do not lend unequivocal support to an endogenous or exogenous transmission of the bacteria present in BV. For women undergoing gynaecological surgery such as therapeutic abortion, the relative risk of postoperative infection is clearly elevated (approx. 2.3-2.8). A weaker association exists between BV and pelvic inflammatory disease. Data on treatment of BV as a way of reducing preterm delivery are inconclusive and do not support recommendations for general treatment of BV during pregnancy. The discrepant associations between BV and preterm birth found in recent studies may be explained by variations in immunological response to BV. Genetic polymorphism in the cytokine response--both regarding the TNF alleles and in interleukin production--could make women more or less susceptible to BV, causing different risks of preterm birth. Thus, studies on the vaginal inflammatory response to microbial colonization should be given priority.

The role of human papillomavirus in cervical adenocarcinoma carcinogenesis.

Human papillomavirus (HPV) is considered the single most important co-factor in the development of cervical squamous cell carcinomas. Adenocarcinomas of the cervix are also related to HPV, but the correlation is reported to be less pronounced. In the present study, 131 cervical adenocarcinomas were identified through the Swedish Cancer Registry, examined morphologically and then analysed with sensitive polymerase chain reaction (PCR)-based HPV methods for a study of age-related prevalence of HPV. HPV was identified in 64% of the tumours after PCR amplification of the HPV L1 gene only and in 71% following PCR amplification of both the L1 and E6 genes of HPV. HPV 18 was the most prevalent (52%), followed by HPV 16 (33%) and other types of HPV (15%). The prevalence of HPV was shown to be age-dependent. In women younger than 40 years, HPV was present in 89%, whereas in women 60 years and older, HPV was observed in only 43%. The difference was statistically significant, P<0.005. The HPV-positive adenocarcinomas were represented by an age distribution similar to that of cervical squamous carcinomas with a maximum age, in the 40-49 year old group, whereas the frequency of HPV-negative adenocarcinomas increased with age, typical of most carcinomas occurring in elderly women.

Formation of N-acyl-phosphatidylethanolamines and N-acetylethanolamines: proposed role in neurotoxicity.

The formation of N-acyl-phosphatidylethanolamine (NAPE) and N-acylethanolamine (NAE), including anandamide, in mammals in relation to neurotoxicity is discussed. Data on the characterization of the NAPE-forming N-acyltransferase, the NAPE-hydrolyzing phospholipase D, and the NAE-hydrolyzing amidase are reviewed. We suggest that NAPE and NAE, including anandamide, are formed in neurons in response to the high intracellular calcium concentrations that occur in injured neurons, e.g. due to glutamate excitotoxicity. NAPE may have functions of its own besides being a precursor for NAE. The formation of both of these lipids may serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes, or mediated by activation of cannabinoid receptors. This suggestion implies that NAPE and NAE may have pathophysiological roles in the brain. Whether these lipids also have physiological roles is uncertain.

The subcellular localization of phospholipase D activities in rat Leydig cells.

Rat Leydig cells contain a phospholipase D (PLD), which can be activated by vasopressin and phorbol ester. In order to clarify which Leydig cell organelles that express PLD activity, the subcellular localization of two differently regulated PLD activities was investigated by subcellular fractionation on a 40% (v/v) self-generating Percoll gradient. PLD activities in broken cells were estimated using radiolabeled didecanoylphosphatidylcholine as a substrate. Initial experiments revealed the presence of an oleate Mg2+ -activated PLD and a phosphatidylinositol 4,5-bisphosphate-activated PLD (PIP2-PLD) in the microsomal fraction of Leydig cells. The latter activity could be further stimulated by recombinant nonmyristoylated ADP ribosylating factor 1 (ARF1) plus GTPgammaS. The peak of oleate Mg2+ -PLD activity colocalized with the plasma membrane marker, whereas the highest specific activity of the PIP2-PLD activity was found in fractions with a slightly lower density than those containing the plasma membrane and trans-Golgi marker enzymes. In order to localize phorbol ester-stimulated PLD activity in intact Leydig cells, the cells were prelabeled with [14C]-palmitate and then stimulated for 15 min with 100 nM 4-beta-phorbol-12-myristate-13-acetate (PMA) in the presence of ethanol or butanol. The PLD product [14C]-phosphatidylethanol, expressed as the percentage of total labeled phospholipids in the fraction, was slightly increased in all Percoll fractions and showed a prominent peak in the fractions containing plasma membrane, trans-Golgi, and fractions of slightly lower density. The PMA-induced formation of [14C]-phosphatidylbutanol could be inhibited dose-dependently with brefeldin A suggesting that the activation of PLD by the phorbol ester was mediated by ARF.

Use of Probemix and OmniProbe biotinylated cDNA probes for detecting HPV infection in biopsy specimens from the genital tract.

AIMS: To compare two commercially available pan probes for the identification of human papillomavirus (HPV) DNA expression in histological sections and to type the HPV positive cases. METHODS: 97 formalin fixed, paraffin wax embedded biopsy specimens from the genital tract were tested for HPV positivity with in situ hybridisation using biotinylated cDNA pan probes--Probemix (Enzo) and OmniProbe (Digene). The HPV positive cases were further tested with HPV types 6/11, 16/18, and 31/33/35/51, and the HPV type was related to the histological diagnosis. Formalin fixed, HeLa cells (10-50 HPV 18 copies per cell) and SiHa cells (1-2 HPV 16 copies per cell) were used as reference cell lines. RESULTS: 32% of the specimens gave positive nucleic signals with both Probemix and OmniProbe. Of these, 84% could be further characterised with regard to HPV types 6/11, 16/18, and 31/33/35/51; 4% of all cases were positive with either Probemix or OmniProbe. The concordance of these probes was high, 96% altogether. HeLa cells stained positive but SiHa cells did not. CONCLUSION: There is no difference between Probemix and OmniProbe for the general detection of HPV. The mean detection limit of these probes is about 20 copies a cell.

Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison.

BACKGROUND: The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for triaging women with mild to moderate dysplasia. AIMS: To determine the intermethod agreement between different GP5+/6+ and MY09/11 PCR based protocols for the detection and typing of high risk (HR) HPV DNA in cervical smears and to assess the intramethod reproducibility of the GP5+/6+ PCR enzyme immunoassay (EIA) for HR-HPV detection. METHODS: For the intermethod comparison, crude aliquots of 20 well characterised cervical smears comprising five HPV negative samples, and six and nine samples containing single and multiple HPV infections, respectively, were coded and sent from reference laboratory (A) to three other laboratories. One of these (laboratory B) used the GP5+/6+ PCR-EIA and was provided with standard protocols. Another laboratory (C) used GP5+/6+ PCR combined with sequence analysis and type specific PCR, whereas two laboratories (D and E) used MY09/11 PCR followed by restriction fragment length polymorphism (RFLP) analysis for the detection and typing of HR-HPV. The intramethod agreement of GP5+/6+ PCR-EIA was analysed in a subsequent study with four other laboratories (F to I) on crude aliquots of 50 well characterised cervical smears, consisting of 32 HR-HPV positive and 18 HPV negative samples. Standardised protocols, primers, and probes were also provided by the reference laboratory for HR-HPV detection. RESULTS: In the intermethod comparison, pairwise agreement of the different laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% (kappa values: 0.5 to 1). Typing data revealed a broader range in pairwise agreement rates between 32% and 100%. The highest agreement was found between laboratories A and B using standardised protocols and validated reagents. In the intramethod evaluation, pairwise comparison of the laboratories F to I with reference laboratory A revealed excellent agreement rates from 92% to 100% (kappa values: 0.88 to 1.0) with an overall sensitivity of 97.5% (195/200) and specificity of 99.5% (199/200). CONCLUSIONS: The detection of HR-HPV as a group is highly reproducible with GP5+/6+ PCR-EIA provided that standardised protocols and validated reagents are used.

In situ hybridization for the detection of human papillomavirus (HPV) in gynaecological biopsies. A study of two commercial kits.

Human papillomavirus (HPV) detection in biopsies from the lower genital tract may be requested by clinicians as a complement to ordinary histopathological diagnosis. In the present study, two commercial kits, (Enzo Diagnostics Inc., New York, USA and Biohit, Helsinki, Finland) used for in situ hybridization with biotinylated c-DNA probes were compared and the HPV-expression was evaluated in relation to histopathological findings. The Enzo kit identifies HPV-types 6/11, 16/18, 18 and 31/33/51, whereas the Biohit kit has separate probes for HPV 6, 11, 16, 18, 31 and 33, but none for HPV 51. The usefulness of a general probe (probemix) for the visualization of HPV irrespective of type (Enzo Diagnostics Inc.) was also studied Altogether 226 biopsies from the lower female genital tract were formalin-fixed, paraffin-embedded and processed for routine histopathological grading. Consecutive sections were employed for in situ hybridization. 50 biopsies were subject to double-testing with Enzo and Biohit, whereas 176 were tested with Enzo only. Of the double-tested biopsies, 30% displayed a nuclear staining with the Enzo kit and 28% with the Biohit kit. It is concluded that the probes of these two kits have the same sensitivity in detecting HPV in tissue sections. Condylomata acuminata were HPV-positive in 81%, mostly for types 6/11. Flat condylomas were HPV-positive in 35%. The HPV-positivity of biopsies with low grade SIL (I) was 50% and that of high grade SIL (II and III) was 37%. High grade SIL contained either HPV-types 16/18 or 31/33/51. A correlation was found between the occurrence of koilocytosis and the presence of HPV-DNA. HPV-expression was most easily visualized in condylomata acuminata. In epithelium of normal appearance or with inflammatory alterations HPV-DNA was not seen.

Human papillomavirus. Subclinical and atypical manifestations.

Subclinical HPV infections, together with latent infections, are probably the most likely outcome after exposure to HPV. Subclinical infection is associated with symptoms such as burning, fissuring, and dyspareunia in some patients. Only these patients should be offered treatment. Diagnosing and treating asymptomatic HPV infection cannot be recommended until better knowledge about the infectious potential of the infection in that phase is obtained. Recently, results have been presented showing a median duration of HPV infection of only 8 months, and after 24 months, only 9% of the women studied continued to be infected. This provides the possibility to reassure patients with HPV infection that it is most likely a transient infection, and one should not worry unduly. In light of this knowledge, it seems unwise to diagnose an asymptomatic infection for which no effective treatment is available and for which the natural history and consequences remain unclear; however, if these lesions were found to have the potential to transmit HPV, the patient should be so counseled.