In-vivo efficacy of alcohol-based hand rubs against noroviruses: a novel standardized European test method simulating practical conditions.

BACKGROUND: Non-enveloped viruses are particularly resistant to disinfectants, so it is necessary to use disinfectants with proven virucidal activity in order to prevent and control the spread of viral infections. However, a test such as EN 1500, which uses an internal standard as the reference treatment for determining the bactericidal efficacy of hand rubs, is still lacking. This study aimed to establish a European standard for testing the in-vivo efficacy of hand rubs against non-enveloped viruses. METHODS: The concentration and mode of application of ethanol as the reference were determined, and compared with the efficacies of two commonly used hand rubs. The hands of volunteers were contaminated with murine norovirus strain S99. RESULTS: 70% wt/wt ethanol (2 x 3 mL in 2 x 30 s) was used as the internal reference treatment. The commercial ethanol-based hand rub was able to reduce the titre of murine norovirus significantly in 30 s, whereas a hand rub based on ethanol and propan-2-ol was significantly less effective compared with the reference. CONCLUSION: This study established a possible standard for testing the in-vivo efficacy of hand rubs against non-enveloped viruses using murine norovirus, a low contamination volume technique and ethanol as the internal reference. These findings need to be confirmed in European ring trials.

Evaluation of the substitution of poliomyelitis virus for testing virucidal activities of instrument and surface disinfection.

The Global Polio Eradication initiative has the goal to eradicate poliomyelitis worldwide. This means that poliomyelitisvirus type 1 strain LSc 2ab (PV-1) can no longer be used for the evaluation of virucidal activity of chemical disinfectants. This study evaluated murine parvovirus ATCC VR 1346 (minute virus of mice) as suitable surrogate for PV-1 when testing virucidal activity of biocides in instrument and surface disinfectants. Suspension testing in different laboratories with two commercially available active biocidal substances based on glutaraldehyde (0.01-0.25%) and peracetic acid (0.005-0.1%) with an exposure time of 30 min was performed. Both pathogens showed comparable susceptibility and dose-dependent reduction of virus titres following German and European Guidelines.

Virucidal efficacy of different formulations for hand and surface disinfection targeting SARS CoV-2.

In the ongoing SARS CoV-2 pandemic, effective disinfection measures are needed, and guidance based on the methodological framework of the European Committee for Standardization (CEN) may enable the choice of effective disinfectants on an immediate basis. This study aimed to elucidate whether disinfectants claiming 'virucidal activity against enveloped viruses' as specified in the European Standard EN 14476 as well as in the German Association for the Control of Viral Diseases/Robert Koch Institute (DVV/RKI) guideline are effectively inactivating SARS-CoV-2. Two commercially available formulations for surface disinfection and one formulation for hand disinfection were studied regarding their virucidal activity. Based on the data of this study the enveloped SARS-CoV-2 is at least equally susceptible compared to the standard test virus vaccinia used in the EN 14476 and DVV/RKI guidelines. Thus, chemical disinfectants claiming 'virucidal activity against enveloped viruses' based on the EN 14476 and DVV/RKI guidelines will be an effective choice to target enveloped SARS-CoV-2 as a preventive measure.

Interlaboratory reproducibility of a test method following 4-field test methodology to evaluate the susceptibility of Clostridium difficile spores.

BACKGROUND: Sporicidal surface disinfection is recommended to control transmission of Clostridium difficile in healthcare facilities. EN 17126 provides a method to determine the sporicidal activity in suspension and has been approved as a European standard. In addition, a sporicidal surface test has been proposed. AIM: To determine the interlaboratory reproducibility of a test method for evaluating the susceptibility of a C. difficile spore preparation to a biocidal formulation following the 4-field test (EN 16615 methodology). METHODS: Nine laboratories participated. C. difficile NCTC 13366 spores were used. Glutaraldehyde (1% and 6%; 15 min) and peracetic acid (PAA; 0.01% and 0.04%; 15 min) were used to determine the spores' susceptibility in suspension in triplicate. FINDINGS: One-percent glutaraldehyde revealed a mean decimal log10 reduction of 1.03 with variable results in the nine laboratories (0.37-1.49) and a reproducibility of 0.38. The effect of 6% glutaraldehyde was stronger (mean: 2.05; range: 0.96-4.29; reproducibility: 0.86). PAA revealed similar results. An exemplary biocidal formulation based on 5% PAA was used at 0.5% (non-effective concentration) and 4% (effective concentration) to determine the sporicidal efficacy (4-field test) under clean conditions in triplicate with a contact time of 15 min. When used at 0.5% it demonstrated an overall log10 reduction of 2.68 (range: 2.35-3.57) and at 4% of 4.61 (range: 3.82-5.71). The residual contamination on the three primarily uncontaminated test fields was <50 cfu/25 cm2 in one out of nine laboratories (0.5%) and in seven out of nine laboratories (4%). CONCLUSION: The interlaboratory reproducibility seems to be robust.

Intimate partner violence in early adolescence:the role of gender; socioeconomic factors and the school

BACKGROUND:Intimate partner violence (IPV) among adolescents is common worldwide; but our understanding of perpetration; gender differences and the role of social-ecological factors remains limited.OBJECTIVES:To explore the prevalence of physical and sexual IPV perpetration and victimisation by gender; and associated risk and protective factors.METHODS:Young adolescents (N=2 839) from 41 randomly selected public high schools in the Western Cape region of South Africa (SA); participating in the PREPARE study; completed a self-administered questionnaire. RESULTS:The participants' mean age was 13.65 years (standard deviation 1.01); with 19.1% (541/2 839) reporting being victims/survivors of IPV and 13.0% (370/2 839) reporting perpetrating IPV. Girls were less likely to report being a victim/survivor of physical IPV (odds ratio (OR) 0.72; 95% confidence interval (CI) 0.57 - 0.92) and less likely to be a perpetrator of sexual IPV than boys (OR 0.33; 95% CI 0.21 - 0.52). Factors associated with perpetration of physical and sexual IPV were similar and included being a victim/survivor (physical IPV: OR 12.42; 95% CI 8.89 - 17.36; sexual IPV: OR 20.76; 95% CI 11.67 - 36.93); being older (physical IPV: OR 1.26; 95% CI 1.08 - 1.47; sexual IPV: OR 1.36; 95% CI 1.14 - 1.62 ); having lower scores on school connectedness (physical IPV: OR 0.59; 95% CI 0.46 - 0.75; sexual IPV: OR 0.56; 95% CI 0.42 - 0.76) and scoring lower on feelings of school safety (physical IPV: OR 0.66; 95% CI 0.57 - 0.77; sexual IPV: OR 0.50; 95% CI 0.40 - 0.62).CONCLUSIONS:Physical and sexual IPV was commonly reported among young adolescents in SA. Further qualitative exploration of the role of reciprocal violence by gender is needed; and the role of 'school climate'-related factors should be taken into account when developing preventive interventions

Comparison of the novel ResPlex III assay and existing techniques for the detection and subtyping of influenza virus during the influenza season 2006-2007.

Influenza virus is a major cause of disease worldwide. The accurate detection and further subtyping of influenza A viruses are important for epidemiologic surveillance, and subsequent comprehensive characterization of circulating influenza viruses is essential for the selection of an optimal vaccine composition. ResPlex III is a new multiplex reverse transcriptase polymerase chain reaction (RT-PCR)-based method for detecting, typing, and subtyping influenza virus in clinical specimens. The ResPlex III assay was compared with other methods with respect to sensitivity and accuracy, using 450 clinical specimens obtained from subjects throughout Germany during the 2006-2007 influenza season. Samples were analyzed for the presence of influenza virus in Madin-Darby canine kidney (MDCK) cells by rapid cell culture using peroxidase staining and conventional cell culture confirmed by hemagglutination inhibition assay, a rapid diagnostic assay (Directigen Flu A+B test; BD Diagnostic Systems, Heidelberg, Germany), in-house real-time RT-PCR (RRT-PCR), and ResPlex III (Qiagen, Hilden, Germany). ResPlex III had the highest sensitivity for detecting influenza virus in clinical specimens, followed by in-house RRT-PCR (96% compared with ResPlex III). Conventional cell culture in MDCK cells, rapid culture, and quick test assays were substantially less sensitive (55%, 72%, and 39%, respectively). Virus subtyping results were identical using ResPlex III and the standard virological subtyping method, hemagglutination inhibition. ResPlex III is a quick, accurate, and sensitive assay for detecting and typing influenza A and B viruses and subtyping influenza A viruses in clinical specimens, and might be considered for a supplemental role in worldwide seasonal and pandemic influenza surveillance.

[Epidemiological aspects of gastrointestinal infections]. / Epidemiologische Aspekte gastrointestinaler Infektionen.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate seasonal patterns and age-associated trends of the main bacterial, viral, and parasitic enteric pathogens in Southwest Germany. PATIENTS AND METHODS: From January 2002 through December 2008 a total of 99,057 patients were tested for Norovirus, Rotavirus, bacterial pathogens, Cryptosporidium parvum (C. parvum), and Giardia lamblia (G. lamblia). RESULTS: All these pathogens were detected throughout the whole year. But there were distinctive seasonal patterns of activity of the following pathogens being detected: norovirus was detected mainly from September through April. The highest rotovirus activity was observed from December through June. But bacterial pathogens und C. parvum were found mainly from June to November. The percentage of positive results during the months with the highest activity was 10 - 49% for norovirus, 25% - 41% for rotavirus, 14 - 18% for bacterial infection and 3 - 4 % for C. parvum. G. lamblia and adenovirus were found throughout the year in 7 - 15% and 3 - 10% of samples, respectively. Moreover, the detection rate of different pathogens depended on patient age. In infants younger than one year, rotavirus, norovirus and adenovirus were most frequently isolated pathogenes. Stool samples from kindergarden- and school-age children were positive largely for bacterial pathogens such as Salmonella and Campylobacter particularly in late summer or early autum. In patients older than 60 years, norovirus, rotavirus, and toxin producing Clostridium difficile strains were the most common pathogens. CONCLUSIONS: In view of the age and season related frequency of detection of enteric pathogens, a step-by-step diagnosis of gastrointestinal tract infections is recommended. Considering that most pathogens are detected sporadically over the whole year, the analysis of negative samples should be appropriately expanded. The knowledge of seasonal occurrence can also be applied to improve the application of hygienic measures.

Laboratory-based assessment of influenza in German ambulant patients from 1998 to 2008.

BACKGROUND: In Germany, the cost for PCR diagnosis of influenza in ambulant patients was not covered by the national statutory health insurance system until 2008. Therefore, cell culture was the standard method applied for routine diagnosis. We have prospectively compared a 1-day rapid cell culture assay (RCA) with conventional cell culture (CCC) during the influenza seasons from 1997/1998 to 2007/2008 and with real-time PCR analysis during the influenza seasons 2003/2004 and 2006/2007. PATIENTS AND METHODS: This study is based on 4,262 respiratory samples obtained from ambulant patients between January 1998 and May 2008. The RCA was performed in microtiter plates that were stained with monoclonal antibodies to influenza virus A and B 16 h after inoculation. RESULTS: A total of 1,221 specimens were found to be positive by the cell culture methods - 1,143 (93.6%) by the RCA and 1,012 (82.9%) by the CCC. The sensitivity of the RCA and CCC versus PCR was 75.4% (221/293) and 58% (170/293), respectively. The specificity of both cell culture assays versus PCR was 100%. Influenza A represented 79.3% of the cases diagnosed. An increased activity of influenza was observed between January and March, with the rate of influenza-positive cases being highest for kindergarten and school-aged children. CONCLUSION: While PCR is the most sensitive assay for the diagnosis of influenza, the RCA can still be used for diagnosis and surveillance of this disease. Based on our findings and given the known fact that influenza antibodies reach a plateau 2-4 weeks after immunization, the optimal time for vaccination in Germany is from October through November. Kindergarten and school-aged children represent an important reservoir of infection. Consequently, routine immunization should be considered for this age group to prevent the spread of influenza.

Deficiencia somatotrófica en niños: características clínicas y bioquímicas según la etiología en 75 casos / Somatotrophic deficiency in children: report of 75 cases

Somatotrophic deficiency (SDMT) can be due to a deficiency of growth hormone releasing hormone(GHRH), growth hormone (GH) or insulin like growth factor I (IGF-I). Although its clinical features have been thoroughly described, the diagnosis is still controversial. Now there is an effective treatment with GH or IGF-I for these patients. AIM: To analyze the main clinical, etiological and laboratory characteristics of 75 SD patients (44 males), aged 9.4 + 4.5 years, with severe growth retardation. The diagnosis was confirmed by the lack of response to two GH stimulation tests (Clonidine, Glugagon or Insulin) and low levels of IGF-I or insulin-like growth factor binding protein- 3 (IGFBP-3). RESULTS: In 34 patients (46 percent), the cause of DSMT was considered idiopathic (DSMT-I), in 31 (41 percent) there was an organic cause (DSMT-O), most commonly caused by malformations or pituitary tumors and in 10 (13 percent), it was genetic (DSMT-G) (three patients with Laron's Syndrome, five with mutations of GH gene and 2 with probable mutations of Prop-1 and Pit-1 genes). IGF-1 levels, were significantly lower in DSMT-O and DSMT-G thanin DSMT-I (21.2 +/- 46.1, 23.4 +/-30.3 ng/mL and 50.2 +/- 48.3 ng/mL, respectively). The lowest height score corresponded to DSMT-G, compared to DSMT-O and DSMT (­5.7 +/- 0.9, -4.0 +/- 1.6 and ­4.3 +/- 1.2 DS, respectively) CONCLUSIONS: The high percentage of organic and genetic etiologies in our patients can be due to the systematic search of these diseases. DSMT-G (Laron, mutations in GH and Pit-1 genes) had the most severe growth retardation.

Study of GH sensitivity in chilean patients with idiopathic short stature.

We hypothesized that some children with idiopathic short stature in Chile might bear heterozygous mutations of the GH receptor. We selected 26 patients (3 females, 23 males) from 112 patients who consulted for idiopathic short stature at the University of Chile. Their chronological age was 8.3 +/- 1.9, and bone age was 6.1 +/- 1.0 yr. Their height was -3.0 +/- 0.7 SDS; IGF-I, -1.2 +/- 1.1 SD; IGF binding protein 3, -0.7 +/- 2.0 SDS; and GH binding protein, 0.4 +/- 0.8 SDS. Patients were admitted, and blood samples were obtained every 20 min to determine GH concentrations overnight. Coding sequences and intron-exon boundaries of exons 2-10 of GH receptor gene were amplified by PCR and subsequently analyzed through single-strand conformational analysis. Mean serum GH concentration, over 12-h, was 0.20 +/- 0.08 nM; pulse amplitude, 0.40 +/- 0.15 nM; number of peaks, 5.8 +/-1.5 peaks/12 h; peak value of GH during the 12-h sampling, 1.03 +/- 0.53 nM; and area under the curve, 151.4 +/- 56.1 nM/12 h. There were positive correlations between mean GH vs. area under the curve (P < 0.001) and GH peak (P < 0.01). The single-strand conformational analysis of the GH receptor gene showed abnormal migration for exon 6 in 9 patients and for exon 10 in 9 patients, which (by sequence analysis) corresponded to 2 polymorphisms of the GH receptor gene: an A-to-G transition in third position of codon 168 in exon 6 and a C-to-A transversion in the first position of codon 526 in exon 10. We further sequenced all coding exons and intron-exon boundaries in the most affected patients (nos. 6, 9, 11, 14, 15, 16, and 23). This analysis revealed a C-to-T transition in codon 161 of exon 6 in patient 23, which results in an amino acid change (Arg to Cys) in an heterozygous form in the patient and his father. In conclusion, the results of our study suggest that, in Chilean patients with idiopathic short stature, GH receptor gene mutations are uncommon, although we cannot exclude mutations that were missed by single-strand conformational analysis or mutations within introns or in the promoter regions of the GH receptor gene.

Kaposi's sarcoma-associated herpesvirus serology in Europe and Uganda: multicentre study with multiple and novel assays.

A multicentre study was undertaken to define novel assays with increased inter-assay concordance, sensitivity, specificity and predictive value for serological diagnosis of human herpesvirus type 8 (HHV-8) infection. A total of 562 sera from European and Ugandan human immunodeficiency virus (HIV)-infected or uninfected individuals with or without Kaposi's sarcoma (KS) and blood donors were examined under code by 18 different assays in seven European laboratories. Sera from KS patients and all non-KS sera found positive by at least 70%, 80%, or 90% of the assays were considered "true positive." The validity of the assays was then evaluated by univariate logistic regression analysis. Two immunofluorescence assays (IFA) for detection of antibodies against HHV-8 lytic (Rlyt) or latent (LLANA) antigens and two enzyme-linked-immunosorbent assays (ELISA) (M2, EK8.1) for detection of antibodies against HHV-8 structural proteins were found to be highly concordant, specific, and sensitive, with odds ratios that indicated a high predictive value. When used together, the two IFA (Rlyt-LLANA) showed the best combination of sensitivity (89.1%) and specificity (94.9%). The performance of these assays indicate that they may be used for the clinical management of individuals at risk of developing HHV-8 associated tumours such as allograft recipients.

Use of recombinant glycoprotein antigens gB and gH for diagnosis of primary human cytomegalovirus infection during pregnancy.

The major risk factor for intrauterine transmission of human cytomegalovirus (HCMV) is a primary infection during pregnancy. The neutralizing antibody response appeared after an average of 13 weeks after seroconversion and therefore the absence of neutralizing titers in HCMV IgG positive pregnant women is a reliable marker for primary infection. Determination of neutralizing antibody, however, is time-consuming and labor-intensive. For this reason an immunoblot assay for detection of neutralizing antibodies was developed based on the use of recombinant antigens representing neutralizing epitopes of glycoproteins (gp) gB (gpUL55) and gH (gpUL75) of HCMV. In this study, 93.6% of sera of pregnant women with prior infection recognized the gp-specific epitopes corresponding to a nonresponder rate of 6.4% relative to the neutralizing antibody. In primary infection the gp-response in general coincided with the appearance of neutralizing antibody. Intriguingly, lack of HCMV gB-specific antibodies was correlated with a lower risk of intrauterine fetal infection (P < 0.05).

Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system.

BACKGROUND: Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products. OBJECTIVES: to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines. STUDY DESIGN: 73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay). RESULTS: with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24-48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load. CONCLUSIONS: LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.

PA28alphabeta double and PA28beta single transfectant mouse B8 cell lines reveal enhanced presentation of a mouse cytomegalovirus (MCMV) pp89 MHC class I epitope.

PA28 is an interferon-gamma inducible modulator of proteasome function composed of two subunits, i.e. PA28alpha and PA28beta. Previously we showed that stabile overexpression of the PA28alpha subunit alone supported MHC class I antigen presentation of two viral epitopes. However, no information was obtained on the consequences when PA28alpha and PA28beta function in concert or when PA28beta is overexpressed on its own. Here we demonstrate that overexpression of PA28alpha and beta together is similarly efficient in supporting MHC class I antigen presentation of the MCMV pp89 9mer epitope as PA28alpha alone, excluding a potentially potentiating role of PA28beta. Surprisingly, and despite the fact that PA28beta alone was thought to be inactive and to only stabilize PA28 activity, overexpression of PA28beta also resulted in improved antigen presentation. However, by northernblot and immunoprecipitation experiments we show that while PA28alpha is able to act alone the observed effect in the PA28beta and PA28alphabeta transfectant cell lines is due to increased levels of PA28alphabeta complexes.

Near final height in pubertal growth hormone (GH)-deficient patients treated with GH alone or in combination with luteinizing hormone-releasing hormone analog: results of a prospective, randomized trial.

To study the effects of delaying puberty in GH-deficient (GHD) children, we studied 21 GHD (9 boys, 14 girls), treatment-naive, pubertal patients in a prospective, randomized trial. Their chronological age was 14.3 +/- 1.6 yr, and their bone age was 11.3 +/- 1.1 yr (mean +/- SD) at the beginning of the study. Four patients who developed hypogonadotropic hypogonadism were subsequently excluded from the study. Patients were randomly assigned to receive GH + LH-releasing hormone analog (LHRH-A) (n = 7), or GH alone (n = 10). GH and LHRH-A treatment started simultaneously in each patient. GH (Nutropin) was administered at a dose of 0.1 U/kg x day sc, until patients reached a bone age (BA) of 14 yr in girls and 16 yr in boys, and LHRH-A (Lupron depot) was administered at a dose of 300 microg/ kg every 28 days in during 3 yr. We defined GH deficiency as patients with a growth velocity less than 4 cm/yr, BA delay more than 1 yr in relationship to chronological age, GH response to two stimulation tests less than 7 microg/L, associated with low serum insulin-like growth factor I and insulin-like growth factor binding protein 3 levels. Statistical analysis was performed by ANOVA or Kruskall Wallis when variances were not homogeneous. We observed a significant decrease in the rate of BA maturation in the group treated with GH+LHRH-A (1.5 +/- 0.2 yr) compared with the group treated with GH alone (4.2 +/-0.5 yr) during the 3 years of LHRH-A therapy (P < 0.05). This delay in BA maturation produced a significant gain in final height in the group treated with GH+LHRH-A, which reached - 1.3 +/- 0.5 SD score compared with -2.7 +/- 0.3 SD score (P < 0.05) in the group treated with GH alone. These results indicate that delaying puberty with LHRH-A in GHD children during treatment with GH increases final height.

Combination of microneutralization and avidity assays: improved diagnosis of recent primary human cytomegalovirus infection in single serum sample of second trimester pregnancy.

Estimation of IgG avidity index is a classical serological method. Antibodies with low avidity are detectable at a very early stage of infection whereas high avidity antibodies indicate past infection. Recently, it was shown that the neutralization assay can be routinely used as a reliable method for differentiating between acute primary and non-primary infection in a single serum sample because the first neutralizing titers (NT) appeared after an average of 13 weeks (range, 10-17 weeks). A low positive NT titer in the presence of specific IgM antibodies, however, still represents a diagnostic problem especially if blood sampling occurred after the 12th week of gestation. To overcome this problem the combination of NT and IgG avidity tests was evaluated. Human cytomegalovirus (HCMV) IgG avidity indices of 350 serum samples from 227 pregnant women were investigated using 6M urea in the washing buffer. HCMV specific IgG antibodies reached full maturation approximately 20-22 weeks after seroconversion and low IgG avidity is therefore a marker of primary infection. The combined application of the microneutralization and avidity assays was shown to serve as a helpful tool in diagnosis of a recent primary HCMV infection of second trimester pregnancy particularly when previous serological data were not available.