Investigation of travel-related cases in a multinational outbreak: example of the Shiga-toxin producing E. coli outbreak in Germany, May-June 2011.

Early investigation of travel-related cases in an outbreak of an emerging infectious disease can provide useful information to epidemiologists to characterize the exposure, while they may differ in demographic profiles from cases reported in the country where the outbreak has occurred. During the spring 2011 E. coli outbreak in Germany, we proposed a methodological approach to collect a minimal set of demographic and clinical data that are relatively easy to obtain and available at an early stage of an outbreak investigation. Ninety-eight STEC O104 travel-related cases were reported in a survey by seven EU countries, Switzerland, Canada and the USA. We found a mean incubation period (n = 50) of 8·5 days, which confirmed previous estimations communicated by the Robert Koch Institute. No significant association was found between the duration of the incubation period and possible demographic and clinical factors, although the older the age, the shorter the incubation period that was observed. Such approach and observations are informative for further investigations of outbreaks of enterohaemorrhagic E. coli or other emerging infectious diseases.

Initial surveillance of 2009 influenza A(H1N1) pandemic in the European Union and European Economic Area, April-September 2009.

European Union (EU) and European Economic Area (EEA) countries reported surveillance data on 2009 pandemic influenza A(H1N1) cases to the European Centre for Disease Prevention and Control (ECDC) through the Early Warning and Response System (EWRS) during the early phase of the 2009 pandemic. We describe the main epidemiological findings and their implications in respect to the second wave of the 2009 influenza pandemic. Two reporting systems were in place (aggregate and case-based) from June to September 2009 to monitor the evolution of the pandemic. The notification rate was assessed through aggregate reports. Individual data were analysed retrospectively to describe the population affected. The reporting peak of the first wave of the 2009 pandemic influenza was reached in the first week of August. Transmission was travel-related in the early stage and community transmission within EU/EEA countries was reported from June 2009. Seventy eight per cent of affected individuals were less than 30 years old. The proportions of cases with complications and underlying conditions were 3% and 7%, respectively. The most frequent underlying medical conditions were chronic lung (37%) and cardio-vascular diseases (15%). Complication and hospitalisation were both associated with underlying conditions regardless of age. The information from the first wave of the pandemic produced a basis to determine risk groups and vaccination strategies before the start of the winter wave. Public health recommendations should be guided by early capture of profiles of affected populations through monitoring of infectious diseases.

Surveillance of extensively drug-resistant tuberculosis in Europe, 2003-2007.

This paper describes the results of second-line drug (SLD) susceptibility tests among multidrug-resistant tuberculosis (MDR TB) cases reported in 20 European countries aiming to identify extensively drug-resistant tuberculosis (XDR TB) cases. A project on molecular surveillance of MDR TB cases was conducted by EuroTB and the National Institute for Public Health and the Environment (RIVM) from 2005 to 2007. Information on drug susceptibility testing (DST) was provided to this project and case-based data on MDR TB cases were reported on a quarterly basis by 20 countries of the World Health Organization s European Region, including 15 European Union Member States. Data included SLD susceptibility test results, enabling a retrospective description of XDR TB cases notified between 2003 and 2007 .In 18 countries DST was performed for two or more of the SLD included in the XDR TB definition. The proportion of MDR TB isolates tested for SLD varied widely between countries (range 20 to 100 percent). In the 18 countries, 149 (10%) XDR TB cases were reported among MDR TB cases with available DST results for SLD. Sixteen additional MDR TB cases were reported by the MDR TB surveillance system when compared with the number of routinely reported MDR TB cases to EuroTB in ten countries with representative data reported during three consecutive years (2003-2005). To counter the threat of XDR TB in Europe, a standardised approach to XDR TB surveillance and DST for SLD is needed, as well as increased laboratory capacity across European countries.

Experience and lessons from surveillance and studies of the 2009 pandemic in Europe.

Surveillance and studies in a pandemic is a complex topic including four distinct components: (1) early detection and investigation; (2) comprehensive early assessment; (3) monitoring; and (4) rapid investigation of the effectiveness and impact of countermeasures, including monitoring the safety of pharmaceutical countermeasures. In the 2009 pandemic, the prime early detection and investigation took place in the Americas, but Europe needed to undertake the other three components while remaining vigilant to new phenomenon such as the emergence of antiviral resistance and important viral mutation. Laboratory-based surveillance was essential and also integral to epidemiological and clinical surveillance. Early assessment was especially vital because of the many important strategic parameters of the pandemic that could not be anticipated (the 'known unknowns'). Such assessment did not need to be undertaken in every country, and was done by the earliest affected European countries, particularly those with stronger surveillance. This was more successful than requiring countries to forward primary data for central analysis. However, it sometimes proved difficult to get even those analyses from European counties, and information from Southern hemisphere countries and North America proved equally valuable. These analyses informed which public health and clinical measures were most likely to be successful, and were summarized in a European risk assessment that was updated repeatedly. The estimate of the severity of the pandemic by the World Health Organization (WHO), and more detailed description by the European Centre for Disease Prevention and Control in the risk assessment along with revised planning assumptions were essential, as most national European plans envisaged triggering more disruptive interventions in the event of a severe pandemic. Setting up new surveillance systems in the midst of the pandemic and getting information from them was generally less successful. All European countries needed to perform monitoring (Component 3) for the proper management of their own healthcare systems and other services. The information that central authorities might like to have for monitoring was legion, and some countries found it difficult to limit this to what was essential for decisions and key communications. Monitoring should have been tested for feasibility in influenza seasons, but also needed to consider what surveillance systems will change or cease to deliver during a pandemic. International monitoring (reporting upwards to WHO and European authorities) had to be kept simple as many countries found it difficult to provide routine information to international bodies as well as undertaking internal processes. Investigation of the effectiveness of countermeasures (and the safety of pharmaceutical countermeasures) (Component 4) is another process that only needs to be undertaken in some countries. Safety monitoring proved especially important because of concerns over the safety of vaccines and antivirals. It is unlikely that it will become clear whether and which public health measures have been successful during the pandemic itself. Piloting of methods of estimating influenza vaccine effectiveness (part of Component 4) in Europe was underway in 2008. It was concluded that for future pandemics, authorities should plan how they will undertake Components 2-4, resourcing them realistically and devising new ways of sharing analyses.

An increase in newly diagnosed HIV cases reported among men who have sex with men in Europe, 2000-6: implications for a European public health strategy.

OBJECTIVE: To present HIV surveillance data on men who have sex with men (MSM) in the European Union (EU) and European Free Trade Association (EFTA) countries for the period 2000-6. METHODS: Data from three sources, HIV reporting, AIDS reporting and HIV prevalence studies, were collated by EuroHIV and analysed for 27 EU and three EFTA countries. RESULTS: In 2006, 7693 newly diagnosed HIV infections among MSM were reported (56.7 per million men aged 15-64 years). In 23 countries with data for 2000-6, the number of new HIV diagnoses increased by 86% from 3003 to 5571. In 20 countries reporting individual HIV cases between 2000 and 2006, the median age at HIV diagnosis remained unchanged (36 years), whereas the proportion of MSM presenting with an AIDS-defining illness at the time of HIV diagnosis declined from 25% in 2000 to 10% in 2006 (chi2 = 85.7, p<0.001). In 30 countries reporting AIDS, incidence among MSM decreased by 40% from 2422 in 2000 to 1445 in 2006 and the number of deaths decreased by 57% from 876 to 373. Reported HIV prevalence ranged between 8% and 68% among MSM with sexually transmitted infections, between 10% and 18% among those recruited in community settings, but remained <10% in central Europe and Ireland. CONCLUSIONS: Whereas the decreasing rates of AIDS diagnoses and AIDS deaths reflect relatively good access to therapy, the increasing numbers of new HIV diagnoses and relatively high prevalence of HIV among MSM suggest the need for Europe-wide HIV prevention among MSM.

Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) case reporting in the World Health Organization European Region in 2006.

This article presents information on HIV and AIDS case reporting systems as part of a survey on HIV/AIDS surveillance practices in the World Health Organization (WHO) European Region. A standardised questionnaire was sent to the 53 national correspondents of the European Centre for the Epidemiological Monitoring of AIDS(EuroHIV). The HIV and AIDS case reporting section of the questionnaire comprised four parts: data collection system, HIV/AIDS case definition for surveillance, variables collected, and evaluation of surveillance systems). Individual-based data collection systems for HIV case reports have been implemented in 43 of 44 countries in the WHO European Region and for AIDS case reports in all the countries. For HIV case reports, a coded identifier is used in 28 countries, and full names are used in 11 countries. The European AIDS case definition has been adopted in 35 countries(80%). Information on molecular epidemiology is available in 30 countries, and HIV drug resistance is monitored in 11 countries.HIV/AIDS case reporting systems have been evaluated for underreporting in 17 countries and for completeness in 11 countries.This article outlines the future needs for HIV/AIDS surveillance and presents recommendations on how to improve data comparability across European countries in the WHO region.

Infectious risk associated with wastewater reuse: an epidemiological approach applied to the case of Clermont-Ferrand, France.

Wastewater reuse raises the question of health risk and the epidemiological surveys needed. An epidemiological and environmental approach was used to check the security for the exposed populations (surrounding and agricultural population in Clermont-Ferrand. Four information systems were set up: two sentinel systems joining general physicians (15) and pharmacists (7) for the surrounding population and two follow-up surveys among field workers and farmers. Water quality monitoring and a study of aerosols from spray irrigation were performed. No epidemic event was identified with only some case clusters (not related to water exposure) being observed. All the declared cases were benign. The workers' survey underlined a substantial incidence of nettle rashes, itchy skins, sunburns, and cuts. The follow-up study among farmers and their families did not reveal any particular phenomena. The bacteriological quality of treated wastewater throughout conformed with the recommendations of the Superior Council of Public Health of France (1,000 faecal coliforms/100 mL). No faecal bacteria were observed in aerosols with water concentrations equal to 10(3) cfu/100 mL and an exposure time of 20 min. The survey of such an irrigation system, towards potential and actual risks, required the conjunction of different epidemiological information sources and microbiological data.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2(-/-) mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2(-/-) mice, we used suppressive subtractive hybridization between livers from Usf2(-/-) and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2(-/-) hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Deletion of chromosomal region 13q14.3 in childhood acute lymphoblastic leukemia.

Deletion of the 13q14 chromosomal region is frequent in B cell chronic lymphocytic leukemia (B-CLL) and is believed to inactivate a tumor supressor gene (TSG) next to RB1. We studied microsatellite markers spanning the 13q14 chromosomal region in 138 children with acute lymphoblastic leukemia (ALL). Allelic loss was demonstrated in six cases (4.3%). Deletion did not include RB1 in two cases. In five patients, the deleted region overlapped that described in B-CLL. A sixth patient harbored a smaller deletion, slightly more telomeric than minimal deleted regions reported in B-CLL. Apparent differences in the delineation of the minimal deleted region could be due to the fact that the putative TSG is a very large gene, with some deletions affecting only a part of it. Our present findings suggest that at least some of its exons lie within a region of less than 100 kb more telomeric that previously thought.

Efficacy and tolerability of entacapone as adjunctive therapy to levodopa in patients with Parkinson's disease and end-of-dose deterioration in daily medical practice: an open, multicenter study.

Entacapone is a potent, reversible and orally active inhibitor of catechol-O-methyltransferase. This open multicenter study evaluated the efficacy, safety and tolerability of entacapone as adjunct therapy to levodopa/dopa decarboxylase inhibitor (> or = 3 daily doses) in patients with idiopathic Parkinson's disease and end-of-dose motor fluctuations. The 8-week study included 489 patients under conditions of typical daily medical practice. Patients were treated with a 200-mg fixed dose of entacapone administered with each scheduled dose of levodopa to a maximum of 10 doses per day. Other antiparkinsonian medication should have been stable for at least 1 month. The primary efficacy criteria were: (1) Part II (activities of daily living, ADL) of the Unified Parkinson's Disease Rating Scale (UPDRS), (2) the reduction of 'off' time during the daily waking period as assessed by the percentage of patients improving by at least one category at Item 39 of Part IV of the UPDRS. Secondary outcome measures included: (1) the investigator's global assessment of change, (2) quality of life (QoL) was assessed using the Parkinson's Disease Questionnaire (PDQ-39). Adverse events, vital signs and liver enzymes were monitored at weeks 2 and 8. The baseline mean score for ADL was 10.5 (+/-7.04), which decreased to 8.5 (+/-6.37) at the end of the study (p < 0.0001). Compared to baseline, 40.8% of patients experienced a reduction in 'off' time during the waking period; this improvement was highly significant (p < 0.0001). A reduction in the daily dose of levodopa was observed in 35.8% of patients (mean decrease 209 +/- 149 mg). QoL was improved by a mean of 10% in all categories of the PDQ-39 (p < 0.001), except social support and cognition. This improvement was statistically significant (p < 0.001). The dyskinesia score (UPDRS Item 32) was decreased significantly from 2.3 to 2.1 from baseline to end of study (p < 0.001), although 52.7% of patients reported levodopa-induced dyskinesia as an adverse event. There was no case of increased liver enzymes. The study results confirm that the excellent risk/benefit ratio seen in phase III controlled studies can be seen in daily neurological practice. Moreover, the study suggests that the benefits of entacapone are associated with a significant improvement in QoL.

A 5' splice region G-->C mutation in exon 3 of the human beta-spectrin gene leads to decreased levels of beta-spectrin mRNA and is responsible for dominant hereditary spherocytosis (spectrin Guemene-Penfao).

We studied a family with autosomal dominant hereditary spherocytosis (HS) associated with a mild spectrin deficiency. Linkage analysis using two microsatellite markers (D14S63 and D14S271) very close to the beta-spectrin gene (SPTB) showed that HS co-segregated with alleles of these microsatellite markers and the linkage between the marker and HS was statistically significant. The presence of a beta-spectrin protein polymorphism (beta-spectrin Vay; A1880V) in trans of the HS allele was not itself deleterious, but allowed the detection of decreased membrane expression of the spherocytic beta-spectrin allele in two HS-affected subjects. Direct sequencing of the coding exons of the beta-spectrin gene in one affected subject showed the presence of a G-->C transversion at the terminal nucleotide of exon 3, which did not change the leucine codon 100 (CTG-->CTC). The presence of the mutation was confirmed by restriction enzyme digestion at the DNA level in all affected SH members of the family. The G-->C mutation severely reduced the utilization of the 5' splice site and resulted in aberrant mRNA splicing with intron 3 retention.

Analysis of ETV6 and ETV6-AML1 proteins in acute lymphoblastic leukaemia.

The t(12;21) is a recurring chromosomal abnormality in acute lymphoblastic leukaemias (ALLs) which results in the production of an ETV6-AML1 fusion gene. The association between t(12;21) and the deletion of the untranslocated allele of ETV6 is among the most frequent abnormalities observed in B-lineage ALLs in children. In order to study the proteins encoded by ETV6 and ETV6-AML1, we raised polyclonal antibodies directed against a recombinant peptide corresponding to the junctional region of ETV6-AML1. Cell lysates from various leukaemic cell lines, and from children with B- and T-lineage ALLs, were studied by Western blot. Two isoforms of ETV6 protein were detected in normal bone marrow cells and in leukaemic cells without 12p alteration: a major form (apparent m.w. 63 kD) and a minor one (apparent m.w. 53 kD). In the REH cell line, which expresses the ETV6-AML1 fusion transcript and no normal ETV6 mRNA, the ETV6 isoforms were absent and two new bands were detected corresponding to ETV6-AML1 protein products (apparent m.w. 95 and 105 kD). A similar pattern was obtained with blast cells from patients with a t(12;21) and a deletion of ETV6. In two patients with a t(12;21) but no deletion of ETV6, four bands were detected corresponding to both the normal ETV6 and ETV6-AML1 proteins, suggesting that in these cases the second ETV6 allele was not inactivated. Surprisingly, the expression pattern of ETV6 differed widely from patient to patient. In three out of 13 patients without t(12;21), the relative intensity of the bands corresponding to ETV6 isoforms in blast cells from patients was completely different from normal cells, with a marked predominance of the 53 kD isoform. The pattern of ETV6 expression was normal in bone marrow from the same patients during remission. These finding suggest that ETV6 abnormalities are not restricted to patients with translocations or deletions involving this gene.

Epidemiological studies of spectrin mutations related to hereditary elliptocytosis and spectrin polymorphisms in Benin.

We studied an African population in Benin and discovered an unexpectedly high frequency (1.6%) of hereditary elliptocytosis (HE) among the 1447 subjects studied. In approximately two-thirds of HE individuals we identified molecular defects, primarily those in erythrocyte alpha-spectrin (dupL154, L260P and L207P mutations), as well as a novel mutation of erythrocyte beta-spectrin (beta-W2061R mutation). We also identified the genetic basis of a previously identified protein polymorphism of the alpha III domain of spectrin (R1331I mutation). The genetic background of HE in the African population was studied using a number of polymorphisms of the alpha-spectrin gene, including the alpha III domain polymorphism. These studies suggest that the HE mutations appear to have originated from separate genetic backgrounds in this population.

Search for the candidate genes in dominant hereditary spherocytosis using linkage analysis.

Hereditary spherocytosis (HS) is an inherited hemolytic anemia characterized by the presence of dense spherocytic red cells. In HS patients, red cell membrane protein gel electrophoresis has identified different subsets of abnormalities: isolated spectrin deficiency, combined spectrin and ankyrin deficiency, band 3 deficiency. To direct the search for the molecular defect in 9 families with dominant HS, we developed microsatellite markers specific for the membrane protein encoding genes possibly involved in HS (alpha- and beta-spectrin, ankyrin and band 3 genes) and genotyped each family. In 5 families with isolated spectrin deficiency, the beta-spectrin gene was designated as candidate. In one family with combined spectrin/ankyrin deficiency, only the ankyrin gene was not excluded, whereas in the 3 HS families with band 3 deficiency, only the band 3 gene was not excluded. This work allowed development of a reliable methodology to search for candidate genes in HS and showed the frequent involvement of the beta-spectrin gene in HS with isolated spectrin deficiency.

Mutation in the iron responsive element of the L ferritin mRNA in a family with dominant hyperferritinaemia and cataract.

The synthesis of ferritin, the iron-storing molecule, is regulated at the translational level by iron through interaction between a cytoplasmic protein, iron regulatory protein (IRP), and a conserved nucleotide motif present in the 5' non-coding region of all ferritin mRNAs--the iron responsive element (IRE). This region forms a stem-loop structure and when the supply of iron to the cells is limited, the IRP is bound to IRE and represses ferritin synthesis. Ferritin is composed of a 24-subunit protein shell surrounding an iron core. The two types of subunit, H and L, are encoded by two genes located on chromosomes 11q13 and 19q13.1, respectively. Both genes are ubiquitously expressed but transcriptional regulation mediates tissue-specific changes in the H/L mRNA ratio and isoferritin profiles. We now report the identification of a single point mutation in the IRE of the L-ferritin mRNA in members from a family affected with dominantly inherited hyperferritinaemia and cataract. This mutation consists of an A to G change in the highly conserved CAGUGU motif that constitutes the IRE loop and mediates the high-affinity interaction with the IRP. We show that this mutation abolishes the binding of IRP in vitro and leads to a high constitutive, poorly regulated L-ferritin synthesis in cultured lymphoblastoid cells established from affected patients. This is, to our knowledge, the first mutation affecting the IRP-IRE interaction and the iron-mediated regulation of ferritin synthesis. We suggest that excess production of ferritin in tissues is responsible for the hyperferritinaemia and that intracellular accumulation of ferritin leads to cataract.

Loss of heterozygosity in the chromosomal region 12p12-13 is very common in childhood acute lymphoblastic leukemia and permits the precise localization of a tumor-suppressor gene distinct from p27KIP1.

Abnormalities of the short arm of chromosome 12 are relatively common in hematologic malignancies and deletions of the region. 12p12-13 are found in approximately 5% of the patients with acute lymphoblastic leukemia (ALL). As a potent inhibitor of cyclin-dependent kinases, p27KIP1 prevents the progression of the cell cycle and the gene encoding p27KIP1 represents a potential tumor-suppressor gene. Its recent assignment to the chromosomal region (12p12.3) prompted us to study the p27KIP1 gene in a series of 61 children with ALL. Microsatellite polymorphic markers flanking the p27KIP1 gene were analyzed to detect losses of heterozygosity (LOH). Eleven patients displayed LOH for at least one of the markers. The deleted are encompassed the p27KIP1 gene locus in 10 cases, but inactivation of the remaining allele by deletion, translocation, or mutation was never observed. In addition, in 1 patient, the p27KIP1 gene was situated outside of the region of LOH. Thus, p27KIP1 does not seem to be the target gene of 12p12-13 alterations. However, this study indicates that 12p12-13 alterations at the molecular level, which are present in about 27% of the children with B-lineage ALL, are much more common than had previously been reported by usual chromosome analysis. Moreover, LOH mapping allowed us to better define the location of a putative tumor-suppressor gene implicated in these malignancies and should therefore help in identifying this gene.