Shiga toxin (Stx)-producing Escherichia coli (STEC) from patients with hemolytic-uremic syndrome (HUS), patients with diarrhea without HUS, or asymptomatic subjects were genotyped to assess associations between stx2 variants and clinical manifestations of infection. Neither stx2d nor stx2e was found in 268 STEC isolates from patients with HUS. Of 262 STEC isolates from patients with diarrhea, stx(2d) was found in 41 (15.6%; P<.000001), and stx2e was found in 12 (4.6%; P=.0004). The stx2c genotype frequency was similar among isolates from patients with HUS (3.7%) and diarrhea (5.0%). The frequencies of stx2c, stx2d, and stx2e among 96 STEC isolates from asymptomatic subjects were comparable to those among isolates from patients with diarrhea. None of the 626 STEC isolates contained stx2f. All stx2d-positive or stx2e-positive STEC isolates were eae negative and originated from subjects older than those with STEC isolates with stx2c. stx2c-positive STEC isolates can cause HUS, but the presence of stx2d or stx2e may predict a milder disease with a minimal risk of HUS.

IntroductionTo establish strategic priorities for the German national public health institute (RKI) and guide the institute's mid-term strategic decisions, we prioritized infectious pathogens in accordance with their importance for national surveillance and epidemiological research.MethodsWe used the Delphi process with internal (RKI) and external experts and a metric-consensus approach to score pathogens according to ten three-tiered criteria. Additional experts were invited to weight each criterion, leading to the calculation of a median weight by which each score was multiplied. We ranked the pathogens according to the total weighted score and divided them into four priority groups.Results127 pathogens were scored. Eighty-six experts participated in the weighting; “Case fatality rate” was rated as the most important criterion. Twenty-six pathogens were ranked in the highest priority group; among those were pathogens with internationally recognised importance (e.g., Human Immunodeficiency Virus, Mycobacterium tuberculosis, Influenza virus, Hepatitis C virus, Neisseria meningitides), pathogens frequently causing large outbreaks (e.g., Campylobacter spp.), and nosocomial pathogens associated with antimicrobial resistance. Other pathogens in the highest priority group included Helicobacter pylori, Respiratory Syncytial Virus, Varicella zoster virus and Hantavirus.DiscussionWhile several pathogens from the highest priority group already have a high profile in national and international health policy documents, high scores for other pathogens (e.g., Helicobacter pylori, Respiratory syncytial virus or Hantavirus) indicate a possible under-recognised importance within the current German public health framework. A process to strengthen respective surveillance systems and research has been started. The prioritization methodology has worked well; its modular structure makes it potentially useful for other settings.

Prevalence of and risk factors associated with MRSA-ST398 carriage in 1872 (response 70%) farmers and neighbouring residents in a pig- and poultry-dense area in Germany were investigated using a cross-sectional study and self-sampling nasal swabs. In the population, 1% without occupational livestock contact and 24% with occupational livestock contact tested positive for MRSA-ST398. The group without occupational livestock contact was 3·8 times [95% confidence interval (CI) 1·5-9·3] more likely to be colonized if a household member had livestock contact and 3·2 times (95% CI 1·4-7·4) more likely if they regularly made private farm visits (e.g. to buy eggs or milk). In the group with occupational livestock contact, pig contact had an odds ratio of 7·1 (95% CI 2·9-17·2) for MRSA-ST398 acquisition. This is the first study to associate private farm visits with acquisition of MRSA; more research to explore the exact transmission routes is necessary.

In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min

Experientia 50 (1994), Birkh/iuser Verlag, CH-4010 Basel/Switzerland 797 and/or by colony-, dot or PCR multiplex hybridization, and 5) the lack of reference sequences that may erroneously suggest the taxonomic novelty of a clone. Elucidation of the composition of a bacterial community occurring in a natural sample was attempted by dot blot hybridization of 16S rDNA clone libraries with taxon-specific oligonucleotide probes. The composition changed significantly when the same batch of isolated DNA and the same cloning vector, but two different pairs of amplification primers were used. The distribution of taxon-specific clones was also different from that obtained previously using one of the same primer pairs but a different cloning system. The results indicate that our present knowledge of this approach allows neither the complete qualitative nor the accurate quantitative determination of microbial community compositions. Several procedures to release DNA from acid-fast mycobacteria for polymerase chain reaction-based amplification have been reported. Nevertheless, up to now there is no extraction method available which is simple and reliable enough to allow its application in routine clinical practice. We present a rapid, simple and reliable protocol for the extraction of mycobacterial nucleic acids as template molecules for a subsequent polymerase chain reaction. Samples were suspended in extraction buffer and subjected to several cycles of freezing in liquid nitrogen and heating in a boiling water bath. After the treatment, the rigid cell wall of the mycobacteria was cracked and DNA could be reliably amplified from the supernatant. For the evaluation of this procedure we used serial dilutions of liquid culture. Additionally, PCR was capable of detecting mycobacteria in sputum samples from 13 out of 13 patients with clinically suspected tuberculo: sis which were positive by smear and culture. Amplified DNA products were characterized both by length and direct sequencing. Using PCR primers which hybridize to a conserved sequence that flanks a hypervariable region in the 16S rRNA gene of mycobacteria, we were able to distinguish even between distinct mycobacterial species by determining the nucleotide sequence of the amplification products. In 15 smear-and culture-negative cases without suspected tuberculosis, PCR led to negative results. The routine applicability of this new extraction protocol for nucleic acid from mycobacteria will be further evaluated. Infections caused by the Mycobacterium aviumcomplex (MAC) contribute substantially to morbidity and mortality in patients with AIDS, and the prevalence is increasing. There is an urgent need for methods that can detect this pathogen more rapidly and directly in body fluids. PCR-based detection of Specific detection ofWe have evaluated a PCR amplification method based on the DNA probe sequence by Fries et al. (insert pMAv22) 1. To improve specificity of PCR, a new primer was selected (bases 37-56) and used together with the described primer Mav 22B in order to am...