An outbreak of meticillin-resistant Staphylococcus aureus colonization in a neonatal intensive care unit: use of a case-control study to investigate and control it and lessons learnt.

AIM: To describe the investigation and management of a meticillin-resistant Staphylococcus aureus (MRSA) outbreak on a neonatal intensive care unit (NICU) and the lessons learnt. METHODS: This was an outbreak report and case-control study conducted in a 40-cot NICU in a tertiary referral hospital and included all infants colonized/infected with gentamicin-resistant MRSA. INTERVENTION: Standard infection-control measures including segregation of infants, barrier precautions, enhanced cleaning, assessment of staff practice including hand hygiene, and increased MRSA screening of infants were implemented. Continued MRSA acquisitions led to screening of all NICU staff. A case-control study was performed to assess staff contact with colonized babies and inform the management of the outbreak. FINDINGS: Eight infants were colonized with MRSA (spa type t2068), one of whom subsequently developed an MRSA bacteraemia. MRSA colonization was significantly associated with lower gestational age; lower birthweight and with being a twin. Three nurses were MRSA colonized but only one nurse (45) was colonized with MRSA spa type t2068. Multivariable logistic regression analysis identified being cared for by nurse 45 as an independent risk factor for MRSA colonization. CONCLUSIONS: Lack of accurate recording of which nurses looked after which infants (and when) made identification of the risk posed by being cared for by particular nurses difficult. If this had been clearer, it may have enabled earlier identification of the colonized nurse, avoiding subsequent cases. This study highlights the benefit of using a case-control study, which showed that most nurses had no association with colonized infants.

Virus shedding and environmental deposition of novel A (H1N1) pandemic influenza virus: interim findings.

BACKGROUND: The relative importance of different routes of influenza transmission, including the role of bioaerosols, and ability of masks and/or hand hygiene to prevent transmission, remains poorly understood. Current evidence suggests that infectious virus is not typically released from adults after 5 days of illness, however, little is known about the extent to which virus is deposited by infected individuals into the environment and whether deposited virus has the ability to infect new hosts. Further information about the deposition of viable influenza virus in the immediate vicinity of patients with pandemic influenza is fundamental to our understanding of the routes and mechanisms of transmission. OBJECTIVES: To collect data on patients infected with pandemic H1N1 2009 (swine flu). Primary objectives were to correlate the amount of virus detected in a patient's nose with that recovered from his/her immediate environment, and with symptom duration and severity. Secondary objectives were to describe virus shedding and duration according to major patient characteristics: adults versus children, and those with mild illness (community patients) versus those with more severe disease (hospitalised patients). METHODS: Adults and children, both in hospital and from the community, who had symptoms of pandemic H1N1 infection, were enrolled and visited every day during follow-up for a maximum of 12 days. Symptom data was collected and samples were taken, including nose swabs and swabs from surfaces and objects around patients. Samples of air were obtained using validated sampling equipment. The samples were tested for the presence of pandemic H1N1 virus, using polymerase chain reaction (PCR) to detect virus genome and an immunofluorescence technique to detect viable virus. RESULTS: Forty-three subjects were followed up, and 19 of them were subsequently proven to be infected with pandemic H1N1 virus. The median duration of virus shedding from the 19 infected cases was 6 days when detection was performed by PCR, and 3 days when detection was performed by a culture technique. Over 30% of cases remained potentially infectious for at least 5 days. Only 0.5% of all community and none of the hospital swabs taken revealed virus on surfaces. Five subjects had samples of the air around them collected and virus was detected by PCR from four; some of the air particles in which virus was detected were small enough to be inhaled and deposited deep in the lungs. LIMITATION: Small number of subjects recruited. CONCLUSIONS: The finding that over 30% of infected individuals have infectious virus in their noses for 5 days or more has infection control implications. The data suggest that contact transmission of pandemic influenza via fomites may be less important than previously thought, but transmission via bioaerosols at short range may be possible, meaning that high-level personal protective equipment may be needed by health-care workers when attending patients with pandemic influenza. Further work is being undertaken to consolidate these findings, as they have important potential implications for the protection of health-care workers and the formulation of advice to households, nationally and internationally.

The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2.

Deletion mutation of the RNA 5' leader sequence of simian immunodeficiency virus (SIV) was used to localize the virus packaging signal. Deletion of sequences upstream of the major splice donor (SD) site produced a phenotype most consistent with a packaging defect when analysed by both RNase protection assay and RT-PCR. Sequences downstream of the SD were deleted and produced varying effects but did not affect packaging: a large downstream deletion had little effect on function, whereas a nested deletion produced a profound replication defect characterized by reduced protein production. Secondary structure analysis provided a potential explanation for this. The major packaging signal of SIV appears to be upstream of the SD in a region similar to that of human immunodeficiency virus type 2 (HIV-2) but unlike that of HIV-1; however, the packaging signal of all three viruses are at a similar distance from their respective cap sites. This conserved positioning suggests that it is more important in the virus life cycle than the position of the signal relative to the SD.

Sequences in the 5' leader of Mason-Pfizer monkey virus which affect viral particle production and genomic RNA packaging: development of MPMV packaging cell lines.

We used a series of deletion mutations in the 5' untranslated region of the prototype D type retrovirus, Mason-Pfizer Monkey Virus (MPMV), to analyse RNA encapsidation. A region was identified upstream of the major splice donor which reduced particle production but had a proportionally greater effect on RNA packaging. A small deletion downstream of the splice donor had little effect on RNA production and caused no significant packaging defect. A large deletion encompassing the end of the primer binding site down to the splice donor had a dramatic effect, disrupting viral protein synthesis. Stable cell lines were produced containing packaging-defective virus. These first-generation packaging cell lines were used to package and transfer an MPMV-based vector.

Oligonucleotide mapping of the core genomic RNA dimer linkage in human T-cell leukaemia virus type-1.

We have previously mapped the sequences required for dimerisation of the 5' leader of the human T-cell leukaemia virus type-1 (HTLV-1) genome. The smallest sequence necessary and sufficient for dimer formation, in vitro, was ascertained to be a 37 nucleotide (nt) region downstream of the splice donor and just upstream of the primer binding site. Deletion of a 32 base-pair sequence encompassing this region within the provirus was associated with a minor decrease in infectivity of the virus in an in vitro system. To further map and help elucidate the nature of the dimer linkage, we used RNA and DNA oligonucleotide competition assays to define the nucleotides involved. These experiments revealed that a 14 nt sequence containing a potential stem loop structure, formed from a palindromic sequence, is important for dimer formation. This was confirmed by the ability of this RNA sequence to form heterodimers with larger RNA transcripts from the same region, while sequences lacking this motif could not. RNA transcripts containing the reverse sequence, the same nucleotides in a random arrangement, and complementary DNA oligos, all failed to form heterodimers with the 14 nt sequence. The primary dimer initiation site of HTLV-1 has thus been located to a 14 nt palindrome containing sequence, and dimerisation is shown to be dependent on specific sense-sense RNA interactions.

An accessible analytical approach for investigating what happens between the rounds of a Delphi study.

The Delphi method is used to investigate consensus amongst a panel of experts using repeated rounds of a questionnaire, often in healthcare settings. However, many Delphi studies do not report any investigation into what happens to the stability of consensus or the convergence of agreement between the rounds in the study, which may be of importance. In this paper an accessible analytical approach is outlined using graphical presentations of means and standard deviations to identify what happens between rounds. For Delphi studies where the scale upon which experts are expressing their opinions can be considered to be interval, the mean will represent the group opinion whilst the standard deviation will represent the level of agreement. An example Delphi study from a healthcare setting is used to illustrate the methodology.

A 37 base sequence in the leader region of human T-cell leukaemia virus type I is a high affinity dimerization site but is not essential for virus replication.

Mutagenesis has demonstrated a region in the human T-cell leukaemia virus type I (HTLV-I) 5' leader RNA which, when deleted, abolishes stable RNA dimer formation in vitro. We have further mapped, using both in vitro transcribed and synthesized RNA, this site to a 37 base region, which dimerizes with high affinity. When deleted from an HTLV-I Gag-Pol-expressing plasmid which was co-transfected with an envelope protein expressor to produce virions capable of single round infection, the dimer linkage deletion did not affect viral protein production. In addition, virus infectivity was only slightly reduced, to approximately 75-80% of the wild-type.

Sequences involved in the dimerisation of human T cell leukaemia virus type-1 RNA.

The formation of a genomic RNA dimer appears to be a critical step in the life cycle of all retroviruses. To investigate the site and nucleotide interactions involved in this process, a 531 bp DNA fragment encompassing sequences up- and downstream of the splice donor in human T cell leukaemia virus type 1 (HTLV-1) was inserted into a plasmid vector under the control of the SP6 promoter. RNA transcripts generated in vitro from this template formed dimers which could be dissociated by heating at 60-80 degrees C for 3 min. The physical properties of the dimeric RNA were not consistent with either Watson-Crick base pairing or guanine tetrad formation as being solely responsible for the interaction. Deletion mutagenesis identified a 32 nt sequence required for dimerisation. Computer modelling was carried out in order to identify putative RNA secondary structures within this essential region. A stem-loop structure was identified, the stem of which was conserved among different sequenced isolates of HTLV-1. This sequence also contains a 15 nt palindrome. We sought by disruptive and compensatory mutagenesis to define the possible roles of these two structures in dimer linkage.

Humoral immune responses to Shiga-like toxins and Escherichia coli O157 lipopolysaccharide in hemolytic-uremic syndrome patients and healthy subjects.

Shiga-like-toxin (SLT)-producing Escherichia coli strains, especially serotype O157:H7, are important causes of bloody diarrhea and are associated with the development of hemolytic-uremic syndrome (HUS). Enzyme-linked immunosorbent assays (ELISAs) were developed for the serologic detection of immunoglobulin M (IgM), IgG, and IgA to Shiga toxin (ST) and SLT-I, IgG to SLT-II, and IgM and IgG reactive against E. coli O157 lipopolysaccharide (LPS). Serum samples were collected from 27 HUS patients (25 pediatric and 2 adult) and tested in the ELISAs. Of 27 patients, 10 (37%) were positive for at least one class of antibody to ST/SLT-I. None of the patients were positive for IgG antibody to SLT-II. Twenty-one of the 27 patients (78%) were positive for antibody to E. coli O157 LPS; 19 of 27 (70%) were positive for IgM, and 20 of 27 (74%) were positive for IgG. None of 48 control serum samples were positive in any of the toxin assays, and only 1 of 48 (2%) and 2 of 48 (4%) were positive for IgM and IgG, respectively, to E. coli O157 LPS. Twelve of the 24 patients (50%) from whom stool specimens were collected were positive by culture for E. coli O157. Overall, serology and culture produced confirmation of infection by SLT-producing organisms in 23 of 27 (85%) HUS patients. A combination of ELISA for antibodies to E. coli O157 LPS and culture provided evidence for 22 of 27 (82%) of these patients. The results indicate that while ELISAs for ST/SLT-I and SLT-II antibodies were of limited diagnostic value, the ELISAs for IgM and IgG to E. coli O157 LPS provided valuable and sensitive adjuncts to culture.

Streptococci from primary isolation plates grouped by reverse passive haemagglutination.

Reverse passive haemagglutination, a novel microtitre based assay, was compared with the Streptex (Wellcome UK) latex slide agglutination kit for streptococcal grouping in a diagnostic microbiology laboratory. Three hundred and fifty two extracts from 349 consecutive primary isolation plates were assayed by both methods. Reverse passive haemagglutination gave identical grouping results for 98.0% of the 345 streptococci identified by Streptex, and the kappa coefficient of agreement between the methods for all 352 extracts tested was 0.973. Cross reactions with Listeria spp seen with Streptex were not found by reverse passive haemagglutination. In the reverse passive haemagglutination method 11 streptococci could be grouped on each 96-well plate and most reactions were stable for at least 30 minutes. Reverse passive haemagglutination is more rapid to perform than latex slide agglutination when many organisms are to be grouped, and the patterns of haemagglutination are easily recognised. If the method was taken into routine use in a diagnostic laboratory, the persistence of reverse passive haemagglutination reactions would enable grouping results to be checked for quality control purposes.

Diagnosis and treatment of "verminous aneurysm" formation in the horse.

The clinical manifestations, diagnosis and differential diagnosis of "verminous aneurysm" formation at the root of the cranial mesenteric artery and coeliac artery resulting from Strongylus vulgaris larvae migration are described. Forty-nine of 57 cases were successfully treated with low molecular weight dextran (dextran 70).

Diarrhoea in horses associated with ulceration of the colon and caecum resulting from S vulgaris larval migration.

The clinical manifestations of a diarrhoeic syndrome of horses with ulceration of the mucosae of the colon and caecum are described. Patients could be divided into three groups according to their presenting symptoms and the disease is probably caused by the thrombo-embolism associated with migrating larvae of Strongylus vulgaris. The differential diagnosis, prognosis and treatment are outlined with particular reference to the use of antithrombotic agents.