Monocyte Anisocytosis is Associated with Sepsis in Children with Suspected Infection.

Background: Early, accurate determination of disease severity in an emergency setting is paramount for improving patient outcomes and healthcare costs. Monocyte anisocytosis, quantified as monocyte distribution width (MDW), has been shown to correspond with immune dysregulation. We hypothesize that MDW is broadly associated with illness severity related to sepsis and serious infection in children.

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.

HIV seroprevalence, self-reported STIs and associated risk factors among men who have sex with men: a cross-sectional study in Rwanda, 2015.

Objectives: In many populations, men who have sex with men (MSM) are at a high risk of HIV infection. This study aimed to estimate the burden of HIV, other STIs and risk behaviours among Rwandan MSM.

Methods: In this cross-sectional study, we recruited through peer referral men aged between 18 and 60 years, who reported sex with men at least once in the 12 months prior to the survey.

Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial.

Background: Interleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target.

The environmental deposition of influenza virus from patients infected with influenza A(H1N1)pdm09: Implications for infection prevention and control.

In a multi-center, prospective, observational study over two influenza seasons, we sought to quantify and correlate the amount of virus recovered from the nares of infected subjects with that recovered from their immediate environment in community and hospital settings. We recorded the symptoms of adults and children with A(H1N1)pdm09 infection, took nasal swabs, and sampled touched surfaces and room air. Forty-two infected subjects were followed up.

Low-Density TaqMan® Array Cards for the Detection of Pathogens.

Real-time PCR assays have revolutionised diagnostic microbiology over the past 15 years or more. Adaptations and improvements over that time frame have led to the development of multiplex assays. However, limitations in terms of available fluorophores has meant the number of assays which can be combined has remained in single figures.

New methods for identifying infectious diseases.

Background: The goal of clinical microbiology is to identify the cause of infection, aiding rapid treatment initiation or altering empirically chosen anti-microbial regimens. Automation and molecular techniques have brought about a revolution in the clinical laboratory, ensuring ever faster and more accurate diagnoses. In the last few years however, there have been a number of developments that radically alter the way that microbiology and other diagnostic laboratories are advancing.

HIV-2 genome dimerization is required for the correct processing of Gag: a second-site reversion in matrix can restore both processes in dimerization-impaired mutant viruses.

A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5' ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS.

Use of a human influenza challenge model to assess person-to-person transmission: proof-of-concept study.

Background: Influenza transmission in humans remains poorly understood. In particular, the relative contribution of contact, large droplet, and aerosol transmission is unknown. The aims of this proof-of-concept study were to determine whether an experimentally induced influenza infection is transmissible between humans and whether this would form a viable platform for future studies.

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.

Effectiveness of common household cleaning agents in reducing the viability of human influenza A/H1N1.

Background: In the event of an influenza pandemic, the majority of people infected will be nursed at home. It is therefore important to determine simple methods for limiting the spread of the virus within the home. The purpose of this work was to test a representative range of common household cleaning agents for their effectiveness at killing or reducing the viability of influenza A virus.

Steric block high affinity oligonucleotide analogues: a new tool for mapping RNA-protein binding sites.

Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge.

Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity.

Background: Retroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5' UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity.

Mutation of the Rev-binding loop in the human immunodeficiency virus 1 leader causes a replication defect characterized by altered RNA trafficking and packaging.

An internal RNA loop, located within the packaging signal of human immunodeficiency virus 1, that resembles the Rev-responsive element (RRE) closely was identified previously. Subsequent in vitro studies confirmed that the loop, termed loop A, could bind Rev protein specifically. Its proximity to the major splice donor has suggested a role for Rev-loop A interaction supplementary to or preceding that of the Rev-RRE interaction.

Identification and visualization of the dimerization initiation site of the prototype lentivirus, maedi visna virus: a potential GACG tetraloop displays structural homology with the alpha- and gamma-retroviruses.

Dimerization of retroviral genomic RNA is essential for efficient viral replication and is mediated by structural interactions between identical RNA motifs in the viral leader region. We have visualized, by electron microscopy, RNA dimers formed from the leader region of the prototype lentivirus, maedi visna virus. Characterization by in vitro assays of the domains responsible for this interaction has identified a 20 nucleotide sequence that functions as the core dimerization initiation site.

The retroviral RNA dimer linkage: different structures may reflect different roles.

Retroviruses are unique among virus families in having dimeric genomes. The RNA sequences and structures that link the two RNA molecules vary, and these differences provide clues as to the role of this feature in the viral lifecycles. This review draws upon examples from different retroviral families.

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs.

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs.

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.

Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA.

The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev.

Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal.

The packaging signal (Psi) of the human immunodeficiency virus type 1 (HIV-1) enables encapsidation of the full-length genomic RNA against a background of a vast excess of cellular mRNAs. The core HIV-1 Psi is approximately 109 nucleotides and contains sequences critical for viral genomic dimerisation and splicing, in addition to the packaging signal. It consists of a series of stem-loops (termed SL-1 to SL-4), which can be arranged in a cloverleaf secondary structure.

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.

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.

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.

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.