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. Representativeness was increased using 'seeds' from a variety of sources. Signed informed consent was obtained from all participants. Data on demographics, risk behaviours and self-reported STIs were collected through an interviewer-administered questionnaire. We screened all eligible participants for HIV using the Rwanda-approved protocol for rapid HIV detection. RESULTS: 504 MSM were recruited from five major cities in Rwanda. Participants were mostly young (median age 23 years, range 18-55 years) and unmarried (484/504, 96.0%). Thirteen per cent (65/504) of the participants reported past gonorrhoea and/or syphilis infection. Of 504 MSM, 53 (10.5%) reported they were diagnosed and treated for gonorrhoea in the past 12 months and 24 (4.8%) tested positive for HIV. A high proportion (232/504, 46%) reported receiving payment for sex by a man, with almost half of these reporting on more than three occasions (107/232, 46%). Many reported having had an HIV test within the past 12 months (385/504, 76.4%). In multivariate logistic regression models controlling for age, being paid for sex was associated with higher odds of past STI (OR 3.36 (1.82-6.43]; P<0.001) and testing HIV positive (OR 3.13, P<0.05). CONCLUSION: Further research is needed to understand the high rate of payment for sex in this population, which appears to be a major risk factor for STI including HIV.

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. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs. METHODS AND FINDINGS: To define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = -0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015-0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%-48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the ß chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%-85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only. CONCLUSIONS: The DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2-3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%-50%, with the eventual goal of preventing T1D. TRIAL REGISTRATION: ISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735.

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. The mean duration of virus shedding was 6.2 days by PCR (Polymerase Chain Reaction) and 4.2 days by culture. Surface swabs were collected from 39 settings; 16 (41%) subject locations were contaminated with virus. Overall, 33 of the 671 (4.9%) surface swabs were PCR positive for influenza, of which two (0.3%) yielded viable virus. On illness Day 3, subjects yielding positive surface samples had significantly higher nasal viral loads (geometric mean ratio 25.7; 95% CI 1.75, 376.0, p=0.021) and a positive correlation (r=0.47, p=0.006) was observed between subject nasal viral loads and viral loads recovered from the surfaces around them. Room air was sampled in the vicinity of 12 subjects, and PCR positive samples were obtained for five (42%) samples. Influenza virus shed by infected subjects did not detectably contaminate the vast majority of surfaces sampled. We question the relative importance of the indirect contact transmission of influenza via surfaces, though our data support the existence of super-spreaders via this route. The air sampling results add to the accumulating evidence that supports the potential for droplet nuclei (aerosol) transmission of influenza.

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. This latter limitation has led to the focus tending to be on individual pathogens and their detection. This chapter describes the development of TaqMan® Array Cards (TACs), technology which allows the detection of multiple pathogens (up to 48 targets) from a single nucleic acid extract, utilising small volumes and real-time PCR. This in turn lends itself to a syndromic approach to infectious disease diagnosis. Using the examples of TACs we have developed in our own laboratory, as well as others, we explain the design, optimisation and use of TACs for respiratory, gastrointestinal and liver infections. Refinement of individual assays is discussed as well as the incorporation of appropriate internal and process controls onto the array cards. Finally, specific examples are given of instances where the assays have had a direct, positive impact on patient care.

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. In particular, clinical microbiology will have the opportunity to intervene at the public health level as well as at the individual patient. SOURCES OF DATA, AREAS OF AGREEMENT AND CONTROVERSY: Experts in the new technologies discuss the advances and some of the key literature that has been published to-date. They touch upon both the potential benefits and some of the hurdles that must be overcome before the technologies are embraced fully into the clinical laboratory. GROWING POINTS: This review discusses a number of technologies that may alter the way in which clinical microbiology is used to investigate infectious disease. Diagnostic services in the UK are currently undergoing a process of rationalization, which involves a shift towards laboratory amalgamation, adoption of 24/7 working patterns and greater automation in order to reduce costs. This review explores technologies that are already or are expected to be important in this on-going transition because they simplify or accelerate the complex workflows that are required for pathogen identification.

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. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich ((392)-GGAG-(395)) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the (392)-GGAG-(395) motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.

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. METHODS: In a quarantine facility, healthy volunteers ("donors") were inoculated with A/Wisconsin/67/2005 (H3N2) influenza virus via intranasal drops. On study days 2 and 3 "recipient" volunteers were exposed to donors under close living conditions. Volunteers socialized for 30 hours during a 2-day period. Infection was confirmed by ≥1 positive results from polymerase chain reaction, virus culture, or serology. RESULTS: After inoculation, 4 of 9 donors developed symptoms consistent an influenza-like illness (ILI) and 7 of 9 were proven to be influenza-infected. After exposure, 4 of 15 recipients developed symptoms of ILI and 3 of 15 were proven to be infected. Serum collected within 2 days of study initiation indicated that 1 donor and 3 recipients were seropositive at study initiation. After adjustment for preexposure immunity, the overall secondary attack rate was 25% (3 of 12). CONCLUSIONS: Experimental human exposure studies offer an attractive potential method for answering outstanding questions related to influenza transmission and the evaluation of interventions to reduce it.

Survival of influenza A(H1N1) on materials found in households: implications for infection control.

BACKGROUND: The majority of influenza transmission occurs in homes, schools and workplaces, where many frequently touched communal items are situated. However the importance of transmission via fomites is unclear since few data exist on the survival of virus on commonly touched surfaces. We therefore measured the viability over time of two H1N1 influenza strains applied to a variety of materials commonly found in households and workplaces. METHODOLOGY AND PRINCIPAL FINDINGS: Influenza A/PuertoRico/8/34 (PR8) or A/Cambridge/AHO4/2009 (pandemic H1N1) viruses were inoculated onto a wide range of surfaces used in home and work environments, then sampled at set times following incubation at stabilised temperature and humidity. Virus genome was measured by RT-PCR; plaque assay (for PR8) or fluorescent focus formation (for pandemic H1N1) was used to assess the survival of viable virus. CONCLUSIONS/SIGNIFICANCE: The genome of either virus could be detected on most surfaces 24 h after application with relatively little drop in copy number, with the exception of unsealed wood surfaces. In contrast, virus viability dropped much more rapidly. Live virus was recovered from most surfaces tested four hours after application and from some non-porous materials after nine hours, but had fallen below the level of detection from all surfaces at 24 h. We conclude that influenza A transmission via fomites is possible but unlikely to occur for long periods after surface contamination (unless re-inoculation occurs). In situations involving a high probability of influenza transmission, our data suggest a hierarchy of priorities for surface decontamination in the multi-surface environments of home and hospitals.

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. METHODOLOGY/PRINCIPAL FINDINGS: Plaque assays provided a robust and reproducible method for determining virus viability after disinfection, while a National Standard influenza virus RT-PCR assay (VSOP 25, www.hpa-standardmethods.org.uk) was adapted to detect viral genome, and a British Standard (BS:EN 14476:2005) was modified to determine virus killing. CONCLUSIONS/SIGNIFICANCE: Active ingredients in a number of the cleaning agents, wipes, and tissues tested were able to rapidly render influenza virus nonviable, as determined by plaque assay. Commercially available wipes with a claimed antiviral or antibacterial effect killed or reduced virus infectivity, while nonmicrobiocidal wipes and those containing only low concentrations (<5%) of surfactants showed lower anti-influenza activity. Importantly, however, our findings indicate that it is possible to use common, low-technology agents such as 1% bleach, 10% malt vinegar, or 0.01% washing-up liquid to rapidly and completely inactivate influenza virus. Thus, in the context of the ongoing pandemic, and especially in low-resource settings, the public does not need to source specialized cleaning products, but can rapidly disinfect potentially contaminated surfaces with agents readily available in most homes.

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. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.

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. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation in vivo and its implication in viral replication. RESULTS: Using a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores. CONCLUSION: In addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target.

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. To investigate this further in a replication-competent provirus, loop A was mutated to decrease its affinity for Rev. Impairing the Rev-loop A interaction led to reduced nuclear export of viral genomic RNA. RNA packaging decreased by approximately 30%. Viral protein production and export of virus particles appeared normal; however, the virus was severely replication-deficient. The loop A sequence, which is 98% conserved amongst viral isolates, is implicated in several cis-acting functions critical to virus viability.

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. This region is predicted to form a GACG tetraloop and therefore differs significantly from the kissing loop palindromes utilized to initiate dimerization in primate lentiviruses. The motif is strongly conserved across the ovine and caprine lentiviruses, implying a critical functional role. Furthermore, the proposed GACG tetraloop exhibits marked structural homology with similar structural motifs present in the leader regions of the alpha- and gamma-retroviruses, and the maedi visna virus dimer linkage region is capable of forming heterodimeric species with the Moloney murine leukemia virus Psi domain. This may be indicative of commonality of origin of the two viruses or convergent evolution.

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. Differences and similarities in both secondary and tertiary structure are discussed. The implication of varying roles for the dimer linkage in related viruses is considered.

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. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.

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. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.

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. Using a combination of NMR spectroscopy, UV melting experiments, molecular modeling and phylogenetic analyses, we have explored the structure of two conserved internal loops proximal to the palindromic sequence of SL-1. Internal loop A, composed of six purines, forms a flexible structure that is strikingly similar to the Rev responsive element motif when bound to Rev protein. This result suggests that it may function as a protein-binding site. The absolutely conserved four-purine internal loop B is instead conformationally and thermodynamically unstable, and exhibits multiple conformations in solution. By introducing a double AGG to GGA mutation within this loop, its conformation is stabilised to form a new intra-molecular G:A:G base-triplet. The structure of the GGA mutant explains the relative instability of the wild-type loop. In a manner analogous to SL-3, we propose that conformational flexibility at this site may facilitate melting of the structure during Gag protein capture or genomic RNA dimerisation.