Clonally expanded HIV-1 proviruses with 5'-leader defects can give rise to nonsuppressible residual viremia.

BackgroundAntiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.MethodsWe undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5'-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.ResultsClones carrying proviruses with 5'-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.ConclusionThese findings show that proviruses with 5'-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5'-leader can help in understanding failure to completely suppress viremia.FundingOffice of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

Efficacy of the World Health Organization-recommended handwashing technique and a modified washing technique to remove Clostridium difficile from hands.

BACKGROUND: The efficacy of the World Health Organization (WHO)-recommended handwashing technique against Clostridium difficile is uncertain, and whether it could be improved remains unknown. Also, the benefit of using a structured technique instead of an unstructured technique remains unclear. METHODS: This study was a prospective comparison of 3 techniques (unstructured, WHO, and a novel technique dubbed WHO shortened repeated [WHO-SR] technique) to remove C difficile. Ten participants were enrolled and performed each technique. Hands were contaminated with 3 × 106 colony forming units (CFU) of a nontoxigenic strain containing 90% spores. Efficacy was assessed using the whole-hand method. The relative efficacy of each technique and of a structured (either WHO or WHO-SR) vs an unstructured technique were assessed by Mann-Whitney U test and Wilcoxon signed-rank test. RESULTS: The median effectiveness of the unstructured, WHO, and WHO-SR techniques in log10 CFU reduction was 1.30 (interquartile range [IQR], 1.27-1.43), 1.71 (IQR, 1.34-1.91), and 1.70 (IQR, 1.54-2.42), respectively. The WHO-SR technique was significantly more efficacious than the unstructured technique (P = .01). Washing hands with a structured technique was more effective than washing with an unstructured technique (median, 1.70 vs 1.30 log10 CFU reduction, respectively; P = .007). CONCLUSIONS: A structured washing technique is more effective than an unstructured technique against C difficile.

RNA interference reveals a role for TLR2 and TLR3 in the recognition of Leishmania donovani promastigotes by interferon-gamma-primed macrophages.

Leishmania donovani promastigotes evade the induction of a proinflammatory response during their invasion of naive macrophages. However, their entry into IFN-gamma-primed macrophages is accompanied by the secretion of nitric oxide (NO) and proinflammatory cytokines. In the present study, we addressed the hypothesis that priming with IFN-gamma induces the expression of a receptor that enables mouse macrophages to recognize L. donovani promastigotes. We observed that in IFN-gamma-primed macrophages, L. donovani promastigotes stimulated Interleukin-1 receptor-associated kinase-1 (IRAK-1) activity. We next showed that Toll-like receptor (TLR)3 is barely detectable in naive macrophages but is expressed in IFN-gamma-treated macrophages. Silencing of TLR3, TLR2, IRAK-1 and myeloid differentiation factor 88 (MyD88) expression by RNA interference revealed that both TLR are involved in the secretion of NO and TNF-alpha induced by L. donovani promastigotes. Using L. donovani mutants, we showed that TLR2-mediated responses are dependent on Galbeta1,4Manalpha-PO(4)-containing phosphoglycans, whereas TLR3-mediated responses are independent of these glycoconjugates. Furthermore, our data indicate a participation of TLR2 and TLR3 in the phagocytosis of L. donovani promastigotes and a role for TLR3 in the leishmanicidal activity of the IFN-gamma-primed macrophages. Collectively, our data are consistent with a model where recognition of L. donovani promastigotes depends on the macrophage activation status and requires the expression of TLR3.

Epidemiological survey of human metapneumovirus infection in a large pediatric tertiary care center.

The human metapneumovirus (hMPV) was recently identified and linked to acute respiratory tract infections (ARTI). To assess the clinical importance of this virus in infants and children, we developed a rapid and efficient reverse transcription-PCR-based screening method for a large volume of samples and tested retrospectively a collection of 1,132 respiratory specimens submitted over a full year period to the virology laboratory of a large tertiary care pediatric center in Montreal, Canada. A total of 41 samples from 37 patients were positive by this method. During the winter months of 2001, up to 8% of specimens submitted for respiratory virus testing were hMPV positive. Sequencing data of the hMPV M gene revealed that two genogroups of the virus, each of which can be divided into two subgroups, cocirculated during this time period. A case-controlled study was conducted to compare the symptoms associated with hMPV infection with those involving other etiologic agents causing ARTI. Symptoms most frequently observed in hMPV-positive patients were cough, wheezing, and dyspnea, although the symptomatology could differ substantially from patient to patient. No distinct symptom profile could be associated with hMPV. Three nosocomial cases of hMPV infection were identified. Together, our data suggest that hMPV is a significant cause of symptomatic respiratory tract infections in infants and children. The incidence of the disease and the morbidity associated with the infection justify adding hMPV to the list of common respiratory viruses routinely screened for by clinical laboratories.

Modulation of lipopolysaccharide-induced NF-IL6 activation by protein kinase C-alpha in a mouse macrophage cell line.

We have previously shown that overexpression of a dominant-negative (DN) mutant of protein kinase C-alpha (PKC-alpha) in RAW 264.7 macrophages inhibited lipopolysaccharide (LPS)-induced IL-1alpha, inducible nitric oxide synthase and cyclooxygenase-2 expression. This inhibition was not related to defective NF-kappaB nuclear translocation, suggesting that PKC-alpha might be involved in the modulation of other LPS-inducible transcription factors. In the present study, we have investigated the impact of PKC-alpha on the activation of AP-1 and NF-IL6 in LPS-treated RAW 264.7 macrophages. Electrophoretic mobility shift assays and luciferase reporter constructs revealed that LPS-induced AP-1 transcriptional activity was normal in DN PKC-alpha-overexpressing RAW 264.7 cells. In contrast, LPS-induced DNA-binding and transcriptional activities of NF-IL6 were inhibited in DN PKC-alpha-overexpressing RAW 264.7 cells and correlated with an impairment of NF-IL6 nuclear translocation. Conversely, overexpression of either wild-type PKC-alpha or a constitutively active PKC-alpha mutant significantly enhanced LPS-stimulated NF-IL6-dependent promoter activity. Finally, LPS-induced expression of two genes regulated by NF-IL6, namely IL-1beta and granulocyte colony-stimulating factor, was impaired in DN PKC-alpha-overexpressing RAW 264.7 cells. Taken together, these results suggest that regulation of NF-IL6 activity constitutes one of the mechanisms by which PKC-alpha modulates LPS-induced gene expression in the mouse macrophage cell line RAW 264.7.