Evaluation of Archival HIV DNA in Brain and Lymphoid Tissues.

HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV-env) sequences from a subset of 14 participants. We detected HIV DNA (gag) in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain (P < 0.01). Levels of HIV gag between BG and FC were similar (P > 0.2), while OCC had the lowest levels (P = 0.01). Females had higher HIV DNA levels in tissues than males (gag, P = 0.03; 2-LTR, P = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV-env sequences (n = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. IMPORTANCE HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies.

Long-read amplicon denoising.

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called 'amplicon denoising', this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available.

Inconsistent reversal of HIV-1 latency ex vivo by antigens of HIV-1, CMV, and other infectious agents.

BACKGROUND: A reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-1 infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV-1 and other common pathogens to reverse latency. RESULTS: We obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV-1 and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-1 expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-1 expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. CONCLUSIONS: In this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-1 latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

Efficacy of triphala mouth rinse (aqueous extracts) on dental plaque and gingivitis in children.

AIM: The aim of the present study was to evaluate the efficacy of triphala mouth rinse (aqueous) in the reduction of plaque and gingivitis among children. METHODS: The study was a randomized, double-blinded, controlled trial, with a total of 60 school children (n = 30 in each group; triphala and chlorhexidine groups). Plaque and gingival indices were used to evaluate baseline and follow-up plaque and gingivitis. RESULTS: A total of 57 children completed the study. Both chlorhexidine and triphala groups showed significantly lower mean gingival and plaque index scores at follow up than baseline (P < 0.001). There was no significant difference in the percentage change in the mean gingival index between the two groups (P = 0.826). The percentage change in the mean plaque index was significantly higher in the chlorhexidine group compared to the triphala group (P = 0.048). CONCLUSION: The effectiveness of triphala in the reduction of plaque and gingivitis was comparable to chlorhexidine, and can be used for short-term purposes without potential side-effects. It is a cost-effective alternative in reducing plaque and gingivitis.

Effects of artesunate on parasite recrudescence and dormancy in the rodent malaria model Plasmodium vinckei.

Artemisinin (ART) is the recommended first line therapy for treating uncomplicated and drug-resistant Plasmodium falciparum, the most pathogenic form of malaria. However, treatment failure following ART monotherapy is not uncommon and resistance to this rapidly acting drug has been reported in the Thai-Cambodian border. Recent in vitro studies have shown that following treatment with dihydroartemisinin (DHA), the development of ring-stage parasites is arrested for up to 20 days. These arrested (i.e. dormant) rings could be responsible for the recrudescence of infection that is observed following ART monotherapy. To develop a better understanding of the stage-specific effects of ART and determine if dormancy occurs in vivo, the ART derivative artesunate (AS) was used to treat mice infected with the synchronous rodent malaria parasites P. vinckei petteri (non-lethal) and P. v. vinckei (lethal). Results show that in both the non-lethal and lethal strains, ring-stage parasites are the least susceptible to treatment with AS and that the day of treatment has more of an impact on recrudescence than the total dose administered. Additionally, 24 hrs post-treatment with AS, dormant forms similar in morphology to those seen in vitro were observed. Finally, rate of recrudescence studies suggest that there is a positive correlation between the number of dormant parasites present and when recrudescence occurs in the vertebrate host. Collectively, these data suggest that dormancy occurs in vivo and contributes to recrudescence that is observed following AS treatment. It is possible that this may represent a novel mechanism of parasite survival following treatment with AS.