Murtagh, R. | Search bioRxiv

bioRxiv2020

Sequence evaluation and comparative analysis of novel assays for intact proviral HIV-1 DNA

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon ART-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The Intact Proviral DNA Assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR), is a lower-throughput assay that uses limiting dilution long distance PCR amplification followed by qPCR and near-full length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. ImportanceThe Intact Proviral DNA Assay (IPDA) and Quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 ART-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

microbiology↗

bioRxiv2018

Metagenomic 16S rRNA gene sequencing survey of Borrelia species in Irish samples of Ixodes ricinus ticks

Lyme borreliosis is a systemic infection caused by tick-borne pathogenic borreliae of the Borrelia burgdorferi sensu lato complex or of the more heterogeneous relapsing fever borrelia group. Clinical distinction of the infections due to different borrelia species is difficult. Accurate knowledge of the prevalence and the species of borreliae in the infected ticks in the endemic areas is valuable for formulating appropriate guidelines for proper management of this infectious disease. The purpose of this research was to design a readily implementable protocol to detect the divergent species of borreliae known to exist in Europe, using Irish samples of Ixodes ricinus ticks as the subject for study. Questing I. ricinus nymph samples were taken at six localities within Ireland. The crude DNA of each dried tick was extracted by hot NH4OH and used to initiate a same-nested PCR with a pair of borrelial genus-specific primers to amplify a highly conserved 357/358 bp segment of the 16S rRNA gene for detection and as the template for Sanger sequencing. To distinguish B. garinii from B. burgdorferi and to discriminate the various strains of B. garinii, a second 282 bp segment of the 16S rRNA gene was amplified for Sanger sequencing. A signature segment of the DNA sequence excised from the computer-generated electropherogram was submitted to the GenBank for BLAST alignment analysis. A 100% ID match with the unique reference sequence in the GenBank was required for the molecular diagnosis of the borrelial species or strain. We found the overall rate of borrelial infection in the Irish tick population to be 5%, with a range from 2% to 12% depending on the locations of tick collection. At least 3 species, namely B. garinii, B. valaisiana and B. miyamotoi, are infecting the ticks collected in Ireland. The isolates of B. garinii were confirmed to be strain BgVir, strain Bernie or strain T25. Since antigens for diagnostic serology tests may be species- or even strain-specific, expanded surveillance of the species and strains of the borreliae among human-biting ticks in Ireland is needed to ensure that the antigens used for the serology tests do contain the epitopes matching the antibodies elicited by the borrelial species and strains in the ticks cohabitating in the same environment.

microbiology↗