bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/364570

A Multiplex Droplet Digital PCR Assay for Quantification of HTLV-1c DNA Proviral Load and T-Cells from Blood and Respiratory Exudates Sampled in a Remote Setting

Yurick, D.·Khoury, G.·Clemens, B.·Loh, L.·Pham, H.·Kedzierska, K.·Einsiedel, L.·Purcell, D. F. J.
View original source ↗Export metadata ↓

Abstract

During human T-cell leukemia virus type-1 (HTLV-1) infection the frequency of cells harboring an integrated copy of viral cDNA, the proviral load (PVL), is the main risk factor for progression of HTLV-1-associated diseases. Accurate quantification of provirus by droplet digital PCR (ddPCR) is a powerful diagnostic tool with emerging uses for monitoring viral expression. Current ddPCR techniques quantify HTLV-1 PVL in terms of whole genomic cellular material, while the main target of HTLV-1 infection is the CD4+ and CD8+ T-cell. Our understanding of HTLV-1 proliferation and the amount of viral burden present in different compartments is limited. Recently a sensitive ddPCR assay was applied to quantifying T-cells by measuring loss of germline T-cell receptor genes as method of distinguishing non-T-cell from recombined T-cell DNA. In this study, we demonstrated and validated novel applications of the duplex ddPCR assay to quantify T-cells from various sources of human gDNA extracted from frozen material (PBMCs, bronchoalveolar lavage, and induced sputum) from a cohort of remote Indigenous Australians and then compared the T-cell measurements by ddPCR to the prevailing standard method of flow cytometry. The HTLV-1c PVL was then calculated in terms of extracted T-cell gDNA from various compartments. Because HTLV-1c preferentially infects CD4+ T-cells, and the amount of viral burden correlates with HTLV-1c disease pathogenesis, application of this ddPCR assay to accurately measure HTLV-1c-infected T-cells can be of greater importance for clinical diagnostics, prognostics as well as monitoring therapeutic applications.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Yurick, D., Khoury, G., Clemens, B., Loh, L., Pham, H., Kedzierska, K., Einsiedel, L., Purcell, D. F. J.. 2018-07-08. A Multiplex Droplet Digital PCR Assay for Quantification of HTLV-1c DNA Proviral Load and T-Cells from Blood and Respiratory Exudates Sampled in a Remote Setting. https://doi.org/10.1101/364570

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Lipopolysaccharide and Wzi retain capsular polysaccharide at the Klebsiella pneumoniae cell surface and promote bloodstream infection fitness

Bacterial capsules are formed by individual capsular polysaccharide (CPS) polymers and are the outermost extracellular layers of many Gram-positive and Gram-negative bacteria. Capsules affect several aspects of bacterial biology, including resistance to host immune factors, survival in unfavorable environments and antimicrobial resistance. Research on CPS biology has largely focused on its synthesis and the pathogenic consequences of its presence or absence. Beyond synthesis, our understanding of its retention (attachment) on the cell surface is limited. Klebsiella pneumoniae is an important Gram-negative pathogen and expresses more than 80 capsular serotypes using the common Wzy-dependent CPS biosynthesis system and phenotypically manifests in various forms, making this species an attractive bacterial model to study the underexplored aspects of CPS biology. Here, we identified and characterized bacterial components involved in CPS retention in K. pneumoniae and assessed the importance of CPS retention on bloodstream fitness. Our examination revealed that lipopolysaccharide (LPS) and the outer-membrane protein, Wzi, collectively retain K. pneumoniae CPS on the bacterial surface. We demonstrate that the core-oligosaccharide domain of LPS and the extracellular surface of Wzi likely provide anchoring sites for CPS on the bacterial surface. Furthermore, CPS retention depends on divalent cation availability. Importantly, CPS retention on the bacterial surface is important for survival in serum and bacterial fitness at secondary infection sites in a murine bacteremia model. Together, our findings demonstrate that the functional importance of CPS to bacteria extends beyond synthesis and its overall abundance. Bacterial control over other CPS properties, like cell surface retention, are also important for K. pneumoniae fitness during bacteremia.

microbiology↗
bioRxiv2026

Systematic analysis reveals an Mln1-dependent trade-off between metabolic flexibility and thermotolerance in Cryptococcus neoformans

During infection, fungal pathogens must adapt to multiple environmental challenges, including changes in temperature, nutrient availability, and immune stress. Although transcriptional regulators are central to these adaptations, how individual regulators contribute to distinct environmental responses and how these responses are functionally interconnected remain poorly understood. Here, we investigated the functions of infection-associated transcription factors under distinct host-relevant conditions in Cryptococcus neoformans. Integrating in vivo expression profiling with infectivity data from signature-tagged mutagenesis, we prioritized 24 transcription factors, 12 of which were induced under host-mimicking conditions. Initial functional analyses revealed distinct roles for these transcription factors in adaptation to temperature, carbon, and nitrogen availability. Further characterization of Mln1, a fungal basic helix-loop-helix transcription factor, uncovered an unexpected connection between carbon source adaptation and thermotolerance. Mig1-dependent repression limited MLN1 expression in the presence of glucose, whereas Mln1 accumulated in the nucleus in the presence of trehalose or ethanol, but not glucose. Loss of MLN1 impaired carbon-starvation gene expression and growth on non-preferred carbon sources, particularly trehalose. Conversely, mln1{Delta} cells accumulated more intracellular trehalose and exhibited enhanced thermotolerance, yet showed reduced melanization, titan cell formation, virulence in Drosophila, and brain fungal burden in mice. Together, these results suggest that Mln1 contributes to metabolic flexibility and fungal virulence while differentially influencing thermotolerance. More broadly, these findings highlight a potential physiological trade-off between nutrient utilization and stress resistance, suggesting that these adaptive processes may be interconnected in fungal pathogens.

microbiology↗
bioRxiv2026

The large conjugative plasmid pAB3 integrates into the chromosome of Acinetobacter baumannii ATCC 17978

ATCC 17978 is one of the most utilised lab strains of Acinetobacter baumannii. Current literature suggests ATCC 17978 can have either a sulfamethoxazole-resistant phenotype or have the type VI secretion system (T6SS) switched on depending on the presence/absence of the large conjugative plasmid pAB3, which encodes the antimicrobial resistance gene (ARG) sul2 and T6SS repressors tetR1/tetR2. Here, we show that pAB3 integrates into the ATCC 17978 chromosome, which enables a large inversion resulting in the chromosomal arrangement labelled GS2.24 by Socru. This integration has no transcriptional impact on nearby genes or tetR1/tetR2. We find several instances were a portion of integrated pAB3 containing tetR2 is lost but the rest of the sequence, including sul2, is maintained, suggesting pAB3 integration may serve to make further rearrangements more tractable, enabling a putative T6SS+ sulfamethoxazole-resistant phenotype.

microbiology↗