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BACKGROUND: Strategies toward HIV-1 cure aim to clear, inactivate, reduce, or immunologically control the virus from a pool of latently infected cells such that combination antiretroviral therapy (cART) can be safely interrupted. In order to assess the impact of any putative curative interventions on the size and inducibility of the latent HIV-1 reservoir, robust and scalable assays are needed to precisely quantify the frequency of infected cells containing inducible HIV-1. METHODS: We developed Specific Quantification of Inducible HIV-1 by RT-LAMP (SQuHIVLa), leveraging the high sensitivity and specificity of RT-LAMP, performed in a single reaction, to detect and quantify cells expressing tat/rev HIV-1 multiply spliced RNA (msRNA) upon activation. The LAMP primer/probe used in SQuHIVLa was designed to exclusively detect HIV-1 tat/rev msRNA and adapted for different HIV-1 subtypes. RESULTS: Using SQuHIVLa, we successfully quantify the inducible viral reservoir in CD4+ T cells from people living with HIV-1 subtypes B and C on cART. The assay demonstrates high sensitivity, specificity, and reproducibility. CONCLUSIONS: SQuHIVLa offers a high throughput, scalable, and specific HIV-1 reservoir quantification tool that is amenable to resource-limited settings. This assay poses remarkable potential in facilitating the evaluation of potential interventional strategies toward achieving HIV-1 cure.
HIV infection remains challenging because the virus hides in certain cells, making it invisible to the immune system. This hidden virus forms what is called a latent HIV reservoir. If someone with HIV stops their antiviral therapy, the virus quickly re-emerges. Because of this, researchers are exploring various strategies to eliminate this reservoir and cure HIV. To evaluate these strategies, we need a method to measure the reservoir's size before and after trials. Our study introduces SQuHIVLa, a highly sensitive and specific method for quantifying the latent reservoir. SQuHIVLa could become a vital tool for monitoring HIV patients and assessing treatment effectiveness, bringing us closer to finding a cure.
RESUMO
Fungal endophytes have the capacity to biosynthesize secondary metabolites that are produced by their host plants. In this study, a dilactone terpenoid of C16 architecture was isolated from the fungal endophytes of Kigelia africana, in our attempt to identify anti-Pseudomonas aeruginosa metabolites. Thirty-eight fungal isolates were cultured for biomolecule production over a period of thirty days. Extracts from three (ZF 34, ZF 52 and ZF 91) of the fungi showed good anti-P. aeruginosa activity, with ZF 52 presenting the best MIC of 19.53 µg/mL and was accordingly subjected to chromatographic separation. Based on nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry and single crystal X-ray diffraction (XRD) analyses, the isolated compound was identified as a C16-terpene dilactone, with a structure consistent with that of the known diterpene, CJ-14445. The isolated dilactone showed anti-P. aeruginosa activity with MIC of 0.61 µg/mL, signifying the antibacterial potential of the biomolecule. The bioactive fungal isolate (ZF 52) was identified as Neofusicoccum luteum based on genomic DNA sequencing. This is the first report of the endophyte N. luteum from K. africana and the first reported occurrence of CJ-14445 in the fungus.