Simultaneous Ultrasensitive Subpopulation Staining/Hybridization In Situ (SUSHI) in HIV-1 Disease Monitoring

Abstract

The field of virology is undergoing a revolution as diagnostic tests and new therapies are allowing clinicians to treat, monitor, and predict outcomes of viral diseases. The majority of these techniques, however, destroy the factory of viral production and the information inherent in the reservoir - the cell. In this chapter, we describe a technique that combines cell surface immunophenotyping (to unequivocally identify cell types) and ultrasensitive fluorescence in situ hybridization (U-FISH) for HIV-1 to detect productively infected cells. Identification of virus and host (cells) allows earlier detection of changes in viral production and viral suppression but most importantly allows clinicians to monitor response to anti-viral therapy on a cell-by-cell and tissue-by-tissue basis taking into account the fact that the human body consists of very different, distinct compartments with unique selection pressures exerted on the viral life cycle.

What do we measure when we measure cell-associated HIV RNA

“…This method was shown to be biologically relevant as Derdeyn et al demonstrated a near-perfect correlation between the frequencies of cells from HIV-infected donors that could be stimulated ex vivo to transcribe viral RNA, enumerated by ISH, and cells that could be stimulated to produce infectious virus, measured by a coculture assay [ 136 ]. At about the same time, Patterson and colleagues developed simultaneous ultrasensitive subpopulation staining/hybridization in situ assay (SUSHI), combining cell surface immunophenotyping with fluorescent ISH for US RNA [ 137 , 138 ]. This assay has been used in several studies, which demonstrated the correlation of US RNA+ cell frequencies with ex vivo proliferative responses to HIV CA-p24 and confirmed our data on the clinical relevance of US RNA measurement for predicting the response to ART [ 139 , 140 ].…”

Prevalence and correlates of persistent intracellular HIV transcription in individuals on efavirenz versus atazanavir-based regimens: A prospective cohort study

“…In our prospective study, we used a method which combines immunophenotyping with ultrasensitive-FISH to detect unspliced HIV-1 gag-pol in relevant cell populations in order to compare HIVpt in virologically suppressed patients on efavirenz(EFV) or atazanavir/ritonavir(ATV/r) and a backbone of emtricitabine-tenofovir(FTC-TDF). [ 7 , 10 , 11 ] We followed prospectively our patient cohort for one year and investigated the impact of HIVpt on virological outcomes and CD4+ T-cell recovery. Finally, we tested for differences in the mRNA expression of two widely studied ABC transporters (P-glycoprotein, P-gp/ABCB1 and multidrug resistance-associated protein-1, MRP1/ABCC1) in peripheral blood mononuclear cells(PBMCs) between patients with and without HIVpt by treatment regimen.…”

Μελέτη Της Μεταγραφικής Δραστηριότητας Γονιδίων Που Κωδικοποιούν Πρωτεΐνες - Μεταφορείς Σε Ασθενείς Με HIV Λοίμωξη Υπό Αντιρετροϊκή Αγωγή

“…Τα επίπεδα μη ματισμένου CA-RNA έχουν συσχετιστεί με το χρόνο υποτροπής ιαιμίας σε μια πρόσφατη μελέτη (70), ενώ είχαν καίριο ρόλο στις μελέτες με χρήση παραγόντων αναστροφής της λανθάνουσας λοίμωξης (latency reversal agents) (66).4. Μέθοδοι ανοσοφαινοτυπικού προσδιορισμού/in situ υβριδισμού(Simultaneous ultrasensitive subpopulation staining/hybridization in situ, SUSHI) είναι μια μέθοδος η οποία επιτρέπει την ανίχνευση HIV usRNA σε επίπεδα 10-20cp/κύτταρο στην περίπτωση που 0,3% των κυττάρων είναι θετικά για τον ιό με τη μέθοδο αυτή(71). Νεότερες μέθοδοι στοχεύουν στο συνδυασμό ανίχνευσης ενδοκυττάριου ιικού RNA και ιικών πρωτεϊνών όπως η gag με ενθαρρυντικά αποτελέσματα για τον περιορισμό των ψευδώς θετικών αποτελεσμάτων (53).5.…”