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Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020
(2020)
Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.
The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.
In resource-limited or point-of-care settings, rapid diagnostic tests (RDTs), that aim to simultaneously detect HIV antibodies and p24 capsid (p24CA) antigen with high sensitivity, can pose important alternatives to screen for early infections. We evaluated the performance of the antibody and antigen components of the old and novel version of the Determine™ HIV-1/2 Ag/Ab Combo RDTs in parallel to quantifications in a fourth-generation antigen/antibody immunoassay (4G-EIA), p24CA antigen immunoassay (p24CA-EIA), immunoblots, and nucleic acid quantification. We included plasma samples of acute, treatment-naïve HIV-1 infections (Fiebig stages I–VI, subtypes A1, B, C, F, CRF02_AG, CRF02_AE, URF) or chronic HIV-1 and HIV-2 infections. The tests’ antigen component was evaluated also for a panel of subtype B HIV-1 transmitted/founder (T/F) viruses, HIV-2 strains and HIV-2 primary isolates. Furthermore, we assessed the analytical sensitivity of the RDTs to detect p24CA using a highly purified HIV-1NL4-3 p24CA standard. We found that 77% of plasma samples from acutely infected, immunoblot-negative HIV-1 patients in Fiebig stages II–III were identified by the new RDT, while only 25% scored positive in the old RDT. Both RDTs reacted to all samples from chronically HIV-1-infected and acutely HIV-1-infected patients with positive immunoblots. All specimens from chronically infected HIV-2 patients scored positive in the new RDT. Of note, the sensitivity of the RDTs to detect recombinant p24CA from a subtype B virus ranged between 50 and 200 pg/mL, mirrored also by the detection of HIV-1 T/F viruses only at antigen concentrations tenfold higher than suggested by the manufacturer. The RTD failed to recognize any of the HIV-2 viruses tested. Our results indicate that the new version of the Determine™ HIV-1/2 Ag/Ab Combo displays an increased sensitivity to detect HIV-1 p24CA-positive, immunoblot-negative plasma samples compared to the precursor version. The sensitivity of 4G-EIA and p24CA-EIA to detect the major structural HIV antigen, and thus to diagnose acute infections prior to seroconversion, is still superior.
Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.
Type I interferons (IFNs), including IFN-α, upregulate an array of IFN-stimulated genes (ISGs) and potently suppress Human immunodeficiency virus type 1 (HIV-1) infectivity in CD4(+) T cells, monocyte-derived macrophages, and dendritic cells. Recently, we and others identified ISG myxovirus resistance 2 (MX2) as an inhibitor of HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection, e.g., prior to (or during) reverse transcription, remain to be defined. We show here that the HIV-1 CA mutations N74D and A105T, both of which allow escape from inhibition by MX2 and the truncated version of cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutation P90A, all increase sensitivity to IFN-α-mediated inhibition. Using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, we demonstrate that the IFN-α hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant but modestly increased the IFN-α sensitivity of wild-type virus. Interestingly, the infectivity of wild-type or P90A virus could be rescued from the MX2-independent IFN-α-induced blocks in THP-1 cells by treatment with cyclosporine (Cs) or its nonimmunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFN-α-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shield the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection.
Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.
DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.