Global efforts to monitor and contain the Covid-19 pandemic, caused by the beta-coronavirus SARS-CoV-2, currently rely on RT-qPCR-based diagnostic assays. Yet their high cost, moderate throughput, and dependence on sophisticated equipment limit a broad implementation. Loop-mediated isothermal amplification (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. Here, we established a robust, highly sensitive and versatile RT-LAMP-based SARS-CoV-2 detection assay that is insensitive to carry-over contaminations. Our approach uses a rapid upfront lysis step and hydroxy-naphthol-blue (HNB) for colorimetric detection, which enables the robust identification of Covid-19 infections from a variety of sample types within 30 minutes. By combining RT-LAMP with a simple nucleic acid enrichment method (bead-LAMP), we profoundly increased assay sensitivity to RT-qPCR-like levels, thereby extending applications to large-scale pooled testing. Finally, we developed HomeDip-LAMP for pipette-free SARS-CoV-2 detection for low-resource environments. Our combined optimizations set the stage for implementing RT-LAMP as SARS-CoV-2 diagnostics assay for population-wide and home-based testing.
Purpose: Myelomastocytic leukemia is a term used for patients with advanced myeloid neoplasms, in whom elevated numbers of immature atypical mast cells are found, but criteria for a primary mast cell disease are not met. The origin of mast cells in these patients is presently unknown. Patient and Methods: We have analyzed clonality of mast cells in an 18-year-old patient suffering from acute myeloid leukemia with a complex karyotype including a t(8;21) and mastocytic transformation with a huge increase in immature mast cells and elevated serum tryptase level, but no evidence for a primary mast cell disease/mastocytosis. Results: As assessed by in situ fluorescence hybridization combined with tryptase staining, both the tryptase-negative blast cells and the tryptase-positive mast cells were found to contain the t(8;21)-specific AML1/ETO fusion gene. Myeloablative stem cell transplantation resulted in complete remission with consecutive disappearance of AML1/ETO transcripts, decrease of serum tryptase to normal range, and disappearance of neoplastic mast cells. Conclusion: These data suggest that mast cells directly derive from the leukemic clone in patients with myelomastocytic leukemia.Myelomastocytic leukemia is a term used for patients with advanced myeloid neoplasms, in whom a substantial increase in immature atypical mast cells is found, but the criteria for a primary mast cell disease (mast cell leukemia or systemic mastocytosis) are not fulfilled (1 -5). Typically, patients with myelomastocytic leukemia exhibit an increase in blast cells as well as >10% metachromatic cells in peripheral blood and/or bone marrow smears (3 -5). Blast cells are myeloblasts by morphologic and immunophenotypic criteria. These patients are thus diagnosed to have an advanced myelodysplastic syndrome with excess of blasts, a myeloproliferative disease, or an acute myeloid leukemia (AML; refs. 1 -4). Myelomastocytic leukemia is a rare disease. In fact, <5% of all patients with myelodysplastic syndrome, myeloproliferative disease, or AML are considered to develop a ''mastocytic transformation '' (3, 4).Metachromatic cells in myelomastocytic leukemia are immature, often exhibit a blast-like morphology, and are mast cell lineage cells by electron microscopic and immunophenotypic criteria (CD117 + , tryptase + , CD11b À , and CD123 À ; refs. 3, 4). Mature mast cells may be seen occasionally. As in other patients with myelodysplastic syndrome, myeloproliferative disease, or AML, major signs of dysplasia may be found in erythroid and granulomonocytic cells (1 -3). The bone marrow histology shows a diffuse spread of metachromatic cells (1 -6), whereas multifocal aggregates of tryptase-positive mast cells, typically seen in patients with systemic mastocytosis (6, 7), are not found (1 -6). Other criteria of systemic mastocytosis (6) are also not met. In fact, mast cells are CD2 negative and CD25 negative and do not exhibit transforming mutations at codon 816 of c-kit (1 -6). Thus, a number of diagnostic criteria are available that ...
Spontaneous growth of myeloid colonies (colony-forming unit–granulocyte-macrophage [CFU-GM]) can be observed in methylcellulose cultures containing peripheral blood mononuclear cells (PB-MNCs) and is supposedly caused by the release of colony-stimulating factors (CSF ) by accessory cells. Because of its cytokine synthesis-inhibiting effects on T lymphocytes and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirect modulation of hematopoiesis. We studied the effect of recombinant human IL-10 (rhIL-10) on spontaneous growth of myeloid colonies derived from human PB-MNCs. A total of 10 ng/mL of IL-10 almost completely inhibited spontaneous CFU-GM proliferation (by 95.1%; P < .001, n = 7) in unseparated PB-MNCs. This effect was dose-dependent and specific, because a neutralizing anti–IL-10 antibody was able to prevent IL-10–induced suppression of CFU-GM growth. Spontaneous CFU-GM growth, which required the presence of both monocytes (CD14+ cells) and T lymphocytes (CD3+ cells), was also greatly suppressed by a neutralizing anti–granulocyte-macrophage CSF (GM-CSF ) antibody but was only slightly or not at all inhibited by antibodies against G-CSF or IL-3. Moreover, IL-10–suppressed colony growth could be completely restored by the addition of exogenous GM-CSF. Using semiquantitative polymerase chain reaction, we were able to show that GM-CSF transcripts that spontaneously increased in PB-MNCs within 48 hours of culture were markedly reduced by the addition of IL-10. Inhibiton of GM-CSF production in PB-MNCs by IL-10 was also confirmed at the protein level by measuring GM-CSF levels in suspension cultures. Our findings suggest that autonomous CFU-GM growth, resulting from an interaction of monocytes and T lymphocytes, is mainly caused by endogenous GM-CSF release and can be profoundly suppressed by the addition of exogenous IL-10. Considering the strong inhibitory action of IL-10 on GM-CSF production and spontaneous cell growth in vitro, this cytokine may be useful in myeloid malignancies in which autocrine and/or paracrine mechanisms involving GM-CSF are likely to play a pathogenetic role.
During a pandemic, mitigation as well as protection of system-critical or vulnerable institutions requires massive parallel, yet cost effective testing to monitor the spread of agents such as the current SARS-CoV2 virus. Here we present SARSeq, saliva analysis by RNA sequencing, as an approach to monitor presence of SARS-CoV2 and other respiratory viruses performed on tens of thousands of samples in parallel. SARSeq is based on next generation sequencing of multiple amplicons generated in parallel in a multiplexed RT-PCR reaction. It relies on a two-dimensional unique dual indexing strategy using four indices in total for unambiguous and scalable assignment of reads to individual samples. We calibrated this method using dilutions of synthetic RNA and virions to show sensitivity down to few molecules, and applied it to hundreds of patient samples validating robust performance across various sample types. Double blinded benchmarking to gold-standard quantitative RT-PCR performed in a clinical setting and a human diagnostics laboratory showed robust performance up to a Ct of 36. The false positive rate, likely due to cross contamination during sample pipetting, was estimated at 0.04-0.1%. In addition to SARS-CoV2, SARSeq detects Influenza A and B viruses as well as human rhinovirus and can be easily expanded to include detection of other pathogens. In sum, SARSeq is an ideal platform for differential diagnostic of respiratory diseases at a scale, as is required during a pandemic.
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