Next-generation sequencing technology for diagnosis and efficacy evaluation of a patient with visceral leishmaniasis: A case report.

BACKGROUND: Visceral leishmaniasis (VL) is a parasitic disease caused by Leishmania and transmitted by infected sand flies. VL has a low incidence in China, and its clinical presentation is complex and atypical. This disease is easily misdiagnosed and can become life-threatening within a short period of time. Therefore, early, rapid and accurate diagnosis and treatment of the disease are essential. CASE SUMMARY: A 25-year-old male patient presented with the clinical manifestations of irregular fever, hepatosplenomegaly, increased polyclonal globulin, and pancytopenia. The first bone marrow puncture biopsy did not provide a clear diagnosis. In order to relieve the pressure and discomfort of the organs caused by the enlarged spleen and to confirm the diagnosis, splenectomy was performed, and hemophagocytic syndrome was diagnosed by pathological examination of the spleen biopsy. Following bone marrow and spleen pathological re-diagnosis and metagenomic next-generation sequencing (mNGS) technology detection, the patient was finally diagnosed with VL. After treatment with liposomal amphotericin B, the body temperature quickly returned to normal and the hemocytes recovered gradually. Post-treatment re-examination of the bone marrow puncture and mNGS data showed that Leishmania was not detected. CONCLUSION: As a fast and accurate detection method, mNGS can diagnose and evaluate the efficacy of treatment in suspicious cases of leishmaniasis.

[Effects of Dasatinib on the Expansion, Subsets, Receptor Expression and Cytotoxic Function of NK Cells in Vitro].

OBJECTIVE: To investigate the effect of dasatinib on the expansion of NK cells in vitro, as well as the subsets, receptor expression and cytotoxic function of NK cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult volunteers and cultured with SCGM added IL-2 and IL-15 for expansion of NK cells. In this culture system, dasatinib of different concentrations were added. Cell counting and phenotyping by flow cytometry were used to evaluate the amplification efficiency of NK cells. FCM was used to detect the expression of receptors on the surface of NK cells and the distribution of subsets. Subsequently, degranulation assay and CFSE/7AAD based cytotoxicity assay were used to detect the effects of dasatinib on NK cytotoxicity against leukemia cell line K562 cells. RESULTS: The expansion efficiency of NK cells in vitro could be increased by dasatinib at the concentration range of 5-50 nmol/L, and the expansion efficiency of NK cells reached the peak at 20 nmol/L of dasatinib. The NK cytotoxicity against K562 cells in dasatinib cultured group at the concentration of 20 nmol/L was significantly higher than that in control group. For the cells cultured by disatinib in vitro, the MFI of CD226, NKP46 and NKG2D was up-regulated; the ratio of NKG2A+CD57- subset was down-regulated, while the ratio of NKG2A-CD57+ subset was up-regulated.The degranulation response of NKG2A-CD57+ NK cells to K562 cells was stronger than that of NKG2A+CD57- NK cells. CONCLUSION: The results shows that appropriate dose of dasatinib(20 nmol/L) can increases the amplification efficiency of NK cells, simultaneously up-regulates the expression of NK activating receptors and increases the NKG2A-CD57+ subset, which lead to the enhancement of NK cytotoxicty against leukemia cell lines.

[Gene KIR in match with HLA-Cw impacts on NK cell cytotoxicity].

The aim of this study was to investigate how the killer immune globulin-like inhibition receptor (KIR) in match with HLA-Cw impacts on NK cell activity. Mononuclear cells were isolated in 20 ml peripheral blood from 27 healthy persons by Ficoll-Hypaque and purified by NK cell isolation kit. Target cells were mononuclear cells isolated from bone marrow of 30 de novo AML patients. The KIR expression were detected by flow cytometry with antibodies against CD158a, CD158b. The 2 ml of peripheral blood from healthy persons and AML patients were collected, the DNA was extracted by using PROTRANS method, the HLA-Cw and KIR gene were detected by PCR-SSP typing with sequence specific primers. The NK cell cytotoxicity against AML cells was determined by MTT after combination of KIR with HLA-Cw gene. The results indicated that the purity of NK cells was (90.8 +/- 6.08)%. The less the KIR/HLA-Cw matched, the more activity was shown in NK cells. When no match of NK cell/target cell (KIR/HLA-Cw) there was, the cytotoxicity was (50.66 +/- 8.40)%, 1 or 2 matches showed cytotoxicity of (38.28 +/- 6.71)% and (19.74 +/- 4.15)% (p < 0.001). Expression level of KIRs on NK cells also was related with cytotoxicity level (p < 0.001). It is concluded that the interaction between inhibitory KIR and HLA ligands, and also expression level of KIRs on NK cells both impact significantly on NK cell function, that the less match of KIR/HLA-Cw, and the less expression of KIRs on NK cells result in the stronger NK cell cytotoxicity.