BackgroundConfirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results.Methodology/Principal findingsThe Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%.Conclusions/SignificanceDue to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.

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The HLA-DRB1 gene encodes a protein that is essential for the immune system. This gene is important in organ transplant rejection and acceptance, as well as multiple sclerosis, systemic lupus erythematosus, Addison’s disease, rheumatoid arthritis, caries susceptibility, and Aspirin-exacerbated respiratory disease. The following Homo sapiens variants were investigated: single-nucleotide variants (SNVs), multi-nucleotide variants (MNVs), and small insertions–deletions (Indels) in the HLA-DRB1 gene via coding and untranslated regions. The current study sought to identify functional variants that could affect gene expression and protein product function/structure. ALL target variants available until April 14, 2022, were obtained from the Single Nucleotide Polymorphism database (dbSNP). Out of all the variants in the coding region, 91 nsSNVs were considered highly deleterious by seven prediction tools and instability index; 25 of them are evolutionary conserved and located in domain regions. Furthermore, 31 indels were predicted as harmful, potentially affecting a few amino acids or even the entire protein. Last, within the coding sequence (CDS), 23 stop-gain variants (SNVs/indels) were predicted as high impact. High impact refers to the assumption that the variant will have a significant (disruptive) effect on the protein, likely leading to protein truncation or loss of function. For untranslated regions, functional 55 single-nucleotide polymorphisms (SNPs), and 16 indels located within microRNA binding sites, furthermore, 10 functionally verified SNPs were predicted at transcription factor-binding sites. The findings demonstrate that employing in silico methods in biomedical research is extremely successful and has a major influence on the capacity to identify the source of genetic variation in diverse disorders. In conclusion, these previously functional identified variants could lead to gene alteration, which may directly or indirectly contribute to the occurrence of many diseases. The study’s results could be an important guide in the research of potential diagnostic and therapeutic interventions that require experimental mutational validation and large-scale clinical trials.

Introduction: Protozoa of the genus Leishmania cause a wide range of pathologies from self-healing skin lesions to visceral pathology. The outcome of infection depends on the species of the infecting Leishmania parasite, the quality and quantity of the host immune response and the host genetic background. Objective: This study aimed to determine the differential expression of TL2, TL4, TL9, IFN-γ and IL-10 cytokine genes of Human THP1 cell line following in vitro infection with Leishmania donovani isolates from different clinical forms. Methods: Human THP1 cells were infected by live promastigotes of leishmania donovani isolates from visceral (VL), cutaneous (CL), Post Kala-Azar dermal Leishmaniasis (PKDL) and mucosal Leishmaniasis (ML) Patients. The expression of Tolls like receptor TL2, TL4 and TL9 and the expression of IFN-γ and IL-10 cytokine were measured using Real Time PCR. Results: A significant increase in the expression of TLR 2, TLR4 and TLR9 by THP-1 was detected following infection of THP-1 cells with L. donovani isolates from ML and PKDL patients. A high expression IL-10 mRNA by THP-1 cells was detected in cells infected by Leishmania donovani isolates from mucosal lesions. Conclusion: Leishmania donovani isolates from different clinical forms of Leishmaniasis induce different cytokine responses.

Leishmania, a protozoan parasite, is the causative agent of leishmaniosis. It lives and multiplies within the harsh environment of macrophages. Infection of macrophages by the intracellular protozoan Leishman leads to downregulation of a number of macrophage innate host defense mechanisms, thereby allowing parasite survival and replication. In order to investigate how intracellular parasite manipulates the host cell environment, we undertook a quantitative study of human monocyte-derived macrophages (THP-1) following infection with L. donovani and L. major. In this study we aimed to measure the proliferation rate of human (THP1) macrophage cell line following infection with cutaneous and visceral leishmania isolates. We used sysmex to measure the proliferation rate of human (THP1) cells infected with cutaneous and visceral leishmania isolates after invitro infection of human (THP1) Macrophage cell line by leishmania isolates. We detected L. major significantly increased the proliferation of infected THP1 cell line compared with L. donvani. Our findings indicate that the infection of host cells human macrophage cell lines by L. major stimulates the replication of the cell as a mechanism to enhance the replication of the parasites.

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Background: HLA-DRB1 is the most polymorphic gene in the human leukocyte antigen (HLA) class II, and exon 2 is critical because it encodes antigen-binding sites. This study aimed to detect functional or marker genetic variants of HLA-DRB1 exon 2 in renal transplant recipients (acceptance and rejection) using Sanger sequencing. Methods: This hospital-based case-control study collected samples from two hospitals over seven months. The 60 participants were equally divided into three groups: rejection, acceptance, and control. The target regions were amplified and sequenced by PCR and Sanger sequencing. Several bioinformatics tools have been used to assess the impact of non-synonymous single-nucleotide variants (nsSNVs) on protein function and structure. The sequences data that support the findings of this study with accession numbers (OQ747803-OQ747862) are available in National Center for Biotechnology Information (GenBank database). Results: Seven SNVs were identified, two of which were novel (chr6(GRCh38.p12): 32584356C>A (K41N) and 32584113C>A (R122R)). Three of the seven SNVs were non-synonymous and found in the rejection group (chr6(GRCh38.p12): 32584356C>A (K41N), 32584304A>G (Y59H), and 32584152T>A (R109S)). The nsSNVs had varying effects on protein function, structure, and physicochemical parameters and could play a role in renal transplant rejection. The chr6(GRCh38.p12):32584152T>A variant showed the greatest impact. This is because of its conserved nature, main domain location, and pathogenic effects on protein structure, function, and stability. Finally, no significant markers were identified in the acceptance samples. Conclusion: Pathogenic variants can affect intramolecular/intermolecular interactions of amino acid residues, protein function/structure, and disease risk. HLA typing based on functional SNVs could be a comprehensive, accurate, and low-cost method for covering all HLA genes while shedding light on previously unknown causes in many graft rejection cases.