A patient with fever, chills, and pancytopenia as major clinical manifestations was presented. To investigate the cause, the patient's peripheral blood was collected for pathogen screening using metagenomic next - generation sequencing (mNGS). The DNA sequence of Leishmania donovani was detected, and Leishmania amastigotes were found in bone marrow smears using microscopy. The case was therefore definitively diagnosed as visceral leishmaniasis, and was cured and discharged from hospital following treatment with liposomal amphotericin B for 14 days. This is the first imported case of visceral leishmaniasis since the founding of Shenzhen City in 1979.
Humans ; Fever ; High-Throughput Nucleotide Sequencing ; Leishmania donovani/genetics* ; Leishmaniasis, Visceral/drug therapy*

This study was directed to determine the nucleotide sequence of the ITS (internal transcribed spacer) gene of Leishmania donovani isolates from desert foci (L. d XJ771), hill foci (L. d SC10) and plain foci (L. d SD2), and to find out the differences of the sequences of ITS gene among these three isolates. The specific ITS fragments from nuclear DNA of three Leishmania isolates were amplified by PCR, cloned into PMD18-T vector, and then sequenced by the dideoxy chain termination method. Sequence analysis showed that the amplified DNA fragments of the three isolates were 1 086 bp (L. d XJ771), 1 027 bp (L. d SC10) and 1 028 bp (L. d SD2). There were obvious sequence differences among L. d XJ771, L. d SC10 and L. d SD2. The differences between L. d XJ771 (desert foci isolate) and L. d SC10 (hill foci isolate) were less than the differences between L. d XJ771 (desert foci isolate ) and L. d SD2. (plain foci isolate).
China ; Cloning, Molecular ; DNA, Protozoan ; genetics ; DNA, Ribosomal Spacer ; genetics ; Environment ; Leishmania donovani ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA

Leishmaniasis (Kala-azar) from different endemic regions of China expresses different clinic and epidemiological features, and traditionally is classified as hilly, plain and desert types/foci. We concentrated our review on whether the pathogens from those foci were different at molecular level, if so, whether there are were molecular markers readily identifiable by molecular technologies. This was a review of a 20-year search for such markers by using kinetoplastic DNA (kDNA), nDNA hybridization, PCR-SSCP, RAPD and sequence analysis of SSU rDNA variable regions and LACK gene. The results showed that heterogeneities at molecular level exist in Leishmania isolated from different foci of China, which could be used as markers for different types of Leishmaniasis in China.
China ; DNA Fingerprinting ; DNA, Protozoan ; analysis ; genetics ; Genotype ; Humans ; Leishmania donovani ; classification ; genetics ; isolation & purification ; Leishmaniasis, Visceral ; classification ; parasitology ; Mutation

OBJECTIVETo confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L.d.) isolates from different epidemic foci in China.

METHODSSpecific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM(R)-T Easy Vectors. After that, the specific fragments were sequenced by an automated DNA sequencer.

RESULTSSequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length. All 5 point mutations were located in two unique sequence blocks (UQ-I and UQ-II), and no insertions or deletions were found. The identities of comparison of Leishmania in GeneBank were more than 98%.

CONCLUSIONFive point mutations exist in the SSU rDNA variable region of 5 L.d. isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences of the SSU rDNA variable region exist among L.d. isolates from different foci.

Animals ; DNA, Protozoan ; chemistry ; DNA, Ribosomal ; chemistry ; Humans ; Leishmania donovani ; genetics ; Leishmaniasis, Visceral ; parasitology ; Point Mutation ; Polymerase Chain Reaction

The objective of this study was to construct and express recombinant prokaryotic plasmid pET32a (+)- ast1 in E. coli BL21(DE3). Amastin gene was amplified from genomic DNA of Leishmania Donovani and its transmembran region was predicted by the methods of SOSUI and Tmpred; astl located in N-terminus of amastin gene was amplified and cloned into prokaryotic plasmid pET32a(+), which was named pET32a(+)-ast1, and then rAST1 was expressed in E. coli BL21(DE3). The results of SDS-PAGE and immunobloting assay showed that a fusion protein rAST1 (relative molecular mass about 27 kDa) was able to express in BL21. The recombinant prokaryotic plasmid pET32a(+)- ast1 was successfully constructed, and noted to be efficiently expressed in E. coli BL21(DE3).
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; Genes, Protozoan ; Leishmania donovani ; genetics ; Plasmids ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

The LACK gene from Leishmania, an analogue of the receptor of activated protein kinase C, was discovered recently. In this study, the LACK gene of Leishmania donovani was obtained from the recombinant plasmid T-LACK by PCR. The gene was cloned into eukaryotic expressed plasmid pcDNA3.1(+) to construct recombinant plasmid. This recombinant plasmid then was transfected into the eukaryotic cell COS-7, and the expression of LACK gene in eukaryotic cell was detected by RT-PCR and immunofluorescent staining. Both RT-PCR and immunofluorescent staining of recombinant plasmid transfected COS-7 showed positive reaction, thus indicating that the recombinant plasmid pcDNA3-LACK can express LACK protein in euka ryotic cell COS-7.
Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; COS Cells ; Cloning, Molecular ; DNA, Recombinant ; biosynthesis ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Leishmania donovani ; Plasmids ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vaccines, DNA