Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V⁺) and uninfected (V⁻) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V⁺ compared with V⁻ isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V⁺ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V⁺ and V⁻ isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.
Glucose-6-Phosphate Isomerase ; Glycogen Phosphorylase ; Heat-Shock Proteins ; Host-Parasite Interactions ; Malate Dehydrogenase ; Metabolism ; Polymerase Chain Reaction ; Proteome ; Reticuloendotheliosis virus ; Ribosomal Proteins ; RNA, Double-Stranded ; RNA, Messenger ; Trichomonas vaginalis* ; Trichomonas* ; Triose-Phosphate Isomerase ; Virulence

OBJECTIVETo confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries.

METHODSGenomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank.

RESULTSOf nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3)) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world.

CONCLUSIONGenotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

Amino Acid Sequence ; Animals ; Base Sequence ; DNA, Protozoan ; chemistry ; Genotype ; Giardia lamblia ; classification ; enzymology ; genetics ; Polymerase Chain Reaction ; Restriction Mapping ; Triose-Phosphate Isomerase ; chemistry ; genetics

BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.
ADP Ribose Transferases/genetics ; Bacterial Proteins/*genetics ; Bacterial Toxins/*genetics ; Clostridium difficile/isolation & purification/*metabolism ; DNA, Bacterial/genetics/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Prospective Studies ; Real-Time Polymerase Chain Reaction ; Triose-Phosphate Isomerase/genetics

BACKGROUND: ChromID Clostridium difficile agar (IDCd; bioMerieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). METHODS: A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMerieux SA), and multiplex PCR for tcdA, tcdB, and tpi. RESULTS: The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. CONCLUSIONS: The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.
Agar/*chemistry ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium difficile/genetics/*isolation & purification ; DNA, Bacterial/analysis ; Enterocolitis, Pseudomembranous/diagnosis/microbiology ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Triose-Phosphate Isomerase/genetics

OBJECTIVETo investigate the intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia (G. lamblia).

METHODSTotal genomic DNA of G. lamblia was extracted and partial fragments of the triose phosphate isomerase (tim) gene were amplified by polymerase chain reaction (PCR). All nucleotide sequences were analyzed by using a phylogenetic analysis, which was constructed with parsimony and Neighbor-joining (N-J) methods.

RESULTSA total of 124 variable sites (23% of all sequences detected) was defined, most of which were found at the silent sites of codons. Two similar phylogenetic trees were constructed, subdividing 16 Giardia isolates into two groups.

CONCLUSIONThe genetic diversity of G. lamblia appeared to be little affected by factors of both host and geography, while natural-selection played an important role in DNA molecular evolution level of the tim gene. The tim gene may be considered a very useful genetic marker of the population genetic structure of G. lamblia.

Animals ; Base Sequence ; DNA, Protozoan ; chemistry ; genetics ; Giardia lamblia ; enzymology ; genetics ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Species Specificity ; Triose-Phosphate Isomerase ; genetics

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.
Animals ; China ; Cluster Analysis ; Coinfection/parasitology/veterinary ; Cytoskeletal Proteins/genetics ; DNA, Protozoan/chemistry/genetics ; Dog Diseases/parasitology ; Dogs ; Genotype ; Giardia lamblia/*classification/*genetics/isolation & purification ; Giardiasis/parasitology/*veterinary ; Glutamate Dehydrogenase/genetics ; Molecular Sequence Data ; *Multilocus Sequence Typing ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 18S/genetics ; Triose-Phosphate Isomerase/genetics