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Objective To establish a multiplex nucleic acid assay for rapid detection of Echinococcus multilocularis, E. granulosus and E. shiquicus based on the recombinase-aided isothermal amplification assay (RAA) and to preliminarily assess its diagnostic efficiency. Methods The mitochondrial genomic sequences of E. multilocularis (GenBank accession number: NC_000928), E. granulosus (GenBank accession number: NC_044548) and E. shiquicus (GenBank accession number: NC_009460) were used as target sequences, and three pairs of primers were designed based on the RAA primer design principle and synthesized for the subsequent multiple RAA amplification. The genomic DNA of E. multilocularis, E. granulosus and E. shiquicus at different concentrations and the recombinant plasmids containing the target gene at various concentrations were amplified to evaluate the diagnostic sensitivity of the multiplex RAA assay, and the genomic DNA of E. multilocularis, E. granulosus, E. shiquicus, Taenia multiceps, T. saginata, T. asiatica, Dipylidium caninum, T. hydatigena, Toxocara canis, Fasciola hepatica, T. pisiformis, Mesocestoides lineatus and Cryptosporidiumn canis was detected using the multiplex RAA assay to evaluate its specificity. In addition, the reaction condition of the multiplex RAA assay was optimized, and was then employed to detect the tissues with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples to examine its application values. Results The multiplex RAA assay was effective to specifically amplify the mitochondrial gene fragments of E. multilocularis, E. granulosus and E. shiquicus within 40 min at 39 °C, with sequence lengths of 540, 430 bp and 200 bp, respectively. This multiplex RAA assay showed the minimum detection limits of 2.0, 2.5 pg/μL and 3.1 pg/μL for detection of the genomic DNA of E. multilocularis, E. granulosus and E. shiquicus, and presented the minimum detection limit of 200 copies/μL for detection of the recombinant plasmids containing E. multilocularis, E. granulosus and E. shiquicus target genes. This multiplex RAA assay was effective to simultaneously detect single and multiple infections with E. multilocularis, E. granulosus and E. shiquicus, but failed to amplify the genomic DNA of T. multiceps, T. saginata, T. asiatica, D. caninum, T. hydatigena, T. canis, F. hepatica, T. pisiformis, M. lineatus and C. canis. In addition, the optimized multiplex RAA assay was effective to detect all positive samples from the tissue samples with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples, which was fully consistent with the detection of the single PCR assay. Conclusion A sensitive and specific multiplex nucleic acid assay for rapid detection of E. multilocularis, E. granulosus and E. shiquicus has been successfully established.
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Coenurosis is an important zoonotic helminthic disease caused by the larval stage of the tapeworm Taenia multiceps. This parasite typically infects the brain of the intermediate hosts, including sheep, goat, cattle and even humans. We report a case of T. multiceps infection in a yak confirmed by clinical symptoms, morphological characteristics, and molecular and phylogenetic analyses. The coenurus was thin-walled, whitish, and spherical in shape with a diameter of 10 cm. The parasite species was identified as T. multiceps by PCR amplification and sequencing of the 18S rRNA, cox1 and nad1 genes. Three gene sequences all showed high homology (all above 97%) with the reference sequences from different hosts. Moreover, phylogenetic reconstructions with the 3 published Taenia gene sequences confirmed that the Qinghai yak isolate was closely related to T. multiceps. Although there are advanced diagnosis and treatment methods for coenurosis, early infection is difficult to diagnose. Importantly, the findings of yak infection case should not be ignored due to its zoonotic potential.
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Animals , Cattle , Humans , Brain , Cestoda , China , Diagnosis , Goats , Helminths , Parasites , Polymerase Chain Reaction , Sheep , TaeniaABSTRACT
Objective To analyze the subtypes and characteristics of HIV-1 among men who have sex with men (MSM) in Zhejiang province from the year 2004 to 2011.Methods Blood DNA/RNA was extracted from the MSM HIV -1 infected individuals and then HIV-1 gag and pol fragments were amplified by nested polymerase chain reaction (nested-PCR)or RT-PCR.The positive PCR products were sequenced and the obtained sequences were analyzed by phylogenetic inference.Results A total of 117 HIV-1 infected MSMindividuals were analyzed,covering 21 provinces according to their household registry.Totally,three major strains were identified including CRF01_AE (84.62%,n =99 ),B (5.98%,n=7)and CRF07_BC (5.13%,n=6). Moreover,3 novel 01B recombinant stains,1 CRF59_01B and 1 CRF08_BC were found.According to the neighbor-joining tree by pol fragments,there were more than 30 sub clusters with bootstrap value higher than 70% among the 84 CRF01_AE sequences.Conclusion CRF01_AE is the major HIV-1 infection strain among MSMin Zhejiang province.CRF59_01B and other novel 01B recombinant stains are first reported among MSM individuals in Zhejiang province.
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Objective To analyze the molecular epidemiological characteristics on HIV infectors/AIDS patients (HIV/AIDS) under a follow-up program in Zhejiang province in 2009.Methods 303 cases were randomly sampled.Information on the cases was collected and followed by genomic DNA extraction.Gag gene fragments were amplified by nested PCR,followed by sequencing and bio-informatic analysis.Results The rate of success for sequence acquisition was 74.3％ (225/303).Distributions of HIV subtypes were as follows:CRF01_AE (58.7％),CRF07_BC (13.8％),CRF08_BC (9.8％),B' (15.1％),C (1.8％),G (0.4％) and unassigned BC (unique recombinant form 0.4％).Results from the HIV BLAST analysis showed that the sources of strains with the highest homology involved in 10 provinces/municipalities (Liaoning,Guangxi,Yunnan,Henan,etc.) and five other countries (Thailand,Vietnam,India,South Africa and Libya).The CRF01 _AE phylogenetic tree was divided into four clusters.The sequences of HIV/AIDS with homosexual transmission showed a gathering in cluster 1,and mixing with those infected through heterosexual contact.Conclusion Circulating recombinant forms of HIV seemed to play a dominant role in Zhejiang province.Unique recombinant form and new subtype of HIV were found.People living with HIV under homosexual transmission and heterosexual transmission had a trend of interwoven with each other.Increase of both the diversity and complexity of HIV strains were also noticed in Zhejiang province.
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<p><b>OBJECTIVE</b>To survey the prevalence of drug resistant HIV in Zhejiang province in 2009-2011.</p><p><b>METHODS</b>WHO truncated sequential sampling technique was adopted annually by using 63, 62 and 57 samples of newly diagnosed as HIV positive and aged 16-25 years in Hangzhou, Ningbo and Wenzhou from 2009 to 2011, respectively. RNA was prepared and HIV pol region was amplified by RT-PCR and nested PCR. Pol genetic mutation associated with drug resistance was analyzed.</p><p><b>RESULTS</b>The success rates for sequence acquisition of the survey were 82.5% (52/63), 95.2% (59/62) and 94.7% (54/57) from year 2009 to 2011, respectively, and the main subtype was CRF01_AE (68.5% (37/54)-71.2% (37/52)). A total of 4 surveillance drug-resistance mutation (SDRMs), 2 SDRMs and 2 SDRMs were found by analyzing the 47 sequences each year, sampled from year 2009 to 2010, respectively, indicating that the prevalence of drug resistant HIV stains was moderate in 2009, and low for the next two years (2010-2011). A total of 8 individuals with drug resistant HIV stains found in this study were all infected by sexual transmission, especially in homosexual transmission (6 cases), and the main subtype was CRF01_AE (7 cases). SDRMs for protease inhibitor (PI), nucleotide HIV-reverse transcriptase inhibitor (NRTI) and non-NRTI (NNRTI) (L90M, T215S and Y188L) were all found in one case.</p><p><b>CONCLUSION</b>The prevalence of drug resistant HIV stains in major areas with AIDS epidemic in Zhejiang province was low in 2009-2011.</p>
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Adolescent , Adult , Female , Humans , Male , Young Adult , Anti-HIV Agents , Pharmacology , China , Epidemiology , Drug Resistance, Viral , HIV , Genetics , HIV Infections , VirologyABSTRACT
Objective To analyze the relationship between strain subtypes and transmission of HIV infection on marriage-based immigrant women,their spouses and children in rural area of Zhejiang province.Methods Marriage-based immigrant women with HIV infection,their HIV infected spouses and children in rural area in Zhejiang province,were selected as study objects.Analysis on genetic sequence and epidemiologic information was carried out.Subgenomic gag was amplified by nest-PCR analysis on the whole blood samples.Genetic subtype characterization and the source of HIV strains were analyzed.Relationships on sequences were also examined by phylogenetic tree analysis.Results Genetic sequences of 72 samples from HIV infected marriage-based immigrant women were obtained.The genetic subtypes comprised 21 CRF01_AE (29.2％),12CRF07 BC (16.7％),31 CRF08 BC (43.1％),6 B (8.3％),2 C (2.8％).HIV strains from 45 cases (62.5％) were similar to the prevalent HIV strains in the province where former census of marriagebased immigrant women were registered.In total,there were 26 (70.3％) cases from Yunnan province.84.7％ of the infected women had heterosexual behaviors before settling down in Zhejiang province.Genetic sequences of 17 pairs showed the same subtype between the couples and data from phylogenetic tree analysis supported the assumption of transmission linkage in the family.Conclusion The HIV subtype strains detected in those HIV infected marriage-based immigrant women in the rural area of Zhejiang province characterized with diversity,showing CRF08_BC and CRF01_AE were the main HIV strain subtypes.HIV infection originated mainly fiom Yunnan province and nearby regions.Heterosexual behaviors of the marriage-based immigrant women in the original region where they had their residence registration,seemed to be the primary high risk factors for these women.Surveillance and intervention programs on these marriage-based immigrant women and their family members should be improved.
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Objective To analyse the subtype and transmission of HIV strain in both HIV infected spouses. Methods Reported both HIV infected spouses were selected as objects. Analysis on genetic sequence and high risk behaviors was carried out. Subgenomic gag was amplified by nest-PCR analysis of whole blood samples from objects. Genetic subtype characterization of HIV was identified and pairwise genetic distances were calculated. Sequence relationships were also examined by phylogenetic tree analysis. Results Genetic sequences of 46 pairs (92 cases) were obtained. The genetic subtype comprised 39 CRF01_AE(42.4%), 10 CRF07_BC(10.9%), 18 CRF08_BC (19.6%),18 B (19.6%) ,5 C (5.4%) and 2 CRF02_AG(2.2%). 44 pairs had the same subtype between the two partners, accounted for 95.7% of the total. 33 of the 41 pairs with phylogenetic tree analysis were found having epidemiological linkage in pair wise. Sexual behaviors of out-marriage were the main risk factors of CRF01_AE and CRF08_BC and CRF02_AG strains infection. Blood transmission was associated with B and CRF07_BC. Conclusion The HIV strains subtype detected in HIV infected spouses characterized with diversity. CRF01_AE was the main strain subtype. The main route of transmission to the spouses was through unprotected sexual contacts. Surveillance programs on HIV infected partner together with intervention between the spouses should be improved.
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Objective To study the genotypic drug-resistant mutation among treat-naive or treated patients infected with HIV-1 CRF01_AE in Zhejiang province during 2004-2007. Methods HIV-i pol amplicons (PR+RT) from 13 treated and 43 treat-naive patients were obtained by reverse transcription-polymerase chain reaction (RT-PCR). The sequences were analyzed for genotypic antiretroviral resistance through online tools (http://hivdb.stanford.edu). Results The median count of CD44+ T lymphocytes in 43treat-naive patients was 229 cells/mm3 and the median log10 viral load was 3.41. Some drug-resistant mutations were seen in these samples including amino acid 10, 46, 71, in the genes of protease (PR) and 103, 118, in the genes of reverse transcriptase (RT) whereas twenty-nine resistance mutations in the genes of PR and RT were obtained in the 13 treated patients (8/13, 61.5% ). The high prevalence of drug-resistant mutations was observed in patients who had been receiving HAART (hight active antiretroviral therapy). Among them, cross drug resistance was dominant. Correspondingly, the median counts of CD44+ T lymphocytes and the log10 viral load were 186 cells/mm3 and 3.91. Conclusion There was a low prevalence of genotypic drug-resistant mutations in treat-naive patients, but higher drug-resistant mutation in treated patients. More attention should be paid to the transmission of drug-resistant HIV strains and the antiretroviral therapy recipe should be adjusted correspondingly for the development of ART drugs, intervention as well as clinical therapy programs.
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<p><b>OBJECTIVE</b>Using molecular epidemiology method to characterize human immunodeficiency virus type 1 (HIV-1) subtype CRF01 _ AE strains being prevailed in Zhejiang province.</p><p><b>METHODS</b>Gag fragments of the HIV-1 strains were amplified by nested-polymerase chain reaction (nPCR) from the DNA extracted from whole blood of HIV-1 infected individuals in Zhejiang province. PCR products were sequenced and analyzed by phylogenetic method.</p><p><b>RESULTS</b>81 HIV-1 subtype CRF01 _ AE sequences were identified from the 192 samples that sequenced successfully. As one of the dominant subtypes in Zhejiang, CRF01 _ AE was transmitted mainly by heterosexual or homosexual contact in local residents. In migrants living in Zhejiang, CRF01 _ AE were transmitted mainly by heterosexual contact or injecting drug use. There were three main clusters in the phylogenetic tree which bootstrap value was larger than 60. We named the clusters with group MIX (47 sequences), group SEX (7 sequences) and group MSM (12 sequences) based on the transmission. Pairwise DNA distances in the gag region within the three groups and between CM240 were different (P = 0.000). Data through the analyses of deduced amino acid sequences from the three groups showed that several signature amino acid sites were distinct from the same positions of the subtype reference strains.</p><p><b>CONCLUSION</b>The CRF01 _ AE strain prevailing in Zhejiang province was from several sources, transmitted by more than three different transmission routes, and becoming the main subtypes circulating in homosexual population in this study. More attention needs to be paid to the epidemic characteristic of CRF01 _ AE.</p>