Shao-Hong Chen

Tools to Support Policy Decisions Related to Treatment Strategies and Surveillance of Schistosomiasis Japonica towards Elimination

Background Appropriate diagnostics to monitor disease trends and assess the effectiveness and impact of interventions are essential for guiding treatment strategies at different thresholds of schistosomiasis transmission and for certifying elimination. Field validation of these assays is urgently needed before they can be adopted to support policy decisions of the national programme for control and elimination of schistosomiasis in P.R. China. We compared the efficacy and utility of different immunoassays in guiding control strategies and monitoring the endemic status of S. japonicum infections towards elimination. Methodology/Principal Findings A cross-sectional survey was conducted in seven villages with different transmission intensities settings to assess the performance and utility of three immunoassays, e.g., an indirect hemagglutination assay (IHA_JX), an enzyme linked immunosorbent assay (ELISA_SZ), and a dot immunogold filtration assay (DIGFA_SH). 6,248 individuals aged 6–65 years old who gave consent and supplied their stool and blood samples were included for data analysis. Results showed that ELISA_SZ performed significantly higher sensitivity (95.45%, 95%CI: 92.94–97.97%) than IHA_JX (87.59%, 95%CI: 83.51–91.49%) and DIGFA_SH (79.55%, 95%CI: 74.68–84.41%), especially in subgroups with very low infection intensity. The specificity of ELISA_SZ, IHA_JX, DIGFA_SH in 6–9 year olds with occasional exposure was nearly 90%. DIGFA_SH performed the highest screening efficacy for patients among three assays with overall positive predicative value of 13.07% (95%CI: 11.42–14.72%). We found a positive correlation of antibody positive rate of IHA_JX with results of stool examination in age strata (r = 0.70, P<0.001). Seropositivity of IHA_JX in children aged 6–9 years old showed an excellent correlation with prevalence of schistosome infection in the seven communities (r = 0.77, P<0.05). Conclusions/Significance Studies suggest that ELISA_SZ could be used to guide selective chemotherapy in moderate or low endemic regions. IHA_JX could be used to as a surveillance tool and for certifying elimination of schistosomiasis through monitoring children as a sentinel population.

Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay

The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of Fasciola hepatica and Fasciola gigantica. The LAMP assay is inexpensive, easy to perform and shows rapid reaction, wherein the amplification can be obtained in 45 min under isothermal conditions of 61 °C or 62 °C by employing a set of four species-specific primer mixtures and results can be checked through naked-eye visualization. The optimal assay conditions with no cross-reaction with other closely related trematodes (Clonorchis sinensis, Opisthorchis viverrini, Orientobilharzia turkestanicum and Schistosoma japonicum) as well as within the two Fasciola species were established. The assay was validated by examining F. gigantica DNA in the intermediate host snails and in faecal samples. The results indicated that the LAMP assay is approximately 10(4) times more sensitive than the conventional specific PCR assays. These findings indicate that this Fasciola species-specific LAMP assay may have a potential clinical application for detection and differentiation of Fasciola species, especially in endemic countries.

Evaluation of Immunoassays for the Diagnosis of Schistosoma japonicum Infection Using Archived Sera

Background With a national program initiated recently to reduce transmission of Schistosoma japonicum in the Peoples Republic of China (P.R. China), there is an urgent need for accessible, quality-assured diagnostics for case detection, surveillance, and program monitoring of chemotherapy efficacy and other control interventions in areas of low endemicity. We compared the performance of nine immunodiagnostic tests developed in P.R. China for detection of antibodies against S. japonicum and established their priority for further assessment in field settings. Methodology/Principal Findings Using the Kato-Katz technique as the reference standard, 240 well-characterized archived serum specimens (100 positive and 140 negative) were evaluated in nine immunological tests developed in P.R. China. The enzyme-linked immunoelectrotransfer blot assay (EITB), which uses an adult worm extract of S. japonicum, supplied by the Center of Disease Control and Prevention, USA, was also evaluated. The sensitivity and specificity of each test were determined and the reproducibility of each test was assessed by evaluating operator-to-operator and run-to-run variation. In addition the simplicity of use for the end-user was evaluated. All tests showed good sensitivities ranging from 92.0% (95% confidence interval (CI): 86.7–97.3%) to 98.0% (95% CI: 95.3–100.0%). The test specificities varied from 70.0% (95% CI: 62.4–77.6%) to 97.1% (95% CI: 94.4–99.9%). All tests showed excellent reproducibility with a discordant rate in the range of 0–10.0% for operator-to-operator variation and run-to-run variation. All tests, except one magnetic particle-based enzyme-linked immunosorbent assay, were found to be easy to use, especially the dot immunogold filtration assays. Conclusions/Significance Most evaluated tests had acceptable performance characteristics and could make an impact on the schistosomiasis control programs in P.R. China. Three tests with the highest sensitivity, specificity and greatest ease of use, were selected for further evaluation in field settings.

Characterization of microRNAs in Taenia saginata of zoonotic significance by Solexa deep sequencing and bioinformatics analysis

The beef tapeworm Taenia saginata infects human beings with symptoms ranging from nausea, abdominal discomfort to digestive disturbances and intestinal blockage. In the present study, microRNA (miRNA) expressing profile in adult T. saginata was analyzed using Solexa deep sequencing and bioinformatics analysis. A total of 15.8 million reads was obtained by Solexa sequencing, and 13.3 million clean reads (1.73 million unique sequences) was obtained after removing reads smaller than 18 nt. Ten conserved miRNAs corresponding to 607,382 reads were found when matching the reads against known miRNAs of Schistosoma japonicum in miRBase database. The miR-71 had the most abundant expression in T. saginata, followed by miR-219-5p, but some other common miRNAs such as let-7, miR-40, and miR-103 were not identified in T. saginata. Nucleotide bias analysis found that the known miRNAs showed high bias and the uracil was the dominant nucleotide, particularly at the first and 11th positions which were almost at the beginning and middle of conserved miRNAs. One novel miRNA (Tsa-miR-001) corresponding to ten precursors was identified and confirmed by stem-loop RT-PCR. To our knowledge, this is the first report of miRNA profiles in T. saginata, which will contribute to better understanding of the complex biology of this zoonotic trematode. The reported data of T. saginata miRNAs should provide valuable references for miRNA studies of closed related zoonotic Taenia cestodes such as Taenia solium and Taenia asiatica.

Identification and characterization of microRNAs in Trichinella spiralis by comparison with Brugia malayi and Caenorhabditis elegans.

Trichinella spiralis is an important zoonotic nematode causing trichinellosis which is associated with human diseases such as malaise, anorexia, nausea, vomiting, abdominal pain, fever, diarrhea, and constipation. microRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in the regulation of gene expression. The objective of the present study was to examine the miRNA expression profile of the larvae of T. spiralis by Solexa deep sequencing combined with stem-loop real-time polymerase chain reaction (PCR) analysis. T. spiralis larvae were collected from the skeletal muscle of naturally infected pigs in Henan province, China, by artificial digestion using pepsin. The specific identity of the T. spiralis larvae was confirmed by PCR amplification and subsequent sequence analysis of the internal transcribed spacer of ribosomal DNA. A total of 17,851,693 reads with 2,773,254 unique reads were obtained. Eleven conserved miRNAs from 115 unique xsmall RNAs (sRNAs) and 12 conserved miRNAs from 130 unique sRNAs were found by BLAST analysis against the known miRNAs of Caenorhabditis elegans (ftp://ftp.ncbi.nih.gov/genomes/Caenorhabditis_elegans) and Brugia malayi dataset (http://www.ncbi.nlm.nih.gov/genomeprj?Db=genomeprj&cmd=ShowDetailView&TermToSearch=9549) in miRBase, respectively. One novel miRNA with 12 precursors were identified and certified using the reference genome of B. malayi, while no novel miRNA was found when using the reference genome of C. elegans. Nucleotide bias analysis showed that the uracil was the prominent nucleotide, particularly at the 1st, 6th, 18th, and 23th positions, which were almost at the beginning, middle, and the end of the conserved miRNAs. The identification and characterization of T. spiralis miRNAs provides a new resource to study regulation of genes and their networks in T. spiralis.

Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults.

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.

Chicken egg yolk antibodies (IgY) for detecting circulating antigens of Schistosoma japonicum.

BACKGROUND IgY isolated from egg yolk has been widely used in immunodiagnostic tests, including tests to detect circulating antigen (soluble egg antigen or SEA) of Schistosoma japonicum. RESULTS A sandwich ELISA was established using a combination of anti-S. japonicum SEA-IgY polyclonal antibodies and IgM monoclonal antibodies. To explore sensitivity and specificity of the sandwich ELISA, serum samples from 43 patients infected with S. japonicum were tested. All acute cases and 91.3% of the chronic cases showed a positive reaction. Only 5% of the control sera from healthy persons gave a positive response. Cross-reactions with antibodies to nine other parasites were rare. CONCLUSION The developed immunoassay is reasonably sensitive and specific. It could be used for field research and treatment efficacy assessments.

Multi-host Model-Based Identification of Armillifer agkistrodontis (Pentastomida), a New Zoonotic Parasite from China

Background Pentastomiasis is a rare parasitic infection of humans. Pentastomids are dioecious obligate parasites requiring multiple hosts to complete their lifecycle. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, their systematic position is not uncontested and historically, they have been considered as a separate phylum. Methodology/Principal Findings An appraisal of Armillifer agkistrodontis was performed in terms of morphology and genetic identification after its lifecycle had been established in a multi-host model, i.e., mice and rats as intermediate hosts, and snakes (Agkistrodon acutus and Python molurus) as definitive hosts. Different stages of the parasite, including eggs, larvae and adults, were isolated and examined morphologically using light and electron microscopes. Phylogenetic and cluster analysis were also undertaken, focusing on the 18S rRNA and the Cox1 gene. The time for lifecycle completion was about 14 months, including 4 months for the development of eggs to infectious larvae in the intermediate host and 10 months for infectious larvae to mature in the final host. The main morphological difference between A. armillatus and Linguatula serrata is the number of abdominal annuli. Based on the 18S rRNA sequence, the shortest hereditary distance was found between A. agkistrodontis and Raillietiella spp. The highest degree of homology in the Cox 1 nucleic acid sequences and predicted amino acid sequences was found between A. agkistrodontis and A. armillatus. Conclusion This is the first time that a multi-host model of the entire lifecycle of A. agkistrodontis has been established. Morphologic and genetic analyses supported the notion that pentastomids should be placed into the phylum Arthropoda.

Monoclonal antibodies against excretory/secretory antigens of Angiostrongylus cantonensis.

In the present study, four murine monoclonal antibodies (MAbs) were generated against the excretory/secretory (ES) products of Angiostrongylus cantonensis adult worms; two represented IgG1 and two represented IgM MAbs, and they were designated 12D5, 15F8, 21B7 and 14G10, respectively. Immunoblotting revealed that all of the MAbs predominantly recognized a 98 kDa antigen in the ES products of A. cantonensis adult worms, and no cross reactions were found with the whole worm antigens of some other common parasites, namely, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani, Ascaris lumbricoides, Trichinella spiralis, Anisakis sp., Echinococcus granulosus, Taenia solium, and Spirometra erinacei. Immunolocalization showed that all of the four MAbs reacted with the cuticle of the adult parasite, the external surface of its intestinal canal and reproductive organs, and its egg and first-stage larvae in the lungs of rats experimentally infected with A. cantonensis. The generation and characterization of four specific MAbs against A. cantonensis ES antigens provide foundation for the development of specific immunological diagnostic techniques for human infections with A. cantonensis.

A Protein Microarray for the Rapid Screening of Patients Suspected of Infection with Various Food-Borne Helminthiases

Background Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. Methodology/Principal Findings In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. Conclusions/Significance The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.