Molecular Identification of Ascaridoid Nematodes Causing Larva Migrans

恆溫環形核酸增幅法(LAMP)能快速增幅所需基因片段且具有較高靈敏度。在本研究中，我們利用恆溫環形核酸增幅檢測法用於檢測及鑑定可能引起幼蟲移行症之蛔蟲屬寄生蟲，包括犬蛔蟲及浣熊蛔蟲，並根據寄生蟲核糖體DNA中ITS-2的特異性遺傳標誌設計引子進行檢測。LAMP法在65度穩溫箱中反應60分鐘。增幅後的產物以電泳方式進行鑑定。LAMP法鑑定蛔蟲屬之寄生蟲具有高度特異性，與非標靶基因片段則無陽性反應。DNA的增幅產物檢出時間最快可達到30分鐘。以特定引子利用LAMP法檢測犬蛔蟲、貓蛔蟲及浣熊蛔蟲僅需10 fg、100 pg及1 fg的量即可有效檢出。傳統的PCR檢測在偵測犬蛔蟲及浣熊蛔蟲具有較低的靈敏度，分別需要 10 ng及1 pg的DNA，但是針對貓蛔蟲之靈敏度約為LAMP assay的10倍。本研究結果顯示，在動物或人體組織中所採集的蛔蟲屬幼蟲之種別鑑定方式中，相較於型態學的鑑定，分子鑑定技術提供更為快速且容易。該方法亦可運用於鑑定土壤或糞材中所含有的蛔蟲卵，將可有效防止人類因接觸污染源而遭受感染。Loop-mediated isothermal amplification (LAMP) is a novel technique which rapidly amplifies target DNA with high specificity and efficiency under isothermal conditions. In the present study, we developed a LAMP method for the detection and identification of the larva migrans causing ascaridoid nematodes, Toxocara canis, T. cati and Baylisascaris procyonis, using primer sets designed from species-specific genetic markers in the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA (rDNA) of the nematode species. LAMP reactions were carried out isothermally at 65C for 60 min in a simple incubator. Positive amplification products analyzed in agarose gel electrophoresis were also detected by the addition of SYBR Green I. The LAMP assay for each nematode species was specific and produced negative results when tested against non-target DNA. DNA amplifications were detected as early as 30 min. The LAMP primer sets for T. canis, T. cati and B. procyonis were capable of detecting down to 10 fg, 100 pg and 1 fg of genomic DNA, respectively. Conventional PCR on the other hand, had a comparatively lower sensitivity in detecting T. canis and B. procyonis DNA down to 10 ng and1 pg, respectively, except for T. cati which is 10-fold more sensitive compared to the LAMP assay. These results showed that the molecular diagnostic technique developed in this study would be potentially valuable in the rapid yet simple approach of differentiating and identifying ascaridoid larvae in tissues of humans and animals when morphological methods are difficult or inadequate to attain diagnosis. This would also be useful in the detection of ascaridoid eggs in soil and in feces of dogs and cats in the environment which could serve as source of infection for humans.TABLE OF CONTENTS Page ACKNOWLEDGMENTS …………………………………………………….. i CHINESE ABSTRACT …….………………………………………………… ii ENGLISH ABSTRACT ………………………………………………………. iii TABLE OF CONTENTS …………………………………………………….. iv LIST OF TABLES ……………………………………………………………. vi LIST OF FIGURES ……………………………………………………………. vii LIST OF APPENDICES ………………………………………………………. ix INTRODUCTION ……………………………………………………………… 1 LITERATURE REVIEW ……………………………………………………… 3 1. Larva Migrans …………………………………………………………… 3 1.1. Categories of Larva Migrans …………………………………………….. 3 1.2. Helminths Causing Larva Migrans Syndromes ………………………….. 3 2. Toxocara spp. in Dogs and Cats …………………………………………. 4 2.1. Taxonomy and Morphology ……………………………………………... 4 2.2. Life Cycle ……………………………………………………………….. 5 2.3. Epidemiology ……………………………………………………………. 7 2.4. Clinical Disease in Animals ...……………………………………………. 8 2.5. Clinical Disease in Humans ……………………………………………… 8 2.6. Diagnosis ………………………………………………………………… 10 2.7. Treatment ………………………………………………………………… 11 3. Baylisascaris in Raccoons …………………………………………………. 12 3.1. Taxonomy and Morphology ……………………………………………... 12 3.2. Life Cycle ……………………………………………………………….. 13 3.3. Epidemiology ……………………………………………………………. 14 3.4. Clinical Disease in Animals ……………………………………………… 15 3.5. Clinical Disease in Humans ……………………………………………… 15 3.6. Diagnosis ………………………………………………………………… 16 3.7. Treatment ………………………………………………………………… 17 4. Molecular Diagnosis of Parasitic Diseases ……………………………… 18 5. Loop-mediated Isothermal Amplification (LAMP) Method ………….. 19 5.1. Primer Design …………………………………………………………… 20 5.2. Principle of LAMP Method …………………………………………….. 20 5.3. Using Loop Primers ……………………………………………………... 21 5.4. Principle of Visual Detection ……………………………………………. 22 MATERIALS AND METHODS ……………………………………………… 23 1. Parasites …………………………………………………………………… 23 2. Isolation of Genomic DNA ………………………………………………... 23 3. Oligonucleotide Primers …………………………………………………… 23 4. LAMP Reaction …………………………………………………………… 24 5. Detection of LAMP Products ……………………………………………... 24 6. Sensitivity and Specificity of LAMP ……………………………………... 24 7. PCR and Nested PCR …………………………………………………….. 25 RESULTS ……………………………………………………………………... 26 1. Specificities of the LAMP Assays ………………………………………… 26 2. Sensitivities of the LAMP Assays ………………………………………… 27 3. Earliest Time of Amplification …………………………………………… 28 DISCUSSION …………………………………………………………………. 29 CONCLUSION …………………………………………………………………. 34 REFERENCES …………………………………………………………………. 35 TABLES ………………………………………………………………………… 52 FIGURES ……………………………………………………………………….. 53 APPENDICES ………………………………………………………………….. 8

Does structurally-mature dengue virion matter in vaccine preparation in post-Dengvaxia era?

The unexpectedly low vaccine efficacy of Dengvaxia®, developed by Sanofi Pasteur, and a higher risk of severe diseases after vaccination among dengue-naive children or children younger than 6 years old, have cast skepticism about the safety of dengue vaccination resulting in the suspension of school-based immunization programs in the Philippines. The absence of immune correlates of protection from dengue virus (DENV) infection hampers the development of other potential DENV vaccines. While tetravalent live-attenuated tetravalent vaccines (LATVs), which mimic natural infection by inducing both cellular and humoral immune responses, are still currently favored, developing a vaccine that provides a balanced immunity to all four DENV serotypes remains a challenge. With the recently advanced understanding of virion structure and B cell immune responses from naturally infected DENV patients, two points of view in developing a next-generation dengue vaccine emerged: one is to induce potent, type-specific neutralizing antibodies (NtAbs) recognizing quaternary structure-dependent epitopes by having four components of vaccine strains replicate equivalently; the other is to induce protective and broadly NtAbs against the four serotypes of DENV with a universal vaccine. This article reviews the studies related to these issues and the current knowledge gap that needs to be filled in

Does structurally-mature dengue virion matter in vaccine preparation in post-Dengvaxia era?

The unexpectedly low vaccine efficacy of Dengvaxia®, developed by Sanofi Pasteur, and a higher risk of severe diseases after vaccination among dengue-naive children or children younger than 6 years old, have cast skepticism about the safety of dengue vaccination resulting in the suspension of school-based immunization programs in the Philippines. The absence of immune correlates of protection from dengue virus (DENV) infection hampers the development of other potential DENV vaccines. While tetravalent live-attenuated tetravalent vaccines (LATVs), which mimic natural infection by inducing both cellular and humoral immune responses, are still currently favored, developing a vaccine that provides a balanced immunity to all four DENV serotypes remains a challenge. With the recently advanced understanding of virion structure and B cell immune responses from naturally infected DENV patients, two points of view in developing a next-generation dengue vaccine emerged: one is to induce potent, type-specific neutralizing antibodies (NtAbs) recognizing quaternary structure-dependent epitopes by having four components of vaccine strains replicate equivalently; the other is to induce protective and broadly NtAbs against the four serotypes of DENV with a universal vaccine. This article reviews the studies related to these issues and the current knowledge gap that needs to be filled in

Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies

Background: Dengue virus (DENV) infection remains a global public health concern. Enzyme-linked immunosorbent assays (ELISAs), which detect antibodies targeting the envelope (E) protein of DENV, serve as the front-line serological test for presumptive dengue diagnosis. Very few studies have determined the serostatus by detecting antibodies targeting the nonstructural protein 1 (NS1), which can function as diagnostic biomarkers to distinguish natural immunity from vaccine-induced immunity. Methods: We used community-acquired human serum specimens, with the serostatus confirmed by focus reduction microneutralization test (FRμNT), to evaluate the diagnostic performances of two NS1-based ELISA methods, namely, immunoglobulin G antibody-capture ELISA (NS1 GAC–ELISA) and indirect NS1 IgG ELISA, and compared the results with an E-based virus-like particle (VLP) GAC–ELISA. Results: NS1-based methods had comparable accuracies as VLP GAC–ELISA. Although the sensitivity in detecting anti-NS1 IgM was poor, indirect NS1 IgG ELISA showed similar limits of detection (~1–2 ng/mL) as NS1 GAC–ELISA in detecting anti-NS1 IgG. Combining the results from two or more tests as a composite reference standard can determine the DENV serostatus with a specificity reaching 100%. Conclusion: NS1-based ELISAs have comparable accuracies as VLP GAC–ELISA in determining dengue serostatus, which could effectively assist clinicians during assessments of vaccine eligibility

Nonstructural protein 1-specific immunoglobulin M and G antibody capture enzyme-linked immunosorbent assays in diagnosis of flaviviral infections in humans

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection

LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD FOR THE RAPID IDENTIFICATION OF BAYLISASCARIS PROCYONIS, TOXOCARA. CANIS, AND T. CATI

本發明為提供獨特的引子組，其對線蟲ribosomal DNA之ITS-2(Internal transcribed spacer region 2)具有特異性。利用恆溫環形核酸增幅法(Loop-mediated Isothermal Amplification,LAMP)建立快速診斷法。可鑑別不同的線蟲如浣熊蛔蟲、犬蛔蟲和貓蛔蟲。除了設計對浣熊蛔蟲具有特異性的引子之外，本發明也設計且證實對了犬蛔蟲及貓蛔蟲具有特異性的引子組。因此，本發明的LAMP法可用於區別及鑑定人類及動物組織中ascaridoid屬的幼蟲。也能快速鑑定犬貓糞便中或是環境中如土壤裡所含有的幼蟲及寄生蟲卵

Comparison of E and NS1 antigens capture ELISA to detect dengue viral antigens from mosquitoes

In the absence of an effective vaccine or specific antiviral therapy against dengue infection, the only available control measure remains focusing on the incrimination and reduction of vector (mosquito) populations to suppress virus transmission. Diagnosis of dengue in laboratory can be carried out using several approaches, however, their sensitivity and specificity vary from test-to-test. This study was conducted to evaluate the sensitivity and stability of viral envelope (E) and NS1 antigens detected by ELISA in dengue virus infected mosquitoes

Comparison of E and NS1 antigens capture ELISA to detect dengue viral antigens from mosquitoes

Background & objectives: In the absence of an effective vaccine or specific antiviral therapy against dengue infection, the only available control measure remains focusing on the incrimination and reduction of vector (mosquito) populations to suppress virus transmission. Diagnosis of dengue in laboratory can be carried out using several approaches, however, their sensitivity and specificity vary from test-to-test. This study was conducted to evaluate the sensitivity and stability of viral envelope (E) and NS1 antigens detected by ELISA in dengue virus infected mosquitoes. Methods: An in-house developed E-ELISA to detect dengue E antigens was first characterized by using cross-reactive monoclonal antibody (mAb) 42-3 and rabbit polyclonal antibodies as the capture and detector antibodies, respectively. The sensitivity of E-ELISA was compared with the Platelia Dengue NS1 Ag kit using experimentally infected or field-caught mosquitoes. Results: Our results demonstrated that the E-ELISA was capable of detecting viral antigens with the sensitivity of 69.57, 100, 52.38 and 66.67% for DENV-1 to DENV-4 infected mosquito pools, respectively. This was comparable to the Platelia Dengue NS1 Ag kit, detecting 100% of DENV-1 infected mosquito pools. Among 124 field-collected mosquito pools collected in the vicinity of localized outbreak areas; both E-ELISA and NS1 Ag kit confirmed nine RT-PCR positive samples with sensitivity and concordance rate up to 100%. Interpretation & conclusion: With the future potential of antigen capture ELISA to be used in the resource deprived regions, the study showed that E-ELISA has similar sensitivity and antigen stability as NS1 Ag kit to complement the current established virological surveillance in human. The improvement of the sensitivity in detecting DENV-3/4 will be needed to incorporate this method into routine mosquito surveillance system

Improving dengue viral antigens detection in dengue patient serum specimens using a low pH glycine buffer treatment

Background/Purposes: Early diagnosis of dengue virus (DENV) infection to monitor the potential progression to hemorrhagic fever can influence the timely management of dengue-associated severe illness. Nonstructural protein 1 (NS1) antigen detection in acute serum specimens has been widely accepted as an early diagnostic assay for dengue infection; however, lower sensitivity of the NS1 antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) in secondary dengue viral infection has been reported. Methods: In this study, we developed two forms of Ag-ELISA capable of detecting E-Ag containing virion and virus-like particles, and secreted NS1 (sNS1) antigens, respectively. The temporal kinetics of viral RNA, sNS1, and E-Ag were evaluated based on the in vitro infection experiment. Meanwhile, a panel of 62 DENV-2 infected patients' sera was tested. Results: The sensitivity was 3.042 ng/mL and 3.840 ng/mL for sNS1 and E, respectively. The temporal kinetics of the appearance of viral RNA, E, NS1, and infectious virus in virus-infected tissue culture media suggested that viral RNAs and NS1 antigens could be detected earlier than E-Ag and infectious virus. Furthermore, a panel of 62 sera from patients infected by DENV Serotype 2 was tested. Treating clinical specimens with the dissociation buffer increased the detectable level of E from 13% to 92% and NS1 antigens from 40% to 85%. Conclusion: Inclusion of a low-pH glycine buffer treatment step in the commercially available Ag-ELISA is crucial for clinical diagnosis and E-containing viral particles could be a valuable target for acute DENV diagnosis, similar to NS1 detection