{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T07:09:21Z","timestamp":1785481761039,"version":"3.56.0"},"update-to":[{"DOI":"10.12688\/gatesopenres.13534.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,12,22]],"date-time":"2022-12-22T00:00:00Z","timestamp":1671667200000}}],"reference-count":50,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2022,12,22]],"date-time":"2022-12-22T00:00:00Z","timestamp":1671667200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001796","name":"University of the Sunshine Coast","doi-asserted-by":"publisher","award":["UniversityoftheSunshineCoastResearchScholarship(USCRS)"],"award-info":[{"award-number":["UniversityoftheSunshineCoastResearchScholarship(USCRS)"]}],"id":[{"id":"10.13039\/501100001796","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003550","name":"Queensland Government","doi-asserted-by":"publisher","award":["AdvanceQueenslandIndustryResearchFellowship[AQIRF067-2020-CV]"],"award-info":[{"award-number":["AdvanceQueenslandIndustryResearchFellowship[AQIRF067-2020-CV]"]}],"id":[{"id":"10.13039\/501100003550","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100020593","name":"DMTC Limited","doi-asserted-by":"crossref","award":["Medical Countermeasures Program"],"award-info":[{"award-number":["Medical Countermeasures Program"]}],"id":[{"id":"10.13039\/501100020593","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["gatesopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Gates Open Res"],"abstract":"<ns4:p>The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies\/\u00b5L of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48\u2013100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48\u2013100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from &gt; two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.<\/ns4:p>","DOI":"10.12688\/gatesopenres.13534.2","type":"journal-article","created":{"date-parts":[[2022,12,22]],"date-time":"2022-12-22T10:25:07Z","timestamp":1671704707000},"page":"81","update-policy":"https:\/\/doi.org\/10.12688\/gatesopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":13,"title":["Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes"],"prefix":"10.12688","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9750-8377","authenticated-orcid":false,"given":"Madeeha","family":"Ahmed","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nina M.","family":"Pollak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6767-9850","authenticated-orcid":false,"given":"Leon E.","family":"Hugo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew F.","family":"van den Hurk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jody","family":"Hobson-Peters","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4624-3545","authenticated-orcid":false,"given":"Joanne","family":"Macdonald","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2022,12,22]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","first-page":"138-146","DOI":"10.1016\/j.tim.2013.12.011","article-title":"Global spread of dengue virus types: mapping the 70 year history.","volume":"22","author":"J Messina","year":"2014","journal-title":"Trends Microbiol."},{"key":"ref-2","doi-asserted-by":"publisher","first-page":"e1760","DOI":"10.1371\/journal.pntd.0001760","article-title":"Refining the global spatial limits of dengue virus transmission by evidence-based consensus.","volume":"6","author":"O Brady","year":"2012","journal-title":"PLoS Negl Trop Dis."},{"key":"ref-3","doi-asserted-by":"publisher","first-page":"504-7","DOI":"10.1038\/nature12060","article-title":"The global distribution and burden of dengue.","volume":"496","author":"S Bhatt","year":"2013","journal-title":"Nature."},{"key":"ref-4","doi-asserted-by":"publisher","first-page":"37-43","DOI":"10.1099\/0022-1317-70-1-37","article-title":"Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera.","volume":"70","author":"C Calisher","year":"1989","journal-title":"J Gen Virol."},{"key":"ref-5","doi-asserted-by":"publisher","first-page":"816-20","DOI":"10.1016\/0035-9203(87)90041-1","article-title":"Why dengue haemorrhagic fever in Cuba? 1. 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Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,22]],"date-time":"2022-12-22T21:50:13Z","timestamp":1671745813000},"score":1,"resource":{"primary":{"URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,22]]},"references-count":50,"URL":"https:\/\/doi.org\/10.12688\/gatesopenres.13534.2","relation":{"has-review":[{"id-type":"doi","id":"10.21956\/gatesopenres.15574.r32812","asserted-by":"object"}]},"ISSN":["2572-4754"],"issn-type":[{"value":"2572-4754","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,22]]},"assertion":[{"value":"Indexed","URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/gatesopenres.14799.r32271, Prasad N. Paradkar, Australian Centre for Disease Preparedness, CSIRO Health & Biosecurity, Geelong, Vic, Australia, 15 Aug 2022, version 1, indexed","URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v1#referee-response-32271","order":0,"name":"referee-response-32271","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Joanne Macdonald<\/b>; \n<i>Posted: 06 Dec 2022<\/i>; The authors would like to thank the reviewer very much for their careful reading of the manuscript and encouraging comments.","URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v2#referee-comment-3586","order":1,"name":"referee-comment-3586","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/gatesopenres.14799.r32736, Carlos Enrique Muskus-L\u00f3pez, Program for the Study and Control of Tropical Diseases\u2014PECET, Faculty of Medicine, University of Antioquia, Medellin, Colombia, 21 Nov 2022, version 1, indexed","URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v1#referee-response-32736","order":2,"name":"referee-response-32736","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Joanne Macdonald<\/b>; \n<i>Posted: 06 Dec 2022<\/i>; The authors would like to thank the reviewer for their thorough reading of our manuscript and insightful comments. Our responses are below.&nbsp;  \n<b>1.<\/b> In the introduction, second paragraph, it is mentioned that the methods for the surveillance of dengue in mosquitoes include, virus isolation10\u201312, antigen detection13, and molecular testing using either conventional14\u201317 or reverse-transcription quantitative polymerase chain reaction (RT-qPCR )18\u201320 or isothermal amplification. However, it is mentioned that these methods (including isothermal amplification) are limited by the need for sophisticated equipment and expensive infrastructure, however, the authors contradict themselves later in the text (introduction, Paragraph 3) when they mention that RPA, which is an isothermal amplification method, does not require sophisticated equipment (ref 25), which is true.  \n<b>Response: <\/b>We thank the reviewer for their detailed review of our manuscript. The text in paragraph 1 has now been edited to:&nbsp; \n<i>\u201c<b>Apart from isothermal amplification<\/b>, these techniques<b>&nbsp;<\/b>can be limited in their implementation due to issues with sensitivity or specificity, or because they require sophisticated instruments and laboratory infrastructure that is not readily available in resource-limited countries where DENVs exert their greatest toll.\u201d<\/i> &nbsp; \n<b>2.<\/b> The authors mention that at least five different forward primers, three reverse primers, and two probes were designed for each of the DENV assays and cite table 1. However, table 1 shows only the selected primers and probe combinations out of the several primers and probes initially designed and tested.  \n<b>Response:<\/b> We agree that the citation suggests the table would contain the sequence of all primers and probes. We have now changed the position of the citation to suggest that the table contains only the finalised combinations: \n<i>\u201cAt least five different forward primers, three reverse primers, and two probes were designed for each of the DENV assays and tested in various combinations using standard plasmid DNA and synthetic RNA transcripts to optimize the respective serotype specific DENV RPA-LFD assays<b>, which use selected primer and probe sequences (Table 1).\u201d<\/b><\/i> \n<i>Table 1. <b>Selected p<\/b>rimer and probe sequences for the Dengue 1-4 RT-RPA\/LFD test.<\/i>  \n<b>3. <\/b>It is not clear, why mosquitoes exposed to DENV-2 were infected via feeding on infectious blood meal containing the virus while the rest of the DENV virus were infected via intrathoracic microinjections?  \n<b>Response: <\/b>To clarify why 2 different methods were used to expose mosquitoes to the viruses, we modified the sentence \u201c\n<i>Mosquitoes were exposed to DENV-2 via feeding on an infectious blood meal containing 10<sup>7<\/sup>&nbsp;TCID<sub>50<\/sub>\/mL of virus.<\/i>\u201d To read as \u201c\n<i>Mosquitoes were exposed to DENV-2 via feeding on an infectious blood meal containing 10<sup>7<\/sup>&nbsp;TCID<sub>50<\/sub>\/mL of virus, <b>as part of previously reported experiments (Hall-Mendelin et al. 2021)<\/b><\/i>\u201d. The reference has been added to the reference list.  \n<b>4.<\/b> What do you think is the reason why the analytical sensitivity determined from plasmids is much better than from RNA? And if this could have implications for using this method to detect dengue virus in humans, where the starting material is just RNA?  \n<b>Response:<\/b> We thank the reviewer for the opportunity to further discuss the results of RNA detection compared to plasmid detection. This question has now been addressed in the discussion. \n<i>\u201cOur rapid RT-RPA\/LFD dengue tests were 100% accurate compared to RT-PCR for both individual and mosquito pools containing at least 10<sup>3<\/sup> copies\/&micro;L. <b>We do note that analytical sensitivity determined from plasmids in 15 minutes was tenfold better than detection of RNA transcripts in 20 minutes, suggesting a lower relative efficiency of the mMLV reverse transcriptase generating cDNA from an RNA template. This reduction in sensitivity did not appear to affect detection, although<\/b> DENV-4 was not detected in a single individual mosquito and DENV-1 was not detected in a single pool containing an infected mosquito. Variation in DENV-4 individual mosquito testing could be due to experimental error, whereas the false negative in the DENV-1 pool may be attributed to low viral concentration.\u201d<\/i>  \n<b>5. <\/b>In the title of Figures 2 and 3 (the text in bold), it is mentioned that the RT-RPA\/LFD was run using reverse-transcribed RNA. However, reverse-transcribed RNA literally is cDNA and if I understood well, your starting material in the RT-RPA is just in vitro transcribed RNA from the plasmid, which is different to reverse-transcribed RNA. Of course, during the RT-RPA the RNA is converted in cDNA, just before the amplification.  \n<b>Response:<\/b> We thank the reviewer for this comment. We have now replaced the term \u2018reverse\u2019 transcribed RNA\u2019 with \u2018in vitro\u2019 transcribed RNA in both the figure legends.  \n<b>6.<\/b> In addition, in the legend of this figure 2, synthetic RNA is mentioned. What do you exactly mean with synthetic RNA? The RNA obtained by in vitro transcription? Because nothing is mentioned about synthetic RNA in materials and methods.  \n<b>Response:<\/b> Yes, the term synthetic RNA was used interchangeably for the RNA obtained by in vitro transcription. The term has now been excluded and replaced with in-vitro transcribed RNA as described above to avoid confusion. &nbsp; \n<b>7.<\/b> In the discussion, it is annotated, \u201cthat contrarily, our rapid and simple protocol reduces sample preparation time to only 10 minutes\u201d. This is for preparing how many samples? Maybe 10 minutes might result relative, because if many samples are going to be evaluated (for example in a dengue surveillance study in mosquitoes), this could be time consuming, because of the considerable number of samples to evaluate.  \n<b>Response:<\/b> We appreciate the reviewer for pointing this out. We have addressed the timing of larger sample sizes in the discussion.  \u201c\n<i>By combining our sample preparation with RPA and lateral flow, we describe for the first time a complete method for field-based testing of mosquitoes for detection of the four DENVs. Further work is required to evaluate the performance and operational utility of these tests in field trials in locations where DENVs are circulating. <b>However, we note that even when processing up to 20 individual mosquitoes or pools in parallel with traditional RNA extraction and RT-PCR, we were able to obtain our rapid test results for all 20 samples before a traditional column-based RNA extraction was completed.\u201d <\/b><\/i>  \n<b>Minor suggestions: 1.<\/b> In Materials and methods; in the plasmid section, the fragment size for DENV-3 (KF921921.1) is missing. In contrast, for the other DENV viruses, the size of the segments is indicated in parentheses.  \n<b>Response:<\/b> We appreciate the reviewer for pointing this out. The fragment size has now been added in parentheses.","URL":"https:\/\/gatesopenresearch.org\/articles\/6-81\/v2#referee-comment-3587","order":3,"name":"referee-comment-3587","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by the Bill & Melinda Gates Foundation OPP1140133. The work was also funded by the University of the Sunshine Coast, and M. Ahmed is additionally supported by a University of the Sunshine Coast Research Scholarship (USCRS). J. Hobson-Peters is supported by the Advance Queensland Industry Research Fellowship [AQIRF067-2020-CV]. N. M. Pollak is supported by the DMTC Limited (Australia), Medical Countermeasures Program [Project 10.75]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}