Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C in Aedes aegypti (L.)

Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C in Aedes aegypti (L.) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C in Aedes aegypti (L.) Karina Villanueva-Segura, Gustavo Ponce-Garcia, Beatriz Lopez-Monroy, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.19782/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jun, 2020 Read the published version in Parasites & Vectors → Version 1 posted You are reading this latest preprint version Abstract Background Knock down resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of nonsynonymous mutations in the he voltage-gated sodium channel (VGSC) gene, these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1,016I and F1,534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations. Methods A multiplex PCR was developed to detect the V410L, V1,016I and F1,534C mutations in Ae. aegypti . The validation of the technique was carried out using wild populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies. Results The standardized protocol for multiplex endpoint PCR was highly effective in detecting 12 genotypes in five wild Ae. aegypti populations from Mexico. A complete concordance with AS-PCR was found for the simultaneous detection of the three kdr mutations. Conclusions  Our diagnostic method is highly effective for the simultaneous detection of V410L, V1,016I and F1,534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti . Parasitology Multiplex PCR Aedes aegypti V410L V1 016I F1 534C Figures Figure 1 Figure 2 Background The portfolio of insecticides available for the control of arthropod pathogen vectors is very limited and is unlikely to increase in the near future, mainly due to the high cost of developing new molecules and products. Therefore, the emergence of resistance to commonly used insecticides is a serious threat to our ability to fight diseases transmitted by Aedes aegypti (L.). The development of such resistance is a complex and dynamic process dependent on many factors, so its management requires two types of information: good knowledge of the resistance mechanisms and their monitoring. The characterization of the resistance mechanisms involved allows us to assess and predict their impact on vector control strategies. Having routine monitoring of insecticide resistance in natural vector populations helps us to detect early resistance and improve the effectiveness of operational control strategies. Ae. aegypti is the main vector of dengue, chikungunya and Zika virus in Mexico, and its control depends largely on the use of pesticides that vary in their mode of action and include organophosphates, carbamates and neonicotinoids, but pyrethroids remain the preferred class, due to their fast action, high insecticidal activity and low toxicity to mammals [1, 2]. The target site of pyrethroids is the voltage-gated sodium channel (VGSC) present in the axon membrane of neurons and excitable cells in insects, and these insecticides produce a knockdown effect, that is, instantaneous paralysis in the insect due to prolonged activation and subsequent blockage of the action potentials of these channels [1, 3, 4, 5]. The main mechanisms that confer resistance to pyrethroids are overexpression of detoxifying enzymes and/or insensitivity at the target active site [6], being the mechanism associated with detoxification enzymes identified in Ae. aegypti populations from different regions of Mexico [7, 8, 9], particularly the mechanisms associated with esterases, glutathione S-transferases and mixed-function oxidases. On the other hand, knockdown resistance is conferred principally by nonsynonymous mutations that reduce pyrethroid binding to VGSC. In Mexico, it has been determined that pyrethroid resistance in Ae. aegypti is associated with high frequencies of any of the V1,016I, F1,534C and V410L mutations or combinations thereof. V1,016I was the first to be reported in a population of Ae. aegypti from Isla Mujeres, Quintana Roo, Mexico resistant to permethrin [10]. Subsequently, Ponce-García et al . demonstrated through a retrospective analysis carried out with 78 collections of Ae. aegypti that this replacement was practically absent in samples collected between 1996 and 2001, showing a dramatic increase between 2007 and 2009 [11]. Additionally, Siller et al. reported an increase in allele I1,016 frequency in Ae. aegypti populations in 2009 in several locations in Mexico [12]. Vera-Maloof et al . performed a linkage disequilibrium analysis in populations collected in Mexico between 2000 and 2012 that carried I1,016 and C1,534, and their results suggested that the sequential evolution of both mutations was necessary for pyrethroid resistance to develop [13]. Saavedra-Rodríguez et al . reported the V410L mutation in Ae. aegypti collections obtained during 2002 to 2016 from several locations in Mexico observing a high frequency of V410L in collections previously genotyped with V1,016I and F1,534C [14]. Later, Villanueva-Segura et al . reported high allelic frequencies of L410 in 25 populations of Ae. aegypti occurring in 2018 in eastern and southern Mexico with frequencies of 0.10-0.99 [15]. Currently, there are different PCR-based techniques for the detection of kdr mutations, which offer high sensitivity and specificity. However, when choosing one of these techniques, it is important to consider the economic resources of the laboratory, the training of technical personnel and the time available [16]. This point is even more important when considering the geographic extent that the vector occupies, the quantity of samples to be processed and the dynamics and fluctuation of the kdr resistance in short periods of time. The high-performance techniques available for genotyping kdr mutations, which are characterized by their speed and high sensitivity, are: sequencing of specific regions of VGSC, real-time PCR with TaqMan probes and high-resolution melting (HRM). However, given the need to acquire specialized equipment and supplies and the quantity of samples to be processed, the high cost of these methods constitutes its greatest disadvantage. The above has made AS-PCR one of the most used techniques for this purpose, offering a low cost and low error rate. Despite this, for laboratories in developing or low-budget countries, the use of other techniques based on allele-specific PCR is still recommended, when considering cost and ease of use and performance [17, 18. 19. 20]. Recently, the multiplex PCR technique has proven effective in the identification of two kdr mutations (V1,016G and F1,534C) simultaneously in Ae. aegypti [21]. In addition, it is possible to adapt it to different biochemical assays that not only reveal the amplified products optimally, but also contribute to improving the reliability, cost and performance of resistance monitoring [22]. The aim of this study was to develop a multiplex PCR method to detect three mutations, V410L, V1,016I and F1,534C, in Ae. aegypti in a single reaction. This technique can reduce the cost and time spent to monitor allelic frequencies in many countries where all three mutations co-occur. Materials and Methods Collection sites and laboratory conditions Field collections of Ae. aegypti were carried out in 2018 from three states in Mexico: Nuevo Leon in the northeast with two locations, Monterrey and Guadalupe; Yucatan in the southeast with two locations, Merida and San Antonio Kaua; and Chiapas in the south with one location. Mosquitoes collected in the field were at immature stages, and they were reared to adults under laboratory conditions at 25±4°C and a 12:12 h photoperiod. They were morphologically identified and stored at -20°C until DNA extraction. DNA isolation DNA was isolated from ~30 individual mosquitoes per location using the salt extraction technique [23] and resuspended in 50 mL of water (Cellgro® sterile purified water, molecular biology grade and free of proteases, DNases and RNases). The concentration and quality of each DNA sample was determined on a ThermoScientific NanoDrop 2000 spectrophotometer. Development of the multiplex PCR method The amplification primers used for the variants of loci 410, 1,016 and 1,534 are given in Table 1. The specific oligonucleotides V410fw and L410fw amplify a region of 113 bp, corresponding to the L410 allele (resistant). The specific oligonucleotides V410fw and 410rev amplify a region of 133 bp, corresponding to the V410 allele (susceptible). The specific oligonucleotides I1,016f and I1,016r amplify a region of 82 bp, corresponding to the I1,016 allele (resistant). Oligonucleotides V1,016f and I1,016r amplify a region of 102 bp, corresponding to the V1,016 allele (susceptible). The oligonucleotides c1,534-f and c1,534-r amplify a region of 368 bp with which the specific oligonucleotides Ae1,534F-r and Ae1,534C-f hybridize, resulting in products of 180 bp for the C1,534 allele (resistant) and of 232 bp for the F1,534 allele (susceptible) (Figure 1). Tests for optimization of PCR conditions resulted in the following multiplex PCR protocol. The DNA samples used in the amplification process were in a concentration range of 20-250 ng/mL. The final reaction mixture was 19.12 mL and contained: 1.02X buffer, 1.53 mM MgCl 2 , 0.2 mM dNTPs, oligonucleotides for genotyping 410 at a final reaction concentration of 1.27 pmol/mL, for 1,016 at 1.02 pmol/mL and for 1,534 at 0.82 pmol/mL (Table 1), and also 5 U Taq DNA polymerase. The reaction was carried out in a Multigene Optimax thermal cycler (Labnet International, Edison, NJ, USA). The reaction conditions were as follows: 95°C for 2 min for the initial separation of the DNA strands, followed by 45 cycles of 95°C (30 s), 58.6°C (1 min) and 72°C ( 30 s) and a final extension for 2 min. A PCR tube containing all the components except genomic DNA was run with the primers as a contamination control. Controls were included in each PCR performed, the New Orleans strains was used as susceptible control. After amplification, 4 µl of the products of the PCR reaction mixture were analyzed by horizontal electrophoresis on a 2.5% agarose gel. The electrophoresis conditions were 110 V for 1 h using 1X SB buffer (200 mM sodium borate buffer, pH 8) along with a 25-bp molecular weight marker to determine the size of the fragments and staining with GelRed® (Biotium, Hayward CA, USA). The PCR products were visualized with a transilluminator (UVITEC, Cambridge, UK). At the end of amplification, it was possible to obtain up to 7 PCR products per sample, whose size indicated the genotypic combination for loci 410, 1,016 and 1,534. Validation of the study For the validation of the results obtained in multiplex PCR, AS-PCR was performed according to Saavedra et al. [10], Yanola et al. [24] and Villanueva-Segura et al . [15]. Results Multiplex PCR vs AS-PCR assay Mosquito DNA of each population was used to detect the mutations V410L, V1,016I and F1,534C. Molecular assays were conducted on 147 mosquitoes of the populations analyzed. The genotype L410/L410 (homozygous mutant) was seen as a single band of 113 bp and the homozygous wild-type genotype (V410/V410) as a single band of 133 bp, while the heterozygous genotype (V410/L410) showed both bands. Genotype I1,016/I1,016 (homozygous mutant) was seen as a single band of 82bp and the homozygous wild-type genotype (V1,016/V1,016) as a single band of 102 bp, while the heterozygous genotype (V1,016/I1,016) showed both bands. The homozygous wild-type genotype (F1,534/F1,534) showed a single band of 232 bp and the homozygous mutant genotype (C1,534/C1,534) a single band of 180 bp, while the heterozygous genotype (F1,534/C1,534) had both bands. In the case of the detection of the F1534C mutation, an internal control band of 368 bp is obtained (Figure 2). A total of 12 genotypes were obtained and validated in the populations analyzed, using multiplex PCR and the individual endpoint technique previously reported by Saavedra et al. [10], Yanola et al. [24] and Villanueva-Segura et al . [15], demonstrating that all samples had the same genotype using multiplex PCR as with AS-PCR (Tables 2 and 3) . The analysis of the frequencies of the L410, I1,016 and C1,534 was determined in the 147 mosquitoes of the five selected populations. The frequencies for the allele L410 in the population of Chiapas was 0.57, in Merida 0.60 and in San Antonio Kaua 0.67, while the frequencies were 0.63 and 0.59 for the populations of Guadalupe and Monterrey, respectively. The frequencies for I1,016 in the populations of Merida, San Antonio Kaua, Chiapas, Guadalupe and Monterrey were 0.62, 0.73, 0.53, 0.67 and 0.43, respectively, and the mutation F1534C was fixed in the populations of Merida, San Antonio Kaua and Monterrey and with frequencies of 0.90 and 0.98 for the populations of Chiapas and Guadalupe, respectively (Table 3). In the populations analyzed, co-occurrence of homozygous resistant genotypes was greatest in the population of San Antonio Kaua, followed by the population of Merida, both from the state of Yucatan. The lowest levels of triple homozygous resistant were in the populations of Monterrey (0.04) and Chiapas (0.03). The most frequent genotype was double heterozygous for 410 and 1,016 and homozygous resistant for 1,534 (V410/L410, V1,016/I1,016, C1,534/C1,534) (Table 4). Discussion Performing PCR even today still takes time and effort; however, the objective of developing a multiplex PCR method is mainly to reduce these factors by being able to amplify various alleles in the same reaction. This is difficult when it is time to standardize the method, where each pair of primers included increases the difficulty since it is not enough to match the Tm (melting temperature) and AG (adenosine:guanidine) of these, so extra effort is needed in their design [25]. The optimization of every multiplex PCR method has critical difficulties. The design of the primers is key to a successful PCR, and the presence of more than one pair increases the possibility of dimers and also requires the adjustment of the other PCR components (buffers, dNTPs, MgCl 2 and Taq DNA polymerase)[26]. One of the main utilities of multiplex PCR is the simultaneous detection of multiple genes, such as serogroups of pathogens like with Salmonella , Escherichia coli O157 and Listeria monocytogenes [27, 28], including allele-specific multiplex PCR for detection of multiresistance in Mycobacterium tuberculosis [29]. However, the utility of multiplex PCR is not limited to this; one of the first reports of detection of multiple kdr mutations for a single L1014L/S site by multiplex PCR was performed by Tan et al . [30] for the alleles L/S (TTG/TCG), L/F (TTG/TTT) and L/L (TTG/TTG). Assays have been performed for the detection of V1,016I and F1,534C mutations individually [10, 24], and as multiplex PCR with other mutations such as V1,016G and F1,534C, T1520I and F1,534C [21, 31]. The V410L mutation was first detected in co-occurrence with the V1,016I and F1,534C mutations in Ae. aegypti mosquitoes but only by simplex PCR [32]. The utility of multiplex PCR is also not limited only to the detection of a single resistance mechanism. Kazanidou et al . [33] standardized the detection of polymorphisms by the substitution of five nucleotides in the sodium channel gene and the ace-1 gene ([kdr-w homozygous], [kdr-e homozygous], [kdr heterozygous], ace-1r homozygous, and their hybrids [ace1s/ace-1r, kdrs/kdr-w]) in only one of the samples. In this study, we used a simplex PCR as a confirmation technique to characterize each mutation in the samples used and to validate the multiplex PCR method. Six samples were used among which we could determine up to 27 different genotypes. The results of the test validation showed 100% agreement between multiplex PCR and simplex PCR. These results indicated high sensitivity and specificity values for the new multiplex PCR method developed. This method allows us to determine the frequency of multiple mutations, as well as their co-occurrence, in a single reaction [28]. Our method is the first designed for multiplex genotyping of the three kdr mutations in Ae. aegypti in Mexico, i.e., V410L, V1,016I, and F1,534C. Conclusion The multiplex PCR technique allows simultaneous and reliable detection of the V410L, V1,016I and F1,534C mutations in Ae. aegypti populations in Mexico. This method optimizes the monitoring times of mutant alleles and genotypic frequencies in wild populations of this species. The characteristics of this technique make it an advantageous alternative for countries where the dynamics of pyrethroid resistance in mosquitoes is variable and changing and where resources for this purpose are also limited. Abbreviations Kdr: knockdown resistance VGSC: voltage gated sodium channel AS-PCR: allele-specific PCR HRM: high-resolution melting Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests The authors declare that they have no competing interests Funding This work was financially supported by CONACYT Mexico through the fund Problemas Nacionales, Project No. PN2016-2134. Authors’ contributions AEF conceived the study and funds acquisition; OKVS coordinated the experiments; GPG, EdJMJ, KOZ, FJGS carried out collections of the populations included in the study; LP, EdJMJ, FJGS, KOZ performed the lab work; AEF, JADB, BLM analyzed data; AEF, OKVS, JADB drafted the manuscript; JADB, BLM, GPG provided critical input regarding the findings. All authors read and approved the final version of the manuscript. Acknowledgments Dr. A. Leyva (USA) provided the editing of the manuscript. References Carvalho FD, Moreira LA. Why is Aedes aegypti Linnaeus so Successful as a Species?. Neotrop entomol. 2017; 46(3), 243-255. Secretaria de Salud. Lista de productos recomendados por el CENAPRECE para el combate de insectos vectores de enfermedades a partir de 2019. 2019. https://www.gob.mx/cms/uploads/attachment/file/469289/Lista_de_Insumos_Recomendados_por_el_CENAPRECE.pdf. Last accessed Nov 10, 2019. Narahashi T, Ginsburg KS, Nagata K, Song JH, Tatebayashi H. 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Fernando SD, Hapugoda M, Perera R, Saavedra-Rodriguez K, Black WC 4th, De Silva NK. First report of V1,016G and S989P knockdown resistant (kdr) mutations in pyrethroid-resistant Sri Lankan Aedes aegypti mosquitoes. Parasites & vectors. 2018; 11(1), 526. doi:10.1186/s13071-018-3113-0. Kazanidou A, Nikou D, Grigoriou M, Vontas J, Skavdis J. Short Report: A Multiplex PCR Assay for Simultaneous Genotyping of kdr and ace-1 Loci in Anopheles gambiae . Am. J. Trop. Med. Hyg. 2009; 80(2), pp. 236–238. Tables Table 1. Sequences of the primers used for multiplex PCR. Primer name 5’-3’ Sequence Reference 410 Genotyping Saavedra et al . 2018 V410fw* [LongGCTail]ATCTTCTTGGGTTCGTTCTACCGTG L410fw** [ShortGCTail]ATCTTCTTGGGTTCGTTCTACCATT 410 rev TTCTTCCTCGGCGGCCTCTT 1016 Genotyping Saavedra et al . 2007 L1014f* [LongGCTail]ACAAATTGTTTCCCACCCGCACCGG F1014f** [ShortGCTail]ACAAATTGTTTCCCACCCGCACTGA L1014r TGATGAACCSGAATTGGACAAAAGC 1534 Genotyping Saingamsook et al 2017 c1534-f GCGTACCTGTGTCTGTTCCA c1534-r GGCTTCTTCGAGCCCATCTT Ae1534F-r GCGTGAAGAACGACCCGA Ae1534F-f CCTCTACTTTGTGTTCTTCATCATCTG *Long Tail 26b GCGGGCAGGGCGGCGGGGGCGGGGCC; **Short tail 6b GCGGGC Table 2. Comparison of genotyping results for V410L, V1,016I, F1,534C mutations from multiplex PCR and AS-PCR. Population Multiplex PCR genotyping / AS-PCR LL/II/CC LL/VI/CC LL/VV/CC LL/VI/FC VL/II/CC VL/VI/CC VV/II/CC VV/VI/CC VL/VI/FC LL/VV/FF VV/VI/FC VV/VV/CC VL/VV/CC VL/VV/FC VV/VV/FC LL/II/FC N Guadalupe 5/5 6/6 0/0 1/1 4/4 10/10 1/1 3/3 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 30/30 Merida 9/9 0/0 0/0 0/0 1/1 16/16 0/0 1/1 0/0 0/0 0/0 2/2 1/1 0/0 0/0 0/0 30/30 Chiapas 1/1 4/4 0/0 1/1 2/2 14/14 0/0 1/1 4/4 0/0 1/1 0/0 1/1 0/0 0/0 0/0 29/29 Monterrey 1/1 6/6 4/4 0/0 2/2 9/9 1/1 1/1 0/0 0/0 0/0 4/4 0/0 0/0 0/0 0/0 28/28 San Antonio Kaua 15/15 0/0 0/0 0/0 1/1 9/9 0/0 3/3 0/0 0/0 0/0 2/2 0/0 0/0 0/0 0/0 30/30 VV wild type (susceptible); VL heterozygotes; LL mutant (resistant) VV wild type (susceptible); VI heterozygotes; II mutant (resistant) FF wild type (susceptible); FC heterozygotes; CC mutant (resistanr) N sample size Table 3. Comparison of genotypes for V410L, V1016I, and F1534C mutations for multiplex PCR and AS-PCR. Multiplex PCR / AS-PCR 410 Frequency 1016 Frequency 1534 Frequency Population N VV VL LL L410 VV VI II I1,016 FF FC CC C1,534 Merida 30 3/3 18/18 9/9 0.60 3/3 17/17 10/10 0.62 0/0 0/0 30/30 1.0 San Antonio Kaua 30 5/5 10/10 15/15 0.67 2/2 12/12 16/16 0.73 0/0 0/0 30/30 1.0 Chiapas 29 2/2 21/21 6/6 0.57 1/2 25/25 3/3 0.53 0/0 6/6 23/23 0.90 Guadalupe 30 4/4 14/14 12/12 0.63 0/0 20/20 10/10 0.67 0/0 1/1 29/29 0.98 Monterrey 28 6/6 11/11 11/11 0.59 8/8 16/16 4/4 0.43 0/0 0/0 28/28 1.0 VV wild type (susceptible); VL heterozygotes; LL mutant (resistant) VV wild type (susceptible); VI heterozygotes; II mutant (resistant) FF wild type (susceptible); FC heterozygotes; CC mutant (resistant) N sample size Table 4. Frequency of co-occurrence of the mutations V1016I, F1534C y V410L in populations of Ae. aegypti Genotype frequency Population LL/II/CC LL/VI/CC LL/VV/CC LL/VI/FC VL/II/CC VL/VI/CC VV/II/CC VV/VI/CC VL/VI/FC VV/VI/FC VV/VV/CC VL/VV/CC Merida 0.30 0.00 0.00 0.00 0.03 0.53 0.00 0.03 0.00 0.00 0.07 0.03 San Antonio Kaua 0.50 0.00 0.00 0.00 0.03 0.30 0.00 0.10 0.00 0.00 0.07 0.00 Chiapas 0.03 0.14 0.00 0.03 0.07 0.48 0.00 0.03 0.14 0.03 0.00 0.03 Guadalupe 0.17 0.20 0.00 0.03 0.13 0.33 0.03 0.10 0.00 0.00 0.00 0.00 Monterrey 0.04 0.21 0.14 0.00 0.07 0.32 0.04 0.04 0.00 0.00 0.14 0.00 VV wild type (susceptible); VL heterozygotes; LL mutant (resistant) VV wild type (susceptible); VI heterozygotes; II mutant (resistant) FF wild type (susceptible); FC heterozygotes; CC mutant (resistant) Cite Share Download PDF Status: Published Journal Publication published 25 Jun, 2020 Read the published version in Parasites & Vectors → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-10394","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":271054,"identity":"23981aa8-a291-435f-ab8f-19614a9b652c","order_by":1,"name":"Karina Villanueva-Segura","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karina","middleName":"","lastName":"Villanueva-Segura","suffix":""},{"id":271055,"identity":"4b0075dd-7fcd-4038-be81-afe0217774cf","order_by":2,"name":"Gustavo Ponce-Garcia","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Ponce-Garcia","suffix":""},{"id":271056,"identity":"b1f54a69-4b7f-4e13-ac27-811155b25c6f","order_by":3,"name":"Beatriz Lopez-Monroy","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"","lastName":"Lopez-Monroy","suffix":""},{"id":271057,"identity":"8d5c0ff7-b0e8-4e7a-be4b-133b797c1cb3","order_by":4,"name":"Esteban de J. Mora-Jasso","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Esteban","middleName":"de J.","lastName":"Mora-Jasso","suffix":""},{"id":271058,"identity":"1a3db3eb-23f5-4452-a9f0-8f91d9db3227","order_by":5,"name":"Lucia Perales","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lucia","middleName":"","lastName":"Perales","suffix":""},{"id":271059,"identity":"4fa01962-a13f-4eb0-8a61-f820ccb67651","order_by":6,"name":"Francisco J. Gonzalez-Santillan","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"J.","lastName":"Gonzalez-Santillan","suffix":""},{"id":271060,"identity":"d5a78c71-8cff-4187-8859-cc9ec9c68af1","order_by":7,"name":"Kevin Ontiveros-Zapata","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Ontiveros-Zapata","suffix":""},{"id":271061,"identity":"2fc4ba8b-4167-4761-9eb0-e5f4ce9ff99d","order_by":8,"name":"Jesus A. Davila-Barboza","email":"","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jesus","middleName":"A.","lastName":"Davila-Barboza","suffix":""},{"id":271062,"identity":"7bee07fc-225d-4444-a6aa-a4b38c1c2145","order_by":9,"name":"Adriana E Flores","email":"data:image/png;base64,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","orcid":"","institution":"Universidad Autonoma de Nuevo Leon","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"E","lastName":"Flores","suffix":""}],"badges":[],"createdAt":"2019-12-26 14:41:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.19782/v1","doiUrl":"https://doi.org/10.21203/rs.2.19782/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-020-04193-0","type":"published","date":"2020-06-25T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":313220,"identity":"415722df-73c8-4604-bf9b-8eb817ddc1ef","added_by":"auto","created_at":"2019-12-31 17:05:00","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104577,"visible":true,"origin":"","legend":"Schematic of the AS-PCR assay for detection of the A) V410L, B) V1,016I and C) F1,534C mutations.","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/6bf1e810-f926-42fd-80e5-40d42482af90/v1/Figure 1.jpeg"},{"id":313221,"identity":"02a51149-c2fe-4758-8f8f-7a365e18d7b3","added_by":"auto","created_at":"2019-12-31 17:05:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":233113,"visible":true,"origin":"","legend":"Agarose gel 2.5% electrophoresis of amplified gDNA products in several mosquitoes using multiplex PCR. The arrows indicate a common band of 368 bp, a band of 232 bp corresponds to the susceptible allele F1,534, a band of 180 bp corresponds to the resistant allele C1,534, a band of 133 bp corresponds to the susceptible allele V410, a band of 113 bp corresponds to the resistant allele L410, a band of 102 bp corresponds to the susceptible allele V1,016, a band of 82 bp corresponds to the resistant allele I1,016. Lane 1 shows the molecular weight marker (25pb DNA Ladder). Lane 2 corresponds to a resistant homozygous (C1,534 / C1,534), heterozygous (V410 / L410) and heterozygous (V1,016 / I1,016) individual. Lane 3 shows a resistant homozygous individual (C1,534 / C1,534), resistant homozygous (L410 / L410) and resistant homozygous (I1016 / I1016). Lane 4 corresponds to a resistant homozygous individual (C1,534 / C1,534), heterozygous (V410 / L410) and resistant homozygous (I1,016 / I1,016). Lane 5 corresponds to a resistant homozygous individual (C1,534 / C1,534), susceptible homozygous (V410 / V410) and heterozygous (V1,016 / I1,016). Lane 6 corresponds to a resistant homozygous individual (C1,534 / C1,534), heterozygous (V410 / L410) and heterozygous (V1,016 / I1,016). Lane 7 corresponds to a triple susceptible homozygous individual (V410 / V410, V1,016 / V1,016, F1,534 / F1,534).","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/6bf1e810-f926-42fd-80e5-40d42482af90/v1/Figure 2.jpeg"},{"id":15665688,"identity":"eab1d543-0e89-462e-bf63-a8f0246ec6ba","added_by":"auto","created_at":"2021-11-18 13:32:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":490971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-10394/v1/414d1370-9ea9-4526-9c43-591c43bccdd7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMultiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C in \u003cem\u003eAedes aegypti\u003c/em\u003e (L.)\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eThe portfolio of insecticides available for the control of arthropod pathogen vectors is very limited and is unlikely to increase in the near future, mainly due to the high cost of developing new molecules and products. Therefore, the emergence of resistance to commonly used insecticides is a serious threat to our ability to fight diseases transmitted by \u003cem\u003eAedes aegypti\u003c/em\u003e (L.). The development of such resistance is a complex and dynamic process dependent on many factors, so its management requires two types of information: good knowledge of the resistance mechanisms and their monitoring. The characterization of the resistance mechanisms involved allows us to assess and predict their impact on vector control strategies. Having routine monitoring of insecticide resistance in natural vector populations helps us to detect early resistance and improve the effectiveness of operational control strategies. \u003cem\u003eAe. aegypti\u003c/em\u003e is the main vector of dengue, chikungunya and Zika virus in Mexico, and its control depends largely on the use of pesticides that vary in their mode of action and include organophosphates, carbamates and neonicotinoids, but \u0026nbsp;pyrethroids remain the preferred class, due to their fast action, high insecticidal activity and low toxicity to mammals [1, 2]. The target site of pyrethroids is the voltage-gated sodium channel (VGSC) present in the axon membrane of neurons and excitable cells in insects, and these insecticides produce a knockdown effect, that is, instantaneous paralysis in the insect due to prolonged activation and subsequent blockage of the action potentials of these channels [1, 3, 4, 5].\u003c/p\u003e\n\u003cp\u003eThe main mechanisms that confer resistance to pyrethroids are overexpression of detoxifying enzymes and/or insensitivity at the target active site [6], being the mechanism associated with detoxification enzymes identified in \u003cem\u003eAe. aegypti\u003c/em\u003e populations from different regions of Mexico [7, 8, 9], particularly the mechanisms associated with esterases, glutathione S-transferases and mixed-function oxidases. On the other hand, knockdown resistance is conferred principally by nonsynonymous mutations that reduce pyrethroid binding to VGSC. In Mexico, it has been determined that pyrethroid resistance in \u003cem\u003eAe. aegypti\u003c/em\u003e is associated with high frequencies of any of the V1,016I, F1,534C and V410L mutations or combinations thereof. V1,016I was the first to be reported in a population of \u003cem\u003eAe. aegypti\u003c/em\u003e from Isla Mujeres, Quintana Roo, Mexico resistant to permethrin [10]. Subsequently, Ponce-Garc\u0026iacute;a \u003cem\u003eet al\u003c/em\u003e. demonstrated through a retrospective analysis carried out with 78 collections of \u003cem\u003eAe. aegypti\u003c/em\u003e that this replacement was practically absent in samples collected between 1996 and 2001, showing a dramatic increase between 2007 and 2009 [11]. Additionally, Siller et al. reported an increase in allele I1,016 frequency in \u003cem\u003eAe. aegypti \u003c/em\u003epopulations in 2009 in several locations in Mexico [12]. Vera-Maloof \u003cem\u003eet al\u003c/em\u003e. performed a linkage disequilibrium analysis in populations collected in Mexico between 2000 and 2012 that carried I1,016 and C1,534, and their results suggested that the sequential evolution of both mutations was necessary for pyrethroid resistance to develop [13]. Saavedra-Rodr\u0026iacute;guez \u003cem\u003eet al\u003c/em\u003e. reported the V410L mutation in \u003cem\u003eAe. aegypti\u003c/em\u003e collections obtained during 2002 to 2016 from several locations in Mexico observing a high frequency of V410L in collections previously genotyped with V1,016I and F1,534C [14]. Later, Villanueva-Segura \u003cem\u003eet al\u003c/em\u003e. reported high allelic frequencies of L410 in 25 populations of \u003cem\u003eAe. aegypti\u003c/em\u003e occurring in 2018 in eastern and southern Mexico with frequencies of 0.10-0.99 [15].\u003c/p\u003e\n\u003cp\u003eCurrently, there are different PCR-based techniques for the detection of kdr mutations, which offer high sensitivity and specificity. However, when choosing one of these techniques, it is important to consider the economic resources of the laboratory, the training of technical personnel and the time available [16]. This point is even more important when considering the geographic extent that the vector occupies, the quantity of samples to be processed and the dynamics and fluctuation of the kdr resistance in short periods of time. The high-performance techniques available for genotyping kdr mutations, which are characterized by their speed and high sensitivity, are: sequencing of specific regions of VGSC, real-time PCR with TaqMan probes and high-resolution melting (HRM). However, given the need to acquire specialized equipment and supplies and the quantity of samples to be processed, the high cost of these methods constitutes its greatest disadvantage. The above has made AS-PCR one of the most used techniques for this purpose, offering a low cost and low error rate. Despite this, for laboratories in developing or low-budget countries, the use of other techniques based on allele-specific PCR is still recommended, when considering cost and ease of use and performance [17, 18. 19. 20]. Recently, the multiplex PCR technique has proven effective in the identification of two kdr mutations (V1,016G and F1,534C) simultaneously in \u003cem\u003eAe. \u003c/em\u003eaegypti [21]. In addition, it is possible to adapt it to different biochemical assays that not only reveal the amplified products optimally, but also contribute to improving the reliability, cost and performance of resistance monitoring [22].\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to develop a multiplex PCR method to detect three mutations, V410L, V1,016I and F1,534C, in \u003cem\u003eAe. aegypti\u003c/em\u003e in a single reaction. This technique can reduce the cost and time spent to monitor allelic frequencies in many countries where all three mutations co-occur.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCollection sites and laboratory conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eField collections of \u003cem\u003eAe. aegypti\u003c/em\u003e were carried out in 2018 from three states in Mexico: Nuevo Leon in the northeast with two locations, Monterrey and Guadalupe; Yucatan in the southeast with two locations, Merida and San Antonio Kaua; and Chiapas in the south with one location.\u003c/p\u003e\n\u003cp\u003eMosquitoes collected in the field were at immature stages, and they were reared to adults under laboratory conditions at 25\u0026plusmn;4\u0026deg;C and a 12:12 h photoperiod. They were morphologically identified and stored at -20\u0026deg;C until DNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was isolated from ~30 individual mosquitoes per location using the salt extraction technique [23] and resuspended in 50 mL of water (Cellgro\u0026reg; sterile purified water, molecular biology grade and free of proteases, DNases and RNases). The concentration and quality of each DNA sample was determined on a ThermoScientific NanoDrop 2000 spectrophotometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment of the multiplex PCR method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amplification primers used for the variants of loci 410, 1,016 and 1,534 are given in Table 1.\u003c/p\u003e\n\u003cp\u003eThe specific oligonucleotides V410fw and L410fw amplify a region of 113 bp, corresponding to the L410 allele (resistant). The specific oligonucleotides V410fw and 410rev amplify a region of 133 bp, corresponding to the V410 allele (susceptible). The specific oligonucleotides I1,016f and I1,016r amplify a region of 82 bp, corresponding to the I1,016 allele (resistant). Oligonucleotides V1,016f and I1,016r amplify a region of 102 bp, corresponding to the V1,016 allele (susceptible). The oligonucleotides c1,534-f and c1,534-r amplify a region of 368 bp with which the specific oligonucleotides Ae1,534F-r and Ae1,534C-f hybridize, resulting in products of 180 bp for the C1,534 allele (resistant) and of 232 bp for the F1,534 allele (susceptible) (Figure 1).\u003c/p\u003e\n\u003cp\u003eTests for optimization of PCR conditions resulted in the following multiplex PCR protocol. The DNA samples used in the amplification process were in a concentration range of 20-250 ng/mL. The final reaction mixture was 19.12 mL and contained: 1.02X buffer, 1.53 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.2 mM dNTPs, oligonucleotides for genotyping 410 at a final reaction concentration of 1.27 pmol/mL, for 1,016 at 1.02 pmol/mL and for 1,534 at 0.82 pmol/mL (Table 1), and also 5 U Taq DNA polymerase.\u003c/p\u003e\n\u003cp\u003eThe reaction was carried out in a Multigene Optimax thermal cycler (Labnet International, Edison, NJ, USA). The reaction conditions were as follows: 95\u0026deg;C for 2 min for the initial separation of the DNA strands, followed by 45 cycles of 95\u0026deg;C (30 s), 58.6\u0026deg;C (1 min) and 72\u0026deg;C ( 30 s) and a final extension for 2 min.\u003c/p\u003e\n\u003cp\u003eA PCR tube containing all the components except genomic DNA was run with the primers as a contamination control. Controls were included in each PCR performed, the New Orleans strains was used as susceptible control.\u003c/p\u003e\n\u003cp\u003eAfter amplification, 4 \u0026micro;l of the products of the PCR reaction mixture were analyzed by horizontal electrophoresis on a 2.5% agarose gel. The electrophoresis conditions were 110 V for 1 h using 1X SB buffer (200 mM sodium borate buffer, pH 8) along with a 25-bp molecular weight marker to determine the size of the fragments and staining with GelRed\u0026reg; (Biotium, Hayward CA, USA). The PCR products were visualized with a transilluminator (UVITEC, Cambridge, UK). At the end of amplification, it was possible to obtain up to 7 PCR products per sample, whose size indicated the genotypic combination for loci 410, 1,016 and 1,534.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the validation of the results obtained in multiplex PCR, AS-PCR was performed according to Saavedra et al. [10], Yanola et al. [24] and Villanueva-Segura et al\u003cem\u003e.\u003c/em\u003e [15].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMultiplex PCR vs AS-PCR assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMosquito DNA of each population was used to detect the mutations V410L, V1,016I and F1,534C. Molecular assays were conducted on 147 mosquitoes of the populations analyzed. The genotype L410/L410 (homozygous mutant) was seen as a single band of 113 bp and the homozygous wild-type genotype (V410/V410) as a single band of 133 bp, while the heterozygous genotype (V410/L410) showed both bands. Genotype I1,016/I1,016 (homozygous mutant) was seen as a single band of 82bp and the homozygous wild-type genotype (V1,016/V1,016) as a single band of 102 bp, while the heterozygous genotype (V1,016/I1,016) showed both bands. The homozygous wild-type genotype (F1,534/F1,534) showed a single band of 232 bp and the homozygous mutant genotype (C1,534/C1,534) a single band of 180 bp, while the heterozygous genotype (F1,534/C1,534) had both bands. In the case of the detection of the F1534C mutation, an internal control band of 368 bp is obtained (Figure 2).\u003c/p\u003e\n\u003cp\u003eA total of 12 genotypes were obtained and validated in the populations analyzed, using multiplex PCR and the individual endpoint technique previously reported by Saavedra et al. [10], Yanola et al. [24] and Villanueva-Segura \u003cem\u003eet al\u003c/em\u003e. [15], \u0026nbsp;demonstrating that all samples had the same genotype using multiplex PCR as with AS-PCR (Tables 2 and 3)\u003csup\u003e.\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of the frequencies of the L410, I1,016 and C1,534 was determined in the 147 mosquitoes of the five selected populations. The frequencies for the allele L410 in the population of Chiapas was 0.57, in Merida 0.60 and in San Antonio Kaua 0.67, while the frequencies were 0.63 and 0.59 for the populations of Guadalupe and Monterrey, respectively. The frequencies for I1,016 in the populations of Merida, San Antonio Kaua, Chiapas, Guadalupe and Monterrey were 0.62, 0.73, 0.53, 0.67 and 0.43, respectively, and the mutation F1534C was fixed in the populations of Merida, San Antonio Kaua and Monterrey and with frequencies of 0.90 and 0.98 for the populations of Chiapas and Guadalupe, respectively (Table 3).\u003c/p\u003e\n\u003cp\u003eIn the populations analyzed, co-occurrence of homozygous resistant genotypes was greatest in the population of San Antonio Kaua, followed by the population of Merida, both from the state of Yucatan. The lowest levels of triple homozygous resistant were in the populations of Monterrey (0.04) and Chiapas (0.03). The most frequent genotype was double heterozygous for 410 and 1,016 and homozygous resistant for 1,534 (V410/L410, V1,016/I1,016, C1,534/C1,534) (Table 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePerforming PCR even today still takes time and effort; however, the objective of developing a multiplex PCR method is mainly to reduce these factors by being able to amplify various alleles in the same reaction. This is difficult when it is time to standardize the method, where each pair of primers included increases the difficulty since it is not enough to match the Tm (melting temperature) and AG (adenosine:guanidine) of these, so extra effort is needed in their design [25].\u003c/p\u003e\n\u003cp\u003eThe optimization of every multiplex PCR method has critical difficulties. The design of the primers is key to a successful PCR, and the presence of more than one pair increases the possibility of dimers and also requires the adjustment of the other PCR components (buffers, dNTPs, MgCl\u003csub\u003e2\u003c/sub\u003e and Taq DNA polymerase)[26].\u003c/p\u003e\n\u003cp\u003eOne of the main utilities of multiplex PCR is the simultaneous detection of multiple genes, such as serogroups of pathogens like with \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e O157 and \u003cem\u003eListeria monocytogenes \u003c/em\u003e[27, 28], including allele-specific multiplex PCR for detection of multiresistance in \u003cem\u003eMycobacterium \u003c/em\u003etuberculosis [29]. However, the utility of multiplex PCR is not limited to this; one of the first reports of detection of multiple kdr mutations for a single L1014L/S site by multiplex PCR was performed by Tan \u003cem\u003eet al\u003c/em\u003e. [30] for the alleles L/S (TTG/TCG), L/F (TTG/TTT) and L/L (TTG/TTG). Assays have been performed for the detection of V1,016I and F1,534C mutations individually [10, 24], and as multiplex PCR with other mutations such as V1,016G and F1,534C, T1520I and F1,534C [21, 31]. The V410L mutation was first detected in co-occurrence with the V1,016I and F1,534C mutations in \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes but only by simplex PCR [32].\u003c/p\u003e\n\u003cp\u003eThe utility of multiplex PCR is also not limited only to the detection of a single resistance mechanism. Kazanidou \u003cem\u003eet al\u003c/em\u003e. [33] standardized the detection of polymorphisms by the substitution of five nucleotides in the sodium channel gene and the ace-1 gene ([kdr-w homozygous], [kdr-e homozygous], [kdr heterozygous], ace-1r homozygous, and their hybrids [ace1s/ace-1r, kdrs/kdr-w]) in only one of the samples.\u003c/p\u003e\n\u003cp\u003eIn this study, we used a simplex PCR as a confirmation technique to characterize each mutation in the samples used and to validate the multiplex PCR method. Six samples were used among which we could determine up to 27 different genotypes. The results of the test validation showed 100% agreement between multiplex PCR and simplex PCR. These results indicated high sensitivity and specificity values for the new multiplex PCR method developed. This method allows us to determine the frequency of multiple mutations, as well as their co-occurrence, in a single reaction [28].\u003c/p\u003e\n\u003cp\u003eOur method is the first designed for multiplex genotyping of the three kdr mutations in \u003cem\u003eAe. aegypti\u003c/em\u003e in Mexico, i.e., V410L, V1,016I, and F1,534C.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe multiplex PCR technique allows simultaneous and reliable detection of the V410L, V1,016I and F1,534C mutations in \u003cem\u003eAe. aegypti\u003c/em\u003e populations in Mexico. This method optimizes the monitoring times of mutant alleles and genotypic frequencies in wild populations of this species. The characteristics of this technique make it an advantageous alternative for countries where the dynamics of pyrethroid resistance in mosquitoes is variable and changing and where resources for this purpose are also limited.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eKdr: knockdown resistance\u003c/p\u003e\n\u003cp\u003eVGSC: voltage gated sodium channel\u003c/p\u003e\n\u003cp\u003eAS-PCR: allele-specific PCR\u003c/p\u003e\n\u003cp\u003eHRM: high-resolution melting\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by CONACYT Mexico through the fund Problemas Nacionales, Project No. PN2016-2134.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAEF conceived the study and funds acquisition; OKVS coordinated the experiments; GPG, EdJMJ, KOZ, FJGS carried out collections of the populations included in the study; LP, EdJMJ, FJGS, KOZ performed the lab work; AEF, JADB, BLM analyzed data; AEF, OKVS, JADB drafted the manuscript; JADB, BLM, GPG provided critical input regarding the findings. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. A. Leyva (USA) provided \u0026nbsp;the editing of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCarvalho FD, Moreira LA. Why is Aedes aegypti Linnaeus so Successful as a Species?. Neotrop entomol. 2017; 46(3), 243-255.\u003c/li\u003e\n\u003cli\u003eSecretaria de Salud. Lista de productos recomendados por el CENAPRECE para el combate de insectos vectores de enfermedades a partir de 2019. 2019. https://www.gob.mx/cms/uploads/attachment/file/469289/Lista_de_Insumos_Recomendados_por_el_CENAPRECE.pdf. Last accessed Nov 10, 2019.\u003c/li\u003e\n\u003cli\u003eNarahashi T, Ginsburg KS, Nagata K, Song JH, Tatebayashi H. Ion channels as targets for insecticides. 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DOI: 10.5897/AJMR2013.5452.\u003c/li\u003e\n\u003cli\u003eLavalett L., Miryan Margot S\u0026aacute;nchez, N\u0026eacute;lida M\u0026uacute;\u0026ntilde;oz, Jaime Moreno, Nora Cardona-Castro. Desarrollo y validaci\u0026oacute;n de una reacci\u0026oacute;n en cadena de la polimerasa m\u0026uacute;ltiple para la identificaci\u0026oacute;n de los serogrupos B, C2, D y E de Salmonella ent\u0026eacute;rica. Biom\u0026eacute;dica. 2009; 29: 244-52.\u003c/li\u003e\n\u003cli\u003eGarrido A., Mar\u0026iacute;a-Jos\u0026eacute; Chapela, Bel\u0026eacute;n Rom\u0026aacute;n, Paula Fajardo, Juan M. Vieites, Ana G. Cabado. In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes. 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Pyrethroid-resistance and presence of Two Knockdown Resistance (kdr) mutations, F1,534C and a novel mutation T1520I, in Indian \u003cem\u003eAedes aegypti\u003c/em\u003e. PLoS Negl Trop Dis. 2015; 9(1): e3332.\u003c/li\u003e\n\u003cli\u003eFernando SD, Hapugoda M, Perera R, Saavedra-Rodriguez K, Black WC 4th, De Silva NK. First report of V1,016G and S989P knockdown resistant (kdr) mutations in pyrethroid-resistant Sri Lankan \u003cem\u003eAedes aegypti\u003c/em\u003e mosquitoes. Parasites \u0026amp; vectors. 2018; 11(1), 526. doi:10.1186/s13071-018-3113-0.\u003c/li\u003e\n\u003cli\u003eKazanidou A, Nikou D, Grigoriou M, Vontas J, Skavdis J. Short Report: A Multiplex PCR Assay for Simultaneous Genotyping of kdr and ace-1 Loci in \u003cem\u003eAnopheles gambiae\u003c/em\u003e. Am. J. Trop. Med. Hyg. 2009; 80(2), pp. 236\u0026ndash;238.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 1. Sequences of the primers used for multiplex PCR.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" style=\"width:489.1pt;border-collapse:collapse;border:none;\" width=\"652\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003ePrimer name\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e5’-3’ Sequence\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eReference\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e410 Genotyping\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eSaavedra \u003cem\u003eet al\u003c/em\u003e. 2018\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eV410fw*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e[LongGCTail]ATCTTCTTGGGTTCGTTCTACCGTG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eL410fw**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e[ShortGCTail]ATCTTCTTGGGTTCGTTCTACCATT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e410 rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTTCTTCCTCGGCGGCCTCTT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1016 Genotyping\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eSaavedra \u003cem\u003eet al\u003c/em\u003e. 2007\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eL1014f*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e[LongGCTail]ACAAATTGTTTCCCACCCGCACCGG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eF1014f**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e[ShortGCTail]ACAAATTGTTTCCCACCCGCACTGA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eL1014r\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTGATGAACCSGAATTGGACAAAAGC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1534 Genotyping\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eSaingamsook et al 2017\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003ec1534-f\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGCGTACCTGTGTCTGTTCCA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003ec1534-r\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGGCTTCTTCGAGCCCATCTT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eAe1534F-r\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGCGTGAAGAACGACCCGA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"19.78527607361963%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eAe1534F-f\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 283.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"57.97546012269939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCCTCTACTTTGTGTTCTTCATCATCTG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.75pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\" valign=\"top\" width=\"22.23926380368098%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align: justify;\"\u003e\u003cspan style=\"font-size:16px;line-height:150%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:justify;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e*Long Tail 26b GCGGGCAGGGCGGCGGGGGCGGGGCC; **Short tail 6b GCGGGC\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:16px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\n \u003cbr\u003e\u0026nbsp;\n \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 2. Comparison of genotyping results for V410L, V1,016I, F1,534C mutations from multiplex PCR and AS-PCR.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" style=\"width:737.1pt;border-collapse:collapse;border:none;\" width=\"983\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:64.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"8.655804480651732%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003ePopulation\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"16\" style=\"width:658.1pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"89.30753564154786%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eMultiplex PCR genotyping / AS-PCR\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 3.5pt;height: 15.75pt;vertical-align: bottom;\" valign=\"bottom\" width=\"2.0366598778004072%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:37.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.567928730512249%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/II/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.013363028953229%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/VI/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:43.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.458797327394209%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/VV/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.902004454342984%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/VI/FC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:38.15pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.679287305122495%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVL/II/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.25pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.124721603563474%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVL/VI/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.79064587973274%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/II/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.15pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.23608017817372%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/VI/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.013363028953229%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVL/VI/FC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.23608017817372%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/VV/FF\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.25pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.124721603563474%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/VI/FC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.6815144766147%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/VV/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.570155902004454%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan 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Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:38.15pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.188199389623601%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.25pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.595116988809766%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e9/9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.0pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.289928789420142%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.15pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.696846388606307%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.35pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.493387589013225%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.7pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.696846388606307%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.25pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.595116988809766%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.103763987792472%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e4/4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.35pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.002034587995931%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:43.45pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.90030518819939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.35pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.002034587995931%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:36.35pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"4.883011190233978%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:15.0pt;border:none;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"2.034587995930824%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e28/28\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:64.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"8.646998982706002%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eSan Antonio Kaua\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.0864699898270604%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e15/15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.493387589013225%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:43.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.90030518819939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.391658189216684%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:38.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.188199389623601%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.595116988809766%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e9/9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.289928789420142%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.696846388606307%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e3/3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.493387589013225%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.696846388606307%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.595116988809766%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.103763987792472%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.002034587995931%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:43.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"5.90030518819939%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"6.002034587995931%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:36.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"4.883011190233978%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:15.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.75pt;\" width=\"2.034587995930824%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:11px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e30/30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-42.55pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:white;\"\u003eVV wild type (susceptible); VL heterozygotes; \u0026nbsp;LL mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-42.55pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVV wild type (susceptible); VI heterozygotes; II mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-42.55pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFF wild type (susceptible); FC heterozygotes; CC mutant (resistanr)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-42.55pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eN sample size\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-42.55pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:16px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\n \u003cbr\u003e\u0026nbsp;\n \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:justify;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 3. Comparison of genotypes for V410L, V1016I, and F1534C mutations for multiplex PCR and AS-PCR.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" style=\"border-collapse:collapse;border:none;\" width=\"776\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86.55pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"14.838709677419354%\"\u003e\n \u003cbr\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.6pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.516129032258065%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"12\" style=\"width: 468.65pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"80.64516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eMultiplex PCR / AS-PCR\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"14.838709677419354%\"\u003e\n \u003cbr\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.6pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.516129032258065%\"\u003e\n \u003cbr\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 99.25pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"17.032258064516128%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e410\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFrequency\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 99.2pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"17.032258064516128%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1016\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFrequency\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92.9pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"16%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1534\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.9pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"10.96774193548387%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFrequency\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"14.838709677419354%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family: \u0026quot;Times New Roman\u0026quot;,serif;\"\u003ePopulation\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"4.516129032258065%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eN\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eLL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eL410\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVI\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eII\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eI1,016\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFF\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"5.161290322580645%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"5.935483870967742%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eCC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\" valign=\"top\" width=\"10.96774193548387%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eC1,534\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"14.838709677419354%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eMerida\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.6pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.516129032258065%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e3/3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e18/18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e9/9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.60\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e3/3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e17/17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e10/10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New 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style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"5.935483870967742%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e30/30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.9pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"10.96774193548387%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86.55pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"14.838709677419354%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eSan Antonio Kaua\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.6pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"4.516129032258065%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e5/5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e10/10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New 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style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e2/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e12/12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e16/16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.73\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"5.161290322580645%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.55pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"5.935483870967742%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e30/30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.9pt;border: none;padding: 0in 5.4pt;height: 26.25pt;vertical-align: top;\" valign=\"top\" width=\"10.96774193548387%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e21/21\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e6/6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.57\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e25/25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e3/3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.53\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"5.161290322580645%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e6/6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"5.935483870967742%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e23/23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.9pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"10.96774193548387%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.90\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"14.838709677419354%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eGuadalupe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.6pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.516129032258065%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e4/4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e14/14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e12/12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.63\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e20/20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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Roman\u0026quot;,serif;\"\u003e0.67\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"5.161290322580645%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"5.935483870967742%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e29/29\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.9pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"10.96774193548387%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.98\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"14.838709677419354%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eMonterrey\u003c/span\u003e\u003c/p\u003e\n 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style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e6/6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e11/11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e11/11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.59\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e8/8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e16/16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.064516129032258%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e4/4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"9.806451612903226%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0.43\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"4.903225806451613%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"5.161290322580645%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"5.935483870967742%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e28/28\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"10.96774193548387%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:13px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e1.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-28.35pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVV wild type (susceptible); VL heterozygotes; \u0026nbsp; LL mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-28.35pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVV wild type (susceptible); VI heterozygotes; II mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-28.35pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFF wild type (susceptible); FC heterozygotes; CC mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-28.35pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eN sample size\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003e\n \u003cbr\u003e\u0026nbsp;\n \u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:justify;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eTable 4.\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eFrequency of co-occurrence of the mutations V1016I, F1534C y V410L in populations of \u003cem\u003eAe. aegypti\u003c/em\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" style=\"width:674.2pt;border-collapse:collapse;border:none;\" width=\"899\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77.5pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"11.457174638487208%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height: 200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"12\" style=\"width: 596.7pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"88.5428253615128%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eGenotype frequency \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"11.469933184855234%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003ePopulation\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.6815144766147%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/II/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.2383073496659245%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/VI/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.795100222717149%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/VV/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.12694877505568%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eLL/VI/FC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.904231625835189%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVL/II/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.461024498886414%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVL/VI/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.015590200445434%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/II/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.572383073496659%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/VI/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.2383073496659245%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVL/VI/FC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.461024498886414%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/VI/FC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"8.129175946547884%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVV/VV/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.906458797327394%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eVL/VV/CC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77.5pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"11.469933184855234%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family: \u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eMerida\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.6815144766147%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.2383073496659245%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.795100222717149%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.05pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.12694877505568%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.5pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.904231625835189%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.05pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.461024498886414%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.53\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.5pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.015590200445434%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.05pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.572383073496659%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.05pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.2383073496659245%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.05pt;border: none;padding: 0in 5.4pt;height: 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style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.795100222717149%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.05pt;border: none;padding: 0in 5.4pt;height: 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style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53.55pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.906458797327394%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77.5pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"11.469933184855234%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: 200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family: \u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003eChiapas\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"6.6815144766147%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.05pt;border: none;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.572383073496659%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.05pt;border: none;padding: 0in 5.4pt;height: 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style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.75pt;vertical-align: top;\" valign=\"top\" width=\"7.906458797327394%\"\u003e\n \u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:center;\"\u003e\u003cspan style=\"font-size:12px;line-height:200%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-28.35pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVV wild type (susceptible); VL heterozygotes; \u0026nbsp; LL mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-28.35pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eVV wild type (susceptible); VI heterozygotes; II mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:-28.35pt;line-height:115%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;\"\u003e\u003cspan style=\"font-size:12px;line-height:115%;font-family:\u0026quot;Times New Roman\u0026quot;,serif;\"\u003eFF wild type (susceptible); FC heterozygotes; CC mutant (resistant)\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\u0026quot;Calibri\u0026quot;,sans-serif;text-align:justify;\"\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Multiplex PCR, Aedes aegypti, V410L, V1,016I, F1,534C","lastPublishedDoi":"10.21203/rs.2.19782/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.19782/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eKnock down resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of nonsynonymous mutations in the he voltage-gated sodium channel (VGSC) gene, these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1,016I and F1,534C mutations are common in pyrethroid-resistant \u003cem\u003eAedes aegypti\u003c/em\u003e (L.) populations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA multiplex PCR was developed to detect the V410L, V1,016I and F1,534C mutations in \u003cem\u003eAe. aegypti\u003c/em\u003e. The validation of the technique was carried out using wild populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) \u0026nbsp;and with different levels of genotypic frequencies.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe standardized protocol for multiplex endpoint PCR was highly effective in detecting 12 genotypes in five wild \u003cem\u003eAe. aegypti\u003c/em\u003e populations from Mexico. A complete concordance with AS-PCR was found for the simultaneous detection of the three kdr mutations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cspan class=\"ql-cursor\"\u003e\u003c/span\u003e\u003c/strong\u003eOur diagnostic method is highly effective for the simultaneous detection of V410L, V1,016I and F1,534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against \u003cem\u003eAe. aegypti\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C in Aedes aegypti (L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-12-31 17:04:59","doi":"10.21203/rs.2.19782/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fa37019-ddb2-46fd-9cd9-3d009c294e70","owner":[],"postedDate":"December 31st, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45842,"name":"Parasitology"}],"tags":[],"updatedAt":"2020-06-28T15:01:42+00:00","versionOfRecord":{"articleIdentity":"rs-10394","link":"https://doi.org/10.1186/s13071-020-04193-0","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2020-06-25 12:00:00","publishedOnDateReadable":"June 25th, 2020"},"versionCreatedAt":"2019-12-31 17:04:59","video":"","vorDoi":"10.1186/s13071-020-04193-0","vorDoiUrl":"https://doi.org/10.1186/s13071-020-04193-0","workflowStages":[]},"version":"v1","identity":"rs-10394","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-10394","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}