key: cord-0327274-9bho5izi
authors: Fernandes, Luana Paula; Rocha, Marcele Neves; Duarte, Clara Guerra; Minozzo, João Carlos; do Monte-Neto, Rubens L.; Felicori, Liza F.
title: Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation
date: 2022-02-09
journal: bioRxiv
DOI: 10.1101/2022.02.09.479769
sha: 75617e08b0562944a8d1362d69463352f1fb8d95
doc_id: 327274
cord_uid: 9bho5izi

Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and swab samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP. Highlights * Using 28S primers it was possible to identify L. similis’ DNA with high sensitivity; * LAMP was 62-fold more sensitive than PCR and detected as low as 0.32 pg of DNA; * LAMP detected L. intermedia DNA from hair, serum, and exudate from experimentally-envenomed rabbits; * LAMP presents an excellent potential for identifying Loxosceles spp. envenomation.

Highlights: 1 2 *Using 28S primers it was possible to identify L. similis' DNA with high sensitivity; 3 *LAMP was 62-fold more sensitive than PCR and detected as low as 0.32 pg of DNA ; 4 *LAMP detected L. intermedia DNA from hair, serum, and exudate from experimentally-5 envenomed rabbits; 6 * LAMP presents an excellent potential for identifying Loxosceles spp. envenomation. 7 8 4 1. Intr oduction 1 Accidents with spiders of the genus Loxosceles (brown spider) represent a serious 2 global health problem, mainly due to the morbidity associated with the bite of these 3 animals. The clinical manifestations of these accidents, known as loxoscelism, are 4 considered the most serious among the spider genera. The characteristic of the cutaneous 5 form of loxoscelism is a dermonecrotic lesion that is hard to heal, accompanied by 6 nonspecific systemic symptoms such as nausea and fever [1] [2] [3] . In the cutaneous-visceral 7 loxoscelism, dermonecrotic lesion is usually accompanied by vascular manifestations, such 8 as hemolysis (intravascular or extravascular), which can progress to acute renal failure and, 9 in some cases, to disseminated vascular coagulation considered as the leading cause of 10 death from loxoscelism [4, 5] . 11 The early identification of this envenomation makes it possible to use specific 12 treatments such as serum therapy to prevent the progression of systemic symptoms 13 resulting from loxoscelism. However, this identification occurs in less than 20% of the 14 reported cases, mainly because the bite is not painful and goes unnoticed by the victim. In 15 addition, in the beginning, the lesion can be, for example, misdiagnosed as bacterial 16 infection (Staphylococcus, Mycobacterium, Syphilis, Pseudomonas, Rickettsias), fungal 17 infection (Sporothrix schenckii), viral infection by herpes, leishmaniasis, diabetic ulcers, 18 erythema nodosum and Lyme disease [3, 6] . Facing this scenario, it is necessary to develop 19 a quick and simple method that allows the precise differential diagnosis of loxoscelism. 20 Studies have already explored ELISA-based techniques (sandwich and competition) to 21 detect protein components of Loxosceles venom in animal samples. However, these 22 techniques are time-consuming and present low sensitivity (reviewed by [7] . To increase 23 sensitivity and specificity, DNA-based identification methods, such as PCR (polymerase 24 chain reaction), could also be an alternative, as it has already been pursued to snake 25 envenomation [8, 9] However, PCR requires well-trained staff and bench thermocyclers, 26 limiting its use in the field and resource-limited areas. Portable and miniaturized devices, 27 though, can be an alternative for that. The loop-mediated isothermal amplification (LAMP) 28 method is cost-effective (1.50 USD/test), simple (the isothermal reaction requires a simple 29 heating device), fast (results within 60 min) [10] , and visually detected [11] . Because of 30 this, LAMP has been used to detect parasites, [12] [13] [14] [15] , bacterias, [16, 17] , sexually 31 transmitted diseases [18] [19] [20] , and viruses including SARS-CoV-2 [21-23]. 32 5 Therefore, in this study, LAMP was applied to detect Loxosceles intermedia DNA in 1 serum, exudate, and hair samples collected from experimentally-envenomed rabbits. This 2 is a pioneer study devoted, for the first time, to detect DNA from venomous animals 3 envenomation, different from previous studies where venom protein components or 4 antibodies against venom were evaluated. LAMP pr imer design 30 LAMP primers were designed to target the 28S ribosomal RNA gene from spiders 31 belonging to the genus Loxosceles (Access in GenBank No. EU817786.1). The 28S 32 consensus sequences among L. laeta, L. gaucho, L. intermedia, and L. hirsuta were aligned 33 using MUSCLE, [24] . The regions with low or no identity with sequences from organisms 1 that misdiagnose with Loxosceles (Mycobacterium tuberculosis, M ulcerans, 2 Staphylococcus aureus, Syphilis treponema, Rickettsia rickettsii, Pseudomonas aeruginosa, 3 Chromobacterium violaceum, Sporothrix schenckii, Aspergillus sp, Cryptococcus sp, 4 Leishmania sp, Herpes simplex) were considered [3, 6, 25, 26] . Six LAMP-specific primers 5 (two internal -FIP and BIP, two external -F3 and B3, and two loop primers -LF and LB) 6 were then generated using Primer explorer V5 7 (http://primerexplorer.jp/lampv5e/index.html) and analyzed using Multiple Primer 8 Analyzer from Thermo Scientific. The oligos were purchased from GenOne and validated 9 using L. similis DNA as template (Table 1) . 10 amplification took place in a thermocycler (SimpliAmp Thermal Cycler -Thermo Fisher). 19 The amplification product was evaluated on agarose gels ( Limit of detection (LoD), sensitivity and specificity 23 The sensitivity and specificity for LAMP with DNA from Loxosceles similis was . It was centrifuged again at 22 10,000 g. The pellet was washed with absolute ethanol twice. After drying at room 23 temperature, the DNA was diluted in 50 μL milli-Q water. After extraction, DNA was 24 quantified using NanoDropTM One/One c (Thermo Fisher Scientific). Loxosceles similis DNA can be detected by LAMP, which is 62-fold 5 mor e sensitive than PCR 6 The best LAMP condition selected and used in all of the following reactions were: Figure S1 ). Using this condition, the observed LoD, which is the lowest 9 detectable Loxosceles DNA concentration, was 0.32 pg. This was confirmed by visual LAMP is specific for Loxosceles similis DNA 13 We also observed that the LAMP assay was specific for the L. similis DNA when 14 tested with other organisms DNA that would cause dermonecrotic as a clinical 15 manifestation similar to loxoscelism, such as Rickettsia rickettsii, Rickettsia parkeri, 16 Leishmania braziliensis, Corynebacterium pseudotuberculosis, and Herpes simplex virus-1 1 (Figure 2A) . PCR was also specific for L. similis DNA when the other samples were tested 2 ( Figure 2B ). We also demonstrated that it is possible to identify the spider's DNA in the 3 Loxosceles crude venom (Supplementary Figure S2 ). Detection of Loxosceles DNA in r abbit samples by LAMP assays 1 We were able to detect Loxosceles DNA in all samples (serum, exudate, and hair) 2 collected from 6 different rabbits before and after experimental envenomation in six 3 different time points. Their DNA was extracted and then evaluated by LAMP. For hair and 4 serum samples ( Figure 3A and Figure 3C ), detection could be observed from 1 to 72 h, 5 while detection in exudate samples was observed up to 24 h after envenomation ( Figure   6 3B). We can also observe that 100 % sensitivity was observed in serum samples 1 h and in 7 hair samples 24 h respectively ( Figures 3A and 3C , Table 2 ). short as 15 min [28] . There are already strategies aiming to reduce incubation time by 19 using guanidine chloride or the use of additional primers for regions on the opposite 20 strands and upstream to the inner primers (FIP and BIP) [32-34]not used in this work but 21 that can be used to improve the reaction time. 22 Since LAMP conditions were standardized using the L. similis DNA, we also 23 observed a high sensitivity in this test, in the range of 0.315 pg. 24 Using samples from L. intermedia experimentally-envenomed rabbits, we observed 1 a higher sensitivity in the first 24 h after the envenomation, in which it was possible to 2 detect Loxosceles DNA in the wound hair and serum from 1 to 24 h with the sensitivity 3 ranging from 67 to 100 %. Previous works investigating Loxosceles envenomation using 4 ELISA achieved a sensitivity of around 60 % [35, 36] . 5 In addition, it is possible to improve the sensitivity of the test, since some works 6 have even reported strategies to improve the sensitivity and specificity of LAMP tests. 7 Among these strategies, we can mention the addition of DMSO, TMSO, glycerol, and 8 betaine, which are denaturing agents, help in the separation of DNA strands and facilitate 9 the hybridization of the primers. 10 The specificity of LAMP was evaluated with DNA from organisms that, in humans, 

The authors declare that the research was conducted in the absence of any commercial or 14 financial relationships that could be construed as a potential conflict of interest. 

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