Compatibility of Lignocaine with Equine Rabies Immunoglobulin: An In Vitro Study Supporting Pain Management During Rabies Post-Exposure Prophylaxis

Compatibility of Lignocaine with Equine Rabies Immunoglobulin: An In Vitro Study Supporting Pain Management During Rabies Post-Exposure Prophylaxis | 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 Article Compatibility of Lignocaine with Equine Rabies Immunoglobulin: An In Vitro Study Supporting Pain Management During Rabies Post-Exposure Prophylaxis Zinia Thajudeen Nujum, Prathuish P.R., Devika Gopi, Deepu George Simon, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9124830/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Rabies Immunoglobulin (RIG) infiltration in and around the wound is necessary as part of post-exposure prophylaxis (PEP) in patients with Category III exposure to potentially rabid animals. However, pain from RIG infiltration remains a significant challenge. This unnecessary iatrogenic pain can be avoided/ reduced by the topical application of local anaesthetics like lignocaine, using a lignocaine medicated pad at the site, 20 minutes before administration of RIG. We have earlier demonstrated its effectiveness through a clinical trial. However, concerns about a possible interaction between lignocaine and RIG must be addressed before translation of the practice into policy. This study evaluated the potential impact of lignocaine on the virus-neutralizing activity of equine RIG in vitro. Baby Hamster Kidney (BHK21) cells were first exposed to lignocaine to assess cytotoxicity, with viable cell counts determined using trypan blue exclusion. Modified Rapid Fluorescent Focus Inhibition Test (RFFIT) was then used to compare rabies virus-neutralizing antibody (RVNA) titres of lignocaine-supplemented and untreated RIG. The study found that BHK21 cells remained viable at lignocaine concentrations up to 1/16 dilution, with no cytotoxicity observed at this level. Importantly, RVNA titres showed no significant differences between lignocaine-treated and untreated RIG preparations, demonstrating equivalent virus-neutralizing efficacy. These findings support the use of lignocaine as a local anaesthetic during RIG administration without compromising therapeutic effectiveness. It offers a practical solution to enhance patient comfort during rabies PEP, especially in resource-limited settings. This study underscores the potential for integrating lignocaine into RIG administration protocols to provide compassionate and effective rabies prophylaxis. Iatrogenic pain Lignocaine Lignocaine Medicated Pain Post-exposure prophylaxis Rabies Immunoglobulin Rabies Virus Neutralising Antibody Figures Figure 1 Figure 2 Introduction Rabies is a viral encephalitis caused by an RNA virus (Genus: Lyssavirus , Family: Rhabdoviridae ) (Rupprecht 1996 ) and is practically 100% fatal even today (Baxter 2012 ). India reports about 36% of the total global deaths due to human rabies(Baxter 2012 ). Animal exposures are categorised based on WHO guidelines for initiation of post-exposure prophylaxis (World Health Organisation 2023). Single or multiple transdermal bites or scratches, licks on broken skin, and contamination of mucous membrane with saliva (i.e. licks) are considered Category III severe exposures and require administration of Rabies immunoglobulin (hyperimmune serum) and anti-rabies vaccine (World Health Organisation 2023). RIG is also indicated in Category II exposures in immunocompromised individuals. A significant challenge for both healthcare providers and patients is the iatrogenic pain caused by RIG infiltration into and around the wound (Haradanhalli et al. 2022 ). Addressing this pain is crucial to improving the patient experience during rabies PEP.(Haradanhalli et al. 2022 ) Lignocaine (1–4%) has been proven to be useful for pain relief during suturing of lacerated wounds in children (Lambert and Goldman 2018 ) for topical anesthesia and skin-harvest sites (Jellish et al. 1999 ). Lignocaine binds to the sodium channels on the internal surface of the nerve cell membrane and locks them in the open state, thus preventing depolarisation (Beecham et al. 2025 ). It provides local and topical anesthesia with an onset of action 3 to 5 minutes after application (Beecham et al. 2025 ). We have conducted a clinical trial previously, which showed that the application of 2% Lignocaine Medicated Pad (LMG) effectively reduces pain during RIG (Rabies antiserum IP - Equine) administration at the local site (Nujum et al. 2021 ). Translating this research finding into policy and practice will require understanding the interactions of lignocaine with rabies immunoglobulin at the tissue level. Drug interactions between various medications have been documented to augment the efficacy of treatment with some acting synergistically, while others antagonistically (Boobis et al. 2009 ). Local anesthetics like procaine have been found to decrease the efficacy of drugs like amino salicylic acid and sulfonamides (WebMD 2023). So a concern of the potential interaction between Lignocaine and Equine Rabies Immunoglobulin could limit its use among practitioners. Hence, it is imperative to prove that there is no reduction of the neutralisation of the virus by immunoglobulin due to the local application of lignocaine. This in vitro study was therefore carried out to assess the potential impact of 2% lignocaine on the virus-neutralising efficacy of rabies immunoglobulin (Rabies Antiserum IP - Equine) in cell culture. Materials and Methods The study was designed to evaluate the effect of lignocaine-supplemented hyperimmune serum that may impact performance of RFFIT and RVNA titres when compared to hyperimmune serum and standard rabies immunoglobulin. The study was also aimed to demonstrate the effect of lignocaine on BHK21 cell lines and virus infectivity in BHK21 cells. It was done at the State Institute of Animal Diseases, Palode, Kerala and did not require approval from ethics committee. Commercial preparation of lignocaine, Loxicard with 2% lignocaine was selected for the study as it is devoid of preservatives other than sodium chloride. Two commercially available preparations of Rabies antiserum IP - Equine (300 IU/ml) viz., Equirab and VINS were procured from the pharmacy. Baby Hamster Kidney (BHK2) cell line [ATCC Cat. No. CCL-10] and Challenge Virus Standard (CVS-11 rabies virus strain) were obtained from NIMHANS, Bengaluru. Iscove’s Modified Dulbecco’s medium (IMDM), Cat. No. 17633 and Foetal Calf Serum (FCS), Cat. No. F7524 was procured from Sigma-Aldrich. Rabies FITC conjugate was purchased from Biorad (Cat. No. 3572112) Determining the cytotoxicity of lignocaine An initial investigation was conducted using Loxicard (Lignocaine 2%) to determine the cytotoxicity of the local anesthetic. Baby Hamster Kidney (BHK21) cells were treated against two-fold dilutions of Loxicard in a 96-well cell culture plate containing growth media Iscove’s Modified Dulbeco’s medium (IMDM) and incubated at 37°C for 24 hours. The incubated plates were observed for cell growth. Relative potency and safety of lignocaine on BHK21 cells were determined using direct microscopy and trypan blue exclusion assay. Trypan blue exclusion assay was used to determine the percentage of viable cells having clear cytoplasm and dead cells (blue cytoplasm) after exposure to lignocaine. Briefly, a mixture of 10ul of 0.4% trypan blue and 10ul of cell suspension was incubated at room temperature for three minutes. The mixture was applied to a hemocytometer and quantified under an inverted microscope. Modified Rapid Fluorescent Focus Inhibition Test (RFFIT) The RFFIT is presently the gold standard test used to monitor immune response to vaccination against rabies and for the quality testing of rabies hyperimmune serum (Lyng 1994 ). RFFIT assay performed here is adapted to 96-well tissue culture plates, which were followed in WHO-approved testing centres. The cell-safe concentration of lignocaine was added to two-fold dilutions (1/32 − 1/4096) of Rabies antiserum IP - Equine (300 IU/ml) viz., Equirab and VINS at 7.5 IU/ml and 15 IU/ml in a 96-well cell culture plate and incubated for one hour. Also, commercially available Equine Rabies Immunoglobulin preparation, Equirab calibrated against international reference serum having the potency of 30 IU/ml was used as reference anti-rabies serum (standard rabies immunoglobulin). Challenge virus standard [CVS-11 rabies virus strain] at 100FFD50 concentration was added to the hyperimmune serum lignocaine mixture in each well and the plates were again incubated for one hour. BHK 21 cells with a seeding density of 10/well were added. Reference standards, virus control, and cell control were also maintained. All tests were conducted in triplicate. RVNA neutralises the rabies virus and prevents infection of baby hamster kidney (BHK-21) cells susceptible to the rabies virus. Live virus infecting BHK 21 cells at higher serum dilutions was detected using Fluorescein isothiocyanate (FITC) conjugated Ab to the rabies virus. The serum neutralisation endpoint titre (ED50) is defined as the reciprocal dilution of the highest serum dilution at which 50% reduction in the number of fluorescent positive fields is observed as compared to the virus control. This RVNA titre (50% neutralization, ED50) is converted into IU/ml by calibrating against the reference standard. Results Lignocaine-mediated cell cytotoxicity was observed up to 1/16th dilution of local anesthetic (2% Loxicard). BHK21 cells were observed not adherent to the surface of the 96 well plates in neat and ½ dilution of Loxicard and cell attachment was disrupted upto ⅛th dilution. Wells at 1/16th dilution of Loxicard showed normal cell growth and monolayer was found and observed after 24 hours of incubation (Fig. 1 ). After that, Trypan exclusion studies also showed a similar pattern of cell growth on application of lignocaine. 100% cell death was observed in neat and ½ dilution of lignocaine. From ¼th dilution, the percentage of live cells increased from 20% to 60% at ⅛ th dilution and 1/16th dilution onwards 100% viable cell count was obtained. 1/16th dilution of Loxicard was utilised for modified RFFIT studies. The modified test was validated using 2 IU/ml standard rabies immunoglobulin. The available market preparations of hyperimmune serum (300 IU/ml) were diluted to 3.75 IU/ml, 7.5 IU/ml, and 15 IU/ml in IMDM as higher concentrations of RVNA can impact the traditional RFFIT assay. No difference in RVNA titre was observed between lignocaine-supplemented hyperimmune serum and local anesthetic-devoid serum. (Table 1 .) Fluorescent foci were started from 1/512th dilution (3.75IU/ml), 1/1024 dilution (7.5 IU/ml), and 1/2048 dilution (15 IU/ml), (Fig. 2 .). Similar results were obtained in untreated hyperimmune serum. We could demonstrate similar RVNA titers by repeating the RFFIT assay with different lots of hyperimmune serum using the same test parameters. All virus controls showed 80–100% fluorescent foci, all cell controls remained negative in every assay and standard RIG showed fluorescent foci starting at 1/256th (2 IU/ml). Table 1 Rabies Virus Neutralising Antibody titres in untreated and Lignocaine-treated ERIG Reciprocal of Dilution RVNA Titre Value(IU/ml) Untreated ERIG (2IU/ml) RVNA Titre Value(IU/ml) Lignocaine Treated ERIG (2IU/ml) 128 0.94 0.94 256 1.87 1.87 512 3.75 3.75 1024 7.50 7.50 2048 15.0 15.0 Discussion We have previously demonstrated that lignocaine (2%) impregnated cotton pads can effectively reduce pain during RIG administration (Nujum et al. 2021 ). However, before recommending this practice for policy implementation, we sought to investigate the potential interaction between lignocaine and RIG. The primary objective of this study was to determine whether lignocaine, when used as a local anaesthetic to reduce pain, affects the neutralising antibody activity of RIG. Since an in vivo assessment of this interaction was not feasible, we conducted an in vitro experiment to evaluate the potential impact. Our study demonstrated that lignocaine does not compromise the efficacy or viral neutralisation process of RIG in infected BHK21 cells, as evidenced by unchanged RVNA titers in both control and lignocaine-treated samples. Elective surgeries and anaesthesia are known risk factors for rabies vaccination failure. In emergencies, the potential immunomodulatory effects of anaesthesia and surgery should be considered, as they may hinder ARV efficacy (12,13). Moreover, avoiding local anaesthetics for postoperative analgesia is recommended to aid early detection of neurological deficits (Rewari et al. 2010 ). Although some studies have explored interactions between local anaesthetics and immunoglobulins in general (Hijlstra et al. 1980 ), there is limited research specifically addressing the concurrent use of local anaesthetics with immunoglobulin therapies. To our knowledge, this is the first in vitro study evaluating the interaction between a local anaesthetic and rabies immunoglobulin. There may be concerns that local lignocaine application could dilute RIG. However, studies show that diluting RIG with solvents like normal saline up to 16-fold does not affect its efficacy (Madhusudana et al. 2013 ). RIG is routinely diluted two- to threefold as per national guidelines, especially in children where smaller doses must cover multiple/larger wound areas (ScienceDirect 2023). Thus, dilution from lignocaine is unlikely to be problematic. Moreover, lignocaine is applied locally with minimal systemic absorption. Lidocaine patches, designed for targeted application, deliver only about 3 ± 2% of the applied dose into the bloodstream, with peak plasma concentrations remaining below 0.3 µg/mL — far below levels associated with systemic effects or toxicity (18,19). Lignocaine (2%) impregnated cotton pads have proven effective in significantly reducing pain during RIG infiltration (Nujum et al. 2021 ). This laboratory evidence supports translating these findings into policy. The maximum lignocaine quantity used in the study was 5 mL, making it a highly affordable solution compared to commercial lignocaine patches, which range from Rs. 100 to Rs. 3599 (IndiaMART 2023a). Two percent lignocaine is widely available in 30 mL ampoules for just Rs. 20 to Rs. 30 (IndiaMART 2023b), lignocaine-soaked pads provide a practical, cost-effective strategy to minimize iatrogenic pain during RIG administration, particularly in resource-limited settings. The potential for lignocaine patches to interfere with vaccine efficacy is an important concern which can be dismissed from the evidence that this study provides. Studies assessing antibody responses to DTaP-IPV-Hib and hepatitis B vaccines in patients who received lidocaine-prilocaine (EMLA) patches for pain relief found no adverse impact on immunogenicity (Halperin et al. 2002 ). These findings suggest that local anaesthetic application does not compromise vaccine efficacy in general. Nonetheless, further research is warranted to confirm this in the context of rabies post-exposure prophylaxis (PEP), particularly in patients receiving local anaesthetic patches or applications alongside RIG. Conclusions and Recommendations The absence of a significant difference in RVNA titres between lignocaine-supplemented and untreated hyperimmune serum confirms that lignocaine does not affect eRIG efficacy. This supports its safe use to reduce iatrogenic pain during RIG administration. While these results are encouraging, further studies are needed to assess whether local anaesthetics impact rabies vaccine responses, particularly in PEP scenarios. Local anaesthesia during RIG infiltration is a simple, cost-effective intervention that addresses an often-overlooked aspect of care; preventing unnecessary pain and suffering. This approach can improve patient comfort, ensuring rabies prophylaxis is both effective and compassionate. Declarations Author statements ZTN, MRR and SSA conceptualized and designed the study; PPR, DG and AS were instrumental in getting doing the lab work, ZTN did the initial drafting of paper, MRR RK SSG critically revised the manuscript for intellectual content. All authors read and approved the final manuscript. ZTN and PPR are guarantors of the paper. Declaration by authors The manuscript has been read and approved by all the authors. The requirements for authorship have been met. Each author believes that the manuscript represents honest work. All authors declare that they have no conflict of interest to disclose. Acknowledgements We place on record our gratitude to Dr. Reetha Mani, Professor and Head, Department of Neurovirology, NIMHANS, Bangalore India for giving us valuable guidance for the work . We would like to express our sincere gratitude to our current Professor of School of Public Health Dr. Thomas Mathew Funding: None Competing interests: None Ethics Declaration: Not applicable References Baxter JM (2012) One in a million, or one in thousand: What is the morbidity of rabies in India? J Glob Health 2:010303. Becker DE, Reed KL (2012) Local anesthetics: review of pharmacological considerations. Anesth Prog 59:90–102. Beecham GB, Nessel TA, Goyal A (2025) Lidocaine. In: StatPearls. StatPearls Publishing, Treasure Island. Boobis A, Watelet JB, Whomsley R, Benedetti MS, Demoly P, Tipton K (2009) Drug interactions. Drug Metab Rev 41:486–527. Fescharek R, Franke V, Samuel MR (1994) Do anaesthetics and surgical stress increase the risk of post-exposure rabies treatment failure? Vaccine 12:12–13. Ghodki P, Katikar M, Kulkarni P (2021) Dog bite and antirabies vaccination: what the anaesthesiologist needs to know. Indian J Anaesth 65:632–633. Halperin BA, Halperin SA, McGrath P, Smith B, Houston T (2002) Lidocaine-prilocaine patch and vaccine antibody response in infants. Pediatr Infect Dis J 21:399–405. Haradanhalli RS, Fotedar N, Kumari N, Narayana DHA (2022) Safety and clinical efficacy of human rabies immunoglobulin in post exposure prophylaxis for category III animal exposures. Hum Vaccin Immunother 18:2081024. Hijlstra A, Van Dorp W, Daha MR, Leslie GQ (1980) Effect of lidocaine on processing of immune aggregates by macrophages. Immunology 41:237–244. Jellish WS, Gamelli RL, Furry PA, McGill VL, Fluder EM (1999) Effect of topical local anesthetic application to skin harvest sites for pain management in burn patients undergoing skin grafting. Ann Surg 229:115–120. Lambert C, Goldman RD (2018) Pain management for children needing laceration repair. Can Fam Physician 64:900–902. Lyng J (1994) Calibration of a replacement preparation for the international standard for rabies immunoglobulin. Biologicals 22:249–255. Madhusudana SN, Ashwin BY, Sudarshan S (2013) Feasibility of reducing rabies immunoglobulin dosage for passive immunization against rabies. Hum Vaccin Immunother 9:1914–1917. Navez ML, Monella C, Bösl I, Sommer D, Delorme C (2015) 5% lidocaine medicated plaster for treatment of postherpetic neuralgia. Pain Ther 4:1–15. Nujum ZT, Valamparampil MJ, Rajagopal MR, Rahul A, Siju NS, Prajitha KC (2021) Efficacy of 2% lignocaine medicated pad for pain alleviation during rabies immunoglobulin administration: a randomized controlled trial. Indian J Public Health 65:356–361. Rewari V, Garg R, Trikha A (2010) Rabies vaccine and neuraxial anaesthesia: case study. S Afr J Anaesth Analg 16:32–34. Rupprecht CE (1996) Rabies virus. In: Baron S (ed) Medical Microbiology, 4th edn. University of Texas Medical Branch, Galveston. World Health Organization (2023) Rabies vaccinations and immunization. https://www.who.int . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 04 May, 2026 Reviewers agreed at journal 01 May, 2026 Reviewers invited by journal 20 Apr, 2026 Editor assigned by journal 25 Mar, 2026 Submission checks completed at journal 25 Mar, 2026 First submitted to journal 14 Mar, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9124830","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627077887,"identity":"8a5bdba3-d8f3-4f12-a867-0cc5499225ef","order_by":0,"name":"Zinia Thajudeen Nujum","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYDCCAyBkwCAHZj8gRYsxmJ1ArBYQSGwAkURp4bt9/OHhggK79Plhhx8CbbGT020goEXyXELC4RkGybkbb6cZALUkG5sdIKDF4AzDgcM8Bsy5G2cngLQcSNxGWAtjA1BLfbrh7PQPxGphZgBqOZwgL51DpC2SZ9hAWo4bbpDOKTiQYECEX/jOsD/+zPOnWl5+dvrmDx8q7OQIakG4EKzSgFjlICDfQIrqUTAKRsEoGFEAAFCOR4G8xMUaAAAAAElFTkSuQmCC","orcid":"","institution":"School of Public Health Kerala University of Health Sciences","correspondingAuthor":true,"prefix":"","firstName":"Zinia","middleName":"Thajudeen","lastName":"Nujum","suffix":""},{"id":627077894,"identity":"af066ebc-716a-4ea7-8989-a30f4247cdba","order_by":1,"name":"Prathuish P.R.","email":"","orcid":"","institution":"State Institute of Animal Diseases Palode","correspondingAuthor":false,"prefix":"","firstName":"Prathuish","middleName":"","lastName":"P.R.","suffix":""},{"id":627077895,"identity":"d6ca7fd2-b56e-4c60-a636-2f56145722a8","order_by":2,"name":"Devika Gopi","email":"","orcid":"","institution":"State Institute of Animal Diseases Palode","correspondingAuthor":false,"prefix":"","firstName":"Devika","middleName":"","lastName":"Gopi","suffix":""},{"id":627077897,"identity":"bc94ccef-c00b-492b-baa8-e81dde98bc20","order_by":3,"name":"Deepu George Simon","email":"","orcid":"","institution":"Government Medical College","correspondingAuthor":false,"prefix":"","firstName":"Deepu","middleName":"George","lastName":"Simon","suffix":""},{"id":627077898,"identity":"2fef3e3c-a8b1-45ce-b2ad-6a64ce91b8ff","order_by":4,"name":"K Rajamohanan","email":"","orcid":"","institution":"School of Public Health Kerala University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"K","middleName":"","lastName":"Rajamohanan","suffix":""},{"id":627077899,"identity":"0f08c472-af33-4be5-8a3d-89334af276ac","order_by":5,"name":"Aparna Sujatha","email":"","orcid":"","institution":"State Institute of Animal Diseases Palode","correspondingAuthor":false,"prefix":"","firstName":"Aparna","middleName":"","lastName":"Sujatha","suffix":""},{"id":627077900,"identity":"9a983e1c-b7ab-4a03-ae68-22c385e8734b","order_by":6,"name":"Swapna Susan Abraham","email":"","orcid":"","institution":"State Institute of Animal Diseases Palode","correspondingAuthor":false,"prefix":"","firstName":"Swapna","middleName":"Susan","lastName":"Abraham","suffix":""},{"id":627077902,"identity":"e1142021-69c7-43c9-9e13-617fee5606fe","order_by":7,"name":"M. R. Rajagopal","email":"","orcid":"","institution":"Trivandrum Institute of Palliative Sciences","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"R.","lastName":"Rajagopal","suffix":""},{"id":627077904,"identity":"f304b4c0-2ab8-410e-be80-471015075202","order_by":8,"name":"Sheela Saly","email":"","orcid":"","institution":"State Institute of Animal Diseases Palode","correspondingAuthor":false,"prefix":"","firstName":"Sheela","middleName":"","lastName":"Saly","suffix":""}],"badges":[],"createdAt":"2026-03-14 20:09:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9124830/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9124830/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108384198,"identity":"7617724d-8b62-401c-8c2e-10626bce79ea","added_by":"auto","created_at":"2026-05-04 05:51:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":462520,"visible":true,"origin":"","legend":"\u003cp\u003eLignocaine-Mediated Cytotoxicity in Lignocaine-treated and untreated control of BHK21 cells\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9124830/v1/871e9c0652d4a8028c82ea04.png"},{"id":108384199,"identity":"d9c2b51e-09da-4a6e-9f1c-c8961aaaf83f","added_by":"auto","created_at":"2026-05-04 05:51:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":390523,"visible":true,"origin":"","legend":"\u003cp\u003eRabies Virus Neutralisation shown by fluorescent foci in untreated and Lignocaine-treated ERIG\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9124830/v1/2d18c964bcb90d50cb83650b.png"},{"id":108492354,"identity":"6c227446-8fc2-4694-831d-a6558fb7dec1","added_by":"auto","created_at":"2026-05-05 09:57:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1036590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9124830/v1/1d1d7c36-5545-42f7-ad2e-8cd5e8f235a5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Compatibility of Lignocaine with Equine Rabies Immunoglobulin: An In Vitro Study Supporting Pain Management During Rabies Post-Exposure Prophylaxis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRabies is a viral encephalitis caused by an RNA virus (Genus: \u003cem\u003eLyssavirus\u003c/em\u003e, Family: \u003cem\u003eRhabdoviridae\u003c/em\u003e) (Rupprecht \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and is practically 100% fatal even today (Baxter \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). India reports about 36% of the total global deaths due to human rabies(Baxter \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Animal exposures are categorised based on WHO guidelines for initiation of post-exposure prophylaxis (World Health Organisation 2023). Single or multiple transdermal bites or scratches, licks on broken skin, and contamination of mucous membrane with saliva (i.e. licks) are considered Category III severe exposures and require administration of Rabies immunoglobulin (hyperimmune serum) and anti-rabies vaccine (World Health Organisation 2023). RIG is also indicated in Category II exposures in immunocompromised individuals. A significant challenge for both healthcare providers and patients is the iatrogenic pain caused by RIG infiltration into and around the wound (Haradanhalli et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Addressing this pain is crucial to improving the patient experience during rabies PEP.(Haradanhalli et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eLignocaine (1\u0026ndash;4%) has been proven to be useful for pain relief during suturing of lacerated wounds in children (Lambert and Goldman \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) for topical anesthesia and skin-harvest sites (Jellish et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Lignocaine binds to the sodium channels on the internal surface of the nerve cell membrane and locks them in the open state, thus preventing depolarisation (Beecham et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It provides local and topical anesthesia with an onset of action 3 to 5 minutes after application (Beecham et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). We have conducted a clinical trial previously, which showed that the application of 2% Lignocaine Medicated Pad (LMG) effectively reduces pain during RIG (Rabies antiserum IP - Equine) administration at the local site (Nujum et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Translating this research finding into policy and practice will require understanding the interactions of lignocaine with rabies immunoglobulin at the tissue level.\u003c/p\u003e \u003cp\u003eDrug interactions between various medications have been documented to augment the efficacy of treatment with some acting synergistically, while others antagonistically (Boobis et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Local anesthetics like procaine have been found to decrease the efficacy of drugs like amino salicylic acid and sulfonamides (WebMD 2023). So a concern of the potential interaction between Lignocaine and Equine Rabies Immunoglobulin could limit its use among practitioners.\u003c/p\u003e \u003cp\u003eHence, it is imperative to prove that there is no reduction of the neutralisation of the virus by immunoglobulin due to the local application of lignocaine. This \u003cem\u003ein vitro\u003c/em\u003e study was therefore carried out to assess the potential impact of 2% lignocaine on the virus-neutralising efficacy of rabies immunoglobulin (Rabies Antiserum IP - Equine) in cell culture.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe study was designed to evaluate the effect of lignocaine-supplemented hyperimmune serum that may impact performance of RFFIT and RVNA titres when compared to hyperimmune serum and standard rabies immunoglobulin. The study was also aimed to demonstrate the effect of lignocaine on BHK21 cell lines and virus infectivity in BHK21 cells. It was done at the State Institute of Animal Diseases, Palode, Kerala and did not require approval from ethics committee.\u003c/p\u003e \u003cp\u003eCommercial preparation of lignocaine, Loxicard with 2% lignocaine was selected for the study as it is devoid of preservatives other than sodium chloride. Two commercially available preparations of Rabies antiserum IP - Equine (300 IU/ml) viz., Equirab and VINS were procured from the pharmacy. Baby Hamster Kidney (BHK2) cell line [ATCC Cat. No. CCL-10] and Challenge Virus Standard (CVS-11 rabies virus strain) were obtained from NIMHANS, Bengaluru. Iscove\u0026rsquo;s Modified Dulbecco\u0026rsquo;s medium (IMDM), Cat. No. 17633 and Foetal Calf Serum (FCS), Cat. No. F7524 was procured from Sigma-Aldrich. Rabies FITC conjugate was purchased from Biorad (Cat. No. 3572112)\u003c/p\u003e \u003cp\u003eDetermining the cytotoxicity of lignocaine\u003c/p\u003e \u003cp\u003eAn initial investigation was conducted using Loxicard (Lignocaine 2%) to determine the cytotoxicity of the local anesthetic. Baby Hamster Kidney (BHK21) cells were treated against two-fold dilutions of Loxicard in a 96-well cell culture plate containing growth media Iscove\u0026rsquo;s Modified Dulbeco\u0026rsquo;s medium (IMDM) and incubated at 37\u0026deg;C for 24 hours. The incubated plates were observed for cell growth.\u003c/p\u003e \u003cp\u003eRelative potency and safety of lignocaine on BHK21 cells were determined using direct microscopy and trypan blue exclusion assay. Trypan blue exclusion assay was used to determine the percentage of viable cells having clear cytoplasm and dead cells (blue cytoplasm) after exposure to lignocaine. Briefly, a mixture of 10ul of 0.4% trypan blue and 10ul of cell suspension was incubated at room temperature for three minutes. The mixture was applied to a hemocytometer and quantified under an inverted microscope.\u003c/p\u003e \u003cp\u003eModified Rapid Fluorescent Focus Inhibition Test (RFFIT)\u003c/p\u003e \u003cp\u003eThe RFFIT is presently the gold standard test used to monitor immune response to vaccination against rabies and for the quality testing of rabies hyperimmune serum (Lyng \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). RFFIT assay performed here is adapted to 96-well tissue culture plates, which were followed in WHO-approved testing centres. The cell-safe concentration of lignocaine was added to two-fold dilutions (1/32\u0026thinsp;\u0026minus;\u0026thinsp;1/4096) of Rabies antiserum IP - Equine (300 IU/ml) viz., Equirab and VINS at 7.5 IU/ml and 15 IU/ml in a 96-well cell culture plate and incubated for one hour. Also, commercially available Equine Rabies Immunoglobulin preparation, Equirab calibrated against international reference serum having the potency of 30 IU/ml was used as reference anti-rabies serum (standard rabies immunoglobulin). Challenge virus standard [CVS-11 rabies virus strain] at 100FFD50 concentration was added to the hyperimmune serum lignocaine mixture in each well and the plates were again incubated for one hour. BHK 21 cells with a seeding density of 10/well were added. Reference standards, virus control, and cell control were also maintained. All tests were conducted in triplicate. RVNA neutralises the rabies virus and prevents infection of baby hamster kidney (BHK-21) cells susceptible to the rabies virus. Live virus infecting BHK 21 cells at higher serum dilutions was detected using Fluorescein isothiocyanate (FITC) conjugated Ab to the rabies virus. The serum neutralisation endpoint titre (ED50) is defined as the reciprocal dilution of the highest serum dilution at which 50% reduction in the number of fluorescent positive fields is observed as compared to the virus control. This RVNA titre (50% neutralization, ED50) is converted into IU/ml by calibrating against the reference standard.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eLignocaine-mediated cell cytotoxicity was observed up to 1/16th dilution of local anesthetic (2% Loxicard). BHK21 cells were observed not adherent to the surface of the 96 well plates in neat and \u0026frac12; dilution of Loxicard and cell attachment was disrupted upto ⅛th dilution. Wells at 1/16th dilution of Loxicard showed normal cell growth and monolayer was found and observed after 24 hours of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After that, Trypan exclusion studies also showed a similar pattern of cell growth on application of lignocaine. 100% cell death was observed in neat and \u0026frac12; dilution of lignocaine. From \u0026frac14;th dilution, the percentage of live cells increased from 20% to 60% at ⅛ th dilution and 1/16th dilution onwards 100% viable cell count was obtained. 1/16th dilution of Loxicard was utilised for modified RFFIT studies. The modified test was validated using 2 IU/ml standard rabies immunoglobulin. The available market preparations of hyperimmune serum (300 IU/ml) were diluted to 3.75 IU/ml, 7.5 IU/ml, and 15 IU/ml in IMDM as higher concentrations of RVNA can impact the traditional RFFIT assay.\u003c/p\u003e \u003cp\u003eNo difference in RVNA titre was observed between lignocaine-supplemented hyperimmune serum and local anesthetic-devoid serum. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.) Fluorescent foci were started from 1/512th dilution (3.75IU/ml), 1/1024 dilution (7.5 IU/ml), and 1/2048 dilution (15 IU/ml), (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.). Similar results were obtained in untreated hyperimmune serum. We could demonstrate similar RVNA titers by repeating the RFFIT assay with different lots of hyperimmune serum using the same test parameters. All virus controls showed 80\u0026ndash;100% fluorescent foci, all cell controls remained negative in every assay and standard RIG showed fluorescent foci starting at 1/256th (2 IU/ml).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRabies Virus Neutralising Antibody titres in untreated and Lignocaine-treated ERIG\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReciprocal of Dilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRVNA Titre Value(IU/ml)\u003c/p\u003e \u003cp\u003eUntreated ERIG (2IU/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRVNA Titre Value(IU/ml)\u003c/p\u003e \u003cp\u003eLignocaine Treated ERIG (2IU/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have previously demonstrated that lignocaine (2%) impregnated cotton pads can effectively reduce pain during RIG administration (Nujum et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, before recommending this practice for policy implementation, we sought to investigate the potential interaction between lignocaine and RIG. The primary objective of this study was to determine whether lignocaine, when used as a local anaesthetic to reduce pain, affects the neutralising antibody activity of RIG. Since an in vivo assessment of this interaction was not feasible, we conducted an \u003cem\u003ein vitro\u003c/em\u003e experiment to evaluate the potential impact.\u003c/p\u003e \u003cp\u003eOur study demonstrated that lignocaine does not compromise the efficacy or viral neutralisation process of RIG in infected BHK21 cells, as evidenced by unchanged RVNA titers in both control and lignocaine-treated samples.\u003c/p\u003e \u003cp\u003eElective surgeries and anaesthesia are known risk factors for rabies vaccination failure. In emergencies, the potential immunomodulatory effects of anaesthesia and surgery should be considered, as they may hinder ARV efficacy (12,13). Moreover, avoiding local anaesthetics for postoperative analgesia is recommended to aid early detection of neurological deficits (Rewari et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough some studies have explored interactions between local anaesthetics and immunoglobulins in general (Hijlstra et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1980\u003c/span\u003e), there is limited research specifically addressing the concurrent use of local anaesthetics with immunoglobulin therapies. To our knowledge, this is the first \u003cem\u003ein vitro\u003c/em\u003e study evaluating the interaction between a local anaesthetic and rabies immunoglobulin.\u003c/p\u003e \u003cp\u003eThere may be concerns that local lignocaine application could dilute RIG. However, studies show that diluting RIG with solvents like normal saline up to 16-fold does not affect its efficacy (Madhusudana et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). RIG is routinely diluted two- to threefold as per national guidelines, especially in children where smaller doses must cover multiple/larger wound areas (ScienceDirect 2023). Thus, dilution from lignocaine is unlikely to be problematic.\u003c/p\u003e \u003cp\u003eMoreover, lignocaine is applied locally with minimal systemic absorption. Lidocaine patches, designed for targeted application, deliver only about 3\u0026thinsp;\u0026plusmn;\u0026thinsp;2% of the applied dose into the bloodstream, with peak plasma concentrations remaining below 0.3 \u0026micro;g/mL \u0026mdash; far below levels associated with systemic effects or toxicity (18,19).\u003c/p\u003e \u003cp\u003eLignocaine (2%) impregnated cotton pads have proven effective in significantly reducing pain during RIG infiltration (Nujum et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This laboratory evidence supports translating these findings into policy. The maximum lignocaine quantity used in the study was 5 mL, making it a highly affordable solution compared to commercial lignocaine patches, which range from Rs. 100 to Rs. 3599 (IndiaMART 2023a). Two percent lignocaine is widely available in 30 mL ampoules for just Rs. 20 to Rs. 30 (IndiaMART 2023b), lignocaine-soaked pads provide a practical, cost-effective strategy to minimize iatrogenic pain during RIG administration, particularly in resource-limited settings. The potential for lignocaine patches to interfere with vaccine efficacy is an important concern which can be dismissed from the evidence that this study provides. Studies assessing antibody responses to DTaP-IPV-Hib and hepatitis B vaccines in patients who received lidocaine-prilocaine (EMLA) patches for pain relief found no adverse impact on immunogenicity (Halperin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These findings suggest that local anaesthetic application does not compromise vaccine efficacy in general. Nonetheless, further research is warranted to confirm this in the context of rabies post-exposure prophylaxis (PEP), particularly in patients receiving local anaesthetic patches or applications alongside RIG.\u003c/p\u003e"},{"header":"Conclusions and Recommendations","content":"\u003cp\u003eThe absence of a significant difference in RVNA titres between lignocaine-supplemented and untreated hyperimmune serum confirms that lignocaine does not affect eRIG efficacy. This supports its safe use to reduce iatrogenic pain during RIG administration. While these results are encouraging, further studies are needed to assess whether local anaesthetics impact rabies vaccine responses, particularly in PEP scenarios. Local anaesthesia during RIG infiltration is a simple, cost-effective intervention that addresses an often-overlooked aspect of care; preventing unnecessary pain and suffering. This approach can improve patient comfort, ensuring rabies prophylaxis is both effective and compassionate.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor statements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZTN, MRR and SSA conceptualized and designed the study; PPR, \u0026nbsp;DG and AS were instrumental in getting doing the lab work, ZTN did the initial drafting of paper, MRR RK SSG critically revised the manuscript for intellectual content. All authors read and approved the final manuscript. ZTN and PPR are guarantors of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration by authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript has been read and approved by all the authors. The requirements for authorship have been met. Each author believes that the manuscript represents honest work. All authors declare that they have no conflict of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe place on record our gratitude to Dr. Reetha Mani, Professor and Head, Department of Neurovirology, NIMHANS, Bangalore India for giving us valuable guidance for the work . We would like to express our sincere gratitude to our current Professor of School of Public Health Dr. Thomas Mathew\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e None\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e None\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration:\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaxter JM (2012) One in a million, or one in thousand: What is the morbidity of rabies in India? J Glob Health 2:010303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBecker DE, Reed KL (2012) Local anesthetics: review of pharmacological considerations. Anesth Prog 59:90\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeecham GB, Nessel TA, Goyal A (2025) Lidocaine. In: StatPearls. StatPearls Publishing, Treasure Island.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoobis A, Watelet JB, Whomsley R, Benedetti MS, Demoly P, Tipton K (2009) Drug interactions. Drug Metab Rev 41:486\u0026ndash;527.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFescharek R, Franke V, Samuel MR (1994) Do anaesthetics and surgical stress increase the risk of post-exposure rabies treatment failure? Vaccine 12:12\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhodki P, Katikar M, Kulkarni P (2021) Dog bite and antirabies vaccination: what the anaesthesiologist needs to know. Indian J Anaesth 65:632\u0026ndash;633.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalperin BA, Halperin SA, McGrath P, Smith B, Houston T (2002) Lidocaine-prilocaine patch and vaccine antibody response in infants. Pediatr Infect Dis J 21:399\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaradanhalli RS, Fotedar N, Kumari N, Narayana DHA (2022) Safety and clinical efficacy of human rabies immunoglobulin in post exposure prophylaxis for category III animal exposures. Hum Vaccin Immunother 18:2081024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHijlstra A, Van Dorp W, Daha MR, Leslie GQ (1980) Effect of lidocaine on processing of immune aggregates by macrophages. Immunology 41:237\u0026ndash;244.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJellish WS, Gamelli RL, Furry PA, McGill VL, Fluder EM (1999) Effect of topical local anesthetic application to skin harvest sites for pain management in burn patients undergoing skin grafting. Ann Surg 229:115\u0026ndash;120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLambert C, Goldman RD (2018) Pain management for children needing laceration repair. Can Fam Physician 64:900\u0026ndash;902.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLyng J (1994) Calibration of a replacement preparation for the international standard for rabies immunoglobulin. Biologicals 22:249\u0026ndash;255.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadhusudana SN, Ashwin BY, Sudarshan S (2013) Feasibility of reducing rabies immunoglobulin dosage for passive immunization against rabies. Hum Vaccin Immunother 9:1914\u0026ndash;1917.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavez ML, Monella C, B\u0026ouml;sl I, Sommer D, Delorme C (2015) 5% lidocaine medicated plaster for treatment of postherpetic neuralgia. Pain Ther 4:1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNujum ZT, Valamparampil MJ, Rajagopal MR, Rahul A, Siju NS, Prajitha KC (2021) Efficacy of 2% lignocaine medicated pad for pain alleviation during rabies immunoglobulin administration: a randomized controlled trial. Indian J Public Health 65:356\u0026ndash;361.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRewari V, Garg R, Trikha A (2010) Rabies vaccine and neuraxial anaesthesia: case study. S Afr J Anaesth Analg 16:32\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRupprecht CE (1996) Rabies virus. In: Baron S (ed) Medical Microbiology, 4th edn. University of Texas Medical Branch, Galveston.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization (2023) Rabies vaccinations and immunization. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int\u003c/span\u003e\u003cspan address=\"https://www.who.int\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"antonie-van-leeuwenhoek","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anto","sideBox":"Learn more about [Antonie van Leeuwenhoek](https://www.springer.com/journal/10482)","snPcode":"10482","submissionUrl":"https://submission.nature.com/new-submission/10482/3","title":"Antonie van Leeuwenhoek","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Iatrogenic pain, Lignocaine, Lignocaine Medicated Pain, Post-exposure prophylaxis, Rabies Immunoglobulin, Rabies Virus Neutralising Antibody","lastPublishedDoi":"10.21203/rs.3.rs-9124830/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9124830/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRabies Immunoglobulin (RIG) infiltration in and around the wound is necessary as part of post-exposure prophylaxis (PEP) in patients with Category III exposure to potentially rabid animals. However, pain from RIG infiltration remains a significant challenge. This unnecessary iatrogenic pain can be avoided/ reduced by the topical application of local anaesthetics like lignocaine, using a lignocaine medicated pad at the site, 20 minutes before administration of RIG. We have earlier demonstrated its effectiveness through a clinical trial. However, concerns about a possible interaction between lignocaine and RIG must be addressed before translation of the practice into policy. This study evaluated the potential impact of lignocaine on the virus-neutralizing activity of equine RIG \u003cem\u003ein vitro.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eBaby Hamster Kidney (BHK21) cells were first exposed to lignocaine to assess cytotoxicity, with viable cell counts determined using trypan blue exclusion. Modified Rapid Fluorescent Focus Inhibition Test (RFFIT) was then used to compare rabies virus-neutralizing antibody (RVNA) titres of lignocaine-supplemented and untreated RIG. The study found that BHK21 cells remained viable at lignocaine concentrations up to 1/16 dilution, with no cytotoxicity observed at this level. Importantly, RVNA titres showed no significant differences between lignocaine-treated and untreated RIG preparations, demonstrating equivalent virus-neutralizing efficacy.\u003c/p\u003e \u003cp\u003eThese findings support the use of lignocaine as a local anaesthetic during RIG administration without compromising therapeutic effectiveness. It offers a practical solution to enhance patient comfort during rabies PEP, especially in resource-limited settings. This study underscores the potential for integrating lignocaine into RIG administration protocols to provide compassionate and effective rabies prophylaxis.\u003c/p\u003e","manuscriptTitle":"Compatibility of Lignocaine with Equine Rabies Immunoglobulin: An In Vitro Study Supporting Pain Management During Rabies Post-Exposure Prophylaxis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 05:51:43","doi":"10.21203/rs.3.rs-9124830/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-04T20:14:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47102114765555043235709375204344674169","date":"2026-05-01T18:31:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T01:51:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-26T02:51:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T02:51:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Antonie van Leeuwenhoek","date":"2026-03-14T20:01:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"antonie-van-leeuwenhoek","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anto","sideBox":"Learn more about [Antonie van Leeuwenhoek](https://www.springer.com/journal/10482)","snPcode":"10482","submissionUrl":"https://submission.nature.com/new-submission/10482/3","title":"Antonie van Leeuwenhoek","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1f46caf3-64fc-4701-8f42-27e651c52f83","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-04T20:14:46+00:00","index":37,"fulltext":""},{"type":"reviewerAgreed","content":"47102114765555043235709375204344674169","date":"2026-05-01T18:31:15+00:00","index":35,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T05:51:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 05:51:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9124830","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9124830","identity":"rs-9124830","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}