Assessment of serum Homocysteine levels in congenital heart disease and with rubella infection: A hospital- based case-control study in North Indian population

Assessment of serum Homocysteine levels in congenital heart disease and with rubella infection: A hospital- based case-control study in North Indian population | 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 Assessment of serum Homocysteine levels in congenital heart disease and with rubella infection: A hospital- based case-control study in North Indian population Sumedha Tripathi, Shally Awasthi, Shalini Tripathi, Amita Jain, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4744008/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Circulating homocysteine and Rubella infection are independent risk factors for congenital heart disease (CHD). The primary objective of the study was to assess the association of serum homocysteine levels in cases of CHD and healthy controls and the secondary objective was to assess the association of serum homocysteine levels with rubella infection among CHD cases. Methods This case-control study was conducted in King George’s Medical University, Lucknow. Total 245 echo-graphically confirmed cases of CHD and age-gender matched 245 healthy controls of aged 0–11 months were recruited from the outpatient clinic and wards of paediatric and cardiology department and controls were recruited from immunization clinic of the hospital. Samples were tested for rubella specific IgM and IgG antibody and homocysteine levels using enzyme-linked-immunosorbent-assay. Results From July 2022 - December 2023, 245 cases of CHD and 245 healthy controls were recruited. The mean (SD) age of the cases were 4.40 ± 3.34 months and controls were 4.33 ± 3.63. In cases, 70.20% males were recruited while 68.57% in controls. The mean serum homocysteine levels (µmol/L) in CHD cases were 15.70 ± 7.6 while in controls 9.51 ± 3.1 (p = < 0.0001). Within CHD cases, 7.8% (19/245) were found seropositive for rubella infection. The crude odd ratio of homocysteine levels against seropositive rubella infection was 1.08 (95% CI 1.02–1.14) as compare to seronegative. Conclusion Levels of serum homocysteine in cases of CHD were significantly higher as compared to controls, whereas among CHD cases, those who had rubella seropositive showed significantly higher serum homocysteine levels. congenital heart disease hyperhomocysteinemia rubella infection Introduction Congenital heart disease (CHD) is one or more defects in the heart structure that exists since birth. The etiology of CHDs is highly diversified. During pregnancy, the mother provides the environment to the developing foetus for proper development. Therefore, maternal endogenous (metabolic) and exogenous (environmental) both factors are likely to play an important role in the prevention of CHDs and also of other birth defects, such as neural tube defects and orofacial clefts. Increased levels of serum homocysteine, deficiency of essential micro-elements and vitamins in the preconception period, such as B- vitamins (i.e., folic acid and cobalamin), antenatal rubella infection in the mother, maternal age, pregestational diabetes and one or more genetic defects may be the detrimental to the developing foetus. [ 1 ] CHD include simple to complex and critical malformations such as atrial septal defect (ASD), ventricular septal defect (VSD), patent ductus arteriosus (PDA), pulmonary stenosis, tetralogy of Fallot (TOF) and other types.[ 2 ] CHD is one of the most prevalent heart diseases among children. About 1.23% infants are born with CHD each year globally and in Asian countries is about 1.18% per year [ 3 ]. Over 200,000 children are estimated to be born with CHD in India every year [ 4 ]. According to CRS sentinel surveillance in India the prevalence of CHD among CRS was 72.2% [ 5 ]. The mortality rate among CRS due to CHD is about 34%.[ 6 ] Elevated levels of homocysteine (Hcy) have been considered to be an independent risk factor for CHD development. Homocysteine is the product of the intracellular methionine cycle. Hcy is an amino acid formed exclusively by demethylation of methionine. In Hcy synthesis, methionine is activated by ATP to form S-adenosylmethionine (SAM). SAM acts primarily as a universal methyl donor in the synthesis of methylated compounds such as DNA, RNA. A subproduct of these methylation reactions is S-adenosylhomocysteine (SAH), which is hydrolysed to adenosine and Hcy. Hcy can be remethylated to form methionine by the action of the enzyme methionine synthase which uses N 5’10-methylene- THF-reductase (MTHFR) as a methyl donor. Vitamins B12 and folate are co-factors in this reaction. [ 7 ] Elevated homocysteine concentrations lead to accumulation of SAH. Increased SAH is a potent inhibitor of cellular methyl transferases which during organogenesis can alter gene expression, cell differentiation and apoptosis which are associated with CHD. [ 7 – 10 ] The aim of the study was to assess the association of serum homocysteine levels in congenital heart disease and healthy age-gender matched controls and the secondary objective was to assess the association of serum homocysteine levels with rubella infection in cases of CHD. This work was a part of congenital rubella syndrome surveillance project conducted by Indian council of medical research (ICMR). [ 5 ] Method and Materials Study Population This study was conducted in July 2022 to December 2023, from the outpatient and inpatient ward of the Paediatrics & Cardiology of a tertiary teaching institute of Northern India. This research study protocol was approved by Institutional Ethics Committee (IEC) and written informed consent was taken from legal guardians of all participants of cases and controls before recruitment. Study Design: This study was a hospital based prospective case-control study. In this study CHD cases and age and gender matched healthy controls were recruited of aged 0 to 11 months. Cases were recruited from the wards of paediatrics, cardiology department with clinically confirmed CHD. Cases were confirmed by two-dimensional echocardiography (2D- ECHO) and healthy controls were recruited from the immunization clinic of the hospital. Inclusion and Exclusion Criteria of Cases and controls: Inclusion criteria of cases Clinically confirmed CHD Cases aged 0–11 months, positive family history of heart disease in parents or siblings like ASD, VSD, TOF etc. Exclusion criteria of cases Excluded those cases who had chromosomal abnormalities, preterm PDA or Patent foramen ovale (PFO) and any non-cardiac congenital defect like cataract and glaucoma. Inclusion criteria of controls Age and gender matched healthy controls were recruited from immunization clinic of Paediatric department. Exclusion criteria of controls Those controls who had low birth weight (< 2500grams), have a family history of heart disease were excluded from the study. Sample size calculation From the past study, there were a significant increase of homocysteine levels in children with congenital heart disease as compare to control group [ 11 ]. For an α = 0.05 level of significance and power of 95% and 1:1 case to control. We required a minimum sample size of 11 in each group. Data Collection All the Data were collected on the predesigned questionnaire. Parents were interviewed for the demographical details like age, gender and socioeconomic status. Clinical and laboratory data were obtained from the patient’s medical record of the recruited subjects. Immunization history was recorded from the immunization card. Anthropometric data were also noted. Weight was measured in kilograms (kgs) by electronic weighing machine, height was measured in centimetre (cm) and head circumference was also measured in centimetre. Sample collection One ml of venous blood was withdrawn from the peripheral vein of under aseptic conditions and collected in a plain sterile non-oxalate vial for further serological study. Vial placed for 2 hours or at 2–8°C overnight and centrifuged it for 5 minutes at 10,000 revolution per minute (rpm), collect the supernatant and store at -70°C for further analysis. Laboratory Performance ELISA (Enzyme Linked Immuno Sorbent Assay for Serum Homocysteine): Serum homocysteine levels were quantified by commercially available Human enzyme linked immune sorbent assay kit (Fine test, Wuhan Fine Biotech) which quantitatively measures (in duplicate) the concentration of total HCY in serum. Microplate (96 wells) were pre-coated with Ado HCY and standards and samples were pipetted into the wells. After then immediately add Biotin labelled antibody working solution and incubate for 45 minutes and 37°C. After washing the unbound samples and standards, horseradish peroxidase streptavidin conjugate (SABC) was added and incubate for 30 minutes at 37°C. After second washing TMB substrate was added and incubate for 10–20 minutes at 37°C. then to stop the reaction, the stop solution was added which changes the colour from blue to yellow in proportion to the concentration of HCY the intensity of colour was measured at 450 nm by using ultraviolet spectrophotometer. The concentration of Total HCY in the samples is then determined by comparing the OD of the samples to the standard curve. The concentration of the target substance was inversely proportional to the OD value. ELISA for Rubella antibody detection: All the serum samples were analysed for rubella infection (IgM & IgG antibody detection) by using commercially available Enzyme Linked Immune Sorbent Assay (ELISA) kit of Euroimmun (Germany). To detect rubella IgM and IgG antibodies in serum sample, microtiter wells are pre-coated with rubella antigen. The diluted sample and calibrators were added into the well (in duplicate) and any antibodies specific for the antigen present will bind to the bound antigen. After the removal of unbound antibody, rubella-reactive IgM & IgG antibodies in the sample can be detected using a commercial anti-human IgM & IgG antibody that is conjugated to an enzyme. After washing to remove unbound conjugate, a chromogen/substrate is added. This reacts with the conjugate to produce a colour change from blue to yellow. The colour intensity can be quantified photometrically and is proportional to the level of IgM & IgG antibody bound. Statistical analysis: All the data were double entered in MS Excel and analysed using GraphPad Instat statical software (Version 16). Percentage and number were calculated for categorical data. Mean and SD were calculated for continuous data. Independent t-test were used to analyse for continuous variables while Chi-square test was use for categorical variables. A p-value < 0.05 was taken as statistically significant using two-tailed distribution. Two logistic regression model were performed. The model I was used as multivariable regression model to assess the association of congenital heart disease with independent variable that had univariate association with it. Adjusted odds ratio (OR) with 95% confidence interval (CI) were reported. The second model was used to assess the association of serum homocysteine levels in seropositive and seronegative for rubella infection among CHD cases. The crude OR with 95% CI being reported. Results This study was conducted from July 2022 to December 2023 in King George’s Medical University, Lucknow. In this prospective case-control study 245 clinically confirmed CHD cases and age and gender matched 245 healthy controls were recruited. The mean age of cases was 4.4 ± 3.3 in which 70.20% (172/245) were male and the mean age of controls was 4.33± 3.63 in which 68.57% (168/245) were male. Table 1 shows the demographic characteristics of all cases and controls. Table1: Socio- Demographic characteristics of Congenital heart disease cases and healthy controls: Characteristics of Variables (%) Cases, N=245 Controls, N=245 p-value Gender (Male) 172 (70.2) 168 (68.5) 0.768 Age (in months), (mean ± SD) 4.40 ± 3.34 4.33 ± 3.63 0.816 Birth weight, (kgs) (mean± SD) 2.53 ± 0.50 2.730 ± 0.41 <0.001 Weight, (kgs) (mean ± SD) 4.49 ± 1.80 5.36 ± 3.35 0.007 Body length, (cm) (mean ± SD) 58.27 ± 8.92 60.02 ± 8.52 0.027 Head Circumference, (cm) (mean ± SD) 39.01 ± 4.69 38.54 ± 4.39 0.248 Family Type, (Joint) n (%), 231 (94.8) 215 (87.8) 0.017 Residence, (Rural) n (%), 185 (75.51) 162 (66.12) 0.028 Immunization status BCG* 218 (88.97) 238 (97.14) 0.007 DPT* 65 (26.53) 194 (79.18) <0.001 Measles 2 (0.81) 35 (14.28) <0.001 Rubella 1 (0.40) 30 (12.24) <0.001 Gestational Age, n (%) Full term 235 (95.91) 236 (96.32) 0.815 Socio-economic class, n (%) Upper class 1 (0.40) 00 (00) <0.001 Upper middle class 17 (6.93) 32 (13.06) Upper lower class 107 (43.67) 126 (51.42) Lower middle class 103 (42.04) 80 (32.65) Lower class 17 (6.93) 7 (2.85) Serum homocysteine levels (µmol/L*), n, mean ± SD 245, 15.70 ± 7.60 245, 9.51± 3.1 < 0.001 Abbreviations: bacille calmette - guerin, diphtheria-pertussis-tetanus , micromole per liter Serum homocysteine levels in µmol/L (mean± SD) in cases was 15.70 ± 7.60 and in controls was 9.51 ± 3.1 (p< 0.0001). Univariately mean serum homocysteine levels were significantly higher in cases as compared to healthy controls. Association of CHD with homocysteine level and sociodemographic variable was shown in table 2 . In multivariable logistic regression model, I we found that serum homocysteine levels, birth weight (neonates) and family type (joint) were significantly associated with CHD. Table 2: Association of congenital heart disease with homocysteine level and sociodemographic variable. Characteristics of variables Case/Control ref Model I Adjusted OR (95% CI) p- value Hcy levels (µmol/L) 1.17 (1.12 - 1.22) <0.001 Birth weight (Kg) 0.34 (0.21 - 0.55) <0.001 Family type (Joint) 0.45 (0.22 - 0.95) 0.04 Socio-economic status (Lower class) 2.01 (0.71 - 5.70) 0.19 Abbreviations: Odds ratio, confidence interval, Homocysteine, micro-mole per liter Among CHD cases, 7.8% (19/245) were found to be seropositive and 92.2% (226/245) were seronegative for rubella infection. The mean serum homocysteine levels of rubella seropositive was 20.30 ± 10.53 and in seronegative was 15.31 ± 7.17 (p= 0.006). In regression model II we found that the serum homocysteine level was univariately significantly associated with rubella seropositive infection. The crude odd ratio of homocysteine level against seropositive rubella infection was 1.08 (95% CI 1.02–1.14) as compare to seronegative, as shown in Table3. Table 3: Association of homocysteine level in seropositive and seronegative for rubella infection among congenital heart disease cases. Variable Seropositive/Seronegative ref Model II Crude OR* (95% CI*) p- value Hcy Level (µmol/L) * 1.08 (1.02 – 1.14) 0.008 Abbreviations: Odds ratio, confidence interval, Homocysteine, micro-mole per liter Discussion This case-control study was undertaken with the primary objective to compare serum homocysteine levels in cases of CHD and age-gender-matched healthy controls. The secondary objective to compare serum homocysteine levels in cases which were positive or negative for rubella infection in the serum by ELISA. In this study, the serum homocysteine levels were significantly higher in cases of CHD as compared to healthy controls. In addition to this, the serum homocysteine levels in rubella positive cases were significantly higher as compared to rubella negative cases. Our study demonstrated that a total of 7.75% of CHD cases of aged 0-11 months in the studied population were found to have rubella infection. A similar study was available in India (2021), where 80 infant-mother pairs were investigated for congenital rubella as Etiology for causation of CHD, showed the prevalence of rubella infection was 8.75% in infants, which is close similar to our finding [12]. Among socio-demographic characteristics, birth weight, weight and body length, rural residence, joint families were also significantly associated with CHD cases. Similar findings were reported by B. Vaidyanathan et al.. that birth weight and height were significantly associated with congenital heart disease. [13-17] A population-based retrospective cohort study conducted in Canada found that infants born to mothers living in low-income neighbourhoods were more at risk for developing CHD compared to infants born to mothers living in higher-income neighbourhoods [18]. In rural areas most of the families were joint and due to lack of awareness and low education status, children are at higher risk. According to hooshmand et al .., high levels of homocysteine amino acid are often associated with a wide variety of diseases, including CHD and neuro degenerative disorders like neural tube defects (NTD). In our study we found 5 times higher serum homocysteine levels in CHD cases as compared to controls, which is closely similar to the levels of serum homocysteine in the Turkish population as reported by C. Sanli et al . [11] This indicates an enhanced risk of CHD. Few studies suggested that the novel and traditional cardiovascular risk factor, including endothelial dysfunction may interact with infectious agents to influence the inflammatory and procoagulant environ of atherosclerosis.[19] According to Persian Gulf study Chlamydia. pneumonia and cytomegalovirus showed significant association with increased serum Hcy levels (hyperhomocysteinemia) and several other studies also investigated the association of serum homocysteine levels and viral infections. [20-25] In the current study, we found significant higher levels of serum homocysteine in rubella seropositive cases as compared to rubella seronegative cases among CHD cases. CHD is the result of incomplete development of heart during the first six weeks of pregnancy. According to Thomas et al . homocysteine amino acid acts as teratogenic agent which leads the dysmorphogenesis of the heart and neural tube as well as ventral wall.[26] Malik R showed in their study that the increased levels of maternal serum homocysteine (Hyperhomocysteinemia) are statically significantly associated with increased risk of CHD [27]. Some other studies suggested that various maternal biomarkers of homocysteine pathway disturbing homocysteine metabolism as lower folate and cobalamin levels were noticed mother of CHD children with elevated levels of homocysteine. Similar Study was conducted by Huhta JC. [26, 28, 29]. KE Elizabeth analyses the nutrient- gene interaction, they found significant association between low serum folate, high serum homocysteine and the presence of genetic polymorphism among children and their mothers were noted as a risk of CHD. [30] Based on their study’s results consumption of peri-conceptional folate supplementation with vitamin B12 is the best primary prevention of CHD. Our study has some limitations. We should also measure serum folate levels as folate plays very important role in this pathway and expected to decrease in cases with increase in homocysteine. Genetic analysis could be performed to understand the best knowledge of developmental mechanism of CHD. Strength of the study was we have recruited age and gender matched healthy controls. The methodology was robust with adequate sample size and power of the study. Conclusion Concurrent hyperhomocysteinemia and rubella infection was significantly associated with congenital heart disease in North Indian population. This synergistic association may have important impact on risk stratification and intervention trails. Declarations ACKNOWLEDGEMENTS Authors are highly grateful to the participants for their time and valuable contribution in this research. We are also acknowledging the conductive environment provided by the administration of King George’s Medical University, Lucknow that facilitated implementation of this study. We also acknowledge the contribution of Ms. Neha Verma, for contribution in data analysis of this study and Rishabh Dubey for contribution in sample collection in this study. AUTHOR CONTRIBUTIONS: Sumedha Tripathi (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Methodology [equal], Project administration [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Shally Awasthi (Conceptualization [equal], Funding acquisition [equal], Investigation [equal], Methodology [equal], Project administration [equal], Resources [equal], Supervision [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Shalini Tripathi (Conceptualization [equal], Methodology [equal], Resources [equal, Writing—review & editing [equal]), Amita Jain (Conceptualization [equal], Methodology [equal], Resources [equal]) and Akhil Sharma (Conceptualization [equal], Methodology [equal], Resources [equal]). FUNDING: None CONFLICT OF INTEREST: The authors declare that there is no conflict of interest. CONSENT TO PARTICIPATE: The caregivers/guardians of children signed the written, informed consent for participation in this study. CONSENT FOR PUBLICATION: All authors have read and agreed to the published version of the article. DATA AVAILABILITY: The corresponding author has full control of all data and the data may be made available on request. CODE AVAILABILITY: Not applicable. References Nora JJ (1968) Multifactorial inheritance hypothesis for the etiology of congenital heart diseases: the genetic-environmental interaction. Circulation 38(3):604–617. https://doi.org/10.1161/01.CIR.38.3.604 Rohit M, Shrivastava S (2018) Acyanotic and cyanotic congenital heart diseases. Indian J Pediatr 85:454–460. https://doi.org/10.1007/s12098-017-2454-6 Zimmerman MS, Smith AGC, Sable CA, Echko MM, Wilner LB, Olsen HE, Atalay HT, Awasthi A, Bhutta ZA, Boucher JL, Castro F (2020) Global, regional, and national burden of congenital heart disease, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Child Adolesc Health 4(3):185–200. https://doi.org/10.1016/S2352-4642(19)30402-X Saxena A (2018) Congenital heart disease in India: a status report. Indian Pediatr 55:1075–1082. https://doi.org/10.1007/BF02724185 Shanmugasundaram D, Verma S, Singh K, Dwibedi B, Awasthi S, Mahantesh S, Singh H, Santhanam S, Mondal N, Geetha S, Sreenivasan P (2023) Congenital rubella syndrome surveillance in India, 2016–21: Analysis of five years surveillance data. Heliyon 9(5). 10.1016/j.heliyon.2023.e15965 Murhekar M, Verma S, Singh K, Bavdekar A, Benakappa N et al (2020) Epidemiology of Congenital Rubella Syndrome (CRS) in India, 2016-18, based on data from sentinel surveillance. PLoS Negl Trop Dis 14(2):e0007982. https://doi.org/10.1371/journal.pntd.0007982 Shawky RM, Ramy AR, El-Din SMN, Abd Elmonem SM, Elmonem A, M.A (2018) Abnormal maternal biomarkers of homocysteine and methionine metabolism and the risk of congenital heart defects. Egypt J Med Hum Genet 19(1):7–12 Ganguly P, Alam SF (2015) Role of homocysteine in the development of cardiovascular disease. Nutr J 14(1):1–0. https://doi.org/10.1186/1475-2891-14-6 Clare CE, Brassington AH, Kwong WY, Sinclair KD (2019) One-carbon metabolism: linking nutritional biochemistry to epigenetic programming of long-term development. Annu Rev Anim Biosci 7:263–287. https://doi.org/10.1146/annurev-animal-020518-115206 Hobbs CA, Cleves MA, Melnyk S, Zhao W, James SJ (2005) Congenital heart defects and abnormal maternal biomarkers of methionine and homocysteine metabolism. Am J Clin Nutr 81(1):147–153. https://doi.org/10.1093/ajcn/81.1.147 Sanli C, Oguz D, Olgunturk R, Tunaoglu FS, Kula S, Pasaoglu H, Gulbahar O, Cevik A (2012) Elevated homocysteine and asymmetric dimethyl arginine levels in pulmonary hypertension associated with congenital heart disease. Pediatr Cardiol 33:1323–1331. 10.1007/s00246-012-0321-9 Saniya Gupta S, Verma N, Dash M, Goel A, Rawat, Mini P, Singh MK, Rohit P, Kumar (April 2021) Congenital Rubella: A Salient Cause of Congenital Heart Defects in Infants. J Trop Pediatr 67(2):fmab044. https://doi.org/10.1093/tropej/fmab044 Vaidyanathan B, Nair SB, Sundaram KR, Banu UK, Shivaprakasha K, Rao SG, Kumar RK (2008) Malnutrition in Children with Congenital Heart Disease (CHD): Determinants and Short-term Impact of Corrective Intervention. Indian Pediatr 45(7):541–546 Wogu AF, Loffredo CA, Bebu I, Luta G (2014) Mediation analysis of gestational age, congenital heart defects, and infant birth-weight. BMC Res Notes 7:1–6. 10.1186/1756-0500-7-926 Malik S, Cleves MA, Zhao W, Correa A, Hobbs CA, National Birth Defects Prevention Study (2007) Association between congenital heart defects and small for gestational age. Pediatrics 119(4):e976–e982. 10.1542/peds.2006-2742 Elshazali HO, Elshazali OH, Elshazali H (2017) The relationship between birth weight and congenital heart disease at Ahmed Gasim Cardiac Centre, Bahri, Sudan. Sudan J paediatrics 17(2):49. 10.24911/SJP.2017.2.6 Jacobs EG, Leung MP, Karlberg LJ (2003) Birthweight distribution in southern Chinese infants with symptomatic congenital heart disease. J Paediatr Child Health 39(3):191–196. 10.1046/j.1440-1754.2003.00109.x Miao Q, Dunn S, Wen SW, Lougheed J, Reszel J, Lavin Venegas C, Walker M (2021) Neighbourhood maternal socioeconomic status indicators and risk of congenital heart disease. BMC Pregnancy Childbirth 21:1–21. 10.1186/s12884-020-03512-8 Nabipour I, Vahdat K, Jafari SM, Amiri M, Shafeiae E, Riazi A, Amini AL, Pazoki R, Sanjdideh Z (2007) Correlation of hyperhomocysteinaemia and Chlamydia pneumoniae IgG seropositivity with coronary artery disease in a general population. Heart Lung Circulation 16(6):416–422. 10.1016/j.hlc.2007.02.100 Vahdat K, Ostovar A, Hadavand F, Pourbehi MR Serum Homocysteine Levels Are Associated with Infectious Burden: the Persian Gulf Healthy. 10.13140/2.1.4266.5608 Mustafa M, Hussain S, Qureshi S, Malik SA, Kazmi AR, Naeem M (2012) Study of the effect of antiviral therapy on homocysteinemia in hepatitis C virus-infected patients. BMC Gastroenterol 12:1–8. 10.1186/1471-230X-12-117 Abike F, Engin AB, Dunder I, Tapisiz OL, Aslan C, Kutluay L (2011) Human papilloma virus persistence and neopterin, folate and homocysteine levels in cervical dysplasias. Arch Gynecol Obstet 284:209–214. 10.1007/s00404-010-1650-7 Coria-Ramirez E, Cisneros LN, Treviño-Perez S, Ibarra-Gonzalez I, Casillas-Rodriguez J, Majluf-Cruz A (2010) Effect of Highly Active Antiretroviral Therapy on Homocysteine Plasma Concentrations in HIV-1–Infected Patients. JAIDS J Acquir Immune Defic Syndr 54(5):477–481. 10.1097/QAI.0b013e3181d91088 Bernasconi E, Uhr M, Magenta L, Ranno A, Telenti A, Swiss HIV (2001) Cohort Study. Homocysteinaemia in HIV-infected patients treated with highly active antiretroviral therapy. Aids 15(8):1081–1082. 10.1097/00002030-200105250-00027 Danesh J, Collins R, Peto R (1997) Chronic infections and coronary heart disease: is there a link? lancet 350(9075):430–436. 10.1016/S0140-6736(97)03079-1 Rosenquist TH, Ratashak SA, Selhub J (1996) Homocysteine induces congenital defects of the heart and neural tube: effect of folic acid. Proceedings of the National Academy of Sciences. ;93(26):15227-32. 10.1073/pnas.93.26.15227 Malik RA, Lone MR, Ahmed A, Koul KA, Malla RR (2017) Maternal hyperhomocysteinemia and congenital heart defects: A prospective case control study in Indian population. Indian Heart J 69(1):17–19. 10.1016/j.ihj.2016.07.014 Surmiak P, Baumert M, Paprotny M (2017) Abnormal biomarkers of homocysteine metabolism in neonates with conotruncal heart defects. BioMed Research International. ;2017 Huhta JC, Hernandez-Robles JA (2005) Homocysteine, folate, and congenital heart defects. Fetal Pediatr Pathol 24(2):71–79. https://doi.org/10.1155/2017/7404397 Elizabeth KE, Praveen SL, Preethi NR, Jissa VT, Pillai MR (2017) Folate, vitamin B12, homocysteine and polymorphisms in folate metabolizing genes in children with congenital heart disease and their mothers. Eur J Clin Nutr 71(12):1437–1441. 10.1038/ejcn.2017.135 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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. 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-4744008","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328570777,"identity":"e547fd30-1e45-4c75-874c-220c8adaaf50","order_by":0,"name":"Sumedha Tripathi","email":"","orcid":"","institution":"King George’s medical university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sumedha","middleName":"","lastName":"Tripathi","suffix":""},{"id":328570778,"identity":"e00687f8-6a32-4cc7-8461-408079749953","order_by":1,"name":"Shally Awasthi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYDACCQbGAw8YJOr7QZyEAuK0MBxIYLBhnNkA0mJAvJY0xg0HQDxitMjPbn5wILHtMLPx+dWJHx4YMMjzix3Ar8XgzjEDkBY2sxtvN0sAHWY4c3YCAS1AZSAtPGY3zm4AaUkwuE1Ai/yM9A8gLRLGM85u/kGUFoYbOSBb0gwM+Hu3EWeLwY2cggMJ52wSJG7wbrNIMJAg7BegwzY++FAmkcDff3bzzR8VNvL80oQcBgKMbEBCAqxSggjlYPAHiPkPEKt6FIyCUTAKRhoAAGF0TFdrHRNBAAAAAElFTkSuQmCC","orcid":"","institution":"King George’s medical university","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shally","middleName":"","lastName":"Awasthi","suffix":""},{"id":328570779,"identity":"05b63056-ede4-4410-af54-f89847ae0e7c","order_by":2,"name":"Shalini Tripathi","email":"","orcid":"","institution":"King George’s medical university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shalini","middleName":"","lastName":"Tripathi","suffix":""},{"id":328570780,"identity":"a1874384-e2f5-4550-98c5-93c93bde63fe","order_by":3,"name":"Amita Jain","email":"","orcid":"","institution":"King George’s medical university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amita","middleName":"","lastName":"Jain","suffix":""},{"id":328570781,"identity":"3c8db43a-da63-47a2-a141-08255d3d585e","order_by":4,"name":"Akhil Sharma","email":"","orcid":"","institution":"King George’s medical university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akhil","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2024-07-15 15:41:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4744008/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4744008/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60670049,"identity":"f9e7beab-2ed2-4f9c-a3db-8b909621ef12","added_by":"auto","created_at":"2024-07-19 10:01:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":640563,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4744008/v1/43afb887-6220-4b64-adbf-33bd0fe0158e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of serum Homocysteine levels in congenital heart disease and with rubella infection: A hospital- based case-control study in North Indian population","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital heart disease (CHD) is one or more defects in the heart structure that exists since birth. The etiology of CHDs is highly diversified. During pregnancy, the mother provides the environment to the developing foetus for proper development. Therefore, maternal endogenous (metabolic) and exogenous (environmental) both factors are likely to play an important role in the prevention of CHDs and also of other birth defects, such as neural tube defects and orofacial clefts. Increased levels of serum homocysteine, deficiency of essential micro-elements and vitamins in the preconception period, such as B- vitamins (i.e., folic acid and cobalamin), antenatal rubella infection in the mother, maternal age, pregestational diabetes and one or more genetic defects may be the detrimental to the developing foetus. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] CHD include simple to complex and critical malformations such as atrial septal defect (ASD), ventricular septal defect (VSD), patent ductus arteriosus (PDA), pulmonary stenosis, tetralogy of Fallot (TOF) and other types.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCHD is one of the most prevalent heart diseases among children. About 1.23% infants are born with CHD each year globally and in Asian countries is about 1.18% per year [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Over 200,000 children are estimated to be born with CHD in India every year [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. According to CRS sentinel surveillance in India the prevalence of CHD among CRS was 72.2% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The mortality rate among CRS due to CHD is about 34%.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eElevated levels of homocysteine (Hcy) have been considered to be an independent risk factor for CHD development. Homocysteine is the product of the intracellular methionine cycle. Hcy is an amino acid formed exclusively by demethylation of methionine. In Hcy synthesis, methionine is activated by ATP to form S-adenosylmethionine (SAM). SAM acts primarily as a universal methyl donor in the synthesis of methylated compounds such as DNA, RNA. A subproduct of these methylation reactions is S-adenosylhomocysteine (SAH), which is hydrolysed to adenosine and Hcy. Hcy can be remethylated to form methionine by the action of the enzyme methionine synthase which uses \u003cem\u003eN\u003c/em\u003e5\u0026rsquo;10-methylene- THF-reductase (MTHFR) as a methyl donor. Vitamins B12 and folate are co-factors in this reaction. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eElevated homocysteine concentrations lead to accumulation of SAH. Increased SAH is a potent inhibitor of cellular methyl transferases which during organogenesis can alter gene expression, cell differentiation and apoptosis which are associated with CHD. [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe aim of the study was to assess the association of serum homocysteine levels in congenital heart disease and healthy age-gender matched controls and the secondary objective was to assess the association of serum homocysteine levels with rubella infection in cases of CHD. This work was a part of congenital rubella syndrome surveillance project conducted by Indian council of medical research (ICMR). [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e"},{"header":"Method and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Population\u003c/h2\u003e \u003cp\u003eThis study was conducted in July 2022 to December 2023, from the outpatient and inpatient ward of the Paediatrics \u0026amp; Cardiology of a tertiary teaching institute of Northern India. This research study protocol was approved by Institutional Ethics Committee (IEC) and written informed consent was taken from legal guardians of all participants of cases and controls before recruitment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design:\u003c/h2\u003e \u003cp\u003eThis study was a hospital based prospective case-control study. In this study CHD cases and age and gender matched healthy controls were recruited of aged 0 to 11 months. Cases were recruited from the wards of paediatrics, cardiology department with clinically confirmed CHD. Cases were confirmed by two-dimensional echocardiography (2D- ECHO) and healthy controls were recruited from the immunization clinic of the hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eInclusion and Exclusion Criteria of Cases and controls:\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eInclusion criteria of cases\u003c/h2\u003e \u003cp\u003eClinically confirmed CHD Cases aged 0\u0026ndash;11 months, positive family history of heart disease in parents or siblings like ASD, VSD, TOF etc.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExclusion criteria of cases\u003c/h2\u003e \u003cp\u003eExcluded those cases who had chromosomal abnormalities, preterm PDA or Patent foramen ovale (PFO) and any non-cardiac congenital defect like cataract and glaucoma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInclusion criteria of controls\u003c/h2\u003e \u003cp\u003eAge and gender matched healthy controls were recruited from immunization clinic of Paediatric department.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eExclusion criteria of controls\u003c/h2\u003e \u003cp\u003eThose controls who had low birth weight (\u0026lt;\u0026thinsp;2500grams), have a family history of heart disease were excluded from the study.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSample size calculation\u003c/h2\u003e \u003cp\u003eFrom the past study, there were a significant increase of homocysteine levels in children with congenital heart disease as compare to control group [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For an α\u0026thinsp;=\u0026thinsp;0.05 level of significance and power of 95% and 1:1 case to control. We required a minimum sample size of 11 in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Collection\u003c/h2\u003e \u003cp\u003eAll the Data were collected on the predesigned questionnaire. Parents were interviewed for the demographical details like age, gender and socioeconomic status. Clinical and laboratory data were obtained from the patient\u0026rsquo;s medical record of the recruited subjects. Immunization history was recorded from the immunization card. Anthropometric data were also noted. Weight was measured in kilograms (kgs) by electronic weighing machine, height was measured in centimetre (cm) and head circumference was also measured in centimetre.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eOne ml of venous blood was withdrawn from the peripheral vein of under aseptic conditions and collected in a plain sterile non-oxalate vial for further serological study. Vial placed for 2 hours or at 2\u0026ndash;8\u0026deg;C overnight and centrifuged it for 5 minutes at 10,000 revolution per minute (rpm), collect the supernatant and store at -70\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory Performance\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eELISA (Enzyme Linked Immuno Sorbent Assay for Serum Homocysteine):\u003c/h2\u003e \u003cp\u003eSerum homocysteine levels were quantified by commercially available Human enzyme linked immune sorbent assay kit (Fine test, Wuhan Fine Biotech) which quantitatively measures (in duplicate) the concentration of total HCY in serum. Microplate (96 wells) were pre-coated with Ado HCY and standards and samples were pipetted into the wells. After then immediately add Biotin labelled antibody working solution and incubate for 45 minutes and 37\u0026deg;C. After washing the unbound samples and standards, horseradish peroxidase streptavidin conjugate (SABC) was added and incubate for 30 minutes at 37\u0026deg;C. After second washing TMB substrate was added and incubate for 10\u0026ndash;20 minutes at 37\u0026deg;C. then to stop the reaction, the stop solution was added which changes the colour from blue to yellow in proportion to the concentration of HCY the intensity of colour was measured at 450 nm by using ultraviolet spectrophotometer. The concentration of Total HCY in the samples is then determined by comparing the OD of the samples to the standard curve. The concentration of the target substance was inversely proportional to the OD value.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eELISA for Rubella antibody detection:\u003c/h2\u003e \u003cp\u003eAll the serum samples were analysed for rubella infection (IgM \u0026amp; IgG antibody detection) by using commercially available Enzyme Linked Immune Sorbent Assay (ELISA) kit of Euroimmun (Germany). To detect rubella IgM and IgG antibodies in serum sample, microtiter wells are pre-coated with rubella antigen. The diluted sample and calibrators were added into the well (in duplicate) and any antibodies specific for the antigen present will bind to the bound antigen. After the removal of unbound antibody, rubella-reactive IgM \u0026amp; IgG antibodies in the sample can be detected using a commercial anti-human IgM \u0026amp; IgG antibody that is conjugated to an enzyme. After washing to remove unbound conjugate, a chromogen/substrate is added. This reacts with the conjugate to produce a colour change from blue to yellow. The colour intensity can be quantified photometrically and is proportional to the level of IgM \u0026amp; IgG antibody bound.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eAll the data were double entered in MS Excel and analysed using GraphPad Instat statical software (Version 16). Percentage and number were calculated for categorical data. Mean and SD were calculated for continuous data. Independent t-test were used to analyse for continuous variables while Chi-square test was use for categorical variables. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was taken as statistically significant using two-tailed distribution. Two logistic regression model were performed. The model I was used as multivariable regression model to assess the association of congenital heart disease with independent variable that had univariate association with it. Adjusted odds ratio (OR) with 95% confidence interval (CI) were reported. The second model was used to assess the association of serum homocysteine levels in seropositive and seronegative for rubella infection among CHD cases. The crude OR with 95% CI being reported.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThis study was conducted from July 2022 to December 2023 in King George\u0026rsquo;s Medical University, Lucknow. In this prospective case-control study 245 clinically confirmed CHD cases and age and gender matched 245 healthy controls were recruited. The mean age of cases was 4.4 \u0026plusmn; 3.3 in which 70.20% (172/245) were male and the mean age of controls was 4.33\u0026plusmn; 3.63 in which 68.57% (168/245) were male. \u003cstrong\u003eTable 1\u003c/strong\u003e shows the demographic characteristics of all cases and controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable1: Socio- Demographic characteristics of Congenital heart disease cases and healthy controls:\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics of Variables (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCases, N=245\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003eControls, N=245\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eGender (Male)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e172 (70.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e168 (68.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e0.768\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eAge (in months), (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e4.40 \u0026plusmn; 3.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e4.33 \u0026plusmn; 3.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e0.816\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eBirth weight, (kgs) (mean\u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e2.53 \u0026plusmn; 0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e2.730 \u0026plusmn; 0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eWeight, (kgs) (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e4.49 \u0026plusmn; 1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e5.36 \u0026plusmn; 3.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eBody length, (cm) (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e58.27 \u0026plusmn; 8.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e60.02 \u0026plusmn; 8.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.027\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eHead Circumference, (cm) (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e39.01 \u0026plusmn; 4.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e38.54 \u0026plusmn; 4.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e0.248\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eFamily Type, (Joint) n (%),\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e231 (94.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e215 (87.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.017\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eResidence, (Rural) n (%),\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e185 (75.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e162 (66.12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.028\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eImmunization status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eBCG*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e218 (88.97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e238 (97.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eDPT*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e65 (26.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e194 (79.18)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eMeasles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e2 (0.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e35 (14.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eRubella\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e1 (0.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e30 (12.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGestational Age, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eFull term\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e235 (95.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e236 (96.32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e0.815\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSocio-economic class, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eUpper class\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e1 (0.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e00 (00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eUpper middle class\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e17 (6.93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e32 (13.06)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eUpper lower class\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e107 (43.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e126 (51.42)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eLower middle class\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e103 (42.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e80 (32.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eLower class\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e17 (6.93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e7 (2.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003eSerum homocysteine levels (\u0026micro;mol/L*), n, mean \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e245, 15.70 \u0026plusmn; 7.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e245, 9.51\u0026plusmn; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003eAbbreviations: bacille calmette - guerin, diphtheria-pertussis-tetanus\u003cstrong\u003e,\u003c/strong\u003e micromole per liter\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSerum homocysteine levels in \u0026micro;mol/L (mean\u0026plusmn; SD) in cases was\u0026nbsp;15.70 \u0026plusmn; 7.60 and in controls was 9.51 \u0026plusmn; 3.1 (p\u0026lt; 0.0001). Univariately mean serum homocysteine levels were significantly higher in cases as compared to healthy controls.\u003c/p\u003e\n\u003cp\u003eAssociation of CHD with homocysteine level and sociodemographic variable was shown in \u003cstrong\u003etable 2\u003c/strong\u003e. In multivariable logistic regression model, I we found that serum homocysteine levels, birth weight (neonates) and family type (joint) were significantly associated with CHD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Association of congenital heart disease with homocysteine level and sociodemographic variable.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics of variables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.43594009983361%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase/Control \u003csup\u003eref\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eModel I\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAdjusted OR (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.277870216306155%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep- value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eHcy levels (\u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.43594009983361%\" valign=\"top\"\u003e\n \u003cp\u003e1.17 (1.12 - 1.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.277870216306155%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eBirth weight (Kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.43594009983361%\" valign=\"top\"\u003e\n \u003cp\u003e0.34 (0.21 - 0.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.277870216306155%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eFamily type (Joint)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.43594009983361%\" valign=\"top\"\u003e\n \u003cp\u003e0.45 (0.22 - 0.95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.277870216306155%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eSocio-economic status (Lower class)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.43594009983361%\" valign=\"top\"\u003e\n \u003cp\u003e2.01 (0.71 - 5.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.277870216306155%\" valign=\"top\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: Odds ratio, confidence interval, Homocysteine, micro-mole per liter\u003c/p\u003e\n\u003cp\u003eAmong CHD cases, 7.8% (19/245) were found to be seropositive and 92.2% (226/245) were seronegative for rubella infection. The mean serum homocysteine levels of rubella seropositive was\u0026nbsp;20.30\u0026nbsp;\u0026plusmn; 10.53 and in seronegative was\u0026nbsp;15.31\u0026nbsp;\u0026plusmn; 7.17 (p= 0.006).\u003c/p\u003e\n\u003cp\u003eIn regression model II we found that the serum homocysteine level was univariately significantly associated with rubella seropositive infection. The crude odd ratio of homocysteine level against seropositive rubella infection was 1.08 (95% CI 1.02\u0026ndash;1.14) as compare to seronegative, as shown in Table3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Association of homocysteine level in seropositive and seronegative for rubella infection among congenital heart disease cases.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.74565560821485%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"51.97472353870458%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSeropositive/Seronegative \u003csup\u003eref\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eModel II Crude OR* (95% CI*)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.27962085308057%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep- value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.74565560821485%\" valign=\"top\"\u003e\n \u003cp\u003eHcy Level (\u0026micro;mol/L) *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"51.97472353870458%\" valign=\"top\"\u003e\n \u003cp\u003e1.08 (1.02 \u0026ndash; 1.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.27962085308057%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.008\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003eAbbreviations: Odds ratio, confidence interval, Homocysteine, micro-mole per liter\u003c/sup\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis case-control study was undertaken with the primary objective to compare serum homocysteine levels in cases of CHD and age-gender-matched healthy controls. The secondary objective to compare serum homocysteine levels in cases which were positive or negative for rubella infection in the serum by ELISA. In this study, the serum homocysteine levels were significantly higher in cases of CHD as compared to healthy controls. In addition to this, the serum homocysteine levels in rubella positive cases were significantly higher as compared to rubella negative cases.\u003c/p\u003e\n\u003cp\u003eOur study demonstrated that a total of 7.75% of CHD cases of aged 0-11 months in the studied population were found to have rubella infection. A similar study was available in India (2021), where 80 infant-mother pairs were investigated for congenital rubella as Etiology for causation of CHD, showed the prevalence of rubella infection was 8.75% in infants, which is close similar to our finding [12].\u003c/p\u003e\n\u003cp\u003eAmong socio-demographic characteristics, birth weight, weight and body length, rural residence, joint families were also significantly associated with CHD cases. Similar findings were reported by B. Vaidyanathan \u003cem\u003eet al..\u003c/em\u003e that birth weight and height were significantly associated with congenital heart disease. [13-17] A population-based retrospective cohort study conducted in Canada found that infants born to mothers living in low-income neighbourhoods were more at risk for developing CHD compared to infants born to mothers living in higher-income neighbourhoods\u0026nbsp;[18]. In rural areas most of the families were joint and due to lack of awareness and low education status, children are at higher risk.\u003c/p\u003e\n\u003cp\u003eAccording to hooshmand \u003cem\u003eet al\u003c/em\u003e.., high levels of homocysteine amino acid are often associated with a wide variety of diseases, including CHD and neuro degenerative disorders like neural tube defects (NTD). In our study we found 5 times higher serum homocysteine levels in CHD cases as compared to controls, which is closely similar to the levels of serum homocysteine in the Turkish population as reported by C. Sanli \u003cem\u003eet al\u003c/em\u003e. [11] This indicates an enhanced risk of CHD.\u003c/p\u003e\n\u003cp\u003eFew studies suggested that the novel and traditional cardiovascular risk factor, including endothelial dysfunction may interact with infectious agents to influence the inflammatory and procoagulant environ of atherosclerosis.[19] According to Persian Gulf study \u003cem\u003eChlamydia. pneumonia\u003c/em\u003e and cytomegalovirus showed significant association with increased serum Hcy levels (hyperhomocysteinemia) and several other studies also investigated the association of serum homocysteine levels and viral infections. [20-25] In the current study, we found significant higher levels of serum homocysteine in rubella seropositive cases as compared to rubella seronegative cases among CHD cases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCHD is the result of incomplete development of heart during the first six weeks of pregnancy. According to Thomas \u003cem\u003eet al\u003c/em\u003e. homocysteine amino acid acts as teratogenic agent which leads the dysmorphogenesis of the heart and neural tube as well as ventral wall.[26]\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Malik R showed in their study that the increased levels of maternal serum homocysteine (Hyperhomocysteinemia) are statically significantly associated with increased risk of CHD [27]. Some other studies suggested that various maternal biomarkers of homocysteine pathway disturbing homocysteine metabolism as lower folate and cobalamin levels were noticed mother of CHD children with elevated levels of homocysteine. Similar Study was conducted by Huhta JC. [26, 28, 29]. \u0026nbsp; KE Elizabeth analyses the nutrient- gene interaction, they found significant association between low serum folate, high serum homocysteine and the presence of genetic polymorphism among children and their mothers were noted as a risk of CHD. [30] \u0026nbsp;\u0026nbsp;Based on their study\u0026rsquo;s results consumption of peri-conceptional folate supplementation with vitamin B12 is the best primary prevention of CHD.\u003c/p\u003e\n\u003cp\u003eOur study has some limitations. We should also measure serum folate levels as folate plays very important role in this pathway and expected to decrease in cases with increase in homocysteine.\u0026nbsp;Genetic analysis could be performed to understand the best knowledge of developmental mechanism of CHD.\u003c/p\u003e\n\u003cp\u003eStrength of the study was we have recruited age and gender matched healthy controls. The methodology was robust with adequate sample size and power of the study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eConcurrent hyperhomocysteinemia and rubella infection was significantly associated with congenital heart disease in North Indian population. This synergistic association may have important impact on risk stratification and intervention trails.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are highly grateful to the participants for their time and valuable contribution in this research. We are also acknowledging the conductive environment provided by the administration of King George\u0026rsquo;s Medical University, Lucknow that facilitated implementation of this study. We also acknowledge the contribution of Ms. Neha Verma, for contribution in data analysis of this study and Rishabh Dubey for contribution in sample collection in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSumedha Tripathi (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Methodology [equal], Project administration [equal], Validation [equal], Visualization [equal], Writing\u0026mdash;original draft [equal], Writing\u0026mdash;review \u0026amp; editing [equal]), Shally Awasthi (Conceptualization [equal], Funding acquisition [equal], Investigation [equal], Methodology [equal], Project administration [equal], Resources [equal], Supervision [equal], Validation [equal], Visualization [equal], Writing\u0026mdash;original draft [equal], Writing\u0026mdash;review \u0026amp; editing [equal]), Shalini Tripathi (Conceptualization [equal], Methodology [equal], Resources [equal, Writing\u0026mdash;review \u0026amp; editing [equal]), Amita Jain (Conceptualization [equal], Methodology [equal], Resources [equal]) and Akhil Sharma (Conceptualization [equal], Methodology [equal], Resources [equal]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING:\u003c/strong\u003e None\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST:\u0026nbsp;\u003c/strong\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT TO PARTICIPATE:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe caregivers/guardians of children signed the written, informed consent for participation in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author has full control of all data and the data may be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCODE AVAILABILITY:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNora JJ (1968) Multifactorial inheritance hypothesis for the etiology of congenital heart diseases: the genetic-environmental interaction. Circulation 38(3):604\u0026ndash;617. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/01.CIR.38.3.604\u003c/span\u003e\u003cspan address=\"10.1161/01.CIR.38.3.604\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRohit M, Shrivastava S (2018) Acyanotic and cyanotic congenital heart diseases. Indian J Pediatr 85:454\u0026ndash;460. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12098-017-2454-6\u003c/span\u003e\u003cspan address=\"10.1007/s12098-017-2454-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmerman MS, Smith AGC, Sable CA, Echko MM, Wilner LB, Olsen HE, Atalay HT, Awasthi A, Bhutta ZA, Boucher JL, Castro F (2020) Global, regional, and national burden of congenital heart disease, 1990\u0026ndash;2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Child Adolesc Health 4(3):185\u0026ndash;200. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S2352-4642(19)30402-X\u003c/span\u003e\u003cspan address=\"10.1016/S2352-4642(19)30402-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaxena A (2018) Congenital heart disease in India: a status report. Indian Pediatr 55:1075\u0026ndash;1082. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02724185\u003c/span\u003e\u003cspan address=\"10.1007/BF02724185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShanmugasundaram D, Verma S, Singh K, Dwibedi B, Awasthi S, Mahantesh S, Singh H, Santhanam S, Mondal N, Geetha S, Sreenivasan P (2023) Congenital rubella syndrome surveillance in India, 2016\u0026ndash;21: Analysis of five years surveillance data. Heliyon 9(5). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.heliyon.2023.e15965\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2023.e15965\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurhekar M, Verma S, Singh K, Bavdekar A, Benakappa N et al (2020) Epidemiology of Congenital Rubella Syndrome (CRS) in India, 2016-18, based on data from sentinel surveillance. PLoS Negl Trop Dis 14(2):e0007982. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pntd.0007982\u003c/span\u003e\u003cspan address=\"10.1371/journal.pntd.0007982\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShawky RM, Ramy AR, El-Din SMN, Abd Elmonem SM, Elmonem A, M.A (2018) Abnormal maternal biomarkers of homocysteine and methionine metabolism and the risk of congenital heart defects. Egypt J Med Hum Genet 19(1):7\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanguly P, Alam SF (2015) Role of homocysteine in the development of cardiovascular disease. Nutr J 14(1):1\u0026ndash;0. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1475-2891-14-6\u003c/span\u003e\u003cspan address=\"10.1186/1475-2891-14-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClare CE, Brassington AH, Kwong WY, Sinclair KD (2019) One-carbon metabolism: linking nutritional biochemistry to epigenetic programming of long-term development. Annu Rev Anim Biosci 7:263\u0026ndash;287. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-animal-020518-115206\u003c/span\u003e\u003cspan address=\"10.1146/annurev-animal-020518-115206\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHobbs CA, Cleves MA, Melnyk S, Zhao W, James SJ (2005) Congenital heart defects and abnormal maternal biomarkers of methionine and homocysteine metabolism. Am J Clin Nutr 81(1):147\u0026ndash;153. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ajcn/81.1.147\u003c/span\u003e\u003cspan address=\"10.1093/ajcn/81.1.147\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanli C, Oguz D, Olgunturk R, Tunaoglu FS, Kula S, Pasaoglu H, Gulbahar O, Cevik A (2012) Elevated homocysteine and asymmetric dimethyl arginine levels in pulmonary hypertension associated with congenital heart disease. Pediatr Cardiol 33:1323\u0026ndash;1331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00246-012-0321-9\u003c/span\u003e\u003cspan address=\"10.1007/s00246-012-0321-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaniya Gupta S, Verma N, Dash M, Goel A, Rawat, Mini P, Singh MK, Rohit P, Kumar (April 2021) Congenital Rubella: A Salient Cause of Congenital Heart Defects in Infants. J Trop Pediatr 67(2):fmab044. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/tropej/fmab044\u003c/span\u003e\u003cspan address=\"10.1093/tropej/fmab044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaidyanathan B, Nair SB, Sundaram KR, Banu UK, Shivaprakasha K, Rao SG, Kumar RK (2008) Malnutrition in Children with Congenital Heart Disease (CHD): Determinants and Short-term Impact of Corrective Intervention. Indian Pediatr 45(7):541\u0026ndash;546\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWogu AF, Loffredo CA, Bebu I, Luta G (2014) Mediation analysis of gestational age, congenital heart defects, and infant birth-weight. BMC Res Notes 7:1\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1756-0500-7-926\u003c/span\u003e\u003cspan address=\"10.1186/1756-0500-7-926\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik S, Cleves MA, Zhao W, Correa A, Hobbs CA, National Birth Defects Prevention Study (2007) Association between congenital heart defects and small for gestational age. Pediatrics 119(4):e976\u0026ndash;e982. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1542/peds.2006-2742\u003c/span\u003e\u003cspan address=\"10.1542/peds.2006-2742\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElshazali HO, Elshazali OH, Elshazali H (2017) The relationship between birth weight and congenital heart disease at Ahmed Gasim Cardiac Centre, Bahri, Sudan. Sudan J paediatrics 17(2):49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.24911/SJP.2017.2.6\u003c/span\u003e\u003cspan address=\"10.24911/SJP.2017.2.6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacobs EG, Leung MP, Karlberg LJ (2003) Birthweight distribution in southern Chinese infants with symptomatic congenital heart disease. J Paediatr Child Health 39(3):191\u0026ndash;196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1440-1754.2003.00109.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1440-1754.2003.00109.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao Q, Dunn S, Wen SW, Lougheed J, Reszel J, Lavin Venegas C, Walker M (2021) Neighbourhood maternal socioeconomic status indicators and risk of congenital heart disease. BMC Pregnancy Childbirth 21:1\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12884-020-03512-8\u003c/span\u003e\u003cspan address=\"10.1186/s12884-020-03512-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNabipour I, Vahdat K, Jafari SM, Amiri M, Shafeiae E, Riazi A, Amini AL, Pazoki R, Sanjdideh Z (2007) Correlation of hyperhomocysteinaemia and Chlamydia pneumoniae IgG seropositivity with coronary artery disease in a general population. Heart Lung Circulation 16(6):416\u0026ndash;422. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.hlc.2007.02.100\u003c/span\u003e\u003cspan address=\"10.1016/j.hlc.2007.02.100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVahdat K, Ostovar A, Hadavand F, Pourbehi MR Serum Homocysteine Levels Are Associated with Infectious Burden: the Persian Gulf Healthy. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13140/2.1.4266.5608\u003c/span\u003e\u003cspan address=\"10.13140/2.1.4266.5608\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustafa M, Hussain S, Qureshi S, Malik SA, Kazmi AR, Naeem M (2012) Study of the effect of antiviral therapy on homocysteinemia in hepatitis C virus-infected patients. BMC Gastroenterol 12:1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1471-230X-12-117\u003c/span\u003e\u003cspan address=\"10.1186/1471-230X-12-117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbike F, Engin AB, Dunder I, Tapisiz OL, Aslan C, Kutluay L (2011) Human papilloma virus persistence and neopterin, folate and homocysteine levels in cervical dysplasias. Arch Gynecol Obstet 284:209\u0026ndash;214. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00404-010-1650-7\u003c/span\u003e\u003cspan address=\"10.1007/s00404-010-1650-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoria-Ramirez E, Cisneros LN, Trevi\u0026ntilde;o-Perez S, Ibarra-Gonzalez I, Casillas-Rodriguez J, Majluf-Cruz A (2010) Effect of Highly Active Antiretroviral Therapy on Homocysteine Plasma Concentrations in HIV-1\u0026ndash;Infected Patients. JAIDS J Acquir Immune Defic Syndr 54(5):477\u0026ndash;481. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/QAI.0b013e3181d91088\u003c/span\u003e\u003cspan address=\"10.1097/QAI.0b013e3181d91088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernasconi E, Uhr M, Magenta L, Ranno A, Telenti A, Swiss HIV (2001) Cohort Study. Homocysteinaemia in HIV-infected patients treated with highly active antiretroviral therapy. Aids 15(8):1081\u0026ndash;1082. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/00002030-200105250-00027\u003c/span\u003e\u003cspan address=\"10.1097/00002030-200105250-00027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanesh J, Collins R, Peto R (1997) Chronic infections and coronary heart disease: is there a link? lancet 350(9075):430\u0026ndash;436. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(97)03079-1\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(97)03079-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenquist TH, Ratashak SA, Selhub J (1996) Homocysteine induces congenital defects of the heart and neural tube: effect of folic acid. Proceedings of the National Academy of Sciences. ;93(26):15227-32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.93.26.15227\u003c/span\u003e\u003cspan address=\"10.1073/pnas.93.26.15227\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik RA, Lone MR, Ahmed A, Koul KA, Malla RR (2017) Maternal hyperhomocysteinemia and congenital heart defects: A prospective case control study in Indian population. Indian Heart J 69(1):17\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ihj.2016.07.014\u003c/span\u003e\u003cspan address=\"10.1016/j.ihj.2016.07.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSurmiak P, Baumert M, Paprotny M (2017) Abnormal biomarkers of homocysteine metabolism in neonates with conotruncal heart defects. BioMed Research International. ;2017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuhta JC, Hernandez-Robles JA (2005) Homocysteine, folate, and congenital heart defects. Fetal Pediatr Pathol 24(2):71\u0026ndash;79. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2017/7404397\u003c/span\u003e\u003cspan address=\"10.1155/2017/7404397\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElizabeth KE, Praveen SL, Preethi NR, Jissa VT, Pillai MR (2017) Folate, vitamin B12, homocysteine and polymorphisms in folate metabolizing genes in children with congenital heart disease and their mothers. Eur J Clin Nutr 71(12):1437\u0026ndash;1441. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ejcn.2017.135\u003c/span\u003e\u003cspan address=\"10.1038/ejcn.2017.135\" targettype=\"DOI\" 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":true,"hideJournal":true,"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":"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":"congenital heart disease, hyperhomocysteinemia, rubella infection","lastPublishedDoi":"10.21203/rs.3.rs-4744008/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4744008/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eCirculating homocysteine and Rubella infection are independent risk factors for congenital heart disease (CHD). The primary objective of the study was to assess the association of serum homocysteine levels in cases of CHD and healthy controls and the secondary objective was to assess the association of serum homocysteine levels with rubella infection among CHD cases.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis case-control study was conducted in King George\u0026rsquo;s Medical University, Lucknow. Total 245 echo-graphically confirmed cases of CHD and age-gender matched 245 healthy controls of aged 0\u0026ndash;11 months were recruited from the outpatient clinic and wards of paediatric and cardiology department and controls were recruited from immunization clinic of the hospital. Samples were tested for rubella specific IgM and IgG antibody and homocysteine levels using enzyme-linked-immunosorbent-assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFrom July 2022 - December 2023, 245 cases of CHD and 245 healthy controls were recruited. The mean (SD) age of the cases were 4.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34 months and controls were 4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63. In cases, 70.20% males were recruited while 68.57% in controls. The mean serum homocysteine levels (\u0026micro;mol/L) in CHD cases were 15.70\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6 while in controls 9.51\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 (p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Within CHD cases, 7.8% (19/245) were found seropositive for rubella infection. The crude odd ratio of homocysteine levels against seropositive rubella infection was 1.08 (95% CI 1.02\u0026ndash;1.14) as compare to seronegative.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eLevels of serum homocysteine in cases of CHD were significantly higher as compared to controls, whereas among CHD cases, those who had rubella seropositive showed significantly higher serum homocysteine levels.\u003c/p\u003e","manuscriptTitle":"Assessment of serum Homocysteine levels in congenital heart disease and with rubella infection: A hospital- based case-control study in North Indian population","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 09:53:24","doi":"10.21203/rs.3.rs-4744008/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":"6f0efa94-62ef-423f-a26d-800125a26ff5","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-19T09:53:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-19 09:53:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4744008","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4744008","identity":"rs-4744008","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}