CARD8 polymorphisms among bacterial meningitis patients in North-West Ethiopia

CARD8 polymorphisms among bacterial meningitis patients in North-West Ethiopia | 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 CARD8 polymorphisms among bacterial meningitis patients in North-West Ethiopia Meseret Belayneh, Mesfin Mengesha, Berhane A. Idosa, Surafel Fentaw, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4656131/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Oct, 2024 Read the published version in BMC Infectious Diseases → Version 1 posted 4 You are reading this latest preprint version Abstract Background The severity of infectious disease outcomes is believed to depend on the virulence factors of the pathogen in combination with individuals’ unique immune response. CARD8 has been suggested to act as a regulator of the NLRP3 inflammasome, a major regulator of the innate immune proinflammatory response, and has been suggested to modulate the host response to common inflammatory diseases. In the present study, the C10X genetic polymorphism in the CARD8 gene was investigated in relation to bacterial meningitis. Methods A total of 400 clinically suspected meningitis patients hospitalized at the University of Gondar Comprehensive specialized Hospital were enrolled in the study. Cerebrospinal fluid (CSF) and blood samples were collected from all enrolled patients for laboratory investigations. The collected CSF was cultured, and all the results obtained from the culture were confirmed using direct RT‒PCR. Genotyping of whole-blood samples was performed for CARD8 gene using a TaqMan assay. The results were compared with apparently healthy controls and with PCR-negative meningitis suspected patients. Results Of the included patients, 57% were men and the most common clinical signs and symptoms were fever (81%), headache (80%), neck stiffness (76%), nausea (68%), and vomiting (67%). Microbiology culture identified seven patients with bacterial meningitis caused by Neisseria meningitidis (n = 4) and Streptococcus pneumoniae (n = 3). The RT-PCR revealed 39 positive samples for N. meningitidis (n = 10) and S. pneumoniae (n = 29). A total of 332 whole-blood samples were genotyped, with the following results: 151 (45.5%) C10X heterozygotes, 59 (17.7%) C10X homozygotes and 122 (36.7%) wild-type genotypes. The presence of the C10X polymorphism in the CARD8 gene was more prevalent in suspected meningitis patients than in healthy controls (OR 1.2; 1.00-1.5). Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease. The presence of viable or active bacterial infection was found to be associated with the presence of heterozygous C10X carriers. Conclusions A greater proportion of C10X in the CARD8 gene in confirmed bacterial meningitis patients and clinically diagnosed meningitis patients than in healthy controls. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than were heterozygote gene carriers and healthy controls. Meningitis inflammasomes CARD 8 polymorphisms Neisseria meningitidis Streptococcus pneumoniae BACKGROUND Meningitis is a severe neurological disease leading to morbidity and mortality worldwide, with higher burden in low sociodemographic index regions (1,2). Bacterial meningitis causes death and long-term disability that are substantial in all settings, especially those with the least access to health care. [(2)]. Low-income and middle-income countries account for 98% of the estimated 5–6 million disability-adjusted life years attributed due to meningitis globally, and bacterial meningitis ranks among the top ten causes of death in children younger than 14 years in high-income countries (3). Ethiopia is located in the so-called “African meningitis belt” and the country has been grouped among countries with the highest mortality rate from acute bacterial meningitis in sub-Saharan Africa, having a fatality rate of 22–28% [ 4 ]. Due to fast progression of the disease, a meningococcal infection can be fatal within the first 24 hours following dissemination of the bacteria in the blood stream [(3,5]. The pathogenesis and pathophysiology of bacterial meningitis involve a complex interaction between virulence factors of the pathogens and the host immune response [(6)]. Most of the damage from this infection is believed to result from cytokines released as the host mounts an inflammatory response triggered when innate immune cells detect infection or tissue injury [ 7 ]. The innate immune system provides a first line of defense and is essential for the control of common bacterial infections. However, it cannot always eliminate infectious organisms [ 8 ]. On the other hand, it has been shown that the innate immune response has a major role in neuronal damage, particularly during acute bacterial meningitis [ 8 , 9 ]. In the past few years, the NOD-like receptor (NLR) family has been suggested as intracellular sensors of microbial components and cell injury or stress. Upon activation, the majority of NLRs form multi-protein complexes termed “inflammasome” [ 10 ]. Among them, NACHT-leucine-rich repeat-and PYD-containing protein (NLRP3) is one of the most extensively studied inflammasome to date due to its larger range of activators and aberrant activation in several inflammatory diseases. The NLRP3 inflammasome comprise the NLR-protein: NLRP3 (also known as Cryopyrin or CIAS1) and the adaptor protein: ASC. The assembly of the inflammasome recruits and activates caspase-1 that cleaves and produces interleukin-1β (IL-1β) and other members of the IL-1family, from their inactive pro-forms. IL-1β is a proinflammatory cytokine known as one of the most potent mediators of inflammation [ 7 – 9 ], which exerts effector mechanism in clearance of bacterial pathogens and removal of damaged cells. IL-1β is also known to be produced during severe infection, including meningococcal sepsis and meningitis, causing leukocytosis and fever [ 11 ]. Despite this proinflammatory involvement, the excessive cytokine levels seem rather to stage for tissue injury and organ failure than protection, and high levels of IL-1β correlates with severity and mortality [ 12 , 13 ]. Although the excessive cytokine level may result from several possible reasons, the genetic variation in the inflammasome activation cascade involved in the activation of IL-1β has been in focus of studies during the last recent years. Caspase recruitment domain-containing protein 8 (CARD8), also known as TUCAN, has been suggested to act as a specific suppressor of NLRP3/ASC/procaspase-1 protein assembly, thereby inhibiting NLRP3-inflammasome activation [ 14 ]. More specifically, CARD8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1β expression and nuclear factor (NF)-κB activation [ 15 , 16 ]. The C10X SNP polymorphism (rs2043211) in the CARD8 gene, introduces a premature stop codon, which results in the expression of a severely truncated, non-functional protein. The variant CARD8 is unable to suppress NF-κB activity, which leads to loss of CARD8-mediated inhibition of caspase-1, resulting in high constitutive levels of pro-IL-1β [ 17 ]. The effect of C10X polymorphism has been studied in relation to several different diseases [ 18 ], including non-infectious autoimmune disorders, like rheumatoid arthritis [ 19 ], in neurological disorders [ 20 ], and suggested aggravating the atherosclerotic process by promoting inflammation [ 21 ] as well as in promoting susceptibility and severity of infectious diseases, involving bacteraemia and extrapulmonary tuberculosis [ 22 , 23 ]. A link between human polymorphisms regarding the gene IL1RN (encoding IL-1 receptor antagonist; IL-1Ra) per se [ 24 ] or in combination with IL1B [ 25 ] has been found with severity in meningococcal disease, but there are few studies done on the effect of polymorphisms in genes encoding inflammasome proteins in bacterial meningitis. It has been previously shown that both priming and licensing of the NLRP3 inflammasome are activated by Neisseria meningitidis [ 26 ], one major causative agent of meningitis; data fortifying the possibility of inflammasome polymorphisms in susceptibility and/or severity of bacterial meningitis. The present study investigates the presence of C10X polymorphism in the CARD8 gene in patients with bacterial meningitis and its impact in modulating the clinical manifestations in clinically diagnosed and laboratory confirmed meningitis patients. MATERIALS AND METHODS Study area The study was conducted at the University of Gondar Specialized Hospital (UoGSH), Gondar town, Ethiopia. The hospital has more than 700 inpatient beds and it is one of the largest teaching hospitals in the Amhara National Regional State. The facility provides services for more than seven million inhabitants living in the Amhara and neighboring regions such as Tigray and Benshangul Gumuz. Study design and population This hospital based cross-sectional study was conducted from October 2015 to October 2019 just prior to the start of the COVID-19 pandemic. Patients with suspected meningitis who have had clinical and/or laboratory evaluations suggestive for bacterial meningitis were enrolled in the study. Four hundred patients were assumed to enroll in the study to achieve a power of 80% at the 5% significance level to detect a 15% difference from the controls [ 22 ], including a 5% drop-out rate. All age groups who fulfilled the enrollment criteria were included in the study. Sociodemographic characteristics and clinical data collection Sociodemographic characteristics were collected using a pretested and standardized questionnaire. Clinical evaluation was assessed using a clinical decision standard set by the International Classification of Diseases–Clinical Modification code 320.9 and the Bacterial Meningitis Score. The definition of meningitis in suspected patients was based on the sudden onset of fever, headache, a stiff neck, episodes of seizure, and/or other symptoms, such as nausea, vomiting, photophobia, altered mental status, and coma. Two consultant internist physicians from the University of Gondar hospital monitored and evaluated the patients’ clinical conditions during recruitment of the suspected patients. Collection of Biological samples Four hundred CSF samples were collected for microbiological culture through lumbar puncture. The CSF samples were taken by experienced physicians through puncturing between the 3rd and 4th lumbar vertebrae, and CSF was collected in a sterile tube for further testing. The necessary aseptic techniques were applied. Blood samples (5ml) were collected from 336 study participants in heparin-containing tubes and stored at -20 o C freezer until further use. Microbiology examination of CSF samples Color and turbidity were macroscopically inspected for all CSF samples immediately upon collection. Microscopic examination was done using Gram-stain technique, and all the collected CSF samples were inoculated on blood agar, chocolate agar and MacConkey agar culture media plates. Following inoculation, culture media were incubated aerobically at 37°C overnight. Blood and chocolate agar cultures were incubated in a 5% CO 2 -enriched atmosphere for 72 hours. Bacterial growth was examined daily, and isolates were identified using standard microbiological methods considering colony morphology, Gram’s stain reaction, and biochemical test results. All specimens were collected following the standard operating procedure set for specimen collection. To ensure the accuracy of the laboratory method, five percent (5%) of the prepared culture media were randomly selected and incubated aerobically for 24 hours at 37°C to check the sterility of the prepared culture media. Real-time PCR, genotyping and CARD8 polymorphisms DNA was extracted from whole blood using a fully automated Mega Bio extraction method. Confirmatory testing was done using real-time PCR (RT-PCR) using a Bio-Rad cfx96 real-time detection system with PerfeCTa qPCR ToughMix (Quanta Biosciences, Gaithersburg, MD). The three most common bacterial pathogens causing acute bacterial meningitis, namely N. meningitidis , S. pneumoniae , and Haemophilus influenzae type B, were targeted. The three bacteria were typed at the genetic level using the superoxide dismutase enzyme (Sod C) for N. meningitidis , the lytA enzyme-encoding gene for S. pneumoniae , and the hpd protein D gene for H. influenzae type B. Single nucleotide polymorphism (SNP) genotyping was performed to detect the polymorphism of C10X (rs2043211) in the CARD8 gene. The analysis was performed using a Taqpath SNP genotyping assay on a Bio-Rad cfx96 RT‒PCR detection system followed by allelic discrimination to evaluate the frequencies of the different alleles, as previously described [ 22 ]. Briefly, 2 µL of genomic DNA was amplified in a final 10 µL reaction volume containing 5 µL of TaqPath ProAmp Genotyping Master Mix, 0.5 mL of 20x Assay TaqPath SNP Genotyping Mix, and predesigned primers and probes. For the TaqPath amplification cycles, a < 35 cycle cutoff was used. Data previously collected from healthy blood donors were used as controls for CARD8 genotyping [ 20 ]. Data analysis Data were cleaned manually and checked for completeness. Data were entered into Epi-data, version 4.6 and analyzed using Statistical Package for Social Sciences (SPSS) computer software, version 25.0. Binomial and multinomial logistic regression were utilized to assess factors associated with CARD8 polymorphisms. Ethical considerations Ethical approval was obtained from the Ethical Review Committee of the University of Gondar. Written informed consent was obtained from each study participant and guardians. RESULTS Sociodemographic and clinical characteristics A total of 400 hospitalized patients suspected of meningitis were included in the present study and compared to 1,964 apparently healthy controls [ 22 ]. Of the total 400 participants enrolled in the study, 332 (83%) patients have shown laboratory results eligible for genotyping. Accordingly, 144 (43%) women and 188 (57%) men were included though the distribution varied between the different age groups (Table 1 ). The median ages of patients and healthy controls were 35 (IQR: 25–45) years and 25 (22–32) years, respectively. The age and gender distribution of patients included in the present study is shown in Table 1 . Table 1 Socio-demographic characteristics and distribution of CARD 8 polymorphism among suspected meningitis patients. Variables Allelic frequency Age Homozygote wild (CC) N = 122 Heterozygote (CX) N = 151 Homozygote polymorphic (XX) N = 59 C X Age 1–10, N = 7 2 (28.6%) 3 (43%) 2 (28.6%) 7 (50%) 7 (50%) 11–19, N = 34 13 (38.2%) 16 (47%) 5 (15%) 42 (62%) 36 (38%) 20–29, N = 84 34 (40.5%) 40 (47.6%) 10 (12%) 107 (64%) 60 (36%) 30–39, N = 87 31(35.6%) 36 (41.4%) 20 (23%) 98 (56%) 76 (44%) 40–49, N = 38 13 (34.2%) 18 (47.4%) 7 (18.4%) 43 (58%) 32 (42%) 50–59, N = 45 13 (29%) 24 (53.4%) 8 (17.8%) 50 (56%) 40 (44%) 60–69, N = 28 14 (50%) 9 (32%) 5 (18%) 36 (66%) 19 (34%) > 69, N = 9 2 (22%) 5 (55.6%) 2 (22%) 9 (50%) 9 (50%) Sex Male, N = 188 70 (37%) 92 (49%) 26 (14%) 229 (62%) 144 (38%) Female, N = 144 52 (36%) 59 (41%) 33 (23%) 163(57%) 125 (43%) Total 332 The most observed clinical manifestations were fever, headache, neck stiffness, nausea, and vomiting, followed by altered mental status and consciousness, high respiratory rate and cough. Breathing difficulty, photophobia, and seizures were less common (Table 2 ). Table 2 Clinical manifestations and distribution the CARD8 polymorphism among meningitis suspected patients CARD8 gene variants Clinical manifestation Homozygote wild N = 122 Heterozygote N = 151 OR (95% CI) a Homozygote polymorphic N = 59 OR (95% CI) b Total N Fever 88 (33%) 134 (50%) 3.04 (1.60–5.78) 45 (17%) 1.24 (0.61–2.55) 267 Headache 88 (33%) 131 (50%) 2.53 (1.37–4.68) 45 (17%) 1.24 (0.61–2.55) 264 Neck stiffness 83 (34%) 125 (50%) 2.26 (1.27–3.99) 40 (16%) 0.99 (0.51–1.92) 248 Nausea 76 (34%) 106 (48%) 1.43 (0.86–2.36) 40 (18%) 1.27 (0.66–2.45) 222 Vomiting 73 (33%) 110 (50%) 1.80 (1.08–2.99) 38 (17%) 1.21 (0.64–2.31) 221 Cough 34 (34%) 46 (47%) 1.13 (0.67–1.92) 19 (19%) 1.23 (0.63–2.41) 99 High respiratory rate 31 (33%) 46 (50%) 1.28 (0.75–2.19) 16 (17%) 1.09 (0.54–2.21) 93 Altered consciousness 38 (37%) 45 (43%) 0.94 (0.56–1.57) 21 (20%) 1.22 (0.63–2.35) 104 Altered mental status 35 (36%) 43 (45%) 0.99 (0.56–1.57) 18 (19%) 1.01 (0.55–2.15) 96 Photophobia 13 (45%) 13 (45%) 0.78 (0.35–1.77) 3 (10%) 0.51 (0.14–1.88) 29 Seizure 10 (38%) 13 (50%) 1.05 (0.44–2.49) 3 (12%) 0.68 (0.18–2.60) 26 Breathing difficulty 29 (42%) 28 (41%) 0.73 (0.41–1.31) 12 (17%) 0.27 (0.13–0.57) 69 OR = odds ratio, CI = confidence interval, a association between clinical manifestations of heterozygote gene carriers and homozygote wild gene carriers, b association between clinical manifestations of homozygote polymorphic gene carriers and homozygote wild gene carriers CARD 8 polymorphism among clinically suspected patients for bacterial meningitis Genotyping was performed on whole blood samples collected from 332 eligible patients. All genotypes were found to be in Hardy‒Weinberg equilibrium (1.14, p > 0.05). Among patients with suspected meningitis, the heterozygote (CX) genotype prevailed (n = 151; 46%), followed by the wild-type genotype (CC) (n = 122; 37%), whereas few patients had homozygote (XX) C10X genotype (n = 59; 18%) (Table 3 ). A similar genotype distribution was observed among healthy blood donors (CC: 37.4%, CX: 47.7%, and XX: 15.1%). A slight difference in the distribution of the homozygous C10X genotype was detected between patients with suspected meningitis and healthy blood donors (OR = 1.2 (0.85–1.5)). The frequency of the variant allele (X) was significantly different between the patients and healthy controls; the allele frequencies were 43.5% and 39.0%, respectively (OR = 1.2 (1.00-1.5)) (Table 3 ). Table 3 CARD 8 polymorphism among meningitis suspected patients and healthy controls Genotype All meningitis suspected cases N (%) Healthy controls N (%) OR (95%CI) N (%) CC 122 (36.7) 734 (37.4) 1 CX 151 (45.5) 934 (47.7) 0.97 XX 59 (17.7) 296 (15.1) 1.2 (0.85–1.5) Allelic frequency C 273 (56.5) 2402 (61.2) 1 X 210 (43.5) 1526 (39) 1.2 (1.00-1.5) Total 332 1964 OR = odds ratio, CI = confidence interval Our data revealed a significantly higher frequency of fever, headache, neck stiffness and vomiting in patients with the heterozygous C10X genotype than in patients with the wild- genotype, with odds ratios of 3.04 (1.60–5.78) for fever, 2.53 (1.37–4.68) for headache, 2.26 (1.27–3.99) for neck stiffness and 1.80 (1.08–2.99) for vomiting (Table 2 ). Similarly, higher frequent manifestations such as fever, headache, nausea, vomiting, high respiratory rate, and altered mental status and cautiousness were more common among patients with homozygous C10X gene than wild type gene carriers. CARD8 polymorphisms among laboratory-confirmed meningitis patients Out of the 400 CSF samples cultured for bacterial growth, only 7 samples showed bacterial growth for N. meningitidis (n = 4) and S. pneumoniae (n = 3) . On the other hand, 39 samples became positive for N. meningitidis (n = 10) and S. pneumoniae (n = 29) using RT‒PCR. However, CARD8 polymorphism was done on 25 RT-PCT positive samples ( N. meningitidis (n = 8) and S. pneumoniae (n = 17)), and 14 positive samples were excluded from the analysis due to incomplete data. The C10X polymorphism was compared among laboratory-confirmed meningitis patients (n = 25), laboratory negative clinically suspected patients (n = 307) and healthy controls (n = 1,964). The homozygote C10X polymorphism was more prevalent among laboratory-confirmed meningitis patients [6/25 (24%)] and laboratory negative clinically suspected patients [53/307 (17%)] than in healthy blood donor controls [n = 296/1964 (15%)]; odds ratios of 1.6 (0.53-5) and 1.8 (0.64–5.5), respectively) (Table 4 ). Similarly, heterozygous C10X polymorphisms prevailed among laboratory-confirmed and laboratory negative but clinically suspected meningitis patients (44% and 45.6%, respectively), than healthy controls [OR: 1.12 (0.43–2.87) and 1.08 (0.43–2.7)], respectively (Table 4 ). Table 4 Distribution of CARD 8 gene polymorphism among laboratory confirmed meningitis patients, laboratory negative but clinically suspected meningitis patients and healthy controls Laboratory confirmed meningitis patients N (%) N = 25 Lab negative/clinically suspected meningitis patients N (%) N = 307 OR (95%CI) a Healthy control N (%) N = 1964 OR (95% CI) b Genetic distribution CC 8 (32) 114 (37) 1 734 (37.4) 1 CX 11 (44) 140 (45.6) 1.12 (0.43–2.87) 934 (47.6) 1.08 (0.43–2.7) XX 6 (24) 53 (17) 1.6 (0.53-5) 296 (15.1) 1.8 (0.64–5.4) Allelic frequency C 27 (54) 368 (60) 1 2402 (61) 1 X 23 (46) 246 (40) 1.27 (0.7–2.3) 1526 (39) 1.34 (0.76–2.4) OR = odds ratio, CI = confidence interval, a association between laboratory confirmed bacterial meningitis cases with healthy controls, b association between no bacteria identified, clinically diagnosed meningitis suspected cases with healthy controls Further analysis of our data revealed that 67% of the culture-confirmed patients and 37% of the PCR-confirmed patients were heterozygous C10X carriers, with the ORs of 3.1 (0.36-29) and 0.8 (0.3–2.3), respectively compared to healthy controls (Table 5 ). Similarly, the allele frequency of the X variant was greater in culture-confirmed meningitis patients and in PCR-confirmed patients, than healthy controls, respectively [OR:1.6 (0.5-5) and 1.3 (0.7–2.4)], respectively (Table 5 ). Table 5 Genetic distributions of CARD 8 polymorphism among culture positive meningitis patients, PCR positive but culture negative meningitis patients, and Healthy controls Culture positive meningitis patients N = 6 OR (95% CI) a Clinically diagnosed meningitis patients N = 307 OR (95% CI) b PCR positive culture negative patients N = 19 OR (95% CI) c Healthy control N = 1964 Genetic distribution CC 1 (16%) 1 114 (37%) 1 7 (37%) 1 734 (37%) CX 4 (67%) 3.3 (0.36-29) 140 (46%) 3.14 (0.36-29) 7 (37%) 0.8 (0.3–2.3) 934 (48%) XX 1 (16%) 2.1 (0.13-35) 53 (17%) 2.5 (0.2-5) 5 (26%) 1.8 (0.6–5.6) 296 (15%) Allelic frequency C 6 (50) 1 368 (60) 1 21 (55) 1 2402 (61%) X 6 (50) 1.5 (0.5–4.7) 246 (40) 1.6 (0.5-5) 17 (45) 1.3 (0.7–2.4) 1526 (39%) OR, odds ratio, CI, confidence interval a association between culture positive meningitis patients with lab negative but clinically suspected meningitis patients b association between culture positive and healthy control c association between culture negative PCR positive patients with healthy control Species-specific analysis was performed on samples identified by PCR. As a result, the genotype distributions of C10X polymorphisms and homozygous C10X carriers were significantly greater in S. pneumoniae -positive patients than in healthy controls [OR: 1.8 (0.9–3.5) and 3.1 (0.8–12)], respectively (Table 6 ). Table 6 Genetic distribution of CARD8 polymorphism among meningitis patients with Neisseria meningitidis and Streptococcus pneumoniae , laboratory negative but clinically suspected meningitis patients, and healthy controls Neisseria meningitidis N = 8 OR (95% CI) a Streptococcus pneumoniae N = 17 OR (95% CI) b No bacteria clinical diagnosed N = 307 OR (95% CI) c Healthy controls N = 1964 Genetic distribution CC 4 (50%) 1 4 (24%) 1 114 (37%) 1 734 CX 3 (38%) 0.5 (0.13–0.6) 8 (47%) 1.6 (0.5–5.5) 140 (46%) 1.6 (0.5–5.2) 934 XX 1 (13%) 0.61 (0.1–5.6) 5 (29%) 2.7 (0.7–10.4) 53 (17%) 3.1 (0.8–12) 296 Allelic frequency C 11 (69%) 1 16 (47%) 1 368 (60%) 1 2402 (61%) X 5 (31%) 0.71 (0.25-.1) 18 (53%) 1.7 (0.8–3.4) 246 (40) 1.8 (0.9–3.5) 1526 (39%) OR, odds ratio, CI, confidence interval. a association between those infected with Neisseria meningitidis and healthy controls b association between those infected with Streptococcus pneumoniae and no bacterial identified clinically diagnosed meningitis suspected cases c association between those infected with Streptococcus pneumoniae and healthy controls DISCUSSION Compared to those in the healthy control population, C10X polymorphic gene carriers were more prevalent in our cohort of meningitis patients. A slightly greater percentage of female patients were found to be polymorphic gene carriers than male patients, with percentages of 43% and 39%, respectively. The present study showed that fever, headache, neck stiffness and vomiting were the dominant clinical characteristics. Moreover, there was statistically significant difference in manifestation of these clinical symptoms among heterozygous gene carriers compared to wild-type gene carriers, which indicates the strong association heterozygote carriership with development of the above-mentioned clinical manifestations. The odds of fever, headache nausea, vomiting, cough, high respiratory rate, altered cautiousness and altered mental status were greater among homozygous, CARD8 (C10X), gene carriers than wild-type gene carriers, which indicates the potential association of homozygote carriership with the mentioned clinical manifestations, which agreed with a previous study [ 22 ]. The development of clinical manifestations is mediated by induction of pro-inflammatory cytokines which is regulated by NLRP3 inflammasomes which activates caspase 1 (CASP1) through caspase recruitment domains (CARD). The clinical manifestation in turn is influenced by the genetic make-up of these components of the host [ 27 , 28 ], which might explain the enhanced clinical manifestation in polymorphic gene carrier observed in this study. Other studies indicated that single nucleotide polymorphisms (SNPs) in different components, or regulators, of this pathway have been associated with enhanced clinical manifestations in different infective conditions including hepatitis C, TB and other bacterial infections. [ 22 , 27 ]. The implication that a loss-of-function mutation in the CARD 8 gene, which makes it unable to inhibit caspase-1 activation, could be associated with increased NLRP3 inflammasome activity [ 29 ] was observed in the present study. The wild-type gene was found to be protective against bacterial meningitis in the present study. Our finding agrees with a study conducted by Dennis et al. to identify the effect of genetic variation on susceptibility to bacterial infections, which showed overrepresentation of the wild-type allele in healthy controls [ 30 ] in vitro. On the other hand, some findings have not been robustly replicated, suggesting that the effect of relatively frequent C10X polymorphisms might be modest or that associations with additional factors are needed to significantly drive disease outcomes [ 17 ]. The data also showed that the heterozygote type of the C10X polymorphism was more dominant among meningitis patients and blood donors, followed by homozygous wild-type gene carriers. The homozygote polymorphic gene carriers were found to be more susceptible to bacterial meningitis than the heterozygote gene carriers and healthy controls. This finding is supported by a previous report by A. Idosa et al., who reported that homozygote carriers are more susceptible to infectious disease than heterozygote carriers are, indicating that healthy C10X homozygous individuals might have a greater risk of developing an infection when exposed to infectious agents [ 22 ]. The current study also demonstrated that the proportion of C10X gene polymorphisms was relatively greater among patients with identified pathogens than among clinically diagnosed patients and healthy controls. Furthermore, C10X-heterozygote carriers were more prevalent among culture-positive meningitis (67%) than those with suspected nonpathogen-identified meningitis (37%) and healthy controls (47%), with odds ratios of 4 and 3.1, respectively. Previously, Idosa et al. reported that the polymorphism of C10X in the CARD8 gene was significantly more prevalent in patients with bacteremia than in healthy controls and that the prevalence of heterozygote gene distribution was greater among bacteremia patients than among those with negative blood cultures and healthy controls [ 22 ]. The C10X polymorphism causes a nonsense allele, resulting in reduced expression of functional CARD8, which in turn is suggested to result in a loss of its inhibitory effect on caspase 1[ 29 , 30 ]. There are also reports on the polymorphism of CARD8 (C10X) that has been associated with an increased risk of inflammation. Polymorphism of CARD8 contributes to the development of some noninfectious diseases, such as inflammatory bowel disease, gout, rheumatoid arthritis and Alzheimer’s disease, and contributes to susceptibility to and poor disease outcomes in patients with infectious diseases [ 22 , 31 ]. A loss-of-function mutation in the CARD8 gene is reported to render CARD8 unable to inhibit caspase-1 activation, which could also be associated with increased NLRP3 inflammasome activity [ 23 ]. Sarkar A et al reported that caspase-1-deficient mice are protected from diseases involving excessive inflammation, such as bacterial-induced sepsis, and are markedly resistant to the lethal effects of endotoxin [ 32 ]. The C10X genotype has been found to be associated with chronic consequences on human health by resulting in severe autoimmune and autoinflammatory disease, probably due to the long-term effect of an excessive caspase-1 response [ 33 ]. The functional consequences of the C10X polymorphism in infectious diseases do not seem to clear way of mechanism with the involvement of different gene variants. G.V. Paramel et al. reported the discovery of possible isoforms of the CARD8 gene, such as T47, in which its transcription begins downstream of C10X; hence, not affecting the isoform could affect the functional consequences of the C10X polymorphism [ 18 ]. As the review stated, the high rate of homozygous patients with loss-of-function polymorphisms, which appear to be human knockouts, might reflect partial rescue of CARD8 function by alternative splicing, leading to an almost functional full-length protein. Patients who are homozygous for the C10X genotype might therefore have a functional CARD8 protein due to the presence of the T47 isoform [ 15 ]. Therefore, to clarify the effect of C10X polymorphisms and gene variants during bacterial meningitis, or other infectious diseases a detailed study on the mechanistic effect of polymorphic genes and the impact of possible isoforms present is needed. The limitations of this study were the small number of laboratory-confirmed meningitis patients which influenced the statistical analysis and limit to do sub-group analysis and it was not managed to measure IL-1β due to logistical and resource constraints. Conclusions and recommendations The greater proportion of polymorphic C10X in the CARD8 gene in confirmed and suspected meningitis patients than in healthy controls was indicative of a robust inflammatory response due to the polymorphic C10X gene. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than were heterozygote gene carriers and healthy controls. This indicates that the polymorphic C10X gene might not provide better survival conditions by generating a vigorous inflammatory response and that the increased release of proinflammatory cytokines does not always play a protective role unless it is directed by control mechanisms. Detailed studies on the cellular and molecular mechanisms of polymorphic gene variants of the CARD 8 gene will be mandatory to obtain clear insight into its impact on susceptibility to and severity of infectious diseases. Abbreviations CARD8 caspase recruitment domain-containing protein 8 AAU Addis Ababa University EPHI Ethiopian Public Health Institute AHRI Armauer Hanssen Research Institute NLRP3 nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 CSF cerebrospinal fluid PCR polymerase chain reaction DNA deoxyribonucleic acid IL-1β interleukin-1beta SNP single nucleotide polymorphisms UoGSH University of Gondar Specialized Hospital RA rheumatoid arthritis Declarations Acknowledgments We greatly acknowledge staffs from the University of Gondar Hospital, Armauer Hansen Research Institute (AHRI), Ethiopian Public Health Institute (EPHI), Örebro University, Sweden, for the unlimited and continuous support provided during the study. We thank the Department of Medical Microbiology/University of Gondar, Addis Ababa University, Orebro University for financial support. Conflict of interest The authors declare that there is no competing interests. Funding The research was supported through the research seed money given to support PhD program from the University of Gondar. The laboratory work was done at AHRI and EPHI through in-kind support obtained from the two institutions. Sample collection materials were provided through the in-kind support from Örebro University, Sweden. Ethics Ethical approval was obtained from the Ethical Review Committee of the University of Gondar. Written informed consent was obtained from each study participant and guardians. Availability of data and materials All data supporting the findings of this study are available within the paper. Consent for publication All authors have reviewed the manuscript and given their consent for its publication. Authors contribution MB-conception and design, acquisition of data, analysis and wrote manuscript; MM, BA, MA, SF, ÖG support the laboratory work and training; ZT, and BM -statistical analysis; AP, EA, ES, OS and BG involve in the conception and design of the research and editing the final manuscript. References Qu C, Wang Y, Wang X, He R, Cao H, Liu B, et al. Global Burden and Its Association with Socioeconomic Development Status of Meningitis Caused by Specific Pathogens over the Past 30 years: A Population-Based Study. Neuroepidemiology. 2023;57(5):316–35. Chalupka AN, Talmor D. The economics of sepsis. Crit Care Clin. 2012;28(1):57–76. vi. Rudd KE, Kissoon N, Limmathurotsakul D, Bory S, Mutahunga B, Seymour CW, et al. The global burden of sepsis: barriers and potential solutions. Crit Care. 2018;22(1):232. Meningitis [Internet]. [cited 2024 Jun 21]. https://www.who.int/news-room/fact-sheets/detail/meningitis . Amare AT, Kebede ZT, Welch HD. Epidemiology of bacterial meningitis in children admitted to Gondar University Hospital in the post pneumococcal vaccine era. Pan Afr Med J [Internet]. 2018 [cited 2024 Jun 21];31. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488968/ . van Deuren M, Brandtzaeg P, van der Meer JWM. Update on Meningococcal Disease with Emphasis on Pathogenesis and Clinical Management. Clin Microbiol Rev. 2000;13(1):144–66. Herold R, Schroten H, Schwerk C. Virulence Factors of Meningitis-Causing Bacteria: Enabling Brain Entry across the Blood-Brain Barrier. Int J Mol Sci. 2019;20(21):5393. Pathogenesis and pathophysiology of bacterial meningitis. - PMC [Internet]. [cited 2024 Jun 21]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC358273/ . Charles A, Janeway J, Travers P, Walport M, Shlomchik MJ. The Immune System in Health and Disease. In: Immunobiology: The Immune System in Health and Disease 5th edition [Internet]. Garland Science; 2001 [cited 2024 Jun 21]. https://www.ncbi.nlm.nih.gov/books/NBK10775/ . Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10(2):417–26. Barichello T, Generoso JS, Singer M, Dal-Pizzol F. Biomarkers for sepsis: more than just fever and leukocytosis—a narrative review. Crit Care. 2022;26(1):14. Dinarello CA. A clinical perspective of IL-1β as the gatekeeper of inflammation. Eur J Immunol. 2011;41(5):1203–17. Amarante-Mendes GP, Adjemian S, Branco LM, Zanetti LC, Weinlich R, Bortoluci KR. Pattern Recognition Receptors and the Host Cell Death Molecular Machinery. Front Immunol. 2018;9:2379. Lopez-Castejon G, Brough D. Understanding the mechanism of IL-1β secretion. Cytokine Growth Factor Rev. 2011;22(4):189–95. Scott AM, Saleh M. The inflammatory caspases: guardians against infections and sepsis. Cell Death Differ. 2007;14(1):23–31. Razmara M, Srinivasula SM, Wang L, Poyet JL, Geddes BJ, DiStefano PS, et al. CARD-8 protein, a new CARD family member that regulates caspase-1 activation and apoptosis. J Biol Chem. 2002;277(16):13952–8. Fontalba A, Martinez-Taboada V, Gutierrez O, Pipaon C, Benito N, Balsa A, et al. Deficiency of the NF-kappaB inhibitor caspase activating and recruitment domain 8 in patients with rheumatoid arthritis is associated with disease severity. J Immunol Baltim Md 1950. 2007;179(7):4867–73. Paramel GV, Sirsjö A, Fransén K. Role of genetic alterations in the NLRP3 and CARD8 genes in health and disease. Mediators Inflamm. 2015;2015:846782. Kastbom A, Johansson M, Verma D, Söderkvist P, Rantapää-Dahlqvist S. CARD8 p.C10X polymorphism is associated with inflammatory activity in early rheumatoid arthritis. Ann Rheum Dis. 2010;69(4):723–6. Ravichandran KA, Heneka MT. Inflammasomes in neurological disorders - mechanisms and therapeutic potential. Nat Rev Neurol. 2024;20(2):67–83. Paramel GV, Folkersen L, Strawbridge RJ, Elmabsout AA, Särndahl E, Lundman P, et al. CARD8 gene encoding a protein of innate immunity is expressed in human atherosclerosis and associated with markers of inflammation. Clin Sci Lond Engl 1979. 2013;125(8):401–7. Abate E, Blomgran R, Verma D, Lerm M, Fredrikson M, Belayneh M, et al. Polymorphisms in CARD8 and NLRP3 are associated with extrapulmonary TB and poor clinical outcome in active TB in Ethiopia. Sci Rep. 2019;9(1):3126. Asfaw Idosa B, Sahdo B, Balcha E, Kelly A, Söderquist B, Särndahl E. C10X polymorphism in the CARD8 gene is associated with bacteraemia. Immun Inflamm Dis. 2014;2(1):13–20. Balding J, Healy C, Livingstone W, White B, Mynett-Johnson L, Cafferkey M, et al. Genomic polymorphic profiles in an Irish population with meningococcaemia: Is it possible to predict severity and outcome of disease? Genes Immun. 2004;4:533–40. Read RC, Cannings C, Naylor SC, Timms JM, Maheswaran R, Borrow R, et al. Variation within genes encoding interleukin-1 and the interleukin-1 receptor antagonist influence the severity of meningococcal disease. Ann Intern Med. 2003;138(7):534–41. Idosa BA, Kelly A, Jacobsson S, Demirel I, Fredlund H, Särndahl E, et al. Neisseria meningitidis-Induced Caspase-1 Activation in Human Innate Immune Cells Is LOS-Dependent. J Immunol Res. 2019;2019:6193186. Valencia BM, Cvejic E, Vollmer-Conna U, Hickie IB, Wakefield D, Li H, et al. The severity of the pathogen-induced acute sickness response is affected by polymorphisms in genes of the NLRP3 inflammasome pathway. Brain Behav Immun. 2021;93:186–93. Khera AV, Chaffin M, Aragam KG, Haas ME, Roselli C, Choi SH, et al. Genome-wide polygenic scores for common diseases identify individuals with risk equivalent to monogenic mutations. Nat Genet. 2018;50(9):1219–24. Mao L, Kitani A, Similuk M, Oler AJ, Albenberg L, Kelsen J, et al. Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease. J Clin Invest. 2018;128(5):1793–806. Ko DC, Shukla KP, Fong C, Wasnick M, Brittnacher MJ, Wurfel MM, et al. A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease. Am J Hum Genet. 2009;85(2):214–27. Cheung MS, Theodoropoulou K, Lugrin J, Martinon F, Busso N, Hofer M. Periodic Fever with Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome Is Associated with a CARD8 Variant Unable To Bind the NLRP3 Inflammasome. J Immunol Baltim Md. 1950. 2017;198(5):2063–9. Mihret W, Sletbakk Brusletto B, Øvstebø R, Siebke Troseid AM, Norheim G, Merid Y, et al. Molecular studies of meningococcal and pneumococcal meningitis patients in Ethiopia. Innate Immun. 2019;25(3):158–67. Cai B, Deitch EA, Ulloa L. Novel insights for systemic inflammation in sepsis and hemorrhage. Mediators Inflamm. 2010;2010:642462. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Oct, 2024 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 04 Jul, 2024 Editor assigned by journal 02 Jul, 2024 Submission checks completed at journal 02 Jul, 2024 First submitted to journal 28 Jun, 2024 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-4656131","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322521416,"identity":"7bb51eba-fd8a-49cf-9958-9a91c2f912e6","order_by":0,"name":"Meseret Belayneh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBCDBAb2BiBlYEG0DoMEHp4DIFqCFC0SCSAGEVp029sfPq6o+JNnL/n86oYfBRIM/O3dCXi1mJ05Y2x45oxBMY90TtnNHqDDJM6c3YBfy40cNsnGNoPEHumctBs8QC0GErkEtNx//vxn4z+gFskzaTf/EKXlBoMZY2MDUIsE+7HbxNlyJsdYsuGYcTHPmRy22zIGEjyE/XL8+MOPDTVyeeztx5/dfPPHRo6/vRe/FiTAYwAmiVUOAuwPSFE9CkbBKBgFIwgAAKDiSDzjodyxAAAAAElFTkSuQmCC","orcid":"","institution":"University of Gondar","correspondingAuthor":true,"prefix":"","firstName":"Meseret","middleName":"","lastName":"Belayneh","suffix":""},{"id":322521418,"identity":"3e2c0fcf-779b-4ee3-b99c-d249db6030b7","order_by":1,"name":"Mesfin Mengesha","email":"","orcid":"","institution":"Armauer Hanssen Research Institute (AHRI)","correspondingAuthor":false,"prefix":"","firstName":"Mesfin","middleName":"","lastName":"Mengesha","suffix":""},{"id":322521419,"identity":"05cf742a-33b1-4467-9ef6-9c57dac0824b","order_by":2,"name":"Berhane A. 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Bacterial meningitis causes death and long-term disability that are substantial in all settings, especially those with the least access to health care. [(2)]. Low-income and middle-income countries account for 98% of the estimated 5\u0026ndash;6\u0026nbsp;million disability-adjusted life years attributed due to meningitis globally, and bacterial meningitis ranks among the top ten causes of death in children younger than 14 years in high-income countries (3).\u003c/p\u003e \u003cp\u003eEthiopia is located in the so-called \u0026ldquo;African meningitis belt\u0026rdquo; and the country has been grouped among countries with the highest mortality rate from acute bacterial meningitis in sub-Saharan Africa, having a fatality rate of 22\u0026ndash;28% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Due to fast progression of the disease, a meningococcal infection can be fatal within the first 24 hours following dissemination of the bacteria in the blood stream [(3,5]. The pathogenesis and pathophysiology of bacterial meningitis involve a complex interaction between virulence factors of the pathogens and the host immune response [(6)]. Most of the damage from this infection is believed to result from cytokines released as the host mounts an inflammatory response triggered when innate immune cells detect infection or tissue injury [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The innate immune system provides a first line of defense and is essential for the control of common bacterial infections. However, it cannot always eliminate infectious organisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. On the other hand, it has been shown that the innate immune response has a major role in neuronal damage, particularly during acute bacterial meningitis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the past few years, the NOD-like receptor (NLR) family has been suggested as intracellular sensors of microbial components and cell injury or stress. Upon activation, the majority of NLRs form multi-protein complexes termed \u0026ldquo;inflammasome\u0026rdquo; [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among them, NACHT-leucine-rich repeat-and PYD-containing protein (NLRP3) is one of the most extensively studied inflammasome to date due to its larger range of activators and aberrant activation in several inflammatory diseases. The NLRP3 inflammasome comprise the NLR-protein: NLRP3 (also known as Cryopyrin or CIAS1) and the adaptor protein: ASC. The assembly of the inflammasome recruits and activates caspase-1 that cleaves and produces interleukin-1β (IL-1β) and other members of the IL-1family, from their inactive pro-forms. IL-1β is a proinflammatory cytokine known as one of the most potent mediators of inflammation [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], which exerts effector mechanism in clearance of bacterial pathogens and removal of damaged cells. IL-1β is also known to be produced during severe infection, including meningococcal sepsis and meningitis, causing leukocytosis and fever [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite this proinflammatory involvement, the excessive cytokine levels seem rather to stage for tissue injury and organ failure than protection, and high levels of IL-1β correlates with severity and mortality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although the excessive cytokine level may result from several possible reasons, the genetic variation in the inflammasome activation cascade involved in the activation of IL-1β has been in focus of studies during the last recent years.\u003c/p\u003e \u003cp\u003eCaspase recruitment domain-containing protein 8 (CARD8), also known as TUCAN, has been suggested to act as a specific suppressor of NLRP3/ASC/procaspase-1 protein assembly, thereby inhibiting NLRP3-inflammasome activation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. More specifically, CARD8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1β expression and nuclear factor (NF)-κB activation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The C10X SNP polymorphism (rs2043211) in the \u003cem\u003eCARD8\u003c/em\u003e gene, introduces a premature stop codon, which results in the expression of a severely truncated, non-functional protein. The variant \u003cem\u003eCARD8\u003c/em\u003e is unable to suppress NF-κB activity, which leads to loss of CARD8-mediated inhibition of caspase-1, resulting in high constitutive levels of pro-IL-1β [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The effect of C10X polymorphism has been studied in relation to several different diseases [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], including non-infectious autoimmune disorders, like rheumatoid arthritis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], in neurological disorders [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and suggested aggravating the atherosclerotic process by promoting inflammation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] as well as in promoting susceptibility and severity of infectious diseases, involving bacteraemia and extrapulmonary tuberculosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA link between human polymorphisms regarding the gene \u003cem\u003eIL1RN\u003c/em\u003e (encoding IL-1 receptor antagonist; IL-1Ra) \u003cem\u003eper se\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] or in combination with \u003cem\u003eIL1B\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] has been found with severity in meningococcal disease, but there are few studies done on the effect of polymorphisms in genes encoding inflammasome proteins in bacterial meningitis. It has been previously shown that both priming and licensing of the NLRP3 inflammasome are activated by \u003cem\u003eNeisseria meningitidis\u003c/em\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], one major causative agent of meningitis; data fortifying the possibility of inflammasome polymorphisms in susceptibility and/or severity of bacterial meningitis. The present study investigates the presence of C10X polymorphism in the \u003cem\u003eCARD8\u003c/em\u003e gene in patients with bacterial meningitis and its impact in modulating the clinical manifestations in clinically diagnosed and laboratory confirmed meningitis patients.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eThe study was conducted at the University of Gondar Specialized Hospital (UoGSH), Gondar town, Ethiopia. The hospital has more than 700 inpatient beds and it is one of the largest teaching hospitals in the Amhara National Regional State. The facility provides services for more than seven million inhabitants living in the Amhara and neighboring regions such as Tigray and Benshangul Gumuz.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and population\u003c/h2\u003e \u003cp\u003eThis hospital based cross-sectional study was conducted from October 2015 to October 2019 just prior to the start of the COVID-19 pandemic. Patients with suspected meningitis who have had clinical and/or laboratory evaluations suggestive for bacterial meningitis were enrolled in the study. Four hundred patients were assumed to enroll in the study to achieve a power of 80% at the 5% significance level to detect a 15% difference from the controls [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], including a 5% drop-out rate. All age groups who fulfilled the enrollment criteria were included in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSociodemographic characteristics and clinical data collection\u003c/h2\u003e \u003cp\u003eSociodemographic characteristics were collected using a pretested and standardized questionnaire. Clinical evaluation was assessed using a clinical decision standard set by the International Classification of Diseases\u0026ndash;Clinical Modification code 320.9 and the Bacterial Meningitis Score. The definition of meningitis in suspected patients was based on the sudden onset of fever, headache, a stiff neck, episodes of seizure, and/or other symptoms, such as nausea, vomiting, photophobia, altered mental status, and coma. Two consultant internist physicians from the University of Gondar hospital monitored and evaluated the patients\u0026rsquo; clinical conditions during recruitment of the suspected patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCollection of Biological samples\u003c/h2\u003e \u003cp\u003eFour hundred CSF samples were collected for microbiological culture through lumbar puncture. The CSF samples were taken by experienced physicians through puncturing between the 3rd and 4th lumbar vertebrae, and CSF was collected in a sterile tube for further testing. The necessary aseptic techniques were applied. Blood samples (5ml) were collected from 336 study participants in heparin-containing tubes and stored at -20\u003csup\u003eo\u003c/sup\u003eC freezer until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMicrobiology examination of CSF samples\u003c/h2\u003e \u003cp\u003eColor and turbidity were macroscopically inspected for all CSF samples immediately upon collection. Microscopic examination was done using Gram-stain technique, and all the collected CSF samples were inoculated on blood agar, chocolate agar and MacConkey agar culture media plates. Following inoculation, culture media were incubated aerobically at 37\u0026deg;C overnight. Blood and chocolate agar cultures were incubated in a 5% CO\u003csub\u003e2\u003c/sub\u003e-enriched atmosphere for 72 hours. Bacterial growth was examined daily, and isolates were identified using standard microbiological methods considering colony morphology, Gram\u0026rsquo;s stain reaction, and biochemical test results.\u003c/p\u003e \u003cp\u003eAll specimens were collected following the standard operating procedure set for specimen collection. To ensure the accuracy of the laboratory method, five percent (5%) of the prepared culture media were randomly selected and incubated aerobically for 24 hours at 37\u0026deg;C to check the sterility of the prepared culture media.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR, genotyping and CARD8 polymorphisms\u003c/h2\u003e \u003cp\u003eDNA was extracted from whole blood using a fully automated Mega Bio extraction method. Confirmatory testing was done using real-time PCR (RT-PCR) using a Bio-Rad cfx96 real-time detection system with PerfeCTa qPCR ToughMix (Quanta Biosciences, Gaithersburg, MD). The three most common bacterial pathogens causing acute bacterial meningitis, namely \u003cem\u003eN. meningitidis\u003c/em\u003e, \u003cem\u003eS. pneumoniae\u003c/em\u003e, and \u003cem\u003eHaemophilus influenzae\u003c/em\u003e type B, were targeted. The three bacteria were typed at the genetic level using the superoxide dismutase enzyme (Sod C) for \u003cem\u003eN. meningitidis\u003c/em\u003e, the lytA enzyme-encoding gene for \u003cem\u003eS. pneumoniae\u003c/em\u003e, and the hpd protein D gene for \u003cem\u003eH. influenzae\u003c/em\u003e type B.\u003c/p\u003e \u003cp\u003eSingle nucleotide polymorphism (SNP) genotyping was performed to detect the polymorphism of C10X (rs2043211) in the \u003cem\u003eCARD8\u003c/em\u003e gene. The analysis was performed using a Taqpath SNP genotyping assay on a Bio-Rad cfx96 RT‒PCR detection system followed by allelic discrimination to evaluate the frequencies of the different alleles, as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, 2 \u0026micro;L of genomic DNA was amplified in a final 10 \u0026micro;L reaction volume containing 5 \u0026micro;L of TaqPath ProAmp Genotyping Master Mix, 0.5 mL of 20x Assay TaqPath SNP Genotyping Mix, and predesigned primers and probes. For the TaqPath amplification cycles, a\u0026thinsp;\u0026lt;\u0026thinsp;35 cycle cutoff was used. Data previously collected from healthy blood donors were used as controls for CARD8 genotyping [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData were cleaned manually and checked for completeness. Data were entered into Epi-data, version 4.6 and analyzed using Statistical Package for Social Sciences (SPSS) computer software, version 25.0. Binomial and multinomial logistic regression were utilized to assess factors associated with CARD8 polymorphisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEthical considerations\u003c/h2\u003e \u003cp\u003eEthical approval was obtained from the Ethical Review Committee of the University of Gondar. Written informed consent was obtained from each study participant and guardians.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSociodemographic and clinical characteristics\u003c/h2\u003e \u003cp\u003eA total of 400 hospitalized patients suspected of meningitis were included in the present study and compared to 1,964 apparently healthy controls [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Of the total 400 participants enrolled in the study, 332 (83%) patients have shown laboratory results eligible for genotyping. Accordingly, 144 (43%) women and 188 (57%) men were included though the distribution varied between the different age groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The median ages of patients and healthy controls were 35 (IQR: 25\u0026ndash;45) years and 25 (22\u0026ndash;32) years, respectively. The age and gender distribution of patients included in the present study is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eSocio-demographic characteristics and distribution of CARD 8 polymorphism among suspected meningitis patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eAllelic frequency\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHomozygote wild (CC)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;122\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeterozygote (CX)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;151\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHomozygote polymorphic (XX)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;59\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;10, N\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (28.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (43%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (28.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u0026ndash;19, N\u0026thinsp;=\u0026thinsp;34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (38.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42 (62%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36 (38%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u0026ndash;29, N\u0026thinsp;=\u0026thinsp;84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34 (40.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40 (47.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e107 (64%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60 (36%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u0026ndash;39, N\u0026thinsp;=\u0026thinsp;87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31(35.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36 (41.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 (23%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e98 (56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76 (44%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u0026ndash;49, N\u0026thinsp;=\u0026thinsp;38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (34.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18 (47.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (18.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43 (58%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32 (42%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u0026ndash;59, N\u0026thinsp;=\u0026thinsp;45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (53.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (17.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50 (56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40 (44%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u0026ndash;69, N\u0026thinsp;=\u0026thinsp;28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36 (66%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19 (34%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;69, N\u0026thinsp;=\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (55.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale, N\u0026thinsp;=\u0026thinsp;188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92 (49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26 (14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e229 (62%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e144 (38%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale, N\u0026thinsp;=\u0026thinsp;144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52 (36%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59 (41%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33 (23%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e163(57%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e125 (43%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e332\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe most observed clinical manifestations were fever, headache, neck stiffness, nausea, and vomiting, followed by altered mental status and consciousness, high respiratory rate and cough. Breathing difficulty, photophobia, and seizures were less common (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical manifestations and distribution the CARD8 polymorphism among meningitis suspected patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eCARD8 gene variants\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical manifestation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHomozygote wild\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;122\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHeterozygote\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;151\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR (95% CI) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHomozygote polymorphic\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;59\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOR (95% CI) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e134 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.04 (1.60\u0026ndash;5.78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.24 (0.61\u0026ndash;2.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e267\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeadache\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.53 (1.37\u0026ndash;4.68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.24 (0.61\u0026ndash;2.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e264\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeck stiffness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83 (34%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.26 (1.27\u0026ndash;3.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.99 (0.51\u0026ndash;1.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNausea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76 (34%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106 (48%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.43 (0.86\u0026ndash;2.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40 (18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.27 (0.66\u0026ndash;2.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e222\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVomiting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.80 (1.08\u0026ndash;2.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.21 (0.64\u0026ndash;2.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e221\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34 (34%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46 (47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.13 (0.67\u0026ndash;1.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (19%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.23 (0.63\u0026ndash;2.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh respiratory rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.28 (0.75\u0026ndash;2.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.09 (0.54\u0026ndash;2.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAltered consciousness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45 (43%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94 (0.56\u0026ndash;1.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.22 (0.63\u0026ndash;2.35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAltered mental status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35 (36%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.99 (0.56\u0026ndash;1.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (19%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.01 (0.55\u0026ndash;2.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhotophobia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.78 (0.35\u0026ndash;1.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.51 (0.14\u0026ndash;1.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeizure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (38%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.05 (0.44\u0026ndash;2.49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.68 (0.18\u0026ndash;2.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBreathing difficulty\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (42%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28 (41%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.73 (0.41\u0026ndash;1.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.27 (0.13\u0026ndash;0.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eOR\u0026thinsp;=\u0026thinsp;odds ratio, CI\u0026thinsp;=\u0026thinsp;confidence interval,\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003e association between clinical manifestations of heterozygote gene carriers and homozygote wild gene carriers,\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003e association between clinical manifestations of homozygote polymorphic gene carriers and homozygote wild gene carriers\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCARD 8\u003c/b\u003e \u003cb\u003epolymorphism among clinically suspected patients for bacterial meningitis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenotyping was performed on whole blood samples collected from 332 eligible patients. All genotypes were found to be in Hardy‒Weinberg equilibrium (1.14, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Among patients with suspected meningitis, the heterozygote (CX) genotype prevailed (n\u0026thinsp;=\u0026thinsp;151; 46%), followed by the wild-type genotype (CC) (n\u0026thinsp;=\u0026thinsp;122; 37%), whereas few patients had homozygote (XX) C10X genotype (n\u0026thinsp;=\u0026thinsp;59; 18%) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A similar genotype distribution was observed among healthy blood donors (CC: 37.4%, CX: 47.7%, and XX: 15.1%). A slight difference in the distribution of the homozygous C10X genotype was detected between patients with suspected meningitis and healthy blood donors (OR\u0026thinsp;=\u0026thinsp;1.2 (0.85\u0026ndash;1.5)). The frequency of the variant allele (X) was significantly different between the patients and healthy controls; the allele frequencies were 43.5% and 39.0%, respectively (OR\u0026thinsp;=\u0026thinsp;1.2 (1.00-1.5)) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCARD 8 polymorphism among meningitis suspected patients and healthy controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll meningitis suspected cases\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHealthy controls\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR (95%CI)\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e122 (36.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e734 (37.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e151 (45.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e934 (47.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59 (17.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e296 (15.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.2 (0.85\u0026ndash;1.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eAllelic frequency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e273 (56.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2402 (61.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e210 (43.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1526 (39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.2 (1.00-1.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eOR\u0026thinsp;=\u0026thinsp;odds ratio, CI\u0026thinsp;=\u0026thinsp;confidence interval\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOur data revealed a significantly higher frequency of fever, headache, neck stiffness and vomiting in patients with the heterozygous C10X genotype than in patients with the wild- genotype, with odds ratios of 3.04 (1.60\u0026ndash;5.78) for fever, 2.53 (1.37\u0026ndash;4.68) for headache, 2.26 (1.27\u0026ndash;3.99) for neck stiffness and 1.80 (1.08\u0026ndash;2.99) for vomiting (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, higher frequent manifestations such as fever, headache, nausea, vomiting, high respiratory rate, and altered mental status and cautiousness were more common among patients with homozygous C10X gene than wild type gene carriers.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCARD8\u003c/b\u003e \u003cb\u003epolymorphisms among laboratory-confirmed meningitis patients\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOut of the 400 CSF samples cultured for bacterial growth, only 7 samples showed bacterial growth for \u003cem\u003eN. meningitidis (n\u0026thinsp;=\u0026thinsp;4)\u003c/em\u003e and \u003cem\u003eS. pneumoniae (n\u0026thinsp;=\u0026thinsp;3)\u003c/em\u003e. On the other hand, 39 samples became positive for \u003cem\u003eN. meningitidis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;10) and \u003cem\u003eS. pneumoniae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;29) using RT‒PCR. However, CARD8 polymorphism was done on 25 RT-PCT positive samples (\u003cem\u003eN. meningitidis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;8) and \u003cem\u003eS. pneumoniae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;17)), and 14 positive samples were excluded from the analysis due to incomplete data.\u003c/p\u003e \u003cp\u003eThe C10X polymorphism was compared among laboratory-confirmed meningitis patients (n\u0026thinsp;=\u0026thinsp;25), laboratory negative clinically suspected patients (n\u0026thinsp;=\u0026thinsp;307) and healthy controls (n\u0026thinsp;=\u0026thinsp;1,964). The homozygote C10X polymorphism was more prevalent among laboratory-confirmed meningitis patients [6/25 (24%)] and laboratory negative clinically suspected patients [53/307 (17%)] than in healthy blood donor controls [n\u0026thinsp;=\u0026thinsp;296/1964 (15%)]; odds ratios of 1.6 (0.53-5) and 1.8 (0.64\u0026ndash;5.5), respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, heterozygous C10X polymorphisms prevailed among laboratory-confirmed and laboratory negative but clinically suspected meningitis patients (44% and 45.6%, respectively), than healthy controls [OR: 1.12 (0.43\u0026ndash;2.87) and 1.08 (0.43\u0026ndash;2.7)], respectively (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of CARD 8 gene polymorphism among laboratory confirmed meningitis patients, laboratory negative but clinically suspected meningitis patients and healthy controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLaboratory confirmed meningitis patients\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;25\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLab negative/clinically suspected meningitis patients\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;307\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOR (95%CI) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHealthy control\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;1964\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR (95% CI) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGenetic distribution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e114 (37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e734 (37.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e140 (45.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.12 (0.43\u0026ndash;2.87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e934 (47.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.08 (0.43\u0026ndash;2.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53 (17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.6 (0.53-5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e296 (15.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.8 (0.64\u0026ndash;5.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAllelic frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27 (54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e368 (60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2402 (61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23 (46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e246 (40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.27 (0.7\u0026ndash;2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1526 (39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.34 (0.76\u0026ndash;2.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eOR\u0026thinsp;=\u0026thinsp;odds ratio, CI\u0026thinsp;=\u0026thinsp;confidence interval,\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003e association between laboratory confirmed bacterial meningitis cases with healthy controls,\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003e association between no bacteria identified, clinically diagnosed meningitis suspected cases with healthy controls\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of our data revealed that 67% of the culture-confirmed patients and 37% of the PCR-confirmed patients were heterozygous C10X carriers, with the ORs of 3.1 (0.36-29) and 0.8 (0.3\u0026ndash;2.3), respectively compared to healthy controls (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similarly, the allele frequency of the X variant was greater in culture-confirmed meningitis patients and in PCR-confirmed patients, than healthy controls, respectively [OR:1.6 (0.5-5) and 1.3 (0.7\u0026ndash;2.4)], respectively (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenetic distributions of CARD 8 polymorphism among culture positive meningitis patients, PCR positive but culture negative meningitis patients, and Healthy controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCulture positive meningitis patients\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003cp\u003e(95% CI) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClinically diagnosed meningitis patients\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;307\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003cp\u003e(95% CI) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePCR positive culture negative patients N\u0026thinsp;=\u0026thinsp;19\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003cp\u003e(95% CI) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHealthy control\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;1964\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGenetic distribution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e114 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e734 (37%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.3 (0.36-29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e140 (46%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.14 (0.36-29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.8 (0.3\u0026ndash;2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e934 (48%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.1 (0.13-35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5 (0.2-5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8 (0.6\u0026ndash;5.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e296 (15%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAllelic frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e368 (60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21 (55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2402 (61%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5 (0.5\u0026ndash;4.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e246 (40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.6 (0.5-5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17 (45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.3 (0.7\u0026ndash;2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1526 (39%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eOR, odds ratio, CI, confidence interval\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ea\u003c/sup\u003e association between culture positive meningitis patients with lab negative but clinically suspected meningitis patients\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003eb\u003c/sup\u003e association between culture positive and healthy control\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ec\u003c/sup\u003e association between culture negative PCR positive patients with healthy control\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSpecies-specific analysis was performed on samples identified by PCR. As a result, the genotype distributions of C10X polymorphisms and homozygous C10X carriers were significantly greater in \u003cem\u003eS. pneumoniae\u003c/em\u003e-positive patients than in healthy controls [OR: 1.8 (0.9\u0026ndash;3.5) and 3.1 (0.8\u0026ndash;12)], respectively (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenetic distribution of CARD8 polymorphism among meningitis patients with \u003cem\u003eNeisseria meningitidis\u003c/em\u003e and \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, laboratory negative but clinically suspected meningitis patients, and healthy controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNeisseria meningitidis\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003cp\u003e(95% CI) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStreptococcus pneumoniae\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;17\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003cp\u003e(95% CI) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo bacteria clinical diagnosed\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;307\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003cp\u003e(95% CI) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHealthy controls\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;1964\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGenetic distribution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (24%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e114 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e734\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (38%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (0.13\u0026ndash;0.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.6 (0.5\u0026ndash;5.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e140 (46%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.6 (0.5\u0026ndash;5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e934\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (13%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.61 (0.1\u0026ndash;5.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.7 (0.7\u0026ndash;10.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e53 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.1 (0.8\u0026ndash;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e296\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAllelic frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (69%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16 (47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e368 (60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2402 (61%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (31%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.71 (0.25-.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.7 (0.8\u0026ndash;3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e246 (40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8 (0.9\u0026ndash;3.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1526 (39%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eOR, odds ratio, CI, confidence interval.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ea\u003c/sup\u003e association between those infected with \u003cem\u003eNeisseria meningitidis\u003c/em\u003e and healthy controls\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003eb\u003c/sup\u003e association between those infected with \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e and no bacterial identified clinically diagnosed meningitis suspected cases\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ec\u003c/sup\u003e association between those infected with \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e and healthy controls\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCompared to those in the healthy control population, C10X polymorphic gene carriers were more prevalent in our cohort of meningitis patients. A slightly greater percentage of female patients were found to be polymorphic gene carriers than male patients, with percentages of 43% and 39%, respectively.\u003c/p\u003e \u003cp\u003eThe present study showed that fever, headache, neck stiffness and vomiting were the dominant clinical characteristics. Moreover, there was statistically significant difference in manifestation of these clinical symptoms among heterozygous gene carriers compared to wild-type gene carriers, which indicates the strong association heterozygote carriership with development of the above-mentioned clinical manifestations. The odds of fever, headache nausea, vomiting, cough, high respiratory rate, altered cautiousness and altered mental status were greater among homozygous, CARD8 (C10X), gene carriers than wild-type gene carriers, which indicates the potential association of homozygote carriership with the mentioned clinical manifestations, which agreed with a previous study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe development of clinical manifestations is mediated by induction of pro-inflammatory cytokines which is regulated by NLRP3 inflammasomes which activates caspase 1 (CASP1) through caspase recruitment domains (CARD). The clinical manifestation in turn is influenced by the genetic make-up of these components of the host [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which might explain the enhanced clinical manifestation in polymorphic gene carrier observed in this study.\u003c/p\u003e \u003cp\u003eOther studies indicated that single nucleotide polymorphisms (SNPs) in different components, or regulators, of this pathway have been associated with enhanced clinical manifestations in different infective conditions including hepatitis C, TB and other bacterial infections. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe implication that a loss-of-function mutation in the CARD 8 gene, which makes it unable to inhibit caspase-1 activation, could be associated with increased NLRP3 inflammasome activity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] was observed in the present study. The wild-type gene was found to be protective against bacterial meningitis in the present study. Our finding agrees with a study conducted by Dennis et al. to identify the effect of genetic variation on susceptibility to bacterial infections, which showed overrepresentation of the wild-type allele in healthy controls [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] in vitro. On the other hand, some findings have not been robustly replicated, suggesting that the effect of relatively frequent C10X polymorphisms might be modest or that associations with additional factors are needed to significantly drive disease outcomes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe data also showed that the heterozygote type of the C10X polymorphism was more dominant among meningitis patients and blood donors, followed by homozygous wild-type gene carriers. The homozygote polymorphic gene carriers were found to be more susceptible to bacterial meningitis than the heterozygote gene carriers and healthy controls. This finding is supported by a previous report by A. Idosa et al., who reported that homozygote carriers are more susceptible to infectious disease than heterozygote carriers are, indicating that healthy C10X homozygous individuals might have a greater risk of developing an infection when exposed to infectious agents [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study also demonstrated that the proportion of C10X gene polymorphisms was relatively greater among patients with identified pathogens than among clinically diagnosed patients and healthy controls. Furthermore, C10X-heterozygote carriers were more prevalent among culture-positive meningitis (67%) than those with suspected nonpathogen-identified meningitis (37%) and healthy controls (47%), with odds ratios of 4 and 3.1, respectively. Previously, Idosa et al. reported that the polymorphism of C10X in the CARD8 gene was significantly more prevalent in patients with bacteremia than in healthy controls and that the prevalence of heterozygote gene distribution was greater among bacteremia patients than among those with negative blood cultures and healthy controls [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe C10X polymorphism causes a nonsense allele, resulting in reduced expression of functional CARD8, which in turn is suggested to result in a loss of its inhibitory effect on caspase 1[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. There are also reports on the polymorphism of CARD8 (C10X) that has been associated with an increased risk of inflammation. Polymorphism of CARD8 contributes to the development of some noninfectious diseases, such as inflammatory bowel disease, gout, rheumatoid arthritis and Alzheimer’s disease, and contributes to susceptibility to and poor disease outcomes in patients with infectious diseases [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A loss-of-function mutation in the \u003cem\u003eCARD8\u003c/em\u003e gene is reported to render \u003cem\u003eCARD8\u003c/em\u003e unable to inhibit caspase-1 activation, which could also be associated with increased NLRP3 inflammasome activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Sarkar A et al reported that caspase-1-deficient mice are protected from diseases involving excessive inflammation, such as bacterial-induced sepsis, and are markedly resistant to the lethal effects of endotoxin [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The C10X genotype has been found to be associated with chronic consequences on human health by resulting in severe autoimmune and autoinflammatory disease, probably due to the long-term effect of an excessive caspase-1 response [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe functional consequences of the C10X polymorphism in infectious diseases do not seem to clear way of mechanism with the involvement of different gene variants. G.V. Paramel et al. reported the discovery of possible isoforms of the CARD8 gene, such as T47, in which its transcription begins downstream of C10X; hence, not affecting the isoform could affect the functional consequences of the C10X polymorphism [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As the review stated, the high rate of homozygous patients with loss-of-function polymorphisms, which appear to be human knockouts, might reflect partial rescue of CARD8 function by alternative splicing, leading to an almost functional full-length protein. Patients who are homozygous for the C10X genotype might therefore have a functional CARD8 protein due to the presence of the T47 isoform [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, to clarify the effect of C10X polymorphisms and gene variants during bacterial meningitis, or other infectious diseases a detailed study on the mechanistic effect of polymorphic genes and the impact of possible isoforms present is needed. The limitations of this study were the small number of laboratory-confirmed meningitis patients which influenced the statistical analysis and limit to do sub-group analysis and it was not managed to measure IL-1β due to logistical and resource constraints.\u003c/p\u003e "},{"header":"Conclusions and recommendations","content":"\u003cp\u003eThe greater proportion of polymorphic C10X in the CARD8 gene in confirmed and suspected meningitis patients than in healthy controls was indicative of a robust inflammatory response due to the polymorphic C10X gene. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than were heterozygote gene carriers and healthy controls. This indicates that the polymorphic C10X gene might not provide better survival conditions by generating a vigorous inflammatory response and that the increased release of proinflammatory cytokines does not always play a protective role unless it is directed by control mechanisms. Detailed studies on the cellular and molecular mechanisms of polymorphic gene variants of the CARD 8 gene will be mandatory to obtain clear insight into its impact on susceptibility to and severity of infectious diseases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCARD8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecaspase recruitment domain-containing protein 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAAU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAddis Ababa University\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEPHI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEthiopian Public Health Institute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAHRI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArmauer Hanssen Research Institute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLRP3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enucleotide-binding domain, leucine-rich\u0026ndash;containing family, pyrin domain\u0026ndash;containing-3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecerebrospinal fluid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-1β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterleukin-1beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSNP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingle nucleotide polymorphisms\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUoGSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUniversity of Gondar Specialized Hospital\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erheumatoid arthritis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe greatly acknowledge staffs from the University of Gondar Hospital, Armauer Hansen Research Institute (AHRI), Ethiopian Public Health Institute (EPHI), \u0026Ouml;rebro University, Sweden, for the unlimited and continuous support provided during the study. We thank the Department of Medical Microbiology/University of Gondar, Addis Ababa University, Orebro University for financial support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported through the research seed money given to support PhD program from the University of Gondar. The laboratory work was done at AHRI and EPHI through in-kind support obtained from the two institutions. Sample collection materials were provided through the in-kind support from \u0026Ouml;rebro University, Sweden.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the Ethical Review Committee of the University of Gondar. Written informed consent was obtained from each study participant and guardians.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed the manuscript and given their consent for its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMB-conception and design, acquisition of data, analysis and wrote manuscript; MM, BA, MA, SF, \u0026Ouml;G support the laboratory work and training; ZT, and BM -statistical analysis; AP, EA, ES, OS and BG involve in the conception and design of the research and editing the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQu C, Wang Y, Wang X, He R, Cao H, Liu B, et al. 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A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease. Am J Hum Genet. 2009;85(2):214\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheung MS, Theodoropoulou K, Lugrin J, Martinon F, Busso N, Hofer M. Periodic Fever with Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome Is Associated with a CARD8 Variant Unable To Bind the NLRP3 Inflammasome. J Immunol Baltim Md. 1950. 2017;198(5):2063\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMihret W, Sletbakk Brusletto B, \u0026Oslash;vsteb\u0026oslash; R, Siebke Troseid AM, Norheim G, Merid Y, et al. Molecular studies of meningococcal and pneumococcal meningitis patients in Ethiopia. Innate Immun. 2019;25(3):158\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai B, Deitch EA, Ulloa L. Novel insights for systemic inflammation in sepsis and hemorrhage. Mediators Inflamm. 2010;2010:642462.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Meningitis, inflammasomes, CARD 8 polymorphisms, Neisseria meningitidis, Streptococcus pneumoniae","lastPublishedDoi":"10.21203/rs.3.rs-4656131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4656131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe severity of infectious disease outcomes is believed to depend on the virulence factors of the pathogen in combination with individuals\u0026rsquo; unique immune response. CARD8 has been suggested to act as a regulator of the NLRP3 inflammasome, a major regulator of the innate immune proinflammatory response, and has been suggested to modulate the host response to common inflammatory diseases. In the present study, the C10X genetic polymorphism in the \u003cem\u003eCARD8\u003c/em\u003e gene was investigated in relation to bacterial meningitis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 400 clinically suspected meningitis patients hospitalized at the University of Gondar Comprehensive specialized Hospital were enrolled in the study. Cerebrospinal fluid (CSF) and blood samples were collected from all enrolled patients for laboratory investigations. The collected CSF was cultured, and all the results obtained from the culture were confirmed using direct RT‒PCR. Genotyping of whole-blood samples was performed for \u003cem\u003eCARD8\u003c/em\u003e gene using a TaqMan assay. The results were compared with apparently healthy controls and with PCR-negative meningitis suspected patients.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf the included patients, 57% were men and the most common clinical signs and symptoms were fever (81%), headache (80%), neck stiffness (76%), nausea (68%), and vomiting (67%). Microbiology culture identified seven patients with bacterial meningitis caused by \u003cem\u003eNeisseria meningitidis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4) and \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3). The RT-PCR revealed 39 positive samples for \u003cem\u003eN. meningitidis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;10) and \u003cem\u003eS. pneumoniae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;29). A total of 332 whole-blood samples were genotyped, with the following results: 151 (45.5%) C10X heterozygotes, 59 (17.7%) C10X homozygotes and 122 (36.7%) wild-type genotypes. The presence of the C10X polymorphism in the \u003cem\u003eCARD8\u003c/em\u003e gene was more prevalent in suspected meningitis patients than in healthy controls (OR 1.2; 1.00-1.5). Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease. The presence of viable or active bacterial infection was found to be associated with the presence of heterozygous C10X carriers.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eA greater proportion of C10X in the \u003cem\u003eCARD8\u003c/em\u003e gene in confirmed bacterial meningitis patients and clinically diagnosed meningitis patients than in healthy controls. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than were heterozygote gene carriers and healthy controls.\u003c/p\u003e","manuscriptTitle":"CARD8 polymorphisms among bacterial meningitis patients in North-West Ethiopia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 08:40:07","doi":"10.21203/rs.3.rs-4656131/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-04T05:51:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-02T23:53:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-02T23:52:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2024-06-28T17:02:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f129d1bd-08d7-4016-9567-a094ef9930df","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:02:21+00:00","versionOfRecord":{"articleIdentity":"rs-4656131","link":"https://doi.org/10.1186/s12879-024-09953-2","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2024-10-01 15:57:38","publishedOnDateReadable":"October 1st, 2024"},"versionCreatedAt":"2024-07-25 08:40:07","video":"","vorDoi":"10.1186/s12879-024-09953-2","vorDoiUrl":"https://doi.org/10.1186/s12879-024-09953-2","workflowStages":[]},"version":"v1","identity":"rs-4656131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4656131","identity":"rs-4656131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}