Abstract
Background
The severity of infectious disease outcomes is dependent on the virulence factors of the pathogen and the host immune response. CARD8 is 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 Hospital were enrolled in the study. Cerebrospinal fluid (CSF) and blood samples were collected 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 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 7 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 genotypes. The polymorphic gene carriers among laboratory confirmed, clinically diagnosed meningitis and healthy controls were 23(46%), 246(40%), and 1526(39%), respectively with OR = 1.27 (0.7–2.3) and OR = 1.34 (0.76–2.4). 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 heterozygote gene carriers and healthy controls.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12879-024-09953-2.
Keywords: 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 h 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. Interleukin-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. Interleukin 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 [14, 15], 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 [15]. More specifically, CARD8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1β expression and nuclear factor (NF)-κB activation [16, 17]. 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 [18]. The effect of C10X polymorphism has been studied in relation to several different diseases [19], including non-infectious autoimmune disorders, like rheumatoid arthritis [20], in neurological disorders [21], and suggested aggravating the atherosclerotic process by promoting inflammation [22] as well as in promoting susceptibility and severity of infectious diseases, involving bacteraemia and extrapulmonary tuberculosis [23, 24].
A link between human polymorphisms regarding the gene IL1RN (encoding IL-1 receptor antagonist; IL-1Ra) per se [25] or in combination with IL1B 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, 27], 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 setting, design, and period
A retrospective hospital based unmatched case control study was carried out from October 2015 to October 2019 just prior to the start of the COVID-19 pandemic at University of Gondar Comprehensive Specialized Hospital which is located in Gondar town, Ethiopia. The hospital is one of the largest teaching hospitals in the Amhara National Regional state serving more than seven million people coming from Amhara, Tigray and Benishangul Gumuz regions.
Sample size and sampling technique
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 [23], including a 5% drop-out rate. A convenient sampling technique was used and clinically diagnosed patients with signs and symptoms suggestive for meningitis were recruited from OPD and clinical wards. All age groups willing to participate were included in the study.
Demographic characteristics and clinical data collection
Demographic 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 [28]. 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 (5 ml) were collected from 336 study participants in heparin-containing tubes and stored at -20oC 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% CO2-enriched atmosphere for 72 h [29]. 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 properly labelled and coded following the standard operating procedure set for specimen collection. To ensure the accuracy of the laboratory method, 5% of the prepared culture media were randomly selected and incubated aerobically for 24 h at 37 °C to check the sterility of the prepared culture media [30].
Real-time PCR, genotyping and CARD8 polymorphisms
DNA was extracted from 300 µl of blood using Mega Bio Genomic DNA purification Kit (BIOER TECHNOLOGY, Hangzhou, China), on the Bioner Gene pure profully automated Nucleic Acid Purification System (NPA-32P) (BIOER TECHNOLOGY, Hangzhou, China), according to the manufacturer’s instruction [31]. Confirmatory testing targeting the three most common bacterial pathogens causing acute bacterial meningitis, namely N. meningitides, S. Pneumoniae and Haemophilus influenza type B was performed using a real time PCR (RT-PCR) on a Bio-Rad cfx96 real time detection system (Bio-Rad Laboratories, Inc.,CA, USA) using PerfecCTa qPCRToughMix (Quanta Bioscciences, Gaithersburg, MD USA) [32]. The bacteria were typed at the genetic level using primers and probes targeting the superoxide dismutase enzyme (Sod C) encoding gene for N. meningitides, the lytA enzyme- encoding gene for S. pneumoniae, and hpd protien D genefor 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 (Bio-Rad Laboratories, Inc., CA, USA) followed by allelic discrimination to evaluate the frequencies of the different alleles, as previously described [23]. Briefly, 2 µL of genomic DNA was amplified in a final 10 µL reaction volume containing 5 µL of TaqPath ProAmp Genotyping Master Mix (Applied Biosystems, TX, USA), 0.5 mL of 20x Assay TaqPath SNP Genotyping Mix, and predesigned primers and probes [24, 33]. 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 [23].
Data analysis
The completeness and consistencies of the collected data were checked. The data were imported into (into Epi-data, version 4.6 and analyzed using Statistical Package for Social Sciences (SPSS) computer software, version 25.0.). Further analysis was done suing R statistical software, version 4.4.1. Descriptive statistics were run using frequencies and percentages for all the categorical data and median with IQR for Age variable. To assess factors associated with CARD8 polymorphisms, both unadjusted and adjusted binary logistic regressions were used. The p-values resulted from this regression were considered to identify the candidate determinants for the final model. Hardy–Weinberg equilibrium was also calculated to determine whether a population’s observed genotype frequencies differ significantly from the frequencies expected under Hardy-Weinberg equilibrium in accordance with standard procedures using chi-square analysis. All statistical tests were run with a significance threshold of 5%.
Ethical considerations
The study was conducted in accordance with the Declaration of Helsinki. 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
Demographic 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 [23]. 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.
Demographic characteristics and distribution of CARD 8 polymorphism among suspected meningitis patients
| Variables | Allelic frequency | ||||||
|---|---|---|---|---|---|---|---|
| Age | N (%) | Homozygote wild (CC) N = 122 |
Heterozygote (CX) N = 151 |
Homozygote polymorphic (XX) N = 59 |
C | X | |
| Age | 1–10 | 7 | 2 (28.6%) | 3 (43%) | 2 (28.6%) | 7 (50%) | 7 (50%) |
| 11–19 | 34 | 13 (38.2%) | 16 (47%) | 5 (15%) | 42 (62%) | 36 (38%) | |
| 20–29 | 84 | 34 (40.5%) | 40 (47.6%) | 10 (12%) | 107 (64%) | 60 (36%) | |
| 30–39 | 87 | 31(35.6%) | 36 (41.4%) | 20 (23%) | 98 (56%) | 76 (44%) | |
| 40–49, | 38 | 13 (34.2%) | 18 (47.4%) | 7 (18.4%) | 43 (58%) | 32 (42%) | |
| 50–59 | 45 | 13 (29%) | 24 (53.4%) | 8 (17.8%) | 50 (56%) | 40 (44%) | |
| 60–69 | 28 | 14 (50%) | 9 (32%) | 5 (18%) | 36 (66%) | 19 (34%) | |
| > 69 | 9 | 2 (22%) | 5 (55.6%) | 2 (22%) | 9 (50%) | 9 (50%) | |
| Sex | Male | 188 | 70 (37%) | 92 (49%) | 26 (14%) | 229 (62%) | 144 (39%) |
| Female | 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 N (%) |
Heterozygote N = 151 N (%) |
OR (95% CI) a | Homozygote polymorphic N = 59 N (%) |
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). A slightly greater percentage of female patients were found to be polymorphic gene carriers than male patients, with percentages of 43% and 39%, respectively. 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 (0.85–1.67)), p = 0.293. The frequency of the variant allele (X) was significantly different between the patients and healthy controls; the allele frequencies were 40.5% and 39.0%, respectively (OR = 1.2 (0.906–1.267)), p = 0.417 (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 (%) |
P-Value |
|---|---|---|---|---|
| CC | 122 (37) | 734 (37.4) | 1 | |
| CX | 151 (46) | 934 (47.7) | 0.973(0.752, 1.260) | 0.833 |
| XX | 59 (18) | 296 (15.1) | 1.2(0.850, 1.676) | 0.293 |
| Allelic frequency | ||||
| C | 395 (59.5) | 2402 (61.2) | 1 | |
| X | 269 (40.5) | 1526 (39) | 1.2(0.906, 1.267) | 0.417 |
| 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), p = 0.382 and 1.8 (0.64–5.5), p = 0.254 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), p = 0.811 and 1.08 (0.43–2.7)), p = 0.868 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 = 25) N (%) |
Lab negative/clinically suspected meningitis patients(N = 307) N (%) |
OR (95%CI) a | P-value | Healthy control(N = 1964) N (%) |
OR (95% CI) b | P-value | ||
|---|---|---|---|---|---|---|---|---|
| Genetic distribution | CC | 8 (32.0) | 114 (37.1) | 1 | 734 (37.4) | 1 | ||
| CX | 11 (44.0) | 140 (45.6) | 1.120(0.430, 2.87) | 0.811 | 934 (47.6) | 1.081(0.43, 2.67) | 0.868 | |
| XX | 6 (24.0) | 53 (17.3) | 1.601(0.53, 5.01) | 0.382 | 296 (15.1) | 1.80(0.64, 5.49) | 0.254 | |
| Allelic frequency | C | 27 (54.0) | 368 (59.9) | 1 | 2402 (61.2) | 1 | ||
| X | 23 (46.0) | 246 (40.1) | 1.284(0.713, 2.293) | 0.399 | 1526 (38.8) | 1.341 (0.760, 2.346) | 0.304 | |
OR = odds ratio, CI = confidence interval,
a association between laboratory confirmed bacterial meningitis cases with healthy controls adjusted for Age and Sex,
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), p = 0.306 and 0.8 (0.3–2.3), p = 0.653 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), p = 0.433 and 1.3 (0.7–2.4)), p = 0.460 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) N (%) |
OR (95% CI) a | P-valuea | OR (95% CI) b | P-valueb | PCR positive culture negative patients (N = 19) N (%) |
OR (95% CI) c | P-valuec | ||
|---|---|---|---|---|---|---|---|---|---|
| Genetic distribution | CC | 1 (16.7) | 1 | Ref. | 1 | Ref. | 7(36.8) | 1 | Ref. |
| CX | 4 (67) | 3.266(0.473, 64.480) | 0.294 | 3.143 (0.36, 29.563) | 0.306 | 7(37) | 0.804(0.3, 2.305) | 0.653 | |
| XX | 1 (16.7) | 2.134(0.082, 55.256) | 0.596 | 2.480 0.098 62.845 | 0.521 | 5(26.3) | 1.771(0.521, 5.593) | 0.332 | |
| Allelic frequency | C | 6 (50.0) | 1 | Ref. | 1 | Ref. | 21(55.3) | 1 | Ref. |
| X | 6 (50.0) | 1.494(0.460, 4.850) | 0.494 | 1.604(0.502, 5.040) | 0.433 | 17(44.7) | 1.304(0.702, 2.419) | 0.460 | |
OR, odds ratio, CI, confidence interval
a association between culture positive meningitis patients with lab negative but clinically suspected meningitis patients adjusted for age and sex
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), p = 0.116 and 3.1 (0.8–12)), p = 0.127 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 | P-valuea | Streptococcus pneumoniae (N = 18) | OR (95% CI) b | P-valueb | OR (95% CI) c | P-valuec | ||
|---|---|---|---|---|---|---|---|---|---|
| Genetic distribution | CC | 4 (50.0%) | 1 | Ref. | 4(22.2%) | 1 | Ref. | 1 | Ref. |
| CX | 3 (37.5%) | 0.589(0.116, 2.682) | 0.490 | 9(50.0%) | 1.820(0.576, 6.868) | 0.330 | 1.768(0.573, 6.547) | 0.345 | |
| XX | 1 (12.5%) | 0.620(0.032, 4.212) | 0.669 | 5(27.8%) | 3.107(0.802, 12.040) | 0.127 | 3.100(0.815, 12.598) | 0.093 | |
| Allelic frequency | C | 11 (68.8%) | 1 | Ref. | 17 (47.2%) | 1 | Ref. | 1 | Ref. |
| X | 5 (31.2%) | 0.715(0.225, 1.971) | 0.536 | 19 (52.8%) | 1.820(0.904, 3.505) | 0.116 | 1.759(0.910, 3.429) | 0.092 | |
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 adjusted for age and sex
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 meningitis patients of the present study. 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. This indicates the association of being homozygote gene carrier to manifest the mentioned clinical symptoms, which is similar with a previous study [23].
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 [34, 35], 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 [23, 34].
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 [36] 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 previous study conducted identifying the effect of genetic variation on susceptibility to bacterial infections, which showed overrepresentation of the wild-type allele in healthy controls [37] 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 [18].
The present study has shown the heterozygote 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 which 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 [23].
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 3.3 and 3.1, respectively. This finding is in agreement with a previous study assessing the polymorphism of C10X in the CARD8 gene with bacteremia [23].
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 [36, 37]. 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 [23, 38]. 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 [24]. Another study 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 [39]. 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 [40].
The functional consequences of the C10X polymorphism in infectious diseases do not seem to have clear way of mechanism with the involvement of different gene variants. A previous study conducted on the role of genetic alteration in health and disease 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 [19]. 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 [16]. 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. The focus of the preset study is on CARD8 and the influence of other gene polymorphisms such as NLRP3 with disease outcomes has not been done. Survival analysis was not possible to conduct due to lack of supporting data.
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.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
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.
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
Author contributions
MB-conception and design, acquisition of data, analysis and wrote manuscript. MM, BA, MA, SF, ÖG support the laboratory work and trainingZT, and BM -statistical analysisAP, EA, ES, OS and BG took part in the conception and design of the research and editing the final manuscript.
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.
Data availability
The datasets generated and/or analysed during the current study are available in the NCBI Clinvar database repository, with Clinvar Accession – RCV001514794.4.
Declarations
Ethical approval
The study was conducted in accordance with the Declaration of Helsinki. 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.
Consent for publication
Not applicable.
Clinical trial number
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Olof Säll shared last authorship.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated and/or analysed during the current study are available in the NCBI Clinvar database repository, with Clinvar Accession – RCV001514794.4.