Prevalence, In Silico Analysis and Possible Molecular Drug Therapy for Specific Meningitis Types

1. Introduction

The current study aims to find out the incidence of meningitis in children admitted with fever and fits, as well as the effects of age, gender, residence, and vaccination status on the prevalence of meningitis. It also aims to determine the interaction mode and binding affinity of the most frequently used antibiotics with specific bacterial strains and their specific proteins in order to design new and effective drugs against the disease. For molecular docking analysis, Pneumolysin (5aoe), Niesserial surface protein A(1p4t), Haemophilus influenza Hia Adhesion (3syj), and alpha C protein (1ywm) were selected. These bacterial proteins were docked with various ligands (drug) such as ceftriaxone, vancomycin, and meropenem using PyRx virtual screening tool.

2. MATERIALS AND METHODS

The current cross-sectional study included 100 febrile seizures children, from newborns to 10-year olds, who were admitted to LUHMS Hospital Hyderabad with symptoms such as neck stiffness, fever, and fits. Any cases with coma, brain tumor, and other neurological disorders were excluded from the study. The patients were monitored on an hourly basis and their vitals (temperature, heart rate, respiratory rate) were checked after every 3-4 hours.

The study was conducted with the approval of the ethical committee of the Institute of Biochemistry, University of Sindh (Jamshoro) (Date: 09.06.2023, Ref. number: IOB/368/2023). A questionnaire was designed to record the demographics of the patients including their age, gender, and residence. Vaccination status was also assessed, that is, if they had been vaccinated first or multiple times. Home-based prophylaxis, taken at the time of prodromal symptoms, was recorded. Brudzinski's sign and Kernig's sign were checked which indicated the presence of meningitis by neck stiffness and involuntary bending of knees and hips. CSF was collected with spinal needle from the subarachnoid spaces. In this regard, 1-5 ml of CSF sample was collected from each patient for physical, chemical, and microscopic examination. Oral and written permission was taken from parents/guardians before the collection of CSF samples. CSF collected through lumber puncture was evaluated for color, volume, turbidity, and chemical tests including protein, glucose, and WBCs, specifically. Blood sample was assessed to examine RBCs, WBCs, hemoglobin, hematocrit, and platelets.

Docking was executed to predict the possible mechanism of meningitis. Moreover, 3D structures of bacterial proteins were obtained via PDB (protein data bank). These included Pneumolysin (5aoe), Niesserial surface protein A (1p4t), Haemophilus influenza Hia Adhesion (3syj), and alpha C protein (1ywm). Pubchem was used to obtain the structures of ligands (ceftriaxone, vancomycin, and meropenem). Ligands were downloaded in 2D-SDF format, while 3D protein structures were downloaded in PDB format. Hetero atoms and water groups were removed using discovery visualizer. The PyRx virtual screening tool was used to calculate binding affinities which were saved as CSV files. Protein ligand interactions were identified with the help of discovery studio. Whereas, 2D interactions and 3D interactions were saved as JPG images.

For statistical analysis, frequency and percentage were calculated for age and gender. Chi-square analysis was done using SPSS to find out the association of various factors with meningitis, with a p-value of ˂ 0.05 considered as significant.

3. RESULTS

A total of 100 patients were incorporated in this study, out of which 32 were diagnosed with different types of meningitis. Moreover, 53 patients showed physical signs and symptoms of the disease but their parents didn't allow the lumber puncture, so their meningitis status was reported as suspected (Table 1).

Table 1. Frequency Distribution of Different Factors Contributing to Meningitis

The remaining 15 patients were diagnosed with encephalitis, which is not categorized under the term meningitis. Therefore, the prevalence of confirmed meningitis cases in this study was 32%.

Out of the 100 patients included in the study, 56% were in the age range of 0-24 months, 22% were in the age range of 25-60 months, and 22% were older than 60 months. Further, 86% of affected patients belonged to rural areas and the ratio of boys (57%) was higher as compared to girls (43%).

The case fatality rate in this study was found to be 18% (18/100) in children having fits and fever, whereas it was 9% (3/32) in children diagnosed with meningitis. Furthermore, 95% patients suffered from fever and fits, although lumbar puncture was done only in 32% of cases. An elevated ratio of suspected meningitis (53%) was observed in the current study due to hesitation in performing the lumbar puncture. Surprisingly, the ratio of vaccinated patients was higher (48%) than non-vaccinated (35%) or partially vaccinated (17%) patients. Out of 100 patients, 64 were admitted with fits (˂15 min duration), whereas 36 with fits (˃15 min duration). Most patients were given ceftriaxone along with vancomycin, whereas only 5 were treated with meropenem. Therefore, ceftriaxone was established as the drug of choice for meningitis treatment. Out of the 32 children diagnosed with the disease, 15 (47%) were diagnosed with bacterial, 5 (16%) with viral, and 3 (9%) with TBM, while 9 (28%) patients were reported as partially treated (All results are shown in Table 1).

Patients diagnosed with the disease showed low CSF glucose level and high protein level in their CSF DR, which is an indication of bacterial meningitis (Table 2). While identifying the blood parameters via CBC, a higher neutrophil value (>80%) was seen as an indication of bacterial meningitis (Table 2). Chi square test was performed to find out the association of various factors with the disease. No statistical significance was observed between gender and fits duration. The disease was found to be more prevalent among infant children with statistical significance (p-value = 0.01) (Figure 1). Conversely, vaccination status of children showed no statistical significance.

Table 2. CSF and CBC Parameters

Figure 1. Statistical Association of Various Parameters with Meningitis

**p-value ˂ 0.05

Docking results manifested that ceftriaxone showed the greatest affinity for bacterial proteins, namely Haemophilus influenza Hia Adhesin (3syj) and Pneumolysin (5aoe), with the lowest K_d values of -9 and -8.6, respectively. The lower the K_d value, the better is the binding affinity of a ligand with its target. Binding affinity with Haemophilus influenza Hia Adhesin (3syj) and Pneumolysin (5aoe) increased in the following order: ceftriaxone > vancomycin > meropenem. Although, in the case of Neisserial surface protein A (1p4t), it increased in the following sequence: vancomycin > meropenem > ceftriaxone (Table 3).

Table 3. Binding Affinities of Drug Interactions with Bacterial Proteins

The 2D diagram of Fig 2a shows that ceftriaxone interacted with bacterial protein 3syj by forming hydrogen bonds with threonine 513,493 (Figure 2A). Both ceftriaxone and vancomycin formed hydrogen bonds with arginine 501, glutamic acid 42,522, and asparagine 113,43 of that protein (Figure 2A, 3B). Vancomycin and meropenem showed the highest binding affinity for protein 1p4t, with K_d values of-6.4 and -5.9 respectively, by forming hydrogen bonds with alanine 149 and arginine 90 (Figure 3A, 4A). Ceftriaxone showed its second-highest binding affinity (-8.6) with protein 5aoe by forming a hydrogen bond with glutamic acid 35 (Figure 2B).

Figure 2. 2D and 3D Graphical Representation of Drug Binding Modes in Bacterial Proteins (A) Ceftriaxone with 3syj (B) Ceftriaxone with 5aoe

Figure 3. 2D and 3D Graphical Representation of Drug Binding Modes in Bacterial Proteins (A) Vancomycin with 1p4t (B) Vancomycin with 3syj

Figure 4. 2D and 3D Graphical Representation of Drug Binding Modes in Bacterial Proteins A) Meropenem with 1P4t (B) Meropenem with 3syj

4. DISCUSSION

A febrile seizure is a convulsive disorder that affects young children, usually followed by fever. A similar study was conducted in Karachi and Quetta and the cases were 19% and 38%, respectively. This study was conducted to find the incidence of meningitis in children presenting with fever and fits in Hyderabad, Pakistan. The results align with the understanding that meningitis occurs mostly in children because of their weak immunity [3, 7].

Most patients belonged to rural areas because of limited healthcare access and excessive tobacco usage in the vicinity, which causes upper respiratory tract infection, resulting in pneumonia [8]. A higher ratio of male patients indicates low immunity in them as compared to female patients [3]. This is because the estrogen hormone in women boosts type 1 T helper cell's level, which strengthens the immune system against bacterial pathogens [9]. Deaths are caused by several pathogens including Streptococcus pneumonia and Mycobacterium tuberculosis. These are deadly and known for their high mortality rate [10].

In the current study, the majority of cases were of bacterial meningitis because pathogens such as Neisseria meningitidis, Haemophilus influenza, and Streptococcus pneumonia are responsible for the majority of bacterial meningitis cases [11]. All of these can easily spread through the nasopharyngeal pathway and then move towards blood and meninges after several hours [12]. A higher neutrophil value (>80%) was seen as an indication of bacterial infection. During normal conditions, the central nervous system (CNS) does not have significant neutrophil immune surveillance; however, during bacterial meningitis, there is a huge mobilization of neutrophils across the blood-brainbarrier aimed to eradicate the bacteria [13]. Cryptococcus neoformans causes the dysfunction of the blood-brain barrier and the bacteria enters CSF, where it increases glycolysis resulting in low glucose levels in the CSF of the infected patients [14].During bacterial infection, protein level in CSF rises because of the escalated number of replicating bacteria and body cells fighting the infection, which might be the reason for the high concentration of protein. Previous studies reported higher CSF protein concentrations in bacterial meningitis patients, as compared to viral meningitis patients [15]. In a study about meningitis prevalence in Islamabad, Pakistan, CSF protein levels were also found to increase (> 80 mg/dl) in 85.5% of bacterial meningitis cases[16].However, in the current study, out of 32 patients 31% had a higher protein level and 28% had a lower glucose level. Moreover, 95% of patients experienced fits due to complications such as edema, hydrocephalus, and increased intracranial pressure in the brain tissue [17]. Furthermore, the patients in this study were all children which made it more difficult for the family to allow lumbar puncture. The increased ratio of meningitis in vaccinated patients may be due to their vaccination only by the pentavalent vaccine (available free of cost in Pakistan), instead of the special meningococcal and pneumococcal vaccine [18].

To explore the underlying pathogenesis mechanism of meningitis, the docking interactions of commonly used drugs were analyzed against four important bacterial proteins known to cause meningitis. The lowest kd values were shown by ceftriaxone against Haemophilus influenza Hia Adhesin (3syj) and Pneumolysin (5aoe). Haemophilus influenza Hia Adhesin is a protein of the bacteria Haemophilus influenza. It mediates bacterial attachment to host respiratory epithelial cells, an essential step in the process of colonization [19]. Whereas, Pneumolysin is a key player in pneumococcal meningitis, promoting bacterial invasion. However, in the case of Neisserial surface protein A (1p4t), the lowest kd values were found for vancomycin. Neisserial surface protein A (1p4t) is a meningococci that uses its surface protein known as Neisserial surface protein A (NspA) to bind to a host complement inhibitory protein, that is, factor H (fH). Enhanced complement activation occurs on the bacterial surface and increased complement-dependent killing of meningococci can be seen as a result of the loss of NspA [20]. Figures 2-4 show the interaction sites of selected drugs with amino acids of bacterial protein. Therefore, it can be inferred from the docking kd values that among all drugs, ceftriaxone might be the drug of choice if meningitis is caused by 3syj or 5aoe. However, either vancomycin or meropenem may be preferred over ceftriaxone if meningitis is caused by Neisserial surface protein A (1p4t).

5. Conclusion

The current study revealed 32% prevalence of meningitis with a 9% mortality rate. Bacterial meningitis was mostly found with higher neutrophil count, increased WBC count, low glucose levels, and elevated protein values. Age was found to be a statistically significant factor associated with meningitis, whereas gender, fits duration, and vaccination status didn't seem to affect meningitis prevalence. Molecular docking analysis suggested the use of ceftriaxone for Haemophilus influenzae and pneumococcal meningitis, as well as the use of vancomycin or meropenem for meningococcal meningitis. These predicted protein-drug interactions can be confirmed in future studies via molecular docking stimulations for a better understanding of the mechanism of meningitis.

Conflict of Interest

The author of the manuscript has no financial or non-financial conflict of interest in the subject matter or materials discussed in this manuscript.

Data Availability Statement

The data associated with this study will be provided by the corresponding author upon request.

Funding details

This research did not receive grant from any funding source or agency.