Course:PATH417:2021W2/Case1

Path 417 Cases
Instructor
David Harris Agatha Jassem Ramon KleinGeltink Inna Sekirov
Case Projects
Case 1
Case 2
Case 3
Case 4

Case 1. A Stiff Neck

18-year-old Mary has just moved into the dormitory at her university. One day, her roommate finds her lying in bed under her sheets. She is complaining of fever, chills, bad headache and a stiff neck. She is staying under the covers because the light is hurting her eyes. Her roommate calls 911 and an ambulance takes Mary to the local hospital.

The emergency room physician asks Mary about her recent vaccinations and she reports that she has not had any since she was in elementary school. The physician documents a fever of 39.2°C and low blood pressure. He sends blood and cerebral spinal fluid to the Microbiology Laboratory. She is started immediately on intravenous antibiotics. Mary’s blood and cerebral spinal fluid grow Neisseria meningitidis and she is diagnosed with meningococcal meningitis.

1. The Body System

(i) Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).

The signs described in this case include a fever and low blood pressure. Both of these indicators are detected and measured objectively by a healthcare professional, and therefore are considered signs in this case.

The symptoms described by Mary include fever, chills, bad headache, a stiff neck, and sensitivity to light. The fever, chills, bad headache, and stiff neck are all explicitly reported by Mary, while her behaviour, in this case hiding under the covers to stay away from light, indicates that she is experiencing sensitivity to light. Mary’s fever can be considered both a sign and a symptom in this case. Mary’s subjective experience of a high internal temperature is a symptom, while the clinician’s measurement of her temperature objectively confirms Mary’s high internal temperature, making the fever a sign as well.

A key medical history finding is that Mary has not had any vaccinations done since elementary school. This is significant due to the role of meningococcal vaccines, typically received at age 11-12, in reducing the incidence of meningococcal disease caused by N. meningitidis. With regards to meningococcal meningitis, it is also significant that Mary had recently moved into her University dormitory, because there is a greater incidence of meningococcal disease in young adults who have recently changed residence (2). However, it's unclear if that was reported to the emergency room physician.

Table 1. Typical Biochemical Changes Seen in Cerebrospinal Fluid Secondary to Bacterial Meningitis (6)

The emergency room physician collects samples of blood and cerebral spinal fluid (CSF) to be sent to the laboratory for testing. They do this to determine the presence of meningococcal disease. The signs and symptoms Mary is presenting with are strongly associated with meningitis; however, meningitis can be caused by a number of pathogens, including various types of bacteria, viruses, and fungi. The ‘gold standard’ of meningococcal meningitis diagnosis is microbiological isolation of N. meningitidis from a sterile body fluid, which could be CSF or blood (4).  If N. meningitidis is found in the sample, laboratory technicians will be able to culture (reproduce) the bacteria from the CSF/blood sample (1,5). Indeed, Mary’s CSF samples grow N. meningitidis, confirming that she has meningococcal meningitis. Using serological samples for growth cultures can also be used in determining the serogroup of N. meningitidis causing the infection, which may be used for epidemiological surveillance purposes. CSF also provides information regarding the infection outside of the cell culture. In patients with meningococcal meningitis, the CSF often shows a lower glucose level, an increased number of white blood cells and an increased number of proteins (3). As bacteria break down glucose in the CSF, where they primarily reside, a low CSF/serum glucose ratio can also inform about bacterial infection in CSF (3). White cell counts and Gram staining for bacteria should be available within an hour of receiving the CSF sample in the lab (3) The course of treatment for meningococcal meningitis varies from treatment used for other forms of meningitis, including other forms of bacterial meningitis. Therefore, the physician takes blood and CSF samples and sends them to the lab to determine if N. meningitis specifically is causing Mary’s condition.

There are, however, numerous limitations to using serological cultures in diagnosing meningococcal meningitis. Culturing the bacteria can take days, and growth often fails due to the patient receiving pre-emptive antibiotics or because the specimens have been inappropriately transported. Polymerase Chain Reaction (PCR) tests are used to address these limitations, as PCR can rapidly identify the organism based on genetic factors and is not affected by the prior administration of antibiotics (4).

References

1. Centers for Disease Control and Prevention. (2019, May 31). Meningococcal disease diagnosis and treatment. Centers for Disease Control and Prevention. Retrieved January 21, 2022, from https://www.cdc.gov/meningococcal/about/diagnosis-treatment.html
2. Kvalsvig, A. J. (2003). The immunopathogenesis of meningococcal disease. Journal of Clinical Pathology, 56(>6), 417–422. https://doi.org/10.1136/jcp.56.6.417
3. Goering, R. V., Dockrell, H. M., Zuckerman, M., & Chiodini, P. L. (2019). Chapter 25. In MIM's Microbiology and Immunology (Sixth, pp. 316–318)
4. Mola, S. J., Nield, L. S., & Weisse, M. E. (2008). Treatment and prevention of N meningitidis infection. Infections in Medicine, 25(3), 128-133.
5. NORD (National Organization for Rare Disorders). (2015, May 13). Meningococcal meningitis. Meningococcal Meningitis. Retrieved January 21, 2022, from https://rarediseases.org/rare-diseases/meningococcal-meningitis/#:~:text=Meningococcal%20meningitis%20is%20a%20form,or%20develop%20gradually%20(subacute).
6. Huang FS, Brady RC, Mortensen J. 2019. Bacterial Meningitis, Fever, Headache and A Stiff Neck…Looking Pretty Sick. Introduction to Clinical Infectious Diseases 245-257.

(ii) Which body system is affected? In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.

The emergency room physician has diagnosed Mary with meningococcal meningitis – that is, inflammation of the meninges (lining of the brain and spinal cord) and subarachnoid space due to the bacterium Neisseria meningitidis. Therefore, the body system most affected in Mary’s case is the central nervous system (CNS).

The meninges are composed of three layers - the dura mater (blue, Figure 1), arachnoid mater (purple, Figure 1), and the pia mater (yellow, Figure 1). The space between the arachnoid and pia maters is known as the subarachnoid space and is filled with cerebrospinal fluid (CSF) and blood vessels. During invasive meningococcal disease, it is the subarachnoid space that is readily colonised by N. meningitidis. Subsequently, the inflammatory response mounted against the pathogen is responsible for disturbing the homeostatic structure and function of the meninges and the CNS.

Figure 1. 3D models of meninges (1)

Figure 2. Overview of Neisseria meningitidis transmission, carriage state, invasion and virulence factors of the meningococcal outer membrane (2)

It has been hypothesized that the meninges, previously believed to be limited to a protective role in the CNS, may also affect its physiology and pathology (3). In fact, rather than simply forming a layer over top of the brain, the meninges have been shown to penetrate the neural tissues. They also play a role in maintaining the homeostasis of the surrounding tissue via secretion of neurotrophic factors FGF-2, CXCL12, retinoic acid, and SDF-1 (3). The meninges also protect the CNS by tightly anchoring it to nearby skeletal landmarks, stabilising it and preventing movement. CSF acts to cushion the CNS, providing a medium for it to ‘float’ within the protective space of the meninges (3). It also acts as a transport pipeline, providing essential nutrients to the CNS (4).

Once inside the subarachnoid space, N. meningitidis is able to rapidly multiply in an uncontrolled manner due to the lack of complement factors and immunoglobulins, which are typically unable to cross the blood-brain barrier (5). However, the host may still mount a local immune response against the pathogen, leading to inflammation and influx of leukocytes into the site of infection (5). This early inflammatory response can also contribute to further damage of the blood-brain barrier (BBB), leading to increased infiltration of the pathogen into the favourable environment of the subarachnoid space (6).

Invasion of the subarachnoid space occurs when the pathogen bypasses the BBB. This selective and largely impermeable membrane is composed of brain microvascular endothelial cells (BMECs), and protects us from toxins and inflammatory factors secreted from host immune cells (7). BMECs take part in paracellular/transcellular signalling and maintain the ECM (7). Our BMECs also interact with differentiated brain cells such as astrocytes, pericytes, and neurons to make sure our CNS is functioning properly.

Neisseria meningitidis first attaches to our BMECs using their type IV pili and an adhesion receptor. The type IV pili then recruits and activates the β2-adrenoreceptor which leads to the formation of cortical plaques and promotes further infection (8). This is due to the accumulation of ezrin/ezrin-binding proteins and other molecules that promote actin polymerization and junctional proteins (8). This results in the opening of cell junctions and damage to the BBB, whereupon N. meningitidis is then able to enter through the endothelium (8). Following invasion, N. meningitidis then continues to form colonies after attachment to BMECs despite being pushed by our blood flow (8). Importantly, pathogens and leukocytes may enter through the enlarged openings in the BBB, leading to further inflammation and swelling (8).

In fact, the large-scale inflammation of the meninges seen in bacterial meningitis is in large part due to large-scale cytokine release by resident immune cells which recognize bacterial antigens. This is of particular importance because the CNS is by design an immunologically privileged site, where interactions between the host immune system and potential pathogens are limited by the BBB - precisely to avoid damage and dysfunction caused by an inflammatory immune response.

Components of N. meningitidis are recognized by resident immune cells of the CNS, such as microglia, which release large quantities of inflammatory cytokines in response. Cytokines are hormone-like chemical mediators which stimulate tissues and recruit immune cells to participate in an immune response to a pathogen. Cytokine release is an important event in the pathogenesis of meningococcal disease. Pattern Recognition Receptors (PRRs) on microglia recognize molecular products exclusive to bacteria, such as lipopolysaccharide - a major component of the outer membrane in Gram-negative bacteria such as N. meningitidis. This triggers an intracellular signalling cascade, prompting transcriptional changes and leading to cytokine release by microglia. Cytokine release in meningococcal meningitis is characterised by the release of tumour necrosis factor α (TNF-α), IL-1β, IL-6, IL-8, MCP-1, MIP-α and G-CSF (9). These inflammatory cytokines bind to cells in the surrounding neuronal and immune cells, altering their character and function. For instance, IL-1β and TNF-α released in the innate inflammatory response induce apoptosis in neuronal cells, while IL-6 directly contributes to neural degeneration (10). All of these cytokines promote an increase in the production of factors which are toxic for neurons, such as Reactive Oxygen Species (ROS) and Nitric Oxide (NO). Beyond their neurotoxic effect, factors released by microglia in the innate inflammatory response also play a direct role in altering the permeability of the BBB. Banks et al., found that changes in BBB permeability are mediated by the release of NO and prostaglandins, which are released by microglia upon recognition of lipopolysaccharides (11).

Due to the widespread distribution of the meninges within the CNS, it is not surprising that damage to neurons is seen in nearly 50% of bacterial meningitis survivors (12). This may be because the subarachnoid space lies in close proximity to neural tissues and the ventricular system, which allows for rapid diffusion of toxic bacterial by-products and the inflammatory factors mentioned above to nearby cells. In addition, infiltration of leukocytes into the CSF in response to infection contributes to neuronal damage via the cytotoxic and proinflammatory activities of these cells (12).

While neuronal degeneration as a by-product of the inflammatory response has obvious overarching effects on the functionality of the nervous system, changes in BBB permeability caused by the inflammatory response also play a significant role in disturbing the function of the nervous system. The presence of N. meningitidis in the subarachnoid space, and its subsequent recognition by immune cells, results in increased permeability of both the vasculature and the BBB, leading to edema observed in meningococcal meningitis. This increase in BBB permeability contributes to "vasogenic" cerebral edema (swelling of the brain due to fluid leakage from blood vessels). Large numbers of white blood cells, namely neutrophils, respond to the cytokine gradient and enter the CSF, furthering inflammation of the meninges and leading to "interstitial" edema (swelling due to fluid between the cells) (13). Furthermore, this process causes the walls of the blood vessels themselves to become inflamed (cerebral vasculitis), which alters blood flow in the brain. These processes, caused by inflammation, contribute to intracranial swelling, which disturbs the normal physiological functioning of the nervous system primarily by restricting blood flow to the brain, indirectly causing neuronal cell death. This phenomenon is referred to as cerebral ischemia. In cranial CT scans of bacterial meningitis patients, fluid buildup is seen around and within the ventricles of the brain, which can lead to an increase in intracranial pressure (6). Such a change in intracranial pressure can result in the manifestation of a host of symptoms, such as altered levels of consciousness, cognitive impairment, and other sensory impairments (6, 12).

References

1. Overview of Meningitis - Neurologic Disorders [Internet]. Merck Manuals Professional Edition. [cited 2022 Jan 21]. Available from: https://www.merckmanuals.com/professional/neurologic-disorders/meningitis/overview-of-meningitis?query=Introduction%20to%20Meningitis  
2. Caugant DA, Brynildsrud OB. Neisseria meningitidis: using genomics to understand diversity, evolution and pathogenesis. Nat Rev Microbiol. 2020 Feb;18(2):84–96. Available from: https://www.nature.com/articles/s41579-019-0282-6  
3. Telano LN, Baker S. Physiology, Cerebral Spinal Fluid [Internet]. StatPearls [Internet]. StatPearls Publishing; 2021 [cited 2022 Jan 27]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK519007/
4. Decimo I, Fumagalli G, Berton V, Krampera M, Bifari F. Meninges: from protective membrane to stem cell niche. Am J Stem Cells. 2012 May 28;1(2):92–105. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636743/  
5. Coureuil M, Join-Lambert O, Lécuyer H, Bourdoulous S, Marullo S, Nassif X. Mechanism of meningeal invasion by Neisseria meningitidis. Virulence. 2012 Mar 1;3(2):164–72. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396695/  
6. Hoffman O, Weber RJ. Pathophysiology and Treatment of Bacterial Meningitis. Ther Adv Neurol Disord. 2009 Nov;2(6):1–7. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3002609/  
7. Pong S, Karmacharya R, Sofman M, Bishop JR, Lizano P. The Role of Brain Microvascular Endothelial Cell and Blood-Brain Barrier Dysfunction in Schizophrenia. Complex Psychiatry. 2020 Oct;6(1-2):30-46. Available from: https://pubmed.ncbi.nlm.nih.gov/34883503/
8. Coureuil M, Join-Lambert O, Lecuyer H, Bourdoulous S, Marullo S, Nassif X. Pathogenesis of meningococcemia. Cold Spring Harb Perspect Med. 2013 Jun;3(6):a012393. Available from: https://pubmed.ncbi.nlm.nih.gov/23732856/
9. Doran KS, Fulde M, Gratz N, Kim BJ, Nau R, Prasadarao N, Schubert-Unkmeir A, et al. Host-pathogen interactions in bacterial meningitis. Acta Neuropathol. 2016 Jan;131:185-209. Available from: https://doi.org/10.1007/s00401-015-1531-z
10. Sochoka M, Diniz BS, Leszek J. Inflammatory response in the CNS: Friend or foe? Mol Neurobiol.2016 Nov;54(10):8071-8089. Available from: https://doi.org/10.1007/s12035-016-0297-1
11. Banks WA, Gray AM, Erickson MA, Salameh TS, Damodarasamy M, Sheibani N, Meabon JS, et al. Lipopolysaccharide-induced blood-brain barrier disruption: Roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the Neurovascular unit. J Neuroinflammation. 2015 Nov;12(1). Available from: https://pubmed.ncbi.nlm.nih.gov/26608623/
12. Petersen H, Patel M, Ingason EF, Einarsson EJ, Haraldsson Á, Fransson P-A. Long-Term Effects from Bacterial Meningitis in Childhood and Adolescence on Postural Control. PLoS ONE. 2014 Nov 18;9(11):e112016. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4236047/  
13. Saez-Lorenz X, McCracken GH. Bacterial meningitis in children. Lancet. 2003 Jun;361(9375):2139-2148. Available from: https://pubmed.ncbi.nlm.nih.gov/12826449/

(iii) What antibiotics might have been in the intravenous drip i.e. what is the antibacterial treatment of choice and how do the(se) antibiotic(s) work to rid the body of the organism?

Due to the severity of meningococcal meningitis, clinical practice guidelines endorse empirical treatment – therapy begun on the basis of an educated medical guess – prior to confirmation of the presence of meningococcal meningitis. Given that Mary is started immediately on intravenous antibiotics, it appears that the emergency room physician has opted to begin empirical treatment in this case. The recommended empirical treatment for suspected bacterial meningitis is Vancomycin along with a third or fourth-generation cephalosporin, such as Ceftriaxone, Cefotaxime or Cefepime, as this combination therapy acts against all known potential agents of bacterial meningitis (1, 2). These causative agents (e.g. Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis) represent a mix of Gram-positive and Gram-negative pathogens; as such, the ideal antibiotic regimen should be broadly-acting and be able to disrupt the unique membrane structures that differ across Gram-positive and Gram-negative organisms (4).

Figure 3. Cross-sectional view of the meningococcal cell membrane (3)

Vancomycin is a branched tricyclic non-ribosomal peptide used to treat gram-positive bacterial infections, including treatment resistant Staphylococcus aureus meningitis infections (6). Vancomycin is ineffective against gram-negative bacteria primarily because it cannot cross the outer membrane of the bacterial cell wall which shields the peptidoglycan layer, which Vancomycin acts on to have bactericidal effects. In Mary’s case, we learn that it is the gram-negative N. meningitidis bacteria that causes her symptoms, hence the application of Vancomycin as an antibiotic therapy may appear to be redundant at best. However, we should keep in mind that Mary undergoes immediate, empirical therapy, meaning the antibiotics she receives in her intravenous drip are selected prior to the clinical team knowing exactly what is causing her meningitis. In that scenario, administering a diverse set of antibiotics makes sense in order to provide some therapeutic effect against a range of bacteria which may be causing Mary’s illness. Several gram-positive bacteria, such as S. pneumoniae, may have also been causing Mary’s symptoms, and those bacteria would have been vulnerable to antimicrobial activity from Vancomycin. However, due to the emerging prevalence of Vancomycin-resistant pathogens in community and healthcare settings, Vancomycin is generally recommended as a supplementary treatment in cases occurring in regions where cephalosporin-resistant strains are circulating (7).

Table 2. Empirical antimicrobial therapy for patients with bacterial meningitis based on clinical subgroup (7)

Structurally, Cephalosporins contain a six-member dihydrothiazine ring fused to a characteristic four-membered beta-lactam portion. Cephalosporins are a class of beta-lactam antibiotics which inhibit bacterial cell wall synthesis by binding to penicillin-binding proteins (PBPs).

To understand the mechanism of action of beta-lactam antibiotics, it is first necessary to describe the biosynthesis pathways involved in the generation of bacterial cell walls. In bacteria, the cell wall performs the essential functions of maintaining cell structure and preventing lysis under conditions of osmotic pressure. It is primarily composed of cross-linking units of peptidoglycan, which is itself composed of alternating amino sugar subunits (N-acetylglucosamine and N-acetylmuramic acid) (8). A short peptide sequence – capped by D-alanyl-D-alanine – is attached to N-acetylmuramic acid. The cross-linking reaction which confers peptidoglycan with its rigid structural properties occurs via a transpeptidation reaction, creating covalent oligopeptide bridges between polysaccharide chains. This reaction is catalysed by enzymes known as penicillin-binding proteins (PBPs), which recognize the terminal alanine as a substrate (8).

Antibiotics belonging to the beta-lactam class disrupt the synthesis of peptidoglycan by irreversibly binding to the active site of PBPs and inhibiting the essential transpeptidation reaction (8). This is due to the structural similarity between the beta-lactam ring of beta-lactam antibiotics and the terminal D-alanyl-D-alanine substrate recognized by PBPs (highlighted below).

Figure 4. The mimicry of beta-lactam antibiotics to D-alanyl-D-alanine (D-Ala-D-Ala). The four-member lactam ring in penicillin was highlighted in red (8)

Following cessation of peptidoglycan synthesis, bacterial cell death quickly follows. Without a cell wall, bacteria adopt a spherical shape (‘spheroplast’ or ‘protoplast’ morphology) and become highly sensitive to osmotic changes, resulting in lysis in hypotonic environments (9). Although not fully understood, it is also believed that buildup of peptidoglycan precursors following treatment with beta-lactams can induce autolysins to digest the existing cell wall even further (6). Finally, since the cell wall also plays a role in partitioning of cellular contents during bacterial reproduction, growth and reproduction is inhibited in the presence of beta-lactam antibiotics. Due to their mechanism of attack and overall effect, beta-lactams, including cephalosporins, are considered bactericidal (10). This can prevent the dissemination of pathogens and allow the host immune system to mount an effective response and clear the pathogen from the body.

Most third and fourth-generation cephalosporins can act against both Gram-negative and Gram-positive bacteria, making them an effective treatment against meningococcal meningitis. Because of their mechanism of action, beta-lactam antibiotics need access to the inner, peptidoglycan layer of Gram-negative bacterial cell walls to have a bactericidal effect. Both generations of cephalosporins, therefore, can traverse through the outer membrane of gram-negative bacteria cell walls. Fourth-generation cephalosporins accomplish this by acting as zwitterions, allowing them to cross the amphipathic lipopolysaccharides which the outer membrane of the cell wall is composed of (11).

Penicillin was formerly regarded as the primary drug of choice to treat meningococcemia and meningococcal meningitis (12). Penicillin is also a beta-lactam antibiotic, and acts with a similar mechanism to Cephalosporins to inhibit bacterial survival. However, Cephalosporins have generally become preferred to penicillin for two primary reasons. Firstly, Cephalosporins are more easily able to cross the blood-brain barrier (BBB) relative to penicillin. Crossing the BBB is necessary to access the site of the meningococcal meningitis infection. Cephalosporins can cross the BBB because they bind to several classes of transmembrane transporters that are expressed by cells at the BBB, including peptide transporter 2 (PEPT2) and organic anion transporting polypeptides (OATPs) (13). Penicillin, however, cannot readily cross the BBB as they do not bind to transmembrane transporters at the same rate. Penicillin can cross the BBB when the meninges are acutely inflamed (12), as they may be in meningococcal meningitis, however, its ability to access the site of infection is still sub-optimal relative to cephalosporins.

Secondly, cephalosporins have greater resistance to beta-lactamase enzyme degradation, relative to penicillin. Bacteria may produce the enzyme beta-lactamase, which cleaves the beta-lactam ring, preventing beta-lactam antibiotics from attaching to the penicillin-binding proteins, thereby rendering them ineffective in preventing bacterial cell wall growth. The molecular configuration of cephalosporins, namely Cefotaxime, confers greater resistance to beta-lactamase degradation. In particular, the syn-configuration of the methoxyimino functional group on Cefotaxime is directly responsible for increased stability against beta-lactamase activity (14). This is not to say that cephalosporins are immune to beta-lactamase activity – rather, they are more stable relative to other beta-lactam antibiotics such as penicillin. While N. meningitidis resistance to beta-lactams via beta-lactamase production is rare, beta-lactam producing strains have been observed, making cephalosporins a preferred option in treating meningococcal meningitis.  

In cases where the patient is allergic to beta-lactam antibiotics (common in as high as 10% of the population), alternative antibiotic treatments must be considered. These include meropenem, which has been shown in controlled trials to possess a similar efficacy in the treatment of bacterial meningitis compared to cefotaxime or ceftriaxone (15). In addition, the quinolones moxifloxacin and gatifloxacin are known to penetrate the BBB effectively, with experimental studies supporting them as potential treatments (15, 16). However, with the emerging prevalence of fluoroquinolone-resistant strains of pathogens, these should stand as a last-line treatment.

References

1. Tunkel AR, Hartman BJ, Kaplan SL, Kaufman BA, Roos KL, Scheld WM, Whitley RJ. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis. 2004 Nov;39(9):1267-84. Available from: https://doi.org/10.1086/425368
2. van de Beek D, Cabellos C, Dzupova O, Esposito S, Klein M, Loek AT, Leib SL, et al. Escmid guideline: Diagnosis and treatment of acute bacterial meningitis. Clin Microbiol Infect. 2016 May;22. Available from: https://doi.org/10.1016/j.cmi.2016.01.007
3. Huang FS, Brady RC, Mortensen J. Bacterial Meningitis, Fever, Headache and A Stiff Neck…Looking Pretty Sick. Introduction to Clinical Infectious Diseases. 2019:245-257.
4. Bashir HE, Laundy M, Booy R. Diagnosis and treatment of bacterial meningitis. Arch Dis Child. 2003 Jul;88(7):615–20. Available from: https://adc.bmj.com/content/88/7/615  
5. Edwards JS. Molecular basis of vancomycin resistance transfer in Staphylococcus aureus. Proc Natl Acad Sci USA. 2013 Feb;110(8):2804-2809. Available from: https://doi.org/10.2210/pdb4ht4/pdb
6. Beauduy CE, Winston LG. Beta-Lactam & Other Cell Wall- & Membrane-Active Antibiotics. In: Katzung BG, Vanderah TW, editors. Basic & Clinical Pharmacology [Internet]. 15th ed. New York, NY: McGraw-Hill; 2021 [cited 2022 Jan 21]. Available from: https://accessmedicine.mhmedical.com/content.aspx?aid=1176468841
7. Brouwer MC, Tunkel AR, van de Beek D. Epidemiology, Diagnosis, and Antimicrobial Treatment of Acute Bacterial Meningitis. Clin Microbiol rev. 2010 Jul;23(3):467-492. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901656/
8. Zeng X, Lin J. Beta-lactamase induction and cell wall metabolism in Gram-negative bacteria. Front Microbiol. 2013;4. Available from: https://www.frontiersin.org/article/10.3389/fmicb.2013.00128
9. Cushnie TPT, O’Driscoll NH, Lamb AJ. Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action. Cell Mol Life Sci. 2016 Dec;73(23):4471–92. Available from: https://doi.org/10.1007/s00018-016-2302-2      
10. Sarkar P, Yarlagadda V, Ghosh C, Haldar J. A review on cell wall synthesis inhibitors with an emphasis on glycopeptide antibiotics. Medchemcomm. 2017 Mar;8(3):516-533. Available from: https://doi.org/10.1039/c6md00585c
11. Sweet RL, Gibbs RS. Infectious diseases of the female genital tract. 5th ed. Philadelphia: PA Wolters. c2010.
12. Morse SA. Chapter 14 Neisseria, Moraxella, Kingella and Eikenella. In: Medical Microbiology. 4th ed. University of Texas Medical Branch at Galveston. C1996.
13. Chen X, Loryan I, Payan M, Keep RF, Smith DE, Hammarlund-Udenaes M. Effect of transporter inhibition on the distribution of Cefadroxil in rat brain. Fluids Barriers CNS. 2014 Nov;11(1. Available from: https://doi.org/10.1186/2045-8118-11-25
14. Van TT, Nguyen HN, Snooker PM, Coloe PJ. The antibiotic resistance characteristics of non-typhoidal salmonella enterica isolated from food-producing animals, retail meat and humans in South East Asia. Int J Food Microbiol. 2012 Mar;154(3):98-106. Available from: https://doi.org/10.1016/j.ijfoodmicro.2011.12.032
15. McGill F, Heyderman RS, Michael BD, Degrees S, Beeching NJ, Borrow R, Glennie L, et al. The UK joint specialist societies guideline on the diagnosis and management of acute meningitis and meningococcal sepsis in immunocompetent adults. J Infect. 2016 Apr;72(4):405-438. Available from: https://doi.org/10.1016/j.jinf.2016.01.007
16. Rouphael NG, Stephens DS. Neisseria meningitidis: Biology, Microbiology, and Epidemiology. Methods Mol Biol. 2012;799: 1-20.

(iv) Why did the doctor ask about Mary’s vaccination history? Is this a reportable communicable disease?

The vaccination history is significant because it can help the physician narrow down the exact agents that are responsible for Mary’s infection. Bacterial meningitis is caused by a wide variety of pathogens, but most commonly by Streptococcus pneumoniae and Neisseria meningitidis (1). Meningococcal vaccine is strongly recommended for all age groups, and it has been proven as highly effective in all regions where it is introduced (2). Meningococcal vaccine contains four types of attenuated meningococcal bacteria: serogroups A, C, W-135, and Y (8); this is especially useful in disease prevention in young adult populations. Therefore, the fact that Mary did not receive any vaccinations since elementary school provides rationales as to why she would be infected.

In addition, the specific vaccine against meningococcal meningitis is made of purified polysaccharides from Neisseria meningitidis (10). According to the Center for Disease Control and Prevention (CDC), this vaccine is recommended for people with substantial risk of infection and for college freshmen, especially those who live in dormitories (10). Given that Mary just moved into a dormitory and that she had not been given any vaccinations since childhood, it is reasonable for the physician to suspect that she has a higher likelihood of getting meningococcal meningitis.

Whether meningitis is a reportable communicable disease depends on the specific agent that caused the disease. For viral meningitis, the disease is usually transmissible. On the contrary, fungal meningitis and parasitic meningitis are not considered to be contagious. Bacterial meningitis is usually contagious; more importantly, some bacteria such as Neisseria meningitidis, are particularly contagious (6). Therefore, all cases should be reported to local health authorities immediately for proper identification, treatment, and monitoring. In fact, humans are the only reservoir for N. meningitidis (5). The incidence rate for the disease ranges from 1 to 1000 cases per 100,000 individuals (4). The transmission of the disease often involves prolonged contact with infected individuals, such as exposure to respiratory or salivary secretion due to coughing, sneezing, sharing food, or kissing (3). Evidence shows that household contacts have a 500-fold to 800-fold higher risk of infection than the general population. In such situation, close contacts in the family, usually referred to as “kissing contacts”, should be given single-dose ciprofloxacin for prophylaxis (7). In the United States, Rifampin and Ceftriaxone are also used for prophylaxis (9).

Table 3. CDC recommended vaccines for bacterial infections (10)

References

1. Agamanolis D. 2014. Neuropathology. Meningitis. Retrieved January 19, 2022, from https://neuropathology-web.org/chapter5/chapter5aSuppurative.html
2. Baron S. 1996. Medical Microbiology, 4th edition. Chapter 14 and 11. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK7627/
3. Beek D, Brouwer M, Hasbun R, Koedel U, Whitney CG, Wijdicks E. 2016. Community acquired bacterial meningitis. Nature. Volume 2, 74.
4. Caugant DA, Maiden MC. 2009. Meningococcal carriage and disease – population biology and evolution. Vaccine. 27 (4): B64-B70.
5. Center for Disease Control and Prevention. 2019. Meningococcal Disease. Retrieved from https://www.cdc.gov/meningococcal/about/causes-transmission.html
6. Davis CP, M. D. 2020, July 17. Is meningitis contagious? MedicineNet. Retrieved January 19, 2022, from https://www.medicinenet.com/is_meningitis_contagious/article.html
7. Goering, R. V., Dockrell, H. M., Zuckerman, M., Chiodini, P. L. 2019. Chapter 25. In MIM's Microbiology and Immunology (Sixth, pp. 316–318).
8. Government of Canada. 2015. Meningococcal vaccine: Canadian Immunization Guide, For Health Professionals. Retrieved from https://www.canada.ca/en/public-healthG/services/publications/healthy-living/cmanadian-immunization-guide-part-4-active-vaccines/page-13-meningococcal-vaccine.html
9. Huang FS, Brady RC, Mortensen J. 2019. Bacterial Meningitis, Fever, Headache and A Stiff Neck…Looking Pretty Sick. Introduction to Clinical Infectious Diseases. 245-257.
10. Tortora, G. J., Funke, B. R., & Case, C. L. 2021. Microbiology: An introduction. Pearson education Limited.

2. The Microbiology Laboratory

(i) Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.

Neisseria meningitidis, the bacteria found growing in Mary’s blood and cerebrospinal fluid is a Gram-negative bacteria. There are 13 capsular serogroups for Neisseria meningitidis, and serogroups A, B, C, W-135 and Y are those that are most commonly found to cause meningococcal infection in humans [1]. Although being present in the nose or throat of 1 in every 10 individuals without causing illness, this is the leading cause of bacterial meningitis as well as sepsis [2, 3].  

Other than Neisseria meningitidis, meningitis can also be caused by bacteria such as Escherichia coli, Group B streptococcus, Listeria monocytogenes, Streptococcus pneumoniae, and Haemophilus influenzae. Streptococcus pneumoniae, along with Neisseria meningitidis, are responsible for the majority of meningitis cases affecting patients across all ages in the United States [1]. For infants younger than 30 days, however, Group B streptococcus, Escherichia coli, and Listeria monocytogenes are more commonly the cause of bacterial meningitis [4]. Group B streptococcus accounts for 77% of infections in 0-4 day old patients (early-onset) and 50% of infections in 5-28 day old patients (late-onset) [4].

Streptococcus pneumoniae:

Streptococcus pneumoniae are lancet-shaped, facultatively anaerobic, Gram-positive bacteria that are identified as the causative agent of 58% of the meningitis cases in the U.S. [5]. S. pneumoniae is transmitted between hosts through sneezing and coughing, where it then colonizes the nasopharynx region of the new host [6]. S. pneumoniae can establish infection in hosts that do not have prior immunity to the bacteria, as well as have altered natural barriers/immune systems that would normally fight off the infection [6]. Once in the blood, the bacteria can cross the blood-brain barrier and infect the central nervous system (CNS), leading to inflammation of the CNS. This accounts for the neurological symptoms after an S. pneumoniae infection, such as hearing loss and cognitive impairment [6]. While penicillin was a viable option for treating pneumococcal meningitis in the past, resistance rates to penicillin have been reported to be up to 25-50% in the U.S. [7]. Currently, the serotypes included in the vaccine for S. pneumoniae are successfully controlled, but infection by serotypes that are not covered by the vaccine continue to occur [8]. The incidence of S. pneumoniae causing pneumococcal meningitis decreased from 0.8/100000 people in 1997 to 0.3/100000 people in 2010 after the pneumococcal conjugate vaccine, PCV 7, was introduced in 2000 [9], thus pneumococcal meningitis can be prevented with the administration of a vaccine.

Haemophilus influenzae:

Haemophilus influenzae are Gram-negative, facultatively anaerobic bacteria that may also lead to meningitis [10]. H. influenzae is divided into subgroups based on capsule type, with H. influenza type b (Hib) being the predominant group that causes the infection [10]. Patients under 5 years old and over 65 years old are particularly at risk, as well as immunocompromised individuals such as HIV patients, cancer patients, etc. [10]. Hib transmission is usually through respiratory droplet inhalation and can cross the blood-brain barrier similar to S. pneumoniae. Hib infections are usually treated with antibiotics, however, ampicillin is avoided in treatment due to the observed emerging resistance [11]. Fortunately, the Hib conjugate vaccine is protective against infections and current cases primarily involve non-immunized children [10]. Symptoms of meningitis include fever, headache, stiff neck, nausea, photophobia and altered mental status [12], which align with Mary’s symptoms. People who are not sick but have H. influenzae colony in their nose and throat can spread the bacteria as well [13]. Mary may potentially be infected by H. influenzae as the symptoms for meningitis line up with what Mary is feeling.

Group B streptococcus:

Group B streptococcus (GBS) is a Gram-positive bacteria present in the reproductive and gastrointestinal tract of 35% of healthy women [14]. This bacteria generally resides harmlessly in the bowels, vagina, and sometimes the back of the nose and throat [15]. This could be transmitted to the newborn during birth, which is why meningitis caused by GBS is so common in infants, and most babies with early-onset GBS disease acquired the bacteria this way. GBS is not spread through food and water, and not spread through contact. Also, a person with someone that has GBS will not be infected with GBS [15]. Taking this into account, and also the fact that GBS is not spread through contact, Mary likely does not have GBS. GBS can travel in the blood to the brain by crossing the blood-brain barrier into the cerebral spinal fluid (CSF). 20-30% of infants infected with GBS and developed meningitis have severe outcomes illustrated by neurological defects. Approximately 25% of infants with GBS meningitis have moderate neurological impairments, and only 51% exhibit normal development [14]. Treatment for this condition includes antibiotic administration and CSF culture monitoring.

Escherichia coli:

Escherichia coli is a Gram-negative bacteria that is also a common cause of meningitis in infants [16]. It travels in the blood and crosses the blood-brain barrier to infect the meninges, leading to meningitis. The E. coli infection establishes due to an immature immune system in the newborn. Strains of E. coli that are associated with meningitis have the K1 polysialic capsule which is also found in N. meningitidis [16]. This K1 polysialic capsule is vital in the bacteria’s ability to survive after crossing the blood-brain barrier to successfully establish an infection [16]. Most E. coli are harmless, and are commensal in the intestinal tract. However, there are some strains that are pathogenic and can cause diarrhea or other illness [17]. E. coli are the second most common cause of neonatal meningitis [18], usually during vaginal delivery where the infant gets infected as E. coli is normally present in the birth canal [19]. E. coli meningitis is very rare in adults (less than 2%) [20], thus Mary is unlikely to have E. coli meningitis.

Listeria monocytogenes:

Listeria monocytogenes is a Gram-positive bacteria that accounts for 20% of meningitis in newborns and the elderly [21]. It is commonly transmitted by ingesting contaminated food, such as hot dogs, meats, and cheeses, as well as by vertical transmission from mother to child. Mortality resulting from L. monocytogenes meningitis is reported to be around 15-27% [21]. Since L. monocytogenes meningitis can be found in contaminated food, increasing good hygiene may be helpful in reducing risks to these bacterial pathogens.  Particularly, individuals living in the region of sub-Saharan Africa are at risk of bacterial meningitis as there is a high risk of both meningococcal disease cases and epidemics [22].  This is most likely due to many individuals being malnourished and having a weakened immune system due to the crowded and poor living conditions in these regions [22]. Current treatment includes antibiotic administration such as penicillin and ampicillin [21]. Meningitis is the most frequent result of listeriosis of the CNS, and symptoms include headache, stiff neck, confusion, loss of balance, and convulsions [20]. These symptoms can develop within 3 days to 3 months after ingesting contaminated food [20]. Mary does not experience confusion, loss of balance, and convulsion, thus Mary is likely uninfected by L. monocytogenes.

References:

1. Brouwer MC, Tunkel AR, van de Beek D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clinical Microbiology Reviews. 2010;23(3):467–92.  
2. Meningococcal Disease Causes and Transmission | CDC [Internet]. Cdc.gov. 2022 [cited 28 January 2022]. Available from: https://www.cdc.gov/meningococcal/about/causes-transmission.html
3. Tzeng YL, Stephens DS. Epidemiology and pathogenesis of Neisseria meningitidis. Microbes Infect [Internet]. 2000 [cited 2022 Jan 28];2(6):687–700. Available from: https://pubmed.ncbi.nlm.nih.gov/10884620/
4. Gaschignard J, Levy C, Romain O, Cohen R, Bingen E, Aujard Y, et al. Neonatal bacterial meningitis. Pediatric Infectious Disease Journal. 2011Mar;30(3):212–7.  
5. Thigpen M, Whitney C, Messonnier N, Zell E, Hadler J, Harrison L, et al. Bacterial meningitis in the United States, 1998–2007. The New England Journal of Medicine. 2011May26;364:2016–25.  
6. Mook-Kanamori BB, Geldhoff M, van der Poll T, van de Beek D. Pathogenesis and pathophysiology of pneumococcal meningitis. Clinical Microbiology Reviews. 2011Jul;24(3):557–91.  
7. Appelbaum PC. Resistance among Streptococcus pneumoniae: Implications for Drug Selection. Clinical Infectious Diseases. 2002;34(12):1613–20.  
8. Hsu HE, Shutt KA, Moore MR, Beall BW, Bennett NM, Craig AS, et al. Effect of pneumococcal conjugate vaccine on pneumococcal meningitis. New England Journal of Medicine. 2009;360(3):244–56.  
9. Castelblanco RL, Lee M, Hasbun R. Epidemiology of bacterial meningitis in the USA from 1997 to 2010: a population-based observational study. Lancet Infect Dis [Internet]. 2014 [cited 2022 Jan 21];14(9):813–9. Available from: https://pubmed.ncbi.nlm.nih.gov/25104307/
10. Khattak ZE, Anjum F. Haemophilus influenzae [Internet]. StatPearls [Internet]. U.S. National Library of Medicine; 2021 [cited 2022Jan20]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562176/
11. Khattak ZE, Anjum F. Haemophilus influenzae [Internet]. StatPearls [Internet]. U.S. National Library of Medicine; 2021 [cited 2022Jan20]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562176/
12. Symptoms of Haemophilus influenzae [Internet]. Cdc.gov. 2021 [cited 2022 Jan 22]. Available from: https://www.cdc.gov/hi-disease/about/symptoms.html
13. Haemophilus influenzae: Causes and transmission [Internet]. Cdc.gov. 2021 [cited 2022 Jan 22]. Available from: https://www.cdc.gov/hi-disease/about/causes-transmission.html
14. Hanna M, Noor A. Streptococcus Group B [Internet]. StatPearls [Internet]. U.S. National Library of Medicine; 2021 [cited 2022Jan20]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK553143/
15. People at increased risk and how it spreads [Internet]. Cdc.gov. 2021 [cited 2022 Jan 21]. Available from: https://www.cdc.gov/groupbstrep/about/transmission-risks.html
16. Hoffman JA, Wass C, Stins MF, Kim KS. The capsule supports survival but not traversal of escherichia coli K1 across the blood-brain barrier. Infection and Immunity. 1999;67(7):3566–70.  
17. Questions and answers [Internet]. Cdc.gov. 2019 [cited 2022 Jan 22]. Available from: https://www.cdc.gov/ecoli/general/index.html
18. E. coli meningitis [Internet]. Meningitisnow.org. [cited 2022 Jan 22]. Available from: https://www.meningitisnow.org/meningitis-explained/what-is-meningitis/types-and-causes/e-coli-meningitis/
19. E. Coli Meningitis [Internet]. Meningitis.ca. [cited 2022 Jan 22]. Available from: https://www.meningitis.ca/en/EcoliMeningitis
20. What Causes Meningitis & Septicaemia? [Internet]. Meningitis.org. [cited 2022 Jan 22]. Available from: https://www.meningitis.org/meningitis/causes/e-coli-meningitis
21. Clauss HE, Lorber B. Central nervous system infection with listeria monocytogenes. Current Infectious Disease Reports. 2008;10(4):300–6.  
22. Meningococcal disease in other countries [Internet]. Cdc.gov. 2021 [cited 2022 Jan 28]. Available from: https://www.cdc.gov/meningococcal/global.html

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?

While the symptoms Mary is experiencing are very characteristic of meningitis, including fever, headache, and a stiff neck, microbiology laboratory testing is essential to further confirm a meningitis diagnosis The diagnosis is usually made with a triple approach, where the blood culture, cerebrospinal fluid (CSF) gram stain, and CSF culture are evaluated. This approach is able to correctly diagnose 90% of cases with these symptoms [1]. It is essential to administer intravenous antibiotics immediately after symptom recognition to limit the progression of the infection, but it is also important to take samples of CSF and blood as soon as possible as the CSF and blood soon become sterile after receiving antibiotics, which would lose its diagnostic capabilities [2].  

Fig. 1 Cerebrospinal fluid collected through lumbar puncture. [11]

Cerebral Spinal Fluid:

A CSF fluid sample is obtained through a lumbar puncture to extract a sample prior to or shortly after antibiotic treatment. This should be collected by experienced clinicians only and by aseptic technique.  Furthermore, the patient should be sitting up or lying on their sides, motionless, so that the lumbar vertebrae can be separated allowing for the needle to be inserted in between [3]. The CSF should be clear and colorless, and fluid will drip out into a vial for collection.  In rarer cases, a needle may be inserted under the occipital bone at the base of the brain if a lumbar puncture is not possible [4]. When collecting CSF, it is also important that the tube sent to the Microbiology Laboratory is not contaminated with blood if multiple samples are collected.  This is to avoid contamination which can affect the culture of CSF and cause problems with its analysis [5]. Within an hour of collection, the sample should then be taken, at 20-35 degrees Celsius, into the Microbiology Laboratory for analysis.  If analysis is not possible within the hour, then inoculation into Trans-Isolate (T-I) medium should be done.  This should be incubated with around 5% CO2, at 35-37 degree Celsius, until transportation to the Microbiology Laboratory is possible [3]. The CSF is examined for its cell count, glucose level, Gram stain, cultures, and polymerase chain reaction (PCR) [6].

Blood:

Blood samples are also obtained prior to or shortly after beginning antibiotic treatment. This involves minimal risk for patients and collection can be done through serology tests [7].  About 1-3 ml can be collected and diluted to obtain a blood culture with adequate amount of bacterial growth so that analysis can be done [3].  Within one minute of collection, the blood sample should then be inoculated into culture so that the syringe is not clogged with blood [3].  This should be transported into the Microbiology Laboratory for analysis immediately.  As with the CSF sample, this should be incubated with around 5% CO2, at 35-37 degree Celsius, until transportation to the Microbiology Laboratory is possible [3]. It is used for blood work such as complete blood count (CBC), coagulation studies, and electrolyte levels. Inflammatory markers in the blood, such as C-reactive protein and procalcitonin may also be used to determine if it is aseptic or bacterial meningitis [8].

PCR of the CSF sample is especially important if samples were taken after antibiotic administration and/or no bacteria were observed on the Gram stain. The PCR test can confirm meningitis, as well as the species that caused it, such as Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, or Listeria monocytogenes [9]. PCR is also able to provide fast results to help the diagnosis, whereas CSF and blood cultures may take longer to identify the species.

When collecting CSF and the blood sample, it is crucially important to follow all biosafety guidelines to help reduce risks for transmission of any unknown bacterial or viral agent present.  This includes common procedures such as properly washing hands, wearing gloves, sanitation, transportation of samples, as well as proper procedures in dispensing syringes or needles and reporting any injuries or contaminations immediately [3].

Imaging like CT scans and MRIs can be useful in identifying the symptoms of meningitis including the inflammation and swelling of the brain. CT scans can also be used to look for any chest or sinus infection that is usually associated with meningitis [10].

References:

1. Tunkel AR, Hartman BJ, Kaplan SL, Kaufman BA, Roos KL, Scheld WM, et al. Practice guidelines for the management of bacterial meningitis. Clinical Infectious Diseases. 2004;39(9):1267–84.  
2. Chaudhuri A, Martin PM, Kennedy PG, Andrew Seaton R, Portegies P, Bojar M, et al. EFNS guideline on the management of community-acquired bacterial meningitis: Report of an EFNS task force on acute bacterial meningitis in older children and adults. European Journal of Neurology. 2008;7(649):649–59.  
3. Meningitis Lab Manual: Specimen Collection and Transport | CDC. (2022). Retrieved 19 January 2022, from https://www.cdc.gov/meningitis/lab-manual/chpt05-collect-transport-specimens.html
4. Donohue, M. (2022). Cerebrospinal Fluid Culture: Purpose, Procedure & Risks. Retrieved 19 January 2022, from https://www.healthline.com/health/cerebrospinal-fluid-culture
5. Schwenkenbecher P, Janssen T, Wurster U, Konen FF, Neyazi A, Ahlbrecht J, et al. The influence of blood contamination on cerebrospinal fluid diagnostics. Frontiers in Neurology. 2019;10.  
6. Curtis S, Stobart K, Vandermeer B, Simel DL, Klassen T. Clinical features suggestive of meningitis in children: A systematic review of prospective data. Pediatrics. 2010;126(5):952–60.  
7. Hammitt LL, Murdoch DR, Scott JA, Driscoll A, Karron RA, Levine OS, et al. Specimen collection for the diagnosis of pediatric pneumonia. Clinical Infectious Diseases. 2012;54(suppl_2).  
8. Tacon CL, Flower O. Diagnosis and management of bacterial meningitis in the paediatric population: A Review. Emergency Medicine International. 2012;2012:1–8.  
9. van de Beek D, Cabellos C, Dzupova O, Esposito S, Klein M, Kloek AT, et al. Escmid guideline: Diagnosis and treatment of acute bacterial meningitis. Clinical Microbiology and Infection. 2016;22.  
10. Mayo Clinic Staff. Meningitis [Internet]. Mayo Clinic. Mayo Foundation for Medical Education and Research; 2020. Available from: https://www.mayoclinic.org/diseases-conditions/meningitis/diagnosis-treatment/drc-20350514
11. Mayo Clinic Staff. Spinal tap (lumbar puncture) [Internet]. Mayo Clinic. Mayo Foundation for Medical Education and Research; 2020. Available from: https://www.mayoclinic.org/tests-procedures/lumbar-puncture/about/pac-20394631#dialogId15231611

(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

Once samples have been collected and are transported to the Microbiology Laboratory, they are ready for testing. In many cases, potential bacterial pathogens causing bacterial meningitis can be determined by isolating and culturing the bacteria.  

It is important to use cytological processes to examine the CSF and blood samples for pressure, colour, glucose level, protein count, lactate count, turbidity, and pleocytosis [1]. If the CSF sample displays evidence of these inconsistencies, it can help the lab technicians deduce the cause of infection [2]. For example, biomarkers can be analyzed to see if there are any elevated proteins. A positive blood culture would confirm 40% of meningococcal meningitis cases, 50-90% of Haemophilus influenzae meningitis, and 75% of pneumococcal meningitis [3]. This would be concurrently used to make a diagnosis along with CSF gram stain and culture. Because tests have a margin of error, CSF analysis can produce normal results or negative cultures in a patient with compatible symptoms of meningitis; blood cultures should be performed in conjunction with CSF analysis to help characterize the causative agent to ensure it is the same as what was detected in the CNS [4].

After cytological examination, centrifuging the sample is executed to separate sediment bacteria. The sediment is then mixed well and a drop or two each will be used to prepare a Gram stain as well as for streaking the primary culture media [5].

Growing a culture

A pure culture is ideal to ensure accurate test results and therefore an inspection to check for purity of growth is necessary, and a new colony should be streaked on the media [1].  The culture of CSF is also an important step in identifying the causative bacteria, which can be grown on either a blood agar plate (BAP) or a chocolate agar plate (CAP) [1]. In order to favor the growth of  N. meningitidis, adding a small container of water to the incubator will help increase the humidity and growth (5).

CSF cytological analysis

Observing the color of CSF is important as it is normally clear, but the increased presence of WBCs and RBCs can cause CSF to appear unclear [6].  Increased presence of WBCs and RBCs may indicate infection present. The cell count of WBCs and RBCs are important, as an increased WBC cell count may indicate the presence of bacterial meningitis (99% of patients have WBC count > 100/mm3 and 87% of patients have WBC count > 1000/mm3), while low WBC cell count may indicate viral meningitis [6, 7]. Likewise, an elevated C-reactive protein level and a procalcitonin level higher than 0.5 nanograms/mL also point to bacterial meningitis [8, 7]. Abnormal electrolyte levels in the blood may also be observed but are not specific to meningitis [9].

It is also important to note the differentiation of WBCs. Normal CSF has approximately 70% lymphocytes and 30% monocytes; however, patients with bacterial meningitis may have solitary eosinophils dominating their CSF [6]. Additionally, CSF protein concentration is one of the most important and sensitive indicators of CNS disease. Increased levels of CSF proteins indicate the presence of infections, intracranial hemorrhages, and many other diseases [6]. Finally, a CSF glucose level of 0.4mg/dL is 80% indicative of bacterial meningitis diagnosis for patients older than 2 months [7].

Additional tests can also be performed on the CSF sample.  These tests play an important role in detecting any of the potential bacterial pathogens, such as N. meningitidis, S. pneumoniae, and H. influenzae, all of which can cause bacterial meningitis.

Kovac’s oxidase test – to detect N. meningitidis and H influenzae:

Kovac’s oxidase test is to determine the presence of cytochrome c oxidase. The reagent is tetramethyl-p-phenylenediamine dihydrochloride, which turns purple by compounds that have cytochrome c in their respiratory chain [10]. There are multiple methods to perform this test but the most common is the filter technique. The bacteria will be grown on a blood agar plate (BAP) for 24 hours prior to the test. A strip of filter paper will be submerged in a few drops of Kovac’s oxidase reagent then allowed to dry, and an inoculating loop will transfer a colony from the plate and rubbed onto the treated filter paper [2]. A positive result would be shown by a colour change, whereas a negative result would be shown by no colour change.

Carbohydrate utilization – to detect N. meningitidis:

A carbohydrate utilization test examines a pathogens’ ability to ferment carbohydrates, determined by the acid indicator in the medium. This test is used specifically for the identification of N. meningitidis as it can only oxidize glucose and maltose [2].  In this test, glucose, maltose, lactose, and sucrose are added into four different tubes with cystine trypticase agar (CTA) and phenol red indicator. This indicator turns yellow in the presence of acid. The isolate will first be grown on a BAP for 24 hours prior to the test. Colonies from the plate will be removed via a disposable loop, then stabbed in the upper portion of the CTA sugar medium. Upon incubation for at least 72 hours, the development of colour change to yellow will be examined, which indicates a positive test [10]. Glucose and maltose would oxidize with N. meningitidis resulting in a colour change to yellow, whereas lactose and sucrose will not be oxidized [2].

Slide agglutination serogrouping (SASG) – to detect N. meningitidis and H. influenzae:

SASG tests involve using a serogroup-specific antisera.  If the antisera binds to the bacterial cells then a clump will form and an intensity rating can be determined.  The ratings 3+ and 4+ within one to two minutes corresponds to a positive result whereas a rating of 0, 1+ or 2+ corresponds to a negative result [2].  

Latex agglutination – to detect S. pneumonia:

Latex agglutination is done for quick detection of bacterial antigens in CSF, however, the sensitivity varies between different bacteria [6]. Latex agglutination reaction is carried out when a sample with a specific antigen is mixed with latex particles that have antibodies coating the surface, and then visible precipitate can be observed if agglutination occurs [8]. To set up, the test will use the supernatant aspect of the previous centrifuged sample. The supernatant is heated to 100 degrees Celsius, then add 30-50ul of the liquid to disposable latex reagent [5] . Latex agglutination can be used to detect S. pneumonia in place of the standard quelling reaction [11].  A positive test result is indicated by cells clumping together within 5-10 seconds, whereas a negative test result can be indicated by no appearance of agglutination in the same timeframe.

Gram Stain – to differentiate between Gram-positive and Gram-negative bacteria

A Gram stain is used to differentiate between the two main categories of bacteria: Gram-positive and Gram-negative [12]. This test is based on the property of the cell walls of the bacteria. Preparing Gram staining for the CSF sample requires dividing a glass slide in two and smearing one side with the sample, and smearing the other side with the organism in question [5]. After centrifuging the CSF, a smear of the CSF will be allowed to air-dry in a uniform suspension with sterile water or saline. The smear will then be fixed by flooding with methanol, washed with  water, then flooded with crystal violet ammonium oxalate to stain. Flooding with Gram’s iodine will follow, as the iodine acts as a mordant and binds to the crystal violet dye to the cell wall. The slide will be rinsed with ethanol to decolourise, then counterstained with safranin or carbol-fuchsin. When washed and dried, the smear will be examined under a microscope [10, 13]. If the bacteria is gram-positive, then its thick peptidoglycan layer will trap the violet dye and result in a purple color even after safranin is added. In a gram-negative bacteria, since the violet dye is not trapped, adding safranin will result in a pink or red colour [2]. The sensitivity of gram stains can be greatly influenced, and some laboratory techniques used to stain the CSF can affect the liability of the results [6].

Catalase Test – to detect S. pneumonia:

A catalase test uses the enzyme catalase, which decomposes hydrogen peroxide into water and oxygen gas [11]. This test is used to discriminate between Gram-positive cocci. Specifically, this test can be used to determine whether it is S. pneumonia and Enterococcus, or if the bacteria is Staphylococcus [12]. To perform this test, the isolate will be grown on a BAP for 24 hours prior. A disposable loop will then remove a colony from the dish and place it onto a glass slide. Hydrogen peroxide will be added to the slide and mixed with the bacteria. The catalase enzyme breaks down hydrogen peroxide into water and oxygen, resulting in bubbling by the oxygen within the liquid.  A positive catalase test will display vigorous bubbles in the liquid due to the oxygen given off during the reaction [10]. Absence of bubbling would indicate a negative test. Positive tests means that the bacteria is indeed Staphylococcus and a negative test indicates that the gram-positive bacteria is a streptococci [3].

Optochin test – to detect S. pneumonia:

Optochin tests utilize the chemical ethylhydrocupreine hydrochloride (optochin), and as S. pneumoniae strains are sensitive to this chemical, this test can be conducted to help determine if the bacterial pathogen causing bacterial meningitis is S. pneumoniae [11]. Strains to be tested are grown on a BAP for 24 hours. An isolated colony will then be transferred via a loop and streaked onto half of a new BAP. An optochin disk will be placed within the streaked area then incubated overnight at 35-37 degree Celsius [11]. By measuring the zone of inhibition (the area where growth has ceased around the disk), it can be determined if the bacteria is sensitive or not.  A zone of inhibition of 14 mm or greater would indicate that it is sensitive, identifying that the bacterial pathogen is indeed S. pneumonia [11].

Bile solubility test – to detect S. pneumonia:

The bile solubility test is able to discriminate S. pneumoniae from other streptococci, because it is bile soluble whereas the latter are resistant [11]. The bile solution will first be prepared with sodium deoxycholate (bile) dissolved in sterile water. The isolate of bacteria will be grown on a BAP for 24 hours prior to the test, then a colony will be transferred to a tube with saline in the 0.5-1.0 McFarland standard of turbidity. Once this suspension is mixed and divided, the sodium deoxycholate will be added. These tubes will then be incubated in carbon dioxide, vortex, and observed. If the contents are clear (vs turbid), that is an indication that the bacterium is bile positive [11].

Hemin and nicotinamide-adenine-dinucleotide (NAD) growth factor requirement test – can be used to detect H. influenzae:

H. influenzae can be identified based on its requirement for hemin and NAD. First, H. influenzae can be grown on a plate containing chocolate agar for 24 hours.  The colonies formed will then be used to create a heavy suspension of cells (above 1.0 McFarland standard) and vortexed.  If chocolate agar from the plate is also mixed in the cell suspension, this can result in problems with identifying the bacterial pathogen. A heart infusion or trypticase soy agar plate can then be inoculated with the cell suspension and once dry, the paper strips or disks containing hemin, NAD, and hemin + NAD can be placed [4].  With the use of paper strips or disks that contain either hemin, NAD or hemin + NAD, it can be detected if H. influenzae is present and is the cause of bacterial meningitis.  H. influenzae can only grow around the disk with both hemin and NAD, however H. Haemolyticus grows using the same factors. Therefore, a hemolysis test must be checked on rabbit or horse blood agar to differentiate between the two [14].

Polymerase chain reaction (PCR) test:

Furthermore, a PCR/DNA hybridization test can be done to assist with detecting the bacterial pathogen in the blood or CSF sample that is causing bacterial meningitis.  This is done by targeting a unique sequence of the bacterial pathogen causing bacterial meningitis and using a fluorescent oligonucleotide probe complementary to target the sequence [15].  If the specific bacteria is present in culture, this will be indicated by a corresponding fluorescent signal. PCR has high sensitivity and specificity for many bacteria, and can be done with small volumes of CSF [6]. The sensitivity of PCR is 95-100% for enterovirus, which is a virus that causes enterovirus meningitis. PCR’s sensitivity for tuberculous meningitis is 54-100% [6].

References:

1. Cerebrospinal Fluid (CSF) Analysis: MedlinePlus Medical Test [Internet]. Medlineplus.gov. 2020. Available from: https://medlineplus.gov/lab-tests/cerebrospinal-fluid-csf-analysis/?fbclid=IwAR2r-_Qw5Kcq9EdsONyiASlmnASR0tyaNie0c9ndnoE3wgZkrtfPkoQxrYY

2. Meningitis Lab Manual: ID and Characterization of Neisseria | CDC. 2022. Available from https://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html

3. Brouwer, M.,, Tunkel, A., van de Beek, D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clinical Microbiology Reviews 2010. 23:467–492.

4. Fuglsang-Damgaard, D., Pedersen, G., Schønheyder, H. Positive blood cultures and diagnosis of bacterial meningitis in cases with negative culture of cerebrospinal fluid. Scan J. of Inf Dis. 2008;40(3):229-233.

5. Meningitis Lab Manual: Methods for Diagnosis | CDC. 2022.Available from: https://www.cdc.gov/meningitis/lab-manual/index.html

6. Wanger, A., Chavez, V., Huang, R. Microbiology and molecular diagnosis in pathology: A comprehensive review for board preparation, certification and clinical practice. 2017. Philadelphia, PA: Elsevier Science Publishing.

7. Tunkel, A., Hartman, B., Kaplan, S., et al. Practice guidelines for the management of bacterial meningitis. Clin Inf Dis. 2014. 39:1267–1284.

8. Henry, B., Roy, J., Ramakrishnan, P., et al. Procalcitonin as a serum biomarker for differentiation of bacterial meningitis from viral meningitis in children. Clin Ped. 2015 .55:749–764.

9. Brouwer, M., Tunkel, A., van de Beek, D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Micrb Rev. 2010. 23:467–492.

10. Lammert, J. Techniques in Microbiology: a Student Handbook. San Francisco: Pearson Benjamin Cummings, 2007. pp.152-153.

11. Meningitis Lab Manual: ID, Characterization of Strep pneumoniae | CDC. (2022). Retrieved from https://www.cdc.gov/meningitis/lab-manual/chpt08-id-characterization-streppneumo.html

12. Medlineplus.gov. Gram Stain: MedlinePlus Medical Test. 2021. Available at: <https://medlineplus.gov/lab-tests/gram-stain/#:~:text=A%20Gram%20stain%20is%20a,genitals%2C%20or%20in%20skin%20wounds.&text=When%20the%20stain%20combines%20with,%2C%20they%20are%20Gram%2Dpositive.> [Accessed 18 January 2022].

13. Meningitis Lab Manual: Primary Culture and Presumptive ID | CDC. (2022). Retrieved from https://www.cdc.gov/meningitis/lab-manual/chpt06-culture-id.html

14. Meningitis Lab Manual: ID and Characterization of Hib | CDC. 2022. Retrieved from https://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html

15.  Meningitis Lab Manual: PCR Detection and Characterization | CDC. 2022. Retrieved from https://www.cdc.gov/meningitis/lab-manual/chpt10-pcr.html

(iv) What are the results expected from these tests allowing for the identification of the bacteria named in this case.

The blood and CSF samples that have undergone different tests for identification would indicate the following results for a presence of specific bacteria outlined below:

Figure 1: Left bottom corner on left image shows growth of N. meningitidis in BAP. Left top corner on the left image shows growth of S. pneumoniae in BAP. Left bottom corner on the right image shows growth of N. meningitidis in CAP. Top corner on the right image shows the growth of S. pneumoniae in CAP. The bottom right corner on the right image shows the growth of H. influenzae in CAP (1)

Culture test: This method of analysis can be used to visually determine the growth of N. meningitidis, H. influenzae and S. pneumoniae on blood agar plate (BAP) or chocolate agar plate (CAP). Growth of N. meningitidis can be observed as round, smooth, glistening, moist, convex edged colonies on BAP with no hemolysis (1, 2). On CAP, there would be large, colorless, opaque cultures also without hemolysis or discoloration (1). Growth of S. pneumoniae on BAP can be indicated by small, grey, moist and mucoidal colonies with alpha hemolysis (3). On CAP, there should also be small, grey, and moist culture with alpha hemolysis present (2). For H. influenzae, it is not expected to grow on BAP without supplementation (4). On CAP however, it would appear similar in appearance to  N. meningitidis with large, colorless, grey and opaque cultures (4). There would also be no hemolysis or discoloration (2). If unsure between visual appearance of N. meningitidis and H. influenzae, a further NAD test can be conducted to determine the results.

Gram Stain: Since N. meningitis is a gram-negative bacteria, it would be expected for the sample to turn a pink or red colour with the addition of crystal violet dye and iodine followed by safranin (5). The pink/red colour due to the lack of the thick peptidoglycan layer which functions to trap the violet dye, staining it purple (5).

With S. pneumoniae being a gram-positive bacteria, it has a thick peptidoglycan layer. This would function to trap the violet dye and visualize a purple color even after safranin is added (5). H. influenzae is a gram-negative bacteria, it would be expected to visualize a pink or red colour when safranin is added after the crystal violet dye and iodine are added. This is due to the lack of a thick peptidoglycan layer trapping the violet dye (5). Gram negative bacteria such as H. influenzae only have a thin peptidoglycan wall that is surrounded by a lipopolysaccharide outer membrane (6).

Figure 2: Visualization of Gram-negative (left) and Gram-positive (right) bacteria using gram staining test (7).

CSF sample tests:

Polymerase chain reaction (PCR) test: This form of analysis can be used for testing the presence of N. meningitidis, H. influenzae and S. pneumoniae. If N. meningitidis is in the sample, we would expect that Superoxide dismutase genes, sodC, to be detected (8). If H. influenzae is part of the sample, the presence of bexA should be observed since it can be found in H. influenzae serotypes (8). When S. pneumoniae is present, it would be expected that the specific segment of autolysin gene, lytA, will be detected (8).

Kovac’s oxidase test: The presence of N. meningitis and H. influenzae can both be detected by this test (4). This test functions by detecting the presence of cytochrome oxidase systems. If using the filter paper method, when either N. meningitis or H. influenzae are present in the tested culture, it would be expected to visualize a positive reaction on the filter paper (4). The positive indication would be a color change that turns the testing strip with the sample medium blue, as captured in the figure below (5). If using the plate method, tilt and observe the culturing plates for purple color change instead (4).

Figure 3: Visualization of Kovac’s oxidase test with a negative and positive reaction on filter paper swabbed with sample medium(11).

Carbohydrate utilization: After a positive test result is obtained during the Kovac’s oxidase test, the carbohydrate utilization test can be used to detect the presence of N. meningitis. A positive test for N. meningitis, would be indicated by a colour change to yellow (1). This would occur through a production of acid for glucose and maltose, indicated by the yellowing of the sample. While there must be no change in color for the negative test results for lactose and sucrose as visualized in the figure below (1). 

Figure 4: CTA sugar reactions for N. meningitidis with utilization of glucose (dextrose) and maltose, indicated by acid production (colour changes to yellow), and no change in colour for vials labelled lactose or sucrose (12).

Hemin and nicotinamide-adenin-dinucleotide (NAD) growth factor requirement test: This test can be used to determine the presence of H. influenzae. If H. infleunzae is present, we would expect to see the bacteria grow around the paper strip or disk that has both hemin + NAD (9). For confirmation of H. influenzae’s presence, a pink gram stain should be detected as it is gram negative. There should be a presence of bubbling during the catalase test. One should also see growth around the disk with NAD and hemin, as they are growth factors required for this bacteria (9). There should be no growth of the bacteria around the paper strip or disk containing only either hemin or NAD (9). There should be an absence of hemolysis on horse or rabbit blood agar to discriminate H. influenzae from H. Haemolyticus (4).

Slide agglutination serogrouping (SASG): This test is done is used in the identification of the serotype of N. meningitidis. Ultimately, it would be expected that a rating of 3+ or 4+ is present. This would indicate that strong agglutination of a unique strain of N. meningitidis is present (1).

After a Kovac’s oxidase test is done, it is followed by a test of hemin and NAD growth factor as a growth requirement, and finally serological tests are conducted to identify serotype of H. influenzae, which would need to indicate a rating of 3+ or 4+(4).

Latex agglutination: The presence of N. meningitidis, H. influenzae and S. pneumoniae can be detected by this method of analysis. The isolated usage of this test in determining the presence of  N. meningitis is not supported, it should rather be administered as a secondary test if Gram staining is unable to identify an infectious organism, or if the results indicate potential meningococcal infection (10). Functioning in a similar manner as antigen rapid tests, a positive result can be visually observed by the clumping of cells within 5-10 seconds of starting the test. On the other hand, if there is a negative result, no clumping will be noticed after 5-10 seconds (3). In a study conducted by Finlay et. al, it was found that the Latex agglutination test was only “positive in 60% cases of S. pneumoniae, 93% of H. influenzae type b, and 39% of N. meningitidis infections” (10). Since this method of analysis is not 100% accurate, its results should be cross referenced with other analytical methods.

Figure 5: Negative and positive latex agglutination reactions (13).

Catalase Test: This test is considered as positive if there is visualization of vigorous bubbles in the liquid due to the oxygen given off during the reaction. It would be expected to see a negative test result as there will be no bubbling if the bacteria detected in culture is S. pneumoniae (3).  Bubbling would indicate a positive test result, indicating that the bacteria is of the genus Staphylococcus(3).

Optochin Test: This test is used to detect S. pneumonia, so a zone of inhibition of 14 mm or greater would indicate that the sample bacteria is sensitive, identifying that the bacterial pathogen is indeed S. pneumonia (3).

References:

1. Meningitis Lab Manual: ID and Characterization of Neisseria | CDC. (2022). Retrieved 20 January 2022, from https://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html
2. Meningitis Lab Manual: Primary Culture and Presumptive ID | CDC. (2022). Retrieved 20 January 2022, from https://www.cdc.gov/meningitis/lab-manual/chpt06-culture-id.html
3. Meningitis Lab Manual: ID, Characterization of Strep pneumoniae | CDC. (2022). Retrieved 20 January 2022, from https://www.cdc.gov/meningitis/lab-manual/chpt08-id-characterization-streppneumo.html
4. Meningitis Lab Manual: ID and Characterization of Hib | CDC. (2022). Retrieved 20 January 2022, from https://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html
5. Lammert, J., 2007. Techniques in Microbiology: a Student Handbook. San Francisco: Pearson Benjamin Cummings, pp.152-153
6. Silhavy TJ, Kahne D, Walker S. The bacterial cell envelope. Cold Spring Harb Perspect Biol. 2010;2(5):a000414. doi:10.1101/cshperspect.a000414
7. Encyclopedia, M., 2022. Gram stain: MedlinePlus Medical Encyclopedia Image. [online] Medlineplus.gov. Retrieved 18 January 2022, fromhttps://medlineplus.gov/ency/imagepages/19955.htm
8. Meningitis Lab Manual: PCR Detection and Characterization | CDC. (2022). Retrieved 20 January 2022, from https://www.cdc.gov/meningitis/lab-manual/chpt10-pcr.html
9. Fuglsang-Damgaard, D., Pedersen, G., Schønheyder, H. Positive blood cultures and diagnosis of bacterial meningitis in cases with negative culture of cerebrospinal fluid. Scan J. of Inf Dis. 2008;40(3):229-233.
10. Finlay, F., Witherow, H., and Rudd P., Latex agglutination testing in bacterial meningitis. PMC Labs.1995. 73(2): 160-161
11. Meningitis Lab Manual: ID and Characterization of Hib | CDC. (2016). Retrieved 19 January 2022 from: https://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html
12. Meningitis Lab Manual: ID and Characterization of Hib | CDC. (2016). Retrieved 19 January 2022 from: https://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-hi.html
13. Meningitis Lab Manual: ID and Characterization of Hib | CDC. (2016). Retrieved 19 January 2022 from: https://www.cdc.gov/meningitis/lab-manual/chpt06-id-characterization-hi.html

3. Bacterial Pathogenesis

i. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria

Neisseria meningitidis (N. meningitidis), usually referred to as ‘the meningococcus’, is a gram-negative bacterial pathogen that is considered to be the leading cause of meningitis and other forms of meningococcal disease such as meningococcemia (1). It causes anywhere from 500,000 to 1,200,000 cases of invasive meningococcal disease annually, resulting in approximately 135,000 deaths worldwide (2).

Figure 1: Neisseria meningitidis (N. meningitidis)

Geographical location

Strains of N. meningitidis have been classed based on their specific serotypes, which are the antigenic differences in their porin protein (3). Of the 13 discovered serogroups of N. meningitidis, six serogroups (A, B, C, W-135, X, and Y) can be life threatening to infected patients (4). Outbreaks and pandemics of certain serogroups have occurred in different areas of the world.

The highest incidence of meningococcal disease is found in the ‘meningitis belt’ of sub-Saharan Africa with repeated epidemics every 5-10 years since 1905 (5–7). Historically, serogroup A has accounted for 90% of disease cases and most large-scale epidemics (2) . The “meningitis belt of sub-Saharan Africa” is a region of land stretching from Senegal in the east to Ethiopia in the west that is subject to high endemic rates of meningitis, yearly outbreaks, as well as epidemics every five to ten years (8). In Europe, serogroup A was considered the primary cause of invasive meningococcal disease prior to and during World War I and II, after which, serogroup B increased in prevalence and eventually surpassed serogroup A (2). Historically, serogroup A, and serogroup C to a lesser extent, has been responsible for the majority of N. meningitidis epidemics; however, after World War II, the incidence of serogroup A epidemics quickly decreased in most industrialized countries(2). Following 2010, vaccine introduction eliminated serotype A induced epidemics and recent epidemics have been due to serogroups C and W (6). Serogroup X outbreaks have also been reported in the meningitis belt (6). Invasive meningococcal disease (IMD) incidence is lower in all countries of the world outside the meningitis belt where the disease is endemic in crowded settings like schools, universities, and military establishments and is mainly caused by serogroups B and C (5,7). Infrequently, epidemics of serogroup A meningitis have occurred in China, Nepal, India, and Russia (5,7).

Figure 2: Global major meningococcal serogroups distributions and serogroup B outbreaks (purple)

Climate plays an important role in both the spatial distribution and the seasonality of IMD (7). Absolute humidity and land-cover type are climatic factors correlated to IMD epidemics (7,9). Climate zones not having distinct wet and dry seasons, such as deserts and the humid and forested parts of coastal and central Africa, are less likely to have epidemics than those with contrasting seasons like the semi-arid savanna and grassland found in the meningitis belt and eastern and southern Africa (9). The rate of infection rises during the driest months of the year between January and May while during the rainy season, incidence drops by more than a factor of 100 (9). Low absolute humidity and the dry Harmattan winter winds are the main climatic driver behind the seasonality in the meningitis belt (7,9). These environmental factors cause irritation and damage to the pharyngeal mucosa, which is the primary site of colonization by N. meningitidis (10). As a result, colonizing bacteria are more likely to invade the epithelium and cause infection (11)

Another adaptation that has risen in many geographical areas is capsule switching between serogroups, first identified in serogroups B and C. When two serogroups are co-carriage in an individual through recombinant events may happen, and the capsule gene can transfer between serogroups, known as capsule switching (12). This may be alarming in terms of vaccination, as vaccines to one serogroup with a different genetic capsule due to transformation may result in the vaccine-induced antibodies becoming ineffective or naturally acquired immunity (12).

Host location

Figure 3: Map showing meningitis belt in Africa with average annual attack rates

The highest rates of meningococcal disease caused by N. meningitidis are observed in young children, adolescents and young adults (13), with certain age groups having greater cases of specific N. meningitidis serotype carriage (5). Newborns who are less than 1 year of age are at the highest risk of infection by N. meningitidis given their low natural immunity against this bacterium (2). This cohort is closely followed by adolescents and young adults between the ages of 15 and 25, who exhibit increased rates of infection due to their relatively high carriage rate, as well as their behavior (2).

Furthermore, this type of bacterium cannot live long outside of the host but can remain on objects such as towels, glass, plastic, or hard surfaces for a short time. Since the bacterium needs iron to survive and colonize, it requires taking iron from human proteins such as transferrin and hemoglobin (14). If it is not in an environment with access to iron, it will not survive, which is why this bacterium cannot thrive outside the host.

N. meningitidis is a frequent colonizer of the human nasopharynx, where it spends most of its life as a commensal microorganism exploiting nutrients in mucosae (15,16). Most carrier isolates are shown to lack capsule production which may aid evasion of the human immune defense and hence be selected to survive nasopharyngeal colonization (17). N.meningitidis attaches to nonciliated columnar cells of the nasopharyngeal and oropharyngeal mucosa via type IV pili (Tfp), surface exposed filamentous organelles (3,5,18,19). The human nasopharynx meets the conditions for N. meningitidis survival and growth with  a temperature of ~34°C, high humidity, and sufficient CO₂ (5). N. meningitidis also expresses variable outer membrane adhesion proteins Opa and Opc that mediate bacterial attachment to the host, which can cause lead to pneumonia and sinusitis(5). Hence, respiratory secretions can lead to transmission responsible for disease acquisition.

Figure 4: Adhesion and lifecycle of N. meningitidis in the human nasopharynx

Transmission

Since N. meningitidis is a human pathogen, asymptomatic carriers are the major source of the pathogenic strains (17). Thus, transmission can occur through activities like kissing, sharing eating utensils or food, coughing or sneezing (7). Carriage has been linked to attendance at pubs or clubs, and cigarette smoke or exposure to passive smoke (5,7). Damage to the upper respiratory tract by co-infections (ex. mycoplasma, influenza, and other respiratory viral infections), smoking, very low humidity, drying of mucosal surface, and trauma induced by dust, predisposes to carriage and meningococcal disease (5).

How Mary came into contact with N. meningitidis

Since this bacterium keeps the host alive for transmission, it is highly virulent in crowded places, including universities (16). Mary, who has just moved into her university dormitory, could have come into contact with the bacteria through the sharing of drinking vessels with someone who had the bacterium as a asymptomatic or symptomatic carrier. About 1 in 10 people have these bacteria in the back of their nose and throat without being ill. People spread meningococcal bacteria to other people by sharing respiratory and throat secretions (saliva or spit). Generally, it takes close (for example, coughing or kissing) or prolonged contact to spread these bacteria (20). Due to the crowded nature of dormitories, it is also possible if Mary has been around a carrier that may have sneezed or coughed some respiratory droplets in her vicinity in her dorm space. Considering dorms in university have many students living in close quarters together, this seems feasible.

References

1. Tzeng Y-L, Stephens DS. Epidemiology and pathogenesis of Neisseria meningitidis. Microbes and Infection. 2000 May 1;2(6):687–700.

2. Gabutti G, Stefanati A, Kuhdari P. Epidemiology of Neisseria meningitidis infections: case distribution by age and relevance of carriage. J Prev Med Hyg. 2015 Aug;56(3):E116–20.

3. Morse SA. Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2022 Jan 21]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7650/

4. Rosenstein NE, Perkins BA, Stephens DS, Popovic T, Hughes JM. Meningococcal Disease. New England Journal of Medicine. 2001 May 3;344(18):1378–88.

5. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. The Lancet. 2007 Jun 30;369(9580):2196–210.

6. Meningococcal Disease in Other Countries | CDC [Internet]. 2021 [cited 2022 Jan 28]. Available from: https://www.cdc.gov/meningococcal/global.html

7. Palmgren H. Meningococcal disease and climate. Glob Health Action. 2009 Nov 11;2:10.3402/gha.v2i0.2061.

8. Soeters HM, Diallo AO, Bicaba BW, Kadadé G, Dembélé AY, Acyl MA, et al. Bacterial Meningitis Epidemiology in Five Countries in the Meningitis Belt of Sub-Saharan Africa, 2015–2017. The Journal of Infectious Diseases. 2019 Oct 31;220(Supplement_4):S165 74.

9. Magazine BMS Astrobiology. Climate conditions help forecast meningitis outbreaks [Internet]. Climate Change: Vital Signs of the Planet. [cited 2022 Jan 28]. Available from: https://climate.nasa.gov/news/1054/climate-conditions-help-forecast-meningitis-outbreaks

10. Mueller JE, Gessner BD. A hypothetical explanatory model for meningococcal meningitis in the African meningitis belt. International Journal of Infectious Diseases. 2010 Jul 1;14(7):e553–9.

11. Greenwood B. Manson Lecture: Meningococcal meningitis in Africa. Transactions of The Royal Society of Tropical Medicine and Hygiene. 1999 Jul 1;93(4):341–53.

12. Hill DJ, Griffiths NJ, Borodina E, Virji M. Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clinical Science. 2010 Feb 9;118(9):547–64.

13. Goldschneider I, Gotschlich EC, Artenstein MS. Human Immunity To The Meningococcus: I. The Role of Humoral Antibodies. Journal of Experimental Medicine. 1969 Jun 1;129(6):1307–26.

14. Bennett JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases E-Book. Elsevier Health Sciences; 2019. 5208 p.

15. Coureuil M, Join-Lambert O, Lécuyer H, Bourdoulous S, Marullo S, Nassif X. Mechanism of meningeal invasion by Neisseria meningitidis. Virulence. 2012 Mar 1;3(2):164–72.

16. Soriani M. Unraveling Neisseria meningitidis pathogenesis: from functional genomics to experimental models. F1000Res. 2017 Jul 26;6:1228.

17. Yazdankhah SP, Caugant DAY 2004. Neisseria meningitidis: an overview of the carriage state. Journal of Medical Microbiology. 53(9):821–32.

18. Pathogenic Neisseriae: gonorrhea and meningitis [Internet]. [cited 2022 Jan 28]. Available from: http://textbookofbacteriology.net/neisseria_5.html

19. Quagliarello V. Dissemination of Neisseria meningitidis. New England Journal of Medicine. 2011 Apr 21;364(16):1573–5.

20. Meningococcal Disease Diagnosis and Treatment | CDC [Internet]. 2021 [cited 2022 Jan 28]. Available from: https://www.cdc.gov/meningococcal/about/diagnosis-treatment.html

Figure References

Fig. 1: Meningococcal Disease/Meningitis | HR Portal [Internet]. [cited 2022 Jan 28]. Available from: https://hr.un.org/page/meningococcal-diseasemeningitis

Fig. 2: Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. The Lancet. 2007 Jun 30;369(9580):2196–210.

Fig. 3: Marc LaForce F, Ravenscroft N, Djingarey M, Viviani S. Epidemic meningitis due to Group A Neisseria meningitidis in the African meningitis belt: A persistent problem with an imminent solution. Vaccine. 2009 Jun 24;27:B13–9.

Fig. 4: Charles-Orszag A. Cellular and molecular mechanisms of human endothelial cell plasma membrane remodeling by Neisseria meningitidis [Internet] [Theses]. Université Sorbonne Paris Cité; 2017 [cited 2022 Jan 28]. Available from: https://tel.archives ouvertes.fr/tel-02121588

ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.

Entry

Meningococci is spread through respiratory droplets and oral and nasal secretions (1). Portals of entry include the mouth and nose (2). Acquisition of the bacteria may be transient, result in colonization of the nasopharynx epithelium (carriage), or lead to invasive disease (1). After colonization, N. meningitidis can become a habitual component of the normal microbial flora in humans, in which case the individual is considered an asymptomatic carrier (3). Carriers constitute 8-25% of all infected individuals, and the duration of carriage may range from days to months (3, 4). Bacterial colonization also has the potential to cause invasive disease, and this will usually develop within the first two weeks of bacterial acquisition (4).

Major Adhesins

Figure 5. Neisseria meningitidis type IV pilus assembly (7).

Type IV pili

N. meningitidis attaches to non-ciliated columnar cells of the nasopharyngeal mucosa via Tfp which are surface exposed filamentous organelles that extend beyond the capsule (2, 6). Tfp consists of hetero-multimeric pilin subunits that assemble into helical fibers (7). PilE, the major pilin subunit, constitutes the fiber scaffold and is a source of antigenic variation (7). This variation is generated through recombination between the silent loci, pilS, and the corresponding portion of the pilE gene (7). Pilus fiber contains three minor pilins, ComP, PilV, and PilX, which mediate specific Tfp-dependent functions such as competence (ComP), adhesion/signaling (PilV), and aggregation (PilX) (7). PilC1 also modulates the adhesive properties of the fiber (7). Tfp promotes the initial adhesion onto endothelial cells and the formation of aggregates, preventing the detachment of bacteria from the colony (7). The formation of aggregates is under the control of PilX, a minor pilin component, that connects the pilus fibers of adjacent bacteria (7). Tfp is initially assembled at the inner membrane of the bacterium, after which it is extruded to the outer membrane using secretin PilQ (5). Subsequent retraction of the pilus into the bacterium is facilitated by energy released through the ATPase activity of PilT (5). Retraction of the pilus is a form of bacterial motility known as twitching motility, and it is controlled by PilC (5). Pilus-mediated adhesion induces rearrangements in the cortical cytoskeleton and plasma membrane (8). Once meningococci reach the non-ciliated nasopharyngeal epithelial cells, they begin to form cortical plaque, which fully anchors the organism to the non-ciliated cells allowing the formation of microcolonies and biofilm (6). Cortical plaque induced rearrangements comprise high accumulations of phosphotyrosine, actin, ezrin and a subset of transmembrane glycoproteins at the neisserial attachment sites on the epithelial cells (8). Proliferation of N. meningitidis in contact with host cells increases the production of a transferase that adds a phosphoglycerol to the major pilin PilE (7). This unusual post-translational modification specifically releases Tfp-dependent contacts between bacteria, favoring their detachment from the colony and bacterial dissemination (7).

Figure 6. Neisseria meningitidis interaction with host cells (7).

CD46 is a complement regulator expressed on the apical surface of human epithelial cells and a membrane cofactor protein that acts as a receptor for Tfp (9 ).Primary bacterial attachment on endothelial cells depends on Tfp binding to transmembrane protein CD147 (10). CD147 is a member of the immunoglobulin (Ig) superfamily comprising two Ig-like domains, and is a marker of the brain capillaries N. meningitidis adheres to (10). Major pilin PilE and minor pilin PilV are responsible for the interaction with CD147 (10). The second receptor is the β2-adrenoceptor (β2AR), a member of the G protein-coupled receptor family (10). β2AR is recruited under the bacterial microcolonies at the apical surface of endothelial cells (10). In the context of endothelial cell infection by meningococci, β2AR plays the role of the ‘signaling’ receptor where it responds to circulating catecholamines, controlling vascular homeostasis, and signals via the heterotrimeric Gs protein and β-arrestins (10). β-arrestins are scaffolding proteins involved in many cellular processes such as receptor desensitization, internalization, and signaling (10). Similar to CD147, PilE and PilV interact with the extracellular N-terminal region of β2AR to transduce a signal inside cells (10).

Opacity Associated Adhesion Proteins

Opacity protein A (Opa) and opacity protein C (Opc) are integral proteins found in the outer membrane of N. meningitidis that also mediate adhesion (7,11). Opa is made up of eight transmembrane domains that form a β-barrel, with four surface loops exposed on the extracellular surface of the bacterium (11). Similar to Opa, Opc also forms a β-barrel embedded in the outer membrane of N. meningitidis; however, it contains five, rather than four, surface-exposed loops (11). In humans, Opa recognizes and binds to proteins that are part of the carcinoembryonic antigen-related cell-adhesion molecule (CEACAM) family (11). These include CEACAM1 which is expressed on epithelial cells, endothelial cells, as well as immune cells (11). During inflammation high levels of CEACAM are expressed, facilitating Opa interactions and therefore cellular attachment and invasion (7). The amount of CEACAM present in the environment may result in weaker or stronger bacterial interaction that can lead to invasion of pathogenic N. meningitidis (11). Additionally, Opa may also bind to cell-surface-associated heparan sulfate proteoglycan (HSPGs) which are found on the surface of most human epithelial cells (11). Furthermore, Opc can form a trimolecular complex using vitronectin, a glycoprotein, to interact with integrins and HSPGs on the surface of the epithelial cells to mediate adhesion and internalization (11). Opc is particularly implicated in host-cell invasion of endothelial cells (11). The tight association of Opc to vitronectin and/or fibronectin mediates binding of the bacteria to their cognate receptor, endothelial αVβ3 integrin (vitronectin receptor) and/or α5β1-integrin (fibronectin receptor) on brain-vessel cells (11).

Minor Adhesins

Additionally, N. meningitidis has several minor adhesin proteins, such as NadA (neisserial adhesinA), NhhA (Neisseria hia homologue A), App (adhesion and penetration protein), and MspA (meningococcal serine protease A). NadA binds to human epithelial cells via protein-protein interactions; however, its exact receptor is currently not known (11). Similarly, no receptors have been identified for App, but it is thought to contribute to bacterial adhesion to epithelial cells, as well as bacterial colonization and spreading (11). NhhA is known to facilitate adhesion to epithelial cells by binding to HSPGs and laminin (11). MspA adheres to human brain microvascular endothelial cells (12).

Figure 7. Neisseria meningitidis membrane structure (2).

Other Molecules

Meningococcal LOS/endotoxin plays a role in the adherence of the meningococcus and in activation of the innate immune system (7,8). Meningococcal lipid A is responsible for the biological activity and toxicity of meningococcal endotoxin (7,8).

N. meningitidis expresses two distinct porins, PorA and PorB, through which small hydrophilic nutrients diffuse into the bacterium via cation or anion selection (7). PorB inserts into membranes, induces Ca2+ influx and activates TLR2 and cell apoptosis (7). PorA is a major component of OM vesicle-based vaccines and a target for bactericidal antibodies (7).

Iron metabolism is also central to the fitness and ability of N. meningitidis to out-compete neighborhood bacteria and host defenses, making it essential for growth, colonization, and infection (5,13). N. meningitidis has over 80 genes regulated by the iron responsive repressor Fur (5). Meningococcal iron-acquiring proteins include hemoglobin receptor (HmbR), transferrin binding proteins (TbpA and TbpB), lactoferrin binding protein (HbpA and HbpA B), haemoglobin-haptoglobin complex (HpnA and HpnA B) (5). These proteins bind to human iron-carrying proteins transferrin and lactoferrin, then release and internalize iron into the bacterium (8). HbpA and HbpA B are more important than the other iron-binding proteins, since lactoferrin is the major iron source at the mucosal surface (8).

The regulatory protein CrgA acts both as an autorepressor and an activator of transcription at the mdaB promoter, playing a central role in meningococcal adhesion. Inactivation of the crgA gene, which encodes a transcriptional regulator belonging to the LysR family, decreases meningococcal adhesion to epithelial cells (14). CrgA has been reported to function by binding to the promoters of the crgA and pilC1 genes, as well as the pilE and sia genes, repressing transcription upon adhesion of bacteria to target epithelial cells (8). Thus, CrgA allows variation in Tfp expression depending on meningococcal needs for adhesion.  

For colonization of the human nasopharynx, the micro-organism must adhere to the mucosal surface, utilize locally available nutrients and evade the human immune system (8). N. meningitidis posess IgA1 protease which cleaves human IgA1, the dominant immunoglobulin in secretions (8). IgA1 inhibition thus promotes the adherence and colonization of N. meningitidis in the nasopharynx mucosal lining (8).

Host Defense

When N. meningitidis enter the nasopharynx, they adhere to the epithelium to evade the mucus clearing mechanism of the innate immune system (11). Additionally, ciliated cells promote host defense by pushing out foreign bacteria, but N. meningitidis uses the aforementioned major and minor adhesion proteins to adhere to non-ciliated columnar cells and evade this mechanism (6). N. meningitidis capsules contain sialic acids that allow the bacteria to evade the immune response as sialic acid is a protein common to the respiratory tract of host cells (15). Similarly, the α-chain structures of meningococcal LOS can mimic the human I and i antigens (15). Thus, sialic acid and LOS are examples of host molecular mimicry and immune escape mechanisms (15). Once the bacterium enters the blood and begins to replicate, pattern recognition receptors identify the PAMPS on the bacteria (6). Specifically, TLR4 on macrophages and immune cells will recognize the LOS on N. meningitidis (6). This triggers transduction pathways resulting in chemokine and cytokine release, increasing the number of immune cells in the area to defend against the bacterium (6). This inflammatory response is the host’s defence to prevent the bacteria from spreading further into the bloodstream. This constant inflammatory response being signaled may eventually result in sepsis as the bacteria cannot typically be cleared through the immune response (16). Moreover, environmental and geographical conditions like low humidity, dry winds and high levels of dust in the air injures the barriers of the upper respiratory tract (URT) mucosa, allowing N. meningitidis to more easily penetrate mucosal membranes to access the bloodstream and the meninges (1, 6). Dust storms also force people to stay indoors, where they may transmit the disease more easily to each other via droplets or secretions (17). Concurrent respiratory tract infections that irritate the UPR also increase host susceptibility to infection due to more permeable mucosal barriers (9).

N. meningitidis interaction with host cells. (i) N. meningitidis adheres to microvascular endothelial cells through an interaction between Tfp and CD147. At this stage, cellular junctions remain cohesive. (ii) Following initial adhesion, Tfp (via PilE and PilV) recruit and activate the β2AR that sequesters β-arrestins and ezrin, and induces actin polymerization following the activation of Src kinase and the recruitment of the polarity complex (Par6/Par3/PKCz) by the GTPase Cdc42. Adhesion receptors and adherens junction molecules (such as CD44, ICAM-1, and ErbB2) are recruited to the site of meningococcal adhesion. (iii) This signaling by delocalizing the junctional proteins leads to the opening of the intercellular space. (iv) Pili are responsible for bacterial aggregation, and the addition of a phosphoglycerol to the pilin subunit favors bacterial dissemination.

References

1. Palmgren, H. Meningococcal disease and climate. Global Health Action 2, 2061 (2009).  
2. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and neisseria meningitidis. The Lancet. 2007;369(9580):2196–210.  
3. Gabutti G, Stefanati A, Kuhdari P. Epidemiology of neisseria meningitidis infections: Case distribution by age and relevance of carriage [Internet]. Journal of preventive medicine and hygiene. Pacini Editore SPA; 2015.
4. Schmitz, J.E., & Stratton, C.W. (2015). Chapter 98 – Neisseria meningitidis.
5. Coureuil, M. et al. Mechanism of meningeal invasion Byneisseria Meningitidis. Virulence 3, 164–172 (2012).
6. Bennett JE, Blaser MJ, Dolin R. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Philadelphia, PA: Elsevier; 2020.  
7. Coureuil, M., Bourdoulous, S., Marullo, S. & Nassif, X. Invasive meningococcal disease: A disease of the endothelial cells. Trends in Molecular Medicine 20, 571–578 (2014).
8. Yazdankhah SP, Caugant DA. Neisseria meningitidis: An overview of the carriage state [Internet]. Journal of Medical Microbiology. Microbiology Society; 2004.
9. Borkowski J, Schroten H, Schwerk C. Interactions and signal transduction pathways involved during central nervous system entry by neisseria meningitidis across the blood–brain barriers. International Journal of Molecular Sciences. 2020;21(22):8788.  
10. Le Guennec L, Virion Z, Bouzinba-Ségard H, Robbe-Masselot C, Léonard R, Nassif X, et al. Receptor recognition by meningococcal type IV pili relies on a specific complex N-glycan. Proceedings of the National Academy of Sciences. 2020;117(5):2606–12.  
11. Hill DJ, Griffiths NJ, Borodina E, Virji M. Cellular and Molecular Biology of Neisseria meningitidis colonization and invasive disease. Clinical Science. 2010;118(9):547–64.  
12. Schubert-Unkmeir A. Molecular mechanisms involved in the interaction of neisseria meningitidis with cells of the human blood–cerebrospinal fluid barrier. Pathogens and Disease. 2017;75(2).  
13. Turner DP, Marietou AG, Johnston L, Ho KK, Rogers AJ, Wooldridge KG, et al. Characterization of MSPA, an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in neisseria meningitidis. Infection and Immunity. 2006;74(5):2957–64.  
14. Soriani M. Unraveling neisseria meningitidis pathogenesis: From functional genomics to experimental models. F1000Research. 2017;6:1228.  
15. Ieva R, Alaimo C, Delany I, Spohn G, Rappuoli R, Scarlato V. CRGA is an inducible lysr-type regulator of neisseria meningitidis , acting both as a repressor and as an activator of Gene Transcription. Journal of Bacteriology. 2005;187(10):3421–30.  
16. Stephens, D. S. Biology and pathogenesis of the evolutionarily successful, obligate human bacterium neisseria meningitidis. Vaccine 27, (2009).
17. Climate conditions help forecast meningitis outbreaks – climate change: Vital signs of the planet [Internet]. NASA. NASA; 2014. Available from: https://climate.nasa.gov/news/1054/climate-conditions-help-forecast-meningitis-outbreaks/

iii. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

Neisseria meningitidis is a pathogenic gram-negative bacterium that can cause invasive meningococcal disease after colonizing the mucosal lining (1). Its pathophysiology is a multi-step process that starts at the nasopharyngeal mucosal layer, spreads and survives travelling through the bloodstream until it reaches the blood-brain barrier (BBB) to cause meninges infection and then spreading through the cerebrospinal fluid (CSF) to replicate within the central nervous system (CNS) (2).

Spreading from the Nasopharynx Epithelium into the Bloodstream

Figure 7: Schematic overview of N. meningitidis penetration of the epithelial barrier of the nasopharynx (4)

Once the bacterium adheres tightly to cells in the mucosal lining and forms large aggregates called microcolonies at the attachment site with the help of PilX, they begin to form cortical plaques on the apical side of nasopharynx epithelial cells that do not have mucus covering them (3). In order to form these plaques, the Type IV Pili (Tfp) recruits the B2-adrenergic receptor to induce plaque formation (3). Further host cell and bacteria interactions occur via a cortical network containing ezrin-radixin-moesin (ERM) proteins anchored to the plasma membrane by their PIP2 binding domain and control the organization of the cortical actin cytoskeleton through their C-terminal F-actin binding sites (3). The ERM proteins also bind the cytoplasmic domain of several ERM binding transmembrane receptors such as CD44 and ICAM-1 through their C terminal domain and link the actin cytoskeleton and plasma membrane (3). Following the plaque formation, the bacterium uses its outer membrane ligands, Opa and Opc to bind to the host cell receptor carcinoembryonic antigen-related cell adhesion molecule (CEACAM) and heparan sulfate proteoglycan (HSPG) for the actin polymerization and plaque formation, allowing the internalization of the bacterium (4). Now that the bacterium has invaded the cell, it accumulates in the phagocytic vacuoles of the epithelial cells. Then, these bacteria in the vacuoles can be released into the space below the tight junction (1). Once released, they gain access to the bloodstream via cellular entry of the mucosal nasopharyngeal epithelial cells or crossing the epithelium barrier using extracellular proteins such as fibronectin and vitronectin (4).

This bacterium can regulate its capsule with an on and off phase variation to regulate how much capsule is produced using slipped-strand mispairing or insertions at the transcriptional level. This allows the bacterium to be capsule-deficient when being transmitted and provide tight adherence, as seen there is low biosynthesis of the capsule during early stages of nasopharyngeal colonization (5). However, it can also switch into an encapsulated stage for decimation within the host as the capsule protects the bacterium in the bloodstream and eventually the cerebrospinal fluid (CSF). The manipulation of the capsule allows for the survival of the bacteria in varying environments, allowing it to colonize both the nasopharynx and the bloodstream effectively (5).

Since the capillaries are very close to the mucosal epithelial tissues in the nasopharynx, it is most likely how the bacteria enter the bloodstream. In the blood, only encapsulated meningococci survive as the capsule evades the immune response by preventing phagocytosis and complement-mediated lysis (3). The LOS can entice an inflammatory response through TLR4-LPS signalling (4). But, Meningococci bind to multiple negative regulators of complement such as C4bp, factor H and vitronectin to decrease complement-mediated killing (4). The blood vessel wall becomes leakier with an inflammatory response due to the histamines and cytokines released to recruit immune cells. This can cause an entryway to more bacteria crossing the blood vessel lining to enter the bloodstream easier (4).

Invasion of the Central Nervous System Through The Blood-Brain Barrier

Figure 8: Overview of N. meningitidis invasion through Blood-Brain Barrier via two methods to reach meninges and cause meningococcal infection (4)

Once the bacteria is in the blood, it can spread to other parts of the body. Neisseria meningitidis is known to use the blood as a primary route to the brain to cross the blood-brain barrier. The blood-brain barrier refers to the non-fenestrated endothelium lining of the central nervous system (6). There are two hypothesized ways that the bacteria can cross the blood-brain barrier (BBB) to infect the meninges:  

1. Once the bacteria reach the BBB, it forms a cortical plaque by first using the Tfp to adhere to a single transmembrane protein, CD147, for primary attachment on the endothelial cells (7). After this initial interaction, the pili recruits the β2 -adrenoreceptor to start the cortical plaque formation and recruit β-arrestins to the area to lead to actin polymerization. This leads to the recruitment of the Par3/Par6/PKCz polarity complex, which facilitates the formation of intercellular junctional domains between the bacteria and host endothelial cells (8).
2. This cortical plaque will then allow the bacteria to be transcytosed into the human brain endothelial cells to cross the BBB to access the subarachnoid space (3). The brain microvascular epithelium tissue is damaged by all the cytokine release and inflammatory response, causing the depletion of junctional proteins between the endothelial cells causing disorganization of the endothelial lining and the breaking of the tight junctions, allowing the bacterium to squeeze through the leaky BBB via paracellular transport to reach the meninges (9).

Once the bacteria have passed the BBB, it was initially suggested that the bacteria enter the CSF via the choroid plexus to cross the blood-CSF barrier (3). However, an improved theory suggests the subarachnoid space is the site of entry for bacteria to the CSF as they have a leaky interendothelial structure that can drain the bacteria into the CSF (3). In the CSF, replication is easy due to the lack of immunoglobulins and complement proteins. Then the bacteria will infect the rest of the central nervous system as the CSF is the fluid that encompasses the CNS (3). This disease is then known to cause sepsis and bacterial meningitis as the central nervous system is the secondary site it spreads to.

References

1. Tzeng Y, Stephens D. Epidemiology and pathogenesis of Neisseria meningitidis. Microbes and Infection. 2000;2(6):687-700.

2. Sutherland T, Quattroni P, Exley R, Tang C. Transcellular Passage of Neisseria meningitidis across a Polarized Respiratory Epithelium. Infection and Immunity. 2010;78(9):3832-3847.

3. Coureuil M, Join-Lambert O, Lécuyer H, Bourdoulous S, Marullo S, Nassif X. Mechanism of meningeal invasion byNeisseria meningitidis. Virulence. 2012;3(2):164-172.

4. Hill D, Griffiths N, Borodina E, Virji M. Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clinical Science. 2010;118(9):547-564.

5. Spinosa M, Progida C, Talà A, Cogli L, Alifano P, Bucci C. The Neisseria meningitidis Capsule Is Important for Intracellular Survival in Human Cells. Infection and Immunity. 2007;75(7):3594-3603.

6. Schmitz J, Stratton C. Chapter 98 - Neisseria meningitidis. In: Tang Y, Sussman M, Liu D, Poxton I, Schartzman J, Merritt A, ed. by. Molecular Medical Microbiology. 2nd ed. Amsterdam: Academic Press; 2015. p. 1729-1750.

7. Coureuil M, Bourdoulous S, Marullo S, Nassif X. Invasive meningococcal disease: a disease of the endothelial cells. Trends in Molecular Medicine. 2014;20(10):571-578.

8. Coureuil M, Mikaty G, Miller F, Lécuyer H, Bernard C, Bourdoulous S et al. Meningococcal Type IV Pili Recruit the Polarity Complex to Cross the Brain Endothelium. Science. 2009;325(5936):83-87.

9. Stephens D. Neisseria meningitidis. In: Bennett J, Dolin R, Blaser M, ed. by. MANDELL, DOUGLAS, AND BENNETT'S PRINCIPLES AND PRACTICE OF INFECTIOUS DISEASES [Internet]. 9th ed. Philadelphia: Elsevier; 2019 [cited 17 January 2022]. p. 2585-2607.e7.

iv. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?

8-25% of all individuals infected by N. meningitidis are asymptomatic carriers of the microbe (1). In others, colonization can lead to invasive meningococcal disease which usually develops within the first two weeks (1). Meningococcal septicaemia and meningococcal meningitis are two of the most common forms of meningococcal disease seen in infected individuals. As described in further detail below, a majority of the bacterial damage in affected individuals is caused indirectly through the host response; however, some may result directly from the bacteria as well.

Meningococcal Septicaemia:

Figure 9. Structural diagram of lipooligosaccharides found in the outer membrane of Neisseria meningitidis

Invasion of the bloodstream by N. meningitidis is known as meningococcemia, and it is characterized by rapid proliferation of the bacteria in circulation, which results in high concentrations of meningococcal endotoxins (2). N. meningitidis contains endotoxins in the outer membrane of its cell wall in the form of lipooligosaccharides (LOS) which are composed of three main parts: lipid A, a core oligosaccharide, and highly variable short oligosaccharides (3). Lipid A, which is covalently bound to the core oligosaccharide, consists of hydroxy fatty-acid chains and phosphoethanolamine (PEA) (3). The core oligosaccharide, in turn, contains 3-deoxy-D-manno-oct-2-ulosonic acid, as well as heptose residues which link it to the short oligosaccharide residues of the a-, b-, g-chains (3). Lipooligosaccharides are a neisserial form of lipopolysaccharides (LPS) that differ from enteric LPS in that they don’t contain repeating O-antigen subunits (4). Lipid A, particularly, is responsible for much of the toxicity of meningococcal endotoxin (3).

N. meningitidis releases its endotoxins through blebbing which bind to lipopolysaccharide binding protein (LBP) and are transported to immune cells such as macrophages, monocytes, and neutrophils (2). The aforementioned immune cells contain the CD14/TLR4-MD-2 receptor complex on their cell surface (2). Transfer of lipid A to CD14 leads to the generation of a transmembrane signal, which eventually results in the production of nuclear factor kb, among other cytokines (2). Nuclear factor kb initiates the transcription of genes encoding inflammatory mediators (2). The subsequent release of cytokines, chemokines, nitric oxide, and reactive oxygen species characterize the resulting inflammatory response (5). The inflammatory response, in turn, leads to increased vascular permeability which is largely responsible for the shock and multiorgan failure that is characteristic of meningococcemia (6).

Purpura Fulminans:

Purpura fulminans resulting from meningococcal septicaemia is a life-threatening condition with a mortality rate greater than 50% (7). It is characterized by hemorrhagic infarction of the skin resulting from coagulation abnormalities (7).

(a) Production of Thrombin

As discussed previously, the presence of meningococcal endotoxins within the bloodstream leads to an inflammatory response characterized by the production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1) and interleukin-6 (IL-6) (5). These molecules stimulate monocytic overexpression of active tissue factor (TF) which binds to activated factor VII (VIIa), initiating the extrinsic coagulation cascade (8). Next, TF-VIIa complex cleaves factor X, forming its active form, factor Xa (7). Factor Xa, in conjunction with factor Va, cleaves prothrombin into thrombin (7). Thrombin is considered a key enzyme of coagulation given its ability to cleave fibrinogen into fibrin (8).

Figure 10. Overview of the coagulation cascade

In addition to the extrinsic coagulation cascade, the intrinsic (contact) coagulation pathway is also activated (8). This primarily occurs through the binding of factor XII to a negatively charged surface, such as LOS or the surface of activated platelets (8). Like the extrinsic pathway, the intrinsic pathway also results in thrombin production; therefore, the combined effect of both pathways is exponential production of thrombin (8).

(b) Inhibition of Anticoagulant Pathways

Normally, harmful overactivation of the coagulation cascade is prevented through anticoagulant pathways involving antithrombin III, protein C/activated protein C, and the tissue factor pathway inhibitor (8). However, deficiencies in these molecules due to N. meningitidis infection results in uncontrolled coagulation (8).

Antithrombin III regulates the coagulation cascade through inhibition of thrombin and factors Xa, IXa, VIIa, and XIa (7). However, N. meningitidis infection results in extremely low levels of antithrombin III due to decreased synthesis, alongside increased degradation by activated neutrophils (7). Similarly, protein C regulates the coagulation cascade by binding to protein S and facilitating the inactivation of factors Va and VIIa, both of which are vital to thrombin formation (7). Protein C is activated through interactions with thrombomodulin on the surface of vascular endothelial cells (7). Meningococcal infection, however, results in thrombomodulin downregulation (7). Additional protein C deficiency results from increased consumption, decreased production, and increased loss due to vascular leakage (7). Additionally, tissue factor pathway inhibitor (TFPI) acts as a serine protease inhibitor to inhibit factor Xa and the TF-VIIa complex (7). However, clinical data depicts extremely low levels of TFPI in individuals with N. meningitidis infections (7).

(c) Inhibition of Fibrinolysis

Fibrinolysis is the process by which fibrin clots are destroyed. It begins with the conversion of plasminogen into plasmin, primarily using tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) to do so (8). Meningococcal LOS, however, causes monocytes to produce plasminogen activator inhibitor 1 (PA-1) which inhibits tPA and disrupts the fibrinolysis pathway (8).

Figure 11. Clinical presentation of purpuric lesions in an individual with meningococcal septicaemia

All in all, meningococcal septicaemia results in a profound coagulant response that leads to diffuse intravascular coagulation (DIC) and eventually vascular thrombosis (8). The coagulant response is further promoted by the adhesion of N. meningitidis to endothelial cells of the microvasculature (8). Extensive endothelial colonization results in microvessel thrombosis (8). Thrombosis, in turn, causes red blood cells extravasation, and the skin rash that is characteristic of purpura fulminans (8). Petechiae (minute hemorrhagic spots in the skin) or purpura (hemorrhages into the skin) occur from the first to the third day of illness in 30 to 60 percent of patients with meningococcal disease, with or without meningitis (4). Lesions are sparsely distributed over the body. They tend to occur in crops and on any part of the body; however, the face is usually spared, and involvement of the palms and soles is less common (9). The skin rash may progress from a few ill-defined lesions to a widespread eruption within a few hours (9).

Meningococcal Meningitis:

Meningococcal meningitis is a type of meningococcal disease in which N. meningitidis breaches the blood-brain barrier and enters the subarachnoid space, resulting in inflammation of the meninges (6). Clinical manifestations occur primarily due to the resulting increase in intracranial pressure and brain inflammation (6). Brain inflammation occurs due to an increase in LOS, which interacts with TLR4, activating the local immune response (6). Pro-inflammatory cytokines such as interleukin-10 (IL-10) and tumour necrosis factor alpha (TNF-a) increase vascular permeability of the blood-brain barrier to allow the influx of neutrophils and other immune cells (6). This causes increased blood flow and cerebral oedema, which, in turn, leads to an increase in intracranial pressure (9, 10). This process is responsible for the classic symptoms associated with meningococcal meningitis such as sudden headaches, fever, chills, malaise, and neck stiffness (10). Mary, the patient in this case study, presented with all of the aforementioned symptoms. As the disease progresses, photophobia may also develop which explains Mary’s sensitivity to light (9).

References

1. Gabutti G, Stefanati A, Kuhdari P. Epidemiology of Neisseria meningitidis infections: case distribution by age and relevance of carriage. J Prev Med

Hyg. 2015 Aug;56(3):E116–20.

2. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. The Lancet. 2007 Jun 30;369(9580):2196–210.

3. Rouphael NG, Stephens DS. Neisseria meningitidis: Biology, Microbiology, and Epidemiology. Methods Mol Biol. 2012;799:1–20.

4. Todar K. Pathogenic Neisseriae: gonorrhea and meningitis [Internet]. [cited 2022 Jan 21]. Available from: http://textbookofbacteriology.net/neisseria.html

5. Zughaier SM, Tzeng Y-L, Zimmer SM, Datta A, Carlson RW, Stephens DS. Neisseria meningitidis Lipooligosaccharide Structure-Dependent Activation of the

Macrophage CD14/Toll-Like Receptor 4 Pathway. Infection and Immunity [Internet]. 2004 Jan [cited 2022 Jan 21]; Available from: https://journals.asm.org

/doi/abs/10.1128/IAI.72.1.371-380.2004

6. Pathan N. Pathophysiology of meningococcal meningitis and septicaemia. Archives of Disease in Childhood. 2003 Jul 1;88(7):601–7.

7. Betrosian et al. - 2006 - Purpura Fulminans in Sepsis.pdf [Internet]. [cited 2022 Jan 27]. Available from: https://ovidsp.dc1.ovid.com/ovftpdfs

/FPACKPNMBFKLJC00/fs046/ovft/live/gv025/00000441/00000441-200612000-00006.pdf

8. Lecuyer H, Borgel D, Nassif X, Coureuil M. Pathogenesis of meningococcal purpura fulminans. Pathogens and Disease. 2017 Apr 1;75(3):ftx027.

9. 1. Wijdicks EFM. Acute Bacterial Meningitis [Internet]. The Practice of Emergency and Critical Care Neurology. Oxford University Press; [cited 2022 Jan 28]. Available from: https://oxfordmedicine.com/view/10.1093/med/9780195394023.001.0001/med-9780195394023-chapter-31

10.  Schmitz J, Stratton C. Chapter 98 – Neisseria meningitidis. In 2015.

Figure References

Fig. 9: Kahler CM, Nawrocki KL, Anandan A, Vrielink A, Shafer WM. Structure-Function Relationships of the Neisserial EptA Enzyme Responsible for

Phosphoethanolamine Decoration of Lipid A: Rationale for Drug Targeting. Frontiers in Microbiology [Internet]. 2018 [cited 2022 Jan 28];9. Available from:

https://www.frontiersin.org/article/10.3389/fmicb.2018.01922

Fig. 10: Coagulation - Intrinsic - Extrinsic - Fibrinolysis [Internet]. TeachMePhysiology. [cited 2022 Jan 28]. Available from: https://teachmephysiology.com/immune-system/haematology/coagulation/

Fig. 11: Pitukweerakul S, Sinyagovskiy P, Aung PP. Purpura Fulminans in Acute Meningococcemia. J GEN INTERN MED. 2017 Jul 1;32(7):848–9.

4. The Immune Response

i. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

Innate immunity

N. meningitidis will colonize the mucosal surface of the human nasopharynx, which is the only known natural reservoir for the bacteria. It may cross the mucosal barrier and travel via blood vessels to cause disease.

N. meningitidis is a gram-negative bacteria that spreads through respiratory droplets (1). N. meningitidis enters the body via the nasal passage and colonizes the nasopharynx (1). In order to maintain nasopharyngeal colonization, N. meningitidis must evade several host immune defenses.

The first line of defense that N. meningitidis encounters is the mucus that lines the upper layer of respiratory tract cells (2). One of the functions of mucus is to trap bacteria in order to protect the underlying epithelial cells from bacterial invasion and/or damage (2). Mucociliary clearance (also known as the mucociliary escalator) causes the mucus, with embedded bacteria, to be moved up and out of the respiratory tract and swallowed (2). Besides its utility in physically trapping bacteria, mucus consists of antimicrobial proteins, such as immunoglobulins, lysozymes, and human defensins (3). Among the immunoglobulins is secreted immunoglobulin A, the most abundant antibody class in mucosal secretions, which binds to and neutralizes potential pathogens to trap them and enable their clearance through the mucociliary escalator (4). The mucosal barrier serves as a relatively effective line of defense as suggested by the high proportion of asymptomatic carriers of N. meningitidis (5). Estimates for the rate of N.meningitidis carriage in the population based on throat swabs are around 10%, but other diagnostic techniques suggest that the actual portion of carriers may be even higher – up to 30% (5, 6). Even during epidemics of meningococcal meningitis with carrier rates reaching as high as 95%, the incidence of meningococcal disease remained less than 1% (7). The seemingly low incidence of disease following N. meningitidis colonization is indicative of the effectiveness of host innate immune defenses such as mucociliary clearance (7).

Depicts complement activation, inflammation immune activation, and phagocytosis, among other immunological processes.

The innate immune system is the body’s immediate and non-specific defense mechanism against pathogen invasion, and it is triggered when host pattern recognition receptors (PRRs) are activated by pathogen associated molecular patterns (PAMPs) from N. meningitidis (8). An important class of PRRs are toll-like receptors (TLRs), of which TLR1/2, TLR4, and TLR9 have the ability to recognize N. meningitidis PAMPs (9). Porin B (PorB) is a N. meningitidis outer membrane protein recognized by TLR2/TLR1 complex. TLR4 is activated by meningococcal lipooligosoccharide (LOS), whereby the LOS on the bacterial outer membrane binds to LPS-binding protein (LBP)  forming a complex that interacts with CD14 (4). CD14 facilitates transfer of the LBP-LOS complex to MD-2-TLR4 (4). Lastly, TLR9 is activated by bacterial DNA (10). Once the PAMP has bound to the TLR, an adaptor protein, MyD88 binds to the activated TLR and initiates a signaling cascade that ultimately results in NF-kB being sent to the nucleus to trigger transcription of proinflammatory genes, such as genes encoding tumor necrosis factor alpha (TNF-alpha), IL-1, IL-6, IL-12p40, and cyclooxygenase-2 (11). Additionally, NF-kB can induce transcription of M1 macrophages which produce cytokines (e.g., IL-1, IL-6, IL-12, TNF-alpha) involved in a variety of inflammatory responses, such as triggering apoptosis of stressed cells (11).

Another type of PRR are scavenger receptors, SR-A and MARCO. SR-A is the predominant receptor involved in phagocytosis of non-opsonized N. meningitidis by macrophages (8). MARCO recognizes lipopolysaccharide (LPS) and SR-A recognizes lipids A and LPS. Both SR-A and MARCO are involved in controlling TLR4 production of TNF-alpha, nitric oxide (NO), and IL-6 (8). IL-6 can stimulate differentiation of naïve CD4+ T cells and activate B cells to differentiate into plasma cells (12). TNF-alpha production leads to meningeal inflammation, and the role of NO in bacterial meningitis is still being explored but it has been shown to play a role in vasodilation during the inflammatory response (13).

Another defense mechanism the innate immune system will use to rid the body of N. meningitidis is complement activation (14). The complement cascade has three component pathways: classical, lectin, or alternative (14). The complement pathways lead to insertion of C5b-9 MAC complex into the bacterial cell membrane resulting in cell lysis or opsonization of bacteria (14). The classical pathway initiation requires antibody or C-reactive protein (CRP) to be present on the bacterium (15). N. meningitidis PRRs that are recognized by mannose binding lectin (MBL) and trigger the lectin pathway are: terminal mannose, GlcNAc, fucose (8). The primary effects of complement activation includes the generation of inflammatory factors, recruitment and enhancement of phagocytes, and lysis of bacterial cells. This ultimately leads to fever, another component of the immune response, as a non-ideal environment for the bacterial pathogens is created (8).The alternative pathway is activated by complement components directly binding to N. meningitidis surface (14). The classical pathway is particularly noteworthy for its role in meningococcal infection, as the presence or lack of antibodies specific for N. meningitidis antigens has an impact on the rates of infection in in the late teenage years when teens leave their communities to live in university residences, exposing themselves to new strains which they have not acquired immunity to (5). This is entirely consistent with Mary’s situation as a university student in residence for their first time without a meningococcal vaccination.  

Adaptive immunity

If N. meningitidis evades physical/anatomical barriers and the innate immune system is not sufficient to clear infection, the body’s more specific and long-lasting defense, the adaptive immune system, is activated (16). The adaptive immune system response is subdivided into two categories: humoral and cell-mediated immunity (16). Humoral immunity is comprised of the immune components within body serum (i.e., is mediated by antibodies produced by plasma cells). Whereas cell-mediated immunity is the immune response that is carried out by cells (e.g., T cells, macrophages, natural killer [NK] cells) (16).

Adaptive immunity: Cell-mediated Immunity

When a helper T (Th) cell comes into contact with N. meningitidis antigen (Ag) presented by an antigen-presenting cell (APC) it will either differentiate into a Th1 or Th2 cell depending on which class of MHC the Ag peptide was bound to (17). Th1 and Th2 cells activate the production of different types of cytokines and immunoglobulins (Ig) (12). Th1 mainly initiates secretion of interferon-gamma, and Th2 mainly initiates secretion of IL-4, IL-5, and IL-13 (18). Interferon-gamma can induce macrophage activation and phagocytosis of bacteria (19). IL-4, IL-5, and IL-13 stimulate B cell differentiation and play a role in humoral immunity (20).

After antigen-presenting cells phagocytose an antigen and present to CD4+ Th cells in the lymph nodes, CD4+ Th cells are induced to promote proliferation of CD8+ cytotoxic T cells which respond to destroy infected cells displaying the antigen (5). These CD4+ helper T cells do so through the production of lymphokines, which aid both B cells and T cells in mounting an efficient immune response to a specific antigen (5). CD8+ cytotoxic T cells then lyse infected cells, a process triggered by MHC I-dependent antigen presentation by infected cells (5). This antigen specificity is what makes the adaptive immune response stronger compared to the innate immune response and able to recognize the same antigen in future infections (5).

Dendritic cells (DCs) are also responsible for cell-mediated clearance of N. meningitidis. DCs phagocytose N. meningitidis or become infected by the bacteria which causes DC maturation (21). Mature DCs no longer endocytose pathogens, and instead secrete proinflammatory cytokines, such as IL-1beta, IL-6, and TNF-alpha (21). Additionally, DCs are a type of APC that present antigen to lymphocytes (21).

Adaptive immunity: Humoral Immunity

B cells can be activated in a T cell-dependent or T cell-independent way (interact directly with antigen), and one of their main functions is antibody production (22). If a B cell binds to a T-independent antigen (e.g., lipopolysaccharide) and interacts with a TLR bound to a PAMP it is activated in a T cell-independent way that lasts a short amount of time and does not produce memory B cells (23). Alternatively, T cell-dependent activation involves the naïve B cell processing an antigen and presenting it on MHC II to a Th cell (23). Subsequently, the Th cell’s CD4 co-receptor interacts with the B cell MHC II, which then causes the Th cell to secrete cytokines that activate the B cell to proliferate and differentiate into memory B cells (23). The main antibody produced by B cells in response to N. meningitidis infection is IgG, but IgA and IgM are present as well (20). IgG can activate the classical complement pathway by binding with C1q resulting in bacterial lysis (24).

References

1. Weyand NJ. Neisseria models of infection and persistence in the upper respiratory tract. Pathog Dis. 2017;75(3). Available from: http://dx.doi.org/10.1093/femspd/ftx031
2. Audry M, Robbe-Masselot C, Barnier J-P, Gachet B, Saubaméa B, Schmitt A, et al. Airway mucus restricts Neisseria meningitidis away from nasopharyngeal epithelial cells and protects the mucosa from inflammation. mSphere. 2019;4(6). Available from: http://dx.doi.org/10.1128/mSphere.00494-19
3. Brandtzaeg P. Mucosal immunity: induction, dissemination, and effector functions. Scand J Immunol. 2009;70(6):505–15. Available from: http://dx.doi.org/10.1111/j.1365-3083.2009.02319.x
4. Bennett JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, Ninth Edition. 2020.
5. Kvalsvig AJ, Unsworth DJ. The immunopathogenesis of meningococcal disease. J Clin Pathol. 2003 Jun;56(6):417–22.
6. Piet JR, Brouwer MC, Exley R, van der Veen S, van de Beek D, van der Ende A. Meningococcal Factor H Binding Protein fHbpd184 Polymorphism Influences. Clinical Course of Meningococcal Meningitis. PLoS One. 2012 Oct;7(10):e47973.
7. Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2022 Jan 24]. Available from: ttp://www.ncbi.nlm.nih.gov/books/NBK7627/
8. Johswich K. Innate immune recognition and inflammation in Neisseria meningitidis infection. Pathog Dis. 2017;75(2). Available from: http://dx.doi.org/10.1093/femspd/ftx022
9. Mogensen TH, Paludan SR, Kilian M, Ostergaard L. Live Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis activate the inflammatory response through Toll-like receptors 2, 4, and 9 in species-specific patterns. J Leukoc Biol. 2006;80(2):267–77. Available from: http://dx.doi.org/10.1189/jlb.1105626
10. Koedel U. Toll-like receptors in bacterial meningitis. Curr Top Microbiol Immunol. 2009;336:15–40. Available from: http://dx.doi.org/10.1007/978-3-642-00549-7_2
11. Liu T, Zhang L, Joo D, Sun S-C. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2(1):17023. Available from: http://dx.doi.org/10.1038/sigtrans.2017.23
12. Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol [Internet]. 2014;6(10):a016295. Available from: http://dx.doi.org/10.1101/cshperspect.a016295
13. Burgner D, Rockett K, Kwiatkowski D. Nitric oxide and infectious diseases. Arch Dis Child [Internet]. 1999;81(2):185–8. Available from: http://dx.doi.org/10.1136/adc.81.2.185
14. Schneider MC, Exley RM, Ram S, Sim RB, Tang CM. Interactions between Neisseria meningitidis and the complement system. Trends Microbiol. 2007;15(5):233–40. Available from: http://dx.doi.org/10.1016/j.tim.2007.03.005
15. Sprong T, Roos D, Weemaes C, Neeleman C, Geesing CLM, Mollnes TE, et al. Deficient alternative complement pathway activation due to factor D deficiency by 2 novel mutations in the complement factor D gene in a family with meningococcal infections. Blood. 2006;107(12):4865–70. Available from: http://dx.doi.org/10.1182/blood-2005-07-2820
16. Chaplin DD. Overview of the immune response. J Allergy Clin Immunol. 2010;125(2 Suppl 2):S3-23. Available from: http://dx.doi.org/10.1016/j.jaci.2009.12.980
17. Davenport V, Guthrie T, Findlow J, Borrow R, Williams NA, Heyderman RS. Evidence for naturally acquired T cell-mediated mucosal immunity to Neisseria meningitidis. J Immunol. 2003;171(8):4263–70. Available from: http://dx.doi.org/10.4049/jimmunol.171.8.4263
18. Berger A. Science commentary: Th1 and Th2 responses: what are they? BMJ. 2000;321(7258):424–424. Available from: http://dx.doi.org/10.1136/bmj.321.7258.424
19. Tau G, Rothman P. Biologic functions of the IFN-gamma receptors. Allergy. 1999;54(12):1233–51. Available from: http://dx.doi.org/10.1034/j.1398-9995.1999.00099.x
20. Justiz Vaillant AA, Qurie A. Interleukin. In: StatPearls. StatPearls Publishing; 2021.
21. Kolb-Mäurer A, Kurzai O, Goebel W, Frosch M. The role of human dendritic cells in meningococcal and listerial meningitis. Int J Med Microbiol. 2003;293(4):241–9. Available from: http://dx.doi.org/10.1078/1438-4221-00266
22. Hoffman W, Lakkis FG, Chalasani G. B cells, antibodies, and more. Clin J Am Soc Nephrol. 2016;11(1):137–54. Available from: http://dx.doi.org/10.2215/CJN.09430915
23. Ratajczak W, Niedźwiedzka-Rystwej P, Tokarz-Deptuła B, Deptuła W. Immunological memory cells. Cent Eur J Immunol [Internet]. 2018;43(2):194–203. Available from: http://dx.doi.org/10.5114/ceji.2018.77390
24. Vidarsson G, van Der Pol WL, van Den Elsen JM, Vilé H, Jansen M, Duijs J, et al. Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B. J Immunol. 2001;166(10):6250–6. Available from: http://dx.doi.org/10.4049/jimmunol.166.10.6250
    

ii. Host damage: what damage ensues to the host from the immune response?

Infection and the inflammatory response

An intense inflammatory response has been attributed to many of the clinical manifestations of meningococcal meningitis, which in turn cause the signs and symptoms of meningococcal meningitis.(1) Upon the invasion of a pathogen, inflammation is critical for an immune response that successfully eradicates invading microorganisms – however, this inflammation must be localized to prevent damage to the host’s tissues and organs.(3) A hallmark feature of meningococcal meningitis is excessive inflammation.(3) Once N. meningitidis spreads from the nasopharynx and replicates in the bloodstream, inflammation can become systemic and cause multi-organ failure and death.(2)

N. meningitidis infection starts with its attachment to the pharyngeal mucosal epithelial cells, and the subsequent crossing of the mucosal barrier, prompting recognition of PAMPs by the host’s PRRs, and thus, an immune response. Following infection, several changes in the body are produced, such as local inflammation, fever, and low blood pressure as a result of the cytokine storm and subsequent septic shock (4). These changes are mediated by activation of the complement cascade, the recruitment of effector cells, and the consequent secretion of cytokines by immune cells and epithelial cells. More specifically, elevated levels of potent proinflammatory mediators, complements C3a and C5a, in N. meningitidis infection are associated with different inflammatory disorders and negative disease outcomes (2)

Elevated cytokine and chemokine levels in the blood and cerebrospinal fluid are also associated with poor patient prognosis (2). The cytokine-induced leakage of blood vessels to allow for leukocyte entry and the release of histamine by mast cells, eosinophils, and basophils, will also contribute to vasodilation and low blood pressure of the host (5). Non-specific cytotoxic granules released by neutrophils and macrophages may cause host cell lysis and contribute to host damage as well (4). The progression from localised infection to systemic, and the ultimate invasion of the central nervous system by N. meningitidis results in the denominative inflammation of the meninges, as well as increased permeability of the blood brain barrier, and cerebrospinal fluid pleocytosis, or, increased white blood cell count (5). Permanent neurological sequelae due to the excessive inflammatory response of the neutrophils is seen in half of all survivors of bacterial meningitis (5).

Nervous damage (meninges and CSF)

Bacterial meningitis consists of inflammation in the meninges as a result of N. meningitidis infection (2). This occurs when bacteria passes from the nasopharynx, through the mucosal epithelium and basement membrane, into systemic circulation and penetrates the blood brain barrier (BBB) (6). In particular, meningitis occurs when N. meningitidis enters the subarachnoid space (the cavity in between arachnoid and pia mater), resulting in highly activated leukocytes entering via the cerebrospinal fluid (CSF) and releasing pro-inflammatory cytokines (2). Typically, the inflammation goes beyond the meninges and also affects brain parenchyma, ventricles, and could impact the spinal cord (2). Neuroinflammation results in the release of cytotoxic molecules, such as nitric oxide, which triggers apoptosis of neurons and other brain cells (6). Persistent inflammation in the brain can result in neuronal complications, such as hearing loss and motor deficits (7).

Inflammation can lead to increased pressure in the CSF and brain, causing symptoms such as fever, headache, neck stiffness and drowsiness (8). The presence of inflammatory mediators (including liposaccharides and cytotoxin-engaging receptors, TLRs) within the CSF increases the accessibility of the BBB. Endothelial cells can activate downstream cascades that release white-blood cell precursors and other immune cell responses. This allows neutrophils to move across the BBB.

Vascular damage

If N. meningitidis invades the blood stream it could result in septicemia (9), which can occur in up to 20% of patients (10). Septicemia is a result of hyper-activation of the host immune system, such as the coagulation and complement cascades, resulting in hyperinflammation (11). Damage from sepsis can include purpura fulminans which leads to dermal and soft tissue necrosis (9). Septicemia can rapidly progress into septic shock, leading to multiple organ failure, and potentially death (12). In the brain, N. meningitidis are seen interacting with capillaries of the subarachnoidal space, the brain parenchyma and the choroidal plexuses, and inside brain vessels. When a low or moderate number of meningococci is present in the bloodstream, the bacteria interacting with peripheral capillaries cause only few localized purpuric lesions, whereas the interaction with brain endothelial cells is sufficient to lead to meningeal invasion. Adhesion of the bacteria to the meninges and meningeal cells is critical for N. meningitidis to disseminate through the meningeal spaces.

A principal causal factor of this inflammation is circulating bacterial endotoxin, which has been linked to the severity of meningococcal septicaemia.(1) Endotoxin binds to endotoxin binding protein in the plasma, facilitating the activation of inflammatory cells such as macrophages to initiate an intense inflammatory response.(1) Upon activation, macrophages produce a diverse range of proinflammatory cytokines including TNFalpha and IL-beta.(1) The levels of these cytokines are correlated with disease severity, indicating that macrophage activation by endotoxin is a significant contributor to host damage.(1) Endotoxin also binds to cellular receptors such as CD14 and toll-like receptors, which contribute to the intense inflammatory response characteristic of meningococcal infection that causes vascular damage, inflammation of the meninges, and cerebral edema.(1)

Other damage

In 6-15% of cases of meningococcal meningitis, inflammatory syndromes arise, including arthritis (inflammation of joints), cutaneous vasculitis (inflammation of blood vessels in skin and subcutaneous tissue), iritis (inflammation of iris), episcleritis (inflammation of episcleral tissue), pleuritis (inflammation of pleura) and/or pericarditis (inflammation of pericardium) (13).

References

1. Pathan N. Pathophysiology of meningococcal meningitis and septicaemia. Arch Dis Child. 2003 Jul 1;88(7):601–7.
2. Johswich K. Innate immune recognition and inflammation in Neisseria meningitidis infection. Pathog Dis. 2017 Mar 1;75(2):ftx022.
3. Kvalsvig AJ, Unsworth DJ. The immunopathogenesis of meningococcal disease. J Clin Pathol. 2003 Jun;56(6):417–22.
4. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7627/
5. Doran KS, Fulde M, Gratz N, Kim BJ, Nau R, Prasadarao N, et al. Host-pathogen interactions in bacterial meningitis [Internet]. Acta neuropathologica. Springer Berlin Heidelberg; 2016. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713723/
6. Farmen K, Tofiño-Vian M, Iovino F. Neuronal damage and neuroinflammation, a bridge between bacterial meningitis and neurodegenerative diseases. Front Cell Neurosci [Internet]. 2021;15:680858. Available from: http://dx.doi.org/10.3389/fncel.2021.680858
7. Pfister HW, Feiden W, Einhäupl KM. Spectrum of complications during bacterial meningitis in adults. Results of a prospective clinical study. Arch Neurol [Internet]. 1993;50(6):575–81. Available from: http://dx.doi.org/10.1001/archneur.1993.00540060015010
8. Linder, K. A., & Malani, P. N. (2019). Meningococcal meningitis. Jama, 321(10), 1014-1014.
9. Coureuil M, Join-Lambert O, Lécuyer H, Bourdoulous S, Marullo S, Nassif X. Pathogenesis of meningococcemia. Cold Spring Harb Perspect Med [Internet]. 2013;3(6):a012393–a012393. Available from: http://dx.doi.org/10.1101/cshperspect.a012393
10. Delbaz A, Chen M, Jen FE-C, Schulz BL, Gorse A-D, Jennings MP, et al. Neisseria meningitidis induces pathology-associated cellular and molecular changes in trigeminal Schwann cells. Infect Immun [Internet]. 2020;88(4). Available from: http://dx.doi.org/10.1128/IAI.00955-19
11. Lupu F, Keshari RS, Lambris JD, Coggeshall KM. Crosstalk between the coagulation and complement systems in sepsis. Thromb Res [Internet]. 2014;133 Suppl 1:S28-31. Available from: http://dx.doi.org/10.1016/j.thromres.2014.03.014
12. Rouphael NG, Stephens DS. Neisseria meningitidis: biology, microbiology, and epidemiology. Methods Mol Biol [Internet]. 2012;799:1–20. Available from: http://dx.doi.org/10.1007/978-1-61779-346-2_1
13. Batista RS, Gomes AP, Dutra Gazineo JL, Balbino Miguel PS, Santana LA, Oliveira L, et al. Meningococcal disease, a clinical and epidemiological review. Asian Pac J Trop Med [Internet]. 2017;10(11):1019–29. Available from: http://dx.doi.org/10.1016/j.apjtm.2017.10.004

iii. Bacterial evasion: how does the bacteria attempt to evade these host response elements.

i) Adhesion

While the integrity of the host’s pharyngeal and respiratory epithelium plays an important role in preventing disease, N. meningitidis evades this through adhesion (1). This adhesion is mediated by several proteins, the most important of which is Type IV pilin adhesin, which facilitates the attachment of the bacterium to the epithelial wall of the host’s nasopharynx (1). N. meningitidis’ tendency to use the nasopharyngeal mucosal surface as a reserve puts pressure on N. meningitidis to mutate its surface antigens, driving emergence of new antigenic variants (2). These serotypes may not be recognized by B cells, allowing N. meningitidis to evade targeting by antibodies of the adaptive immune response. This bacteria also produces an IgA protease that functions to inactivate a major mucosal immunoglobulin of humans, immunoglobulin A1 (IgA1), by cleaving it (3).

Once in the blood, Type IV pilin adhesin helps N. meningitidis interact with endothelial cells that line the blood vessels of the blood-brain barrier and brain endothelial cells, to enter the subarachnoid space, causing meningitis (1). Outer membrane proteins, such as Opa and Opc, support adhesion and invasion into host cells (2). The presence of Opc, a beta barrel protein featuring five surface loops, in particular is found to increase the likelihood of meningitis.

ii) Polysaccharide capsule

Another major factor allowing N. meningitidis to evade host immune responses is its polysaccharide capsule, which prevents phagocytosis by the host (1). The evasion of phagocytosis allows N. meningitidis to survive in the bloodstream, and is a hallmark of bacteria that have the ability to infect the central nervous system (2). Some strains of N. meningitidis have also been found to camouflage themselves from the host immune response by having a capsule composed of polysaccharides that are structurally similar to those found on mammalian tissue, giving N. meningitidis another evasion method (4). N. meningitidis is also able to incorporate sialic acid into the capsule, and because sialic acid is common on host cell surfaces, this allow N. meningitidis to evade immune recognition as well (6).

Polysaccharide capsule also plays a role in evading host response by inhibiting complement system (see section iv).

iii) IgA protease production

Some strains of N. meningitidis secrete an IgA protease that cleaves a specific peptide bond in the IgA hinge region (5). This allow N. meningitidis to circumvent the antibacterial effects of IgA, which include inhibiting pathogen adhesion to cells at mucosal surfaces, neutralizing bacteria, and interacting with phagocytes to stimulate pathogen clearance (5).

iv) Inhibition of complement system

One significant way that N. meningitidis can evade host immune response is through inhibition of the complement system (8). N. meningitidis possesses a variety of mechanisms that enable it to evade the complement-mediated killing.

• Polysaccharide capsule

The polysaccharide components of its capsule can block antibody binding to reduce deposition of the opsonin C4b (7). In the classical pathway of complement activation, this prevents the subsequent opsonization of the bacteria by antibodies and phagocytosis by immune cells such as macrophages (7).

• fHbp

fHbp (factor H binding protein), or otherwise known as GNA1870 or rLP2086 interacts with human Factor H as an inhibitor of the alternative complement pathway. The binding of fhbp to Factor H enhances meningococcal serum resistance allowing the bacterium to colonize in the blood (9). Additional bacterial components able to bind factor H and inhibit the alternative complement pathway are: NspA (Neisseria surface protein A), which acts cooperatively with fHbp; PorB2 (Porin B2) which binds not only human fH but also regulates the alternative complement pathway of baby rabbit and infant rat (9).

• Sialylation of lipooligosaccharides

The sialylation of lipooligosaccharides (LOS) increases binding of the fH C-terminus domain to bacterial proteins such as fHbp, increasing the bacteria’s resistance to opsononization(9). This is enabled by the lacto-N-neotetraose (LNnT) carbohydrate on bacterial LOS, which serves as a site of sialylation by either endogenous and exogenous sialic acid.(10) The sialylation of LNnT by N. meningitidis is associated with increased resistance to serum bactericidal activity, suggesting that sialylation of LOS is a potential mechanism for N. meningitidis evasion of host defenses (10).

• Neisserial heparin binding antigen (NHBA)

Neisserial heparin binding antigen (NHBA), an important component of the 4CMenB vaccine, is able to bind heparin in vitro through an Arg-rich region and this property correlates with increased survival of N. meningitidis in human blood. Heparin is known to interact with many complement components such as factor H, C4b-binding protein and vitronectin. Therefore, the establishment of the NHBA–heparin complex on the meningococcal cell surface could recruit complement inhibitors, and in turn prevent complement activation (9).

• Serine protease autotransporter NalP

An additional mechanism to evade the complement system is mediated by NalP, a serine protease autotransporter which cleaves C3 four amino acids upstream from the natural C3 cleavage site and produces shorter C3a-like and longer C3b-like fragments. When the C3b-like fragment is degraded, it results in lower deposition of C3b on the bacterial surface which is then reflected in higher meningococcal serum resistance (9).

• Thermoregulators

N. meningitidis has 3 kinds of thermoregulators in the 5’UTR (untranslated region) of genes involved in N. meningitidis structural proteins (11). One of the results of inflammation is pyrogen release, which causes the hypothalamus to change the thermoregulatory setpoint of the body, leading to increased temperature (11). This increased temperature is sensed by the N. meningitidis thermosensors and causes them to initiate their host evasion response strategies (11). Having the capacity to evade immune response at the peak of adaptive immunity helps with N. meningitidis colonization maintenance (11). Moreover, genes encoding for fhbp, LOS sialylation and capsule biosynthesis are regulated by increase in temperature, suggesting that this signal triggers meningococcal immunoevasion and complement resistance (9).

References:

1) Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7627/

2) Kenneth Todar M. Online Textbook of Bacteriology [Internet]. Immune defense against bacterial pathogens: Innate immunity. Available from: http://textbookofbacteriology.net/innate_6.html

3) Coureuil M, Join-Lambert O, Lécuyer H, Bourdoulous S, Marullo S, Nassif X. Mechanism of meningeal invasion by neisseria meningitidis [Internet]. Virulence. Landes Bioscience; 2012. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396695/

4) Cress BF, Englaender JA, He W, Kasper D, Linhardt RJ, Koffas MAG. Masquerading microbial pathogens: Capsular polysaccharides mimic host-tissue molecules [Internet]. FEMS microbiology reviews. U.S. National Library of Medicine; 2014. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4120193/

5) Vidarsson G, Overbeeke N, Stemerding AM, van den Dobbelsteen G, van Ulsen P, van der Ley P, et al. Working mechanism of immunoglobulin A1 (IgA1) protease: cleavage of IgA1 antibody to Neisseria meningitidis PorA requires de novo synthesis of IgA1 Protease. Infect Immun [Internet]. 2005;73(10):6721–6. Available from: http://dx.doi.org/10.1128/IAI.73.10.6721-6726.2005

6) Mg A. Epidemic risk of nisseria meningitidis capsular switching between Sero-groups: The effect of immunological pressure. MOJ Immunol [Internet]. 2015;2(5). Available from: http://dx.doi.org/10.15406/moji.2015.02.00060

7) John CM, Phillips NJ, Stein DC, Jarvis GA. Innate immune response to lipooligosaccharide: pivotal regulator of the pathobiology of invasive Neisseria meningitidis infections. Pathog Dis [Internet]. 2017 Apr 1 [cited 2022 Jan 18];75(3). Available from: https://academic.oup.com/femspd/article-lookup/doi/10.1093/femspd/ftx030

8) Piet JR, Brouwer MC, Exley R, van der Veen S, van de Beek D, van der Ende A. Meningococcal Factor H Binding Protein fHbpd184 Polymorphism Influences Clinical Course of Meningococcal Meningitis. PLoS One. 2012 Oct;7(10):e47973.

9) Pizza M, Rappuoli R. Neisseria meningitidis: pathogenesis and immunity. Curr Opin Microbiol. 2015 Feb 1;23:68–72.

10) Estabrook MM, Griffiss JM, Jarvis GA. Sialylation of Neisseria meningitidis lipooligosaccharide inhibits serum bactericidal activity by masking lacto-N-neotetraose. Infect Immun. 1997 Nov;65(11):4436–44.

11) Loh E, Kugelberg E, Tracy A, Zhang Q, Gollan B, Ewles H, et al. Temperature triggers immune evasion by Neisseria meningitidis. Nature [Internet]. 2013;502(7470):237–40. Available from: http://dx.doi.org/10.1038/nature12616

iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?

Breach of blood-brain barrier by N. meningitidis and infection of meninges. Depicts the layers and paths affected.

The bacteria may not be completely removed following recovery, as meningococcus colonizes the nasopharyngeal mucosa and is typically carried asymptomatically by approximately 10% of the population at any given time (1). Meningococcus may reside in the reservoir of the nasopharynx for months without causing disease in the host (2). Patients infected with N. meningitidis may fully recover if treated early on. Bacterial meningitis treatment typically involves administering corticosteroids to lessen swelling in the brain, as well as antibiotics targeting N. meningitidis (3). Penicillin is usually the drug of choice to treat meningococcemia and meningococcal meningitis (4). Although penicillin does not penetrate the normal blood-brain barrier, it readily penetrates the blood-brain barrier when the meninges are acutely inflamed (1). For those who are allergic to penicillin, either chloramphenicol or a third-generation cephalosporin such as cefotaxime or ceftriaxone is used (3). Even after administration of antibiotics, there is still a relatively high mortality of 10-15 deaths per 100 infections (5). If antibiotics successfully treat N. meningitidis, patients may still face complications, as a more severe disease, in which inflammation of the brain meninges occurs, is associated with long-term health effects, wherein patients do not fully return to their pre-infection baseline. For instance, 4% of survivors have hearing loss, and 7% of survivors experience other neurological sequelae, such as deficits in memory, learning, and cognition, as well as sensorimotor impairments, seizures, epilepsy, hydrocephalus, or changes in behaviour or personality (6). In addition, septicaemia, or bacteria entering the bloodstream, causing blood poisoning, may lead to gangrene, necessitating amputation.

Infection from N. meningitidis does not provide immunity from future re-infection, though survival of bacterial infections often leads the immune system to be primed and capable of fighting off infection more quickly in instances of reinfection (7). Antigen-specific antibodies and memory cells generated from the first infection form a protective immunity generated for the host in subsequent infections. For example, memory B cells may become activated from subsequent infection, resulting in faster and more efficient production of antibodies targeted against the bacteria.  However, illness has been observed even in individuals with antibody levels generally considered to be protective (8). A proposed mechanism for this comparatively lower level of immunity to future infection is that IgA in mucous secretions actually blocks IgG and IgM since it does not bind complement, preventing bactericidal activity by IgG and IgM (8). Further, studies found that factors such as immunodeficiencies, anatomical problems, parameningeal infections, and other underlying issues play a role in the likelihood of re-infection (9). Particularly, it was found that immunoglobulin, IgG subclass, and complement deficiencies predisposed patients to N. meningitidis reinfection. Additionally, the abilities of the immune system are age dependent, and antibody induction is difficult in infants under 18 months of age, resulting in absence of immunological memory (10). This is significant as rates of meningococcal disease are highest in children under 1 year of age (11).

An effective vaccine against meningococcal B, the 4CMenB vaccine, based on multiple antigens with different functions, was licensed in 2014. The antibodies induced by the 4CMenB vaccine can mediate bacterial killing by activating the classical complement pathway directly or indirectly, by inhibiting the binding of factor H binding protein on the bacterial surface, de-opsonizing the bacteria and interfering with colonization. However, studies found that vaccination with the MenB vaccine induced the generation and activation of memory T cells but failed to maintain the memory B cell population at a stable size and/or function (12).

References:

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