Pneumococcal pneumonia and invasive pneumococcal disease: immunopathogenesis and diagnosis

NP colonization is an essential step towards pneumococcal disease including pneumococcal pneumonia. The link between pneumococcal carriage and disease is usually found in children. A study by Greenberg et al. revealed that children with pneumonia were carriage of S. pneumoniae and the serotypes in NP isolates were concordant with serotypes causing pneumonia (14). The rate of NP carriage in adults is lower than that in children (17). However, contact with children may enhance adult carriage (18).

Mechanism of NP colonization of pneumococcus involves adherence of bacteria to epithelial cell via its receptors and activation of the host immune system. In normal mucosa, the bacterial attachment is inhibited by local innate immunity such as saliva, cough reflex, and mucus layer. Then, the cilia movement will transfer bacteria out of the respiratory tract. The mucosa releases lysozyme, lactoferrin, and surfactant to limit bacterial growth (19,20). To survive from the mucosa barrier, pneumococci also produce several enzymes. They are neuraminidase, β-galactosidase, and β-N-acetylglucosaminidase that degrade mucus and inhibit mucociliary clearance. Autolysin facilitates pneumolysin release to destroy the epithelial cells and reduce cilia movement. Cps and other pneumococcal proteins including Psp-A, Cbp-A, and enolase prevent complement deposition. Cbp-E inhibits neutrophil recruitment by degrading platelet-activating factor (PAF). Furthermore, phosphorylcholine (ChoP), a component of teichoic acid in the bacterial cell wall, binds to PAF receptors, whereas Cbp-A binds to polymeric immunoglobulin receptor (pIgR), which is the secretory component of epithelial cells (Figure 2) (19).

Figure 2

In addition to mucosa that acts as the physical barrier of innate immunity, several cytokines and inflammatory mediators are released after pneumococci were recognized by antigen-presenting cells (APCs). They play a role in the recruitment of leukocytes from the blood vessel into the mucosa of the respiratory tract. Mechanism of systemic innate immunity involves either the production of C-reactive protein or the activation of the complement system which stimulates polymorphonuclear (PMN) cells and macrophages in the opsonization of bacteria. Activation of the adaptive immune system consists of a local and systemic response. Pneumococci initiate mucosa-associated lymphoid tissue to produce immunoglobulin (Ig)-A which prevents bacteria invasion to mucosa membranes. Then, both B cell and T cell are activated as part of the systemic adaptive immune response. Systemic antibodies such as anti-capsular polysaccharide and anti-protein pneumococcal antibody will be produced as the response to pneumococcal colonization. Most of the carriage is asymptomatic until pneumococcal clearance is completed (20,21).

Pneumococcal pneumonia occurs after either bacterial aspiration or inhalation of droplet nuclei is presented (20). Bacterial load, synergism effect between viral and pneumococcal infection, the morphology of Cps, and host immune defence influence the onset of disease (10). S. pneumoniae will be recognized by AM via its receptors including class A scavenger receptor; macrophage receptor with collagenous structure (MARCO) that binds to internal site of pneumococcus, toll-like receptor (TLR)-2 that binds to bacterial cell wall, TLR-4 as the receptor of pneumolysin, and TLR-9 for deoxyribonucleic acid (DNA) of bacterial detection. The AM only has the ability to phagocyte a small number of pneumococci. Furthermore, AM and epithelial cells release cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-8, and chemokine CXC which activate neutrophil response, so they will migrate from the bloodstream to alveoli. After the process of phagocytosis, neutrophils become apoptosis. They will be followed by macrophage apoptosis that declines TNF-α expression; thus, it reduces neutrophil migration. These dead cells are removed by macrophages efferocytosis, and the resolution phase is begun (6,15,21).

Adaptive immunity is also critical to the host response to pneumococci. B cells produce Ig that is required for opsonophagocytosis process. T-CD4⁺ cells may contribute to the optimal response against pneumococci such as improving bacterial clearance by T helper (Th)-17. T-CD8⁺ cells will regulate Th-17 cell number, produce chemokine CXC, and recruit neutrophils. Following the resolution phase, memory T cells still persist in alveoli (6,15,21).

The clinical features of pneumococcal pneumonia are similar to other forms of typical pneumonia. The symptoms are fever, malaise, productive cough, shortness of breath, and pleuritic pain. The sputum may be seen as rusty red colour or contains streaks of blood (15). Gastrointestinal symptoms including nausea, vomiting, and diarrhoea may be found in 15–20% cases of pneumococcal pneumonia. In geriatric patients, pneumococcal pneumonia symptoms are unspecified (22). Sometimes, extrapulmonary signs such as meningitis, sepsis, peritonitis, and mastoiditis emerge earlier than pneumonia signs (15). Radiological findings in pneumococcal pneumonia include infiltrating in one or several segments of a lobe, lobar consolidation, air bronchogram, and pleural effusion (22,23).

Etiological diagnosis of pneumococcal pneumonia may be established through either conventional or molecular methods. Conventional methods include Gram staining, sputum, and blood or pleural fluid culture. Gram stain of sputum could be strongly suggestive of pneumococcal pneumonia if it shows the presence of Gram-positive diplococci and fulfils Bartlett criteria, i.e. <10 epithelial cells and >25 PMN cells. The limitations of Gram staining are inadequate sputum collection, antibiotic administration before specimen collection, and availability of skilled analyst. A sputum culture is a further step for the identification of pneumococcal pneumonia, but the diagnostic sensitivity varies, ranging between 29% and 94%. A false negative result is related to inadequate sampling, delayed processing of specimen, and antibiotic therapy before obtaining the specimen. A false positive result is often found in NP carriage, mainly among children; therefore, the result of sputum culture should be interpreted with Gram staining (24). A definite diagnosis of pneumococcal pneumonia is determined if S. pneumoniae could be isolated from sterile body sites such as blood or pleural fluid. The rate of a positive result in blood or pleural fluid culture is low. Bacteraemia is only found <10% among pneumococcal pneumonia patients. Pneumonia without bacteraemia, autolysis in the stationary phase, prior to an antibiotic, the inadequate sample volume are factors that influence the result of blood and pleural fluid culture. The limitation of a conventional method to detect pneumococcus is a false negative result and it needs several days for culture procedures. The molecular method is developed to discover pneumococci quickly, and by using this method the administration of antibiotic does not impact the result. These tests include teichoic acid (C-polysaccharide) detection, Cps detection, and nucleic acid amplification test (24).

There are two strategies of pneumococci transmigration through the epithelial and endothelial lining. First, ChoP will bind to the PAF receptor which is located in epithelial and endothelial cells. This binding may lead to pneumococcal transportation to host basal membrane thus causing the invasion of pneumococci. Second, the binding of Cbp-A to pIgR may initiate the migration of pneumococci between apical to basal membranes. Pneumococci generate hyaluronidase to degrade extracellular matrix in the basal membrane, and its impairment will provoke paracellular invasion (Figure 3) (19,25).

Figure 3

In the bloodstream, bacteria are recognized by innate immune system such as complement system against the antigen through both classic and alternative pathways. Pneumococci enhance their encapsulation that acts to inhibit complement deposition. Pneumococcus is bound to pattern recognition receptor (PRR) including TLR-2, TLR-4, TLR-9, and nucleotide oligomerization domain (NOD)-1 on APCs. It stimulates APCs to release several cytokines such as TNF-α, IL-1, and IL-6 which activate the inflammatory process. Bacteremia also induces coagulation cascade, in which in a severe stage, it will be demonstrated as disseminated intravascular coagulation (25).

Meningitis occurs when pneumococcus penetrates into the central nervous system. It translocates into the blood– brain barrier through either intracellular or intercellular pathway. Pneumococcus attaches to PAF receptor on brain microvascular endothelium and then migrates to the basolateral surface. The alternative routes are Cbp-A that is bound to pIgR and laminin receptor, a major component in the basal membrane of brain microvascular. All of those routes are known as an intracellular pathway. The intercellular pathway involves interepithelial tight junction damage that leads pneumococcal translocation into CSF. After crossing the endothelial layer, pneumococcus will disrupt and invade the basal membrane through several methods. In the first method, plasminogen is bound to the bacterial surface. The other methods are the binding of pneumococcus with fibronectin, vitronectin, and collagen (Figure 3) (19, 25).

Host immune response will be initiated when pathogen-associated molecular patterns (PAMPs) are recognized by APCs through PRRs. Macrophage in perivascular and meninges, endothelium, astrocyte, and microglial cells will produce pro-inflammatory cytokines including IL-1, TNF-α, and IL-6. These cytokines regulate adhesion factors on endothelial cells which mediate leukocyte recruitment into CSF and cause cerebral oedema. Anti-inflammatory cytokines, i.e. IL-10, IL-6, and transforming growth factor (TGF)-β, also act in the pathogenesis of meningitis. IL-10 will reduce pro-inflammatory cytokines activity and co-stimulatory molecules on macrophage. It also inhibits the ability of neutrophils in phagocytosis.

TGF-β plays a role in the differentiation of T-regulator cell and Th-17, inhibits differentiation and maturation of Th-1 and Th-2 cells, suppresses macrophage activation, and induces microglial cells to produce cytokines. IL-6 will minimize cerebral oedema, impairment of blood–brain barrier, and intracranial pressure. Not only cytokine but also other factors such as chemokines, matrix metalloproteinase, complement system, and coagulation factors are activated in the pathogenesis of pneumococcal meningitis (25).

Parapneumonia pleural effusion or empyema exists when pneumococcus translocates into the mesothelial lining of pleura through the cellular crossing. In animal model, empyema is often caused by either pulmonary infection or direct inoculation. It is rarely led by the invasion of bacteremia. After entering the pleural fluid, pneumococcus activates and releases IL-8, TNF-α, vascular endothelial growth factor, and monocyte chemoattractant protein-1. They induce intra-pleural neutrophilia and fibrin formation in pleura (26).

In summary, S. pneumoniae is a coccus bacterium which causes pneumococcal disease. Cps is the major virulence factor, and pneumococci are divided into 97 serotypes based on their Cps type. Aspiration or inhalation of bacteria may lead to interaction between pathogen and host immune system which initiates pneumonia. If the pneumococci penetrate into a sterile body fluid, it is defined as IPD. Diagnostic procedures for pneumococcal detection are still a challenge since positive results may impact the treatment option and prognosis.