Clinical manifestations

Presentation and course

Streptococcus pneumoniae infections are more common in children and persons over 65 years of age. S pneumoniae is a causative agent for many serious systemic infections, including pneumonia, septicemia, sinusitis, and otitis media (20; 56). The most frequent predisposing and associated conditions for pneumococcal meningitis are pneumonia, sinusitis, or otitis media. Invasive pneumococcal disease is defined as a culture of S pneumoniae from a normally sterile body site (blood or cerebrospinal fluid but not sputum) (17).

Pneumococcal meningitis symptoms typically include fever, headache, nausea, vomiting, irritability, and lethargy proceeding to further clouding of consciousness. Fever may be 103°F or higher. Clinical signs include evidence of meningeal irritation, though this can be lacking in children, the elderly, and the deeply comatose. Focal signs may also appear. The course is frequently fulminant, with rapid neurologic deterioration leading to respiratory arrest and death (23; 98).

Lobar pneumonia and meningitis are the two most serious forms of S pneumoniae infection. Lobar pneumonia affects more people and, therefore, causes more morbidity and mortality, whereas meningitis has a higher mortality rate. S pneumoniae is the most common cause of bacterial meningitis in adults worldwide, and, in many countries, it is most common in children above the newborn period (78; 64). S pneumoniae is also a frequent cause of recurrent bacterial meningitis in individuals with a dural defect.

Finally, as a respiratory pathogen, the most typical coexisting complications will affect the upper or lower airways. Concomitant pneumonia is frequent in patients with S pneumoniae meningitis. Not infrequently, it is possible to obtain a history of productive cough, dyspnea, and constitutional symptoms in the days prior to onset of meningitis-like symptoms. Bacteria and inflammatory exudate accumulate in the alveoli of the lung, allowing a diagnosis of pneumonia to be made when a consolidated area appears on chest x-ray (64). The more severe cases of pneumonia are more likely to be accompanied by bacteremia (70; 102) and, therefore, with meningitis. Meningitis occurs in approximately 8% of patients who become bacteremic with this organism (81).

S pneumoniae is a common etiologic agent of otitis media in both children and adults (87; 28) and is either the first or second leading cause of acute sinusitis (37). These infections can provide a source of meningitis by either hematogenous spread or direct extension. It is reasonable to perform an otologic examination on any patient presenting with fever and altered consciousness (72).

The triad of pneumococcal pneumonia, meningitis, and endocarditis is a rare but serious condition known as Austrian syndrome (45). Alcoholism is the most common predisposing factor, but it is also seen with intravenous drug abuse (08), and the diagnosis of endocarditis should be considered early in every patient with pneumococcal meningitis or bacteremia (55).

Other complications of S pneumoniae include septic arthritis and osteomyelitis (31), both of which are manifestations of bacteremic spread, as is meningitis. Osteomyelitis also tends to involve the vertebral bones (97) and, from there, can extend directly into the central nervous system.

Prognosis and complications

The prognosis of Streptococcus pneumoniae meningitis, as with most bacterial meningitis, relates directly to early diagnosis and initiation of appropriate antibiotic therapy (06). With appropriate therapy instituted early, mortality is improved. Morbidity, however, is high, even with the best possible treatment, and significant, permanent neurologic sequelae are observed in up to a third of the survivors (85; 60; 06; 90). After pneumococcal meningitis adult patients are at greater risk of neurologic and neuropsychologic deficits, impaired daily activities, and poor quality of life (48).

French data for children (5 to 15 years) with a diagnosis of pneumococcal meningitis between 2001 and 2013 were analyzed. Among the 316 children with pneumococcal meningitis, the mortality rate was approximately 10%, and additionally, 23% of cases had severe complications (abscess, coma, hemodynamic failure, cerebral thrombophlebitis, or deafness) (38). In an American retrospective study, in-patient data from 2008 to 2014 were evaluated. During the study period, there were 10,493 hospitalizations due to pneumococcal meningitis. Data collected involved patients from all age groups (children younger than 2 years of age to over 65 years of age). There were 1016 deaths, with case fatality rate ranging from 8.3% to 11.2% (44).

Acute neurologic complications, especially coma, are associated with later behavioral and developmental difficulties, developmental delay, hearing loss, motor defects, and seizures (77). Independent predictors of a poor outcome include low Glasgow coma scale score, presence of a cranial nerve palsy, elevated sedimentation rate (104), advanced age, presence of an underlying chronic illness, presence of associated pneumonia, absence of associated otitis media, seizures, requirement for assisted ventilation, high CSF protein concentration, low CSF glucose concentration, CSF white count below 500 cells/microliter (73; 51), abnormal deep tendon reflexes, presence of stroke or hydrocephalus on imaging, and delay in antibiotic administration (90).

Complications during acute illness are similar to those seen with meningitides of any etiology and include subdural effusion, empyema, ischemic or hemorrhagic stroke, cerebritis, ventriculitis, abscess, and hydrocephalus (36; 64). Hearing loss is the most common long-term neurologic sequela of pneumococcal meningitis, occurring in up to 35% of pediatric survivors (51). The pneumococcal toxin pneumolysin is an important factor involved in ototoxic toxicity (76). Other focal neurologic deficits, such as ataxia and paresis, occur in an additional 16% (73).

Boix-Palop and colleagues noted delayed cerebral vasculopathy in approximately 10% of patients with pneumococcal meningitis (10). Delayed cerebral vasculopathy was considered if these patients had clinical worsening or if they failed to show an expected improvement. Neuroimaging in these patients showed small or large cerebral vessel disease. The patients who had delayed cerebral vasculopathy were severely ill, had a longer duration of illness, and more frequently had unfavorable outcomes. The authors hypothesized that the abrupt withdrawal of corticosteroids led to a cascade of rebound inflammatory changes manifesting as vasculopathy.

Cognitive impairment, particularly psychomotor slowing, is found in approximately 30% of survivors and is stable on formal testing over time after meningitis, despite subjective perception of improvements over time (41).

Clinical vignette

A 45-year-old man with alcoholism was brought by ambulance from a homeless shelter to an emergency room due to alteration in mental status. A few hours earlier, he had told the staff at the shelter that he had a bad headache. He had also vomited once. He then went to rest on his bunk. When staff checked on him, they found him to be confused. In the emergency room, he was noted to have a temperature of 39.1 degrees Celsius. He responded to his first name but was not oriented to place or time. He was given doses of ceftriaxone, vancomycin, and ampicillin. A head CT was unremarkable, and a lumbar puncture was performed. CSF examination demonstrated an opening pressure of 500 mmHg, a white count of 4914 cells/ul with 91% neutrophils, a glucose concentration of 5 mg/dl, and a protein concentration of 279 mg/dl. Gram stain revealed gram positive diplococci, and a presumptive diagnosis of pneumococcal meningitis was made.

The patient was admitted to the neurologic intensive care unit. Triple antibiotic coverage was continued pending definitive speciation and antibiotic sensitivity determination. Intravenous dexamethasone was also added to the regimen. The patient’s condition continued to deteriorate, and he required ventilatory support. A repeat head CT demonstrated worsening cerebral edema. The patient developed signs of cerebral herniation. He was temporarily stabilized, but then required pressor support. He became increasingly bradycardic, and eventually died, 30 hours after initial presentation.

CSF and blood cultures both confirmed the diagnosis of infection with Streptococcus pneumoniae, sensitive to all tested antibiotics.

Biological basis

Etiology and pathogenesis

Streptococcus pneumoniae are the prototypic extracellular bacterial pathogens. In the absence of antibody, they resist phagocytosis and grow extracellularly. They are gram-positive cocci that tend to grow in chains. They are catalase negative and grow better with a source of catalase, such as red blood cells. S pneumoniae produce pneumolysin, which degrades hemoglobin to a green pigment; they are, therefore, surrounded by a green zone on blood-agar growth media, a process termed alpha-hemolysis. Currently, at least 94 distinct serotypes have been identified based on the structure and antigenicity of their polysaccharide capsules. Of these, a limited number of capsular serotypes account for most invasive pneumococcal diseases (32; 35; 13; 64; 101). Invasive disease caused by different serotypes may result in different degrees of host response (86), leading to different pathogenic mechanisms, clinical courses, and outcomes.

Streptococcus pneumoniae causes infection of the upper and lower respiratory tracts by spread from the nasopharynx, which is normally colonized; it causes infection elsewhere, including the central nervous system, via hematogenous spread. In fact, an autopsy study of children who died from bacterial meningitis showed no evidence for direct extension, suggesting that even in cases of otitis media, meningitis may be due to bacteremia (29). The bacteremia may actually contribute to the neurologic injury by affecting cerebral blood flow regulation, blood-brain barrier permeability, and cerebral edema (11; 75).

A key bacterial factor enabling S pneumoniae to cause disease is its capsular polysaccharide. The capsule allows the organism to escape immune surveillance and ingestion by phagocytes. The capsule may have electrochemical forces that repel phagocytes and may mask cell wall constituents, which would otherwise be immunogenic (64). Pneumococcal bacterial genetic variation is a determinant of invasive pneumococcal disease phenotype. Variations in S pneumoniae genome also contribute to determining the clinical phenotype of invasive pneumococcal disease. For example, the presence of pneumococcal gene slaA along with sequence cluster 9 were independent predictors of meningitis. In addition, a set of four pneumococcal genes co-located on a prophage independently predicted 30-day increased mortality (22).

Various bacterial and host factors contribute to pathogenicity of the organism within the central nervous system. In its attempt to fight the infection, the host inflammatory response probably causes most of the pathological damage.

The mechanisms responsible for S pneumoniae spread to the brain and meninges are largely unknown. In an experimental study using 2-photon imaging, the authors demonstrated that pneumococci translocate from the nasopharynx to the dorsal meninges via the cribriform plate within minutes of inhaled delivery of pneumococci in mice (07). Once S pneumoniae enters the central nervous system, bacteria multiply in the CSF. Subsequently, there is bacterial lysis with a release of toxic material in the CSF. The prominent toxic materials released during bacterial lysis include protein pneumolysin, bacterial wall fragments, and capsular components. These toxic materials orchestrate an intense inflammatory reaction in the CSF and brain parenchyma (92; 42). Pneumolysin is generally cytotoxic to all types of host cells, including neuronal cells. Pneumolysin is capable of modulating the host complement system, thereby allowing S penumoniae to multiply and grow (24). Pneumolysin destroys phagocytic cells, activates complement and other cytokines, and induces apoptosis-like programmed cell death in neurons and brain-derived endothelial cells (57; 84). In addition, an altered balance of matrix metalloproteinase (MMP-9) and its tissue inhibitor (TIMP-1) may play a role in disruption of the blood-brain barrier and extent of cortical damage in a rat model of pneumococcal meningitis (88). Upregulation of aquaporin-4 may contribute to increased water permeability across the blood-brain barrier, accounting for the brain edema seen in pneumococcal meningitis (74).

In response to bacterial infection of the central nervous system, neural progenitor cells proliferate and differentiate, although bacterial factors, like cell wall constituents, may trigger immunoreactivity, which can hamper this neurogenesis (39). Work in a murine model of pneumococcal meningitis has demonstrated upregulated expression of brain derived neurotrophic factor (BDNF) and its receptor (TrkB), which is associated with increased density of dentate granule cells in the hippocampal formation (94). BDNF was demonstrated to have neuroprotective effects in bacterial meningitis (54).

Brain histopathology of 31 patients who died of pneumococcal meningitis was assessed. Common pathological observations were inflammation of medium-large arteries, cerebral hemorrhage, cerebritis, arterial and venous thrombosis, infarction, and ventriculitis. Inflammation of arteries leading to obstruction of the vascular lumen was associated with cerebral infarction (30). Deliran and colleagues performed brain autopsy in two patients who died of pneumococcal meningitis and had clinical cerebral venous thrombosis (27). They noted multifocal deep intramural inflammation in the cerebral venous sinus. Bacteria were identified within the intramural inflammation and thrombus. Similar changes were not noted in patients with cerebral venous thrombosis without meningitis and in patients with uncomplicated bacterial meningitis.

Epidemiology

Streptococcus pneumoniae is the leading cause of bacterial meningitis in adults in the United States and in adults and children in other parts of world. In an American retrospective study from 2008 to 2014 there were 10,493 hospitalizations due to pneumococcal meningitis. Overall, pneumococcal meningitis incidence decreased from 0.62 to 0.38 cases per 100,000 over this time (39% decrease). Among children less than 2 years of age, the average annualized pneumococcal meningitis rate decreased by 45% from 2.19 to 1.20 per 100,000. Annual pneumococcal meningitis rates decreased in those 18 to 39 years of age (0.25 to 0.15 cases per 100,000) and 40 to 64 years of age (0.95 to 0.54 cases per 100,000) (44). A decrease in incidence of adult bacterial meningitis, most of which was due to S pneumoniae, has also been noted in Europe. Specifically, the incidence of bacterial meningitis declined from 1.72 cases per 100,000 adults per year from 2007 to 2008 to 0.94 cases per 100,000 adults per year from 2013 to 2014; S pneumoniae caused 1017 (72%) of 1412 episodes (09). In England and Wales, introduction of 3-valent (PCV13) pneumococcal conjugate vaccines in 2010 led to a substantial (48%) reduction in pneumococcal meningitis incidence from 2015 to 2016 (67).

A much higher incidence of pneumococcal meningitis is reported in Africa. From 2000 to 2015, the pooled incidence of invasive pneumococcal disease (including meningitis) in children in Africa was 62.6 per 100,000 person-years (43). From 2000 to 2015, pneumococcal meningitis had a pooled African incidence of 24.7 per 100,000 person-years (43).

Patients with various underlying conditions, such as asplenic states, cancer, alcoholism, malnutrition, diabetes mellitus, HIV, and other chronic diseases, may be at increased risk for developing pneumococcal infection, including meningitis (33; 34; 03). Certain ethnic groups, including Native Americans, Alaskans, and Australian Aboriginals, may have an incidence as much as 10-fold greater than the general population (25; 95; 46). The reasons for this remain unclear. Infants (but not neonates) up to 2 years of age and adults over 65 years are also at increased risk (12).

Transmission can occur due to close contact, such as in daycare centers, military camps, prisons, homeless shelters, and nursing homes (62; 40; 65; 89). The lockdown and COVID-19 containment measures had a favorable impact on the transmission of life-threatening invasive diseases caused by S pneumoniae, H influenzae, and N meningitidis globally. The reported incidence of S pneumoniae infections decreased by 68% at 4 weeks and 82% at 8 weeks after start of the lockdown (March 2020) (14).

Prevention

The Centers for Disease Control and Prevention recommends routine pneumococcal vaccine for the following types of patients: immunocompetent but at increased risk of acquiring pneumococcal infection or having a serious complication, such as patients with chronic pulmonary disease, cardiovascular disease, diabetes, alcoholism, liver and renal disease, CSF leak, cochlear implants, and those older than 65 years of age or younger than 2 years of age; and immunocompromised patients, including those with functional or anatomic asplenia (sickle cell disease), congenital or acquired immunodeficiency, malignancy, leukemia or lymphoma, HIV infection, or organ transplantation. Vaccine may also be reasonable for other populations at risk by virtue of ethnicity (Native Americans) or crowded living conditions (64; 15).

Pneumococcal conjugate vaccines are now part of national immunization programs of many countries (20). There are two types of pneumococcal vaccines currently available: pneumococcal conjugate vaccine (PCV13), which consists of purified capsular polysaccharides from 13 bacterial serotypes, and pneumococcal polysaccharide vaccine (PPSV23), which consists of purified capsular polysaccharides from the 23 serotypes responsible for 90% of invasive pneumococcal infections. The polysaccharides are conjugated to a carrier protein, which makes them more immunogenic and effective in protecting against infection, particularly in young children less than 2 years of age. Furthermore, the vaccine protects against both systemic and mucosal infection and prevents nasopharyngeal colonization, thereby reducing transmission in the community.

Since 2010, pneumococcal conjugate vaccine 13 has been recommended for all children younger than 2 years of age. The Centers for Disease Control and Prevention and the American Academy of Pediatrics recommend the expanded use of PCV13 in children 6 through 18 years of age with increased risk of invasive pneumococcal disease. The 23-valent pneumococcal polysaccharide vaccine is recommended for all adults 65 years of age or older and in younger patients who have a condition that increases risk for invasive pneumococcal disease (05; 16). Both vaccines are well tolerated and highly effective, and they can be administered at the same time as other vaccines (04).

Widespread use of polysaccharide conjugate vaccines has led to the emergence of newer strains that are not included in the vaccine. These strains can cause severe pneumococcal meningitis. Mukerji and Briles have hypothesized that these nonpolysaccharide conjugate vaccine type strains do not survive in the blood but possibly enter the brain by nonhematogenous routes (63). Mukerji and Briles emphasized the need to develop a new vaccine that can cover these strains as well.

Vaccination has led to a significant decline in invasive pneumococcal disease-related deaths. After the introduction of the 7-valent pneumococcal conjugate vaccine in the United Kingdom, deaths from invasive pneumococcal disease declined from 1.25 out of 100,000 children from 2006 to 2007 to 0.60 out of 100,000 children from 2009 to 2010. A further reduction in invasive pneumococcal disease following the introduction of the 13-valent pneumococcal conjugate has been documented (0.39 out of 100,000 children from 2013 to 2014) (68), with a reduction in the rate of pneumococcal meningitis in children younger than 15 years of age and near elimination of cefotaxime-resistant isolates (83).

Pneumococcal meningitis is caused by a variety of capsular serotypes (58; 71). Available vaccines do not protect against meningitis caused by all capsular serotypes (61).

Differential diagnosis

Confusing conditions

The history and examination data obtained from any given case of acute bacterial meningitis can be quite variable. Some findings are typically present, whereas others may be absent. Additionally, the signs and symptoms frequently seen with acute bacterial meningitis, including fever, behavioral or personality changes, and mental status changes can be nonspecific and suggest other diagnoses, including systemic infection or sepsis, viral encephalitis or meningitis, fungal or tuberculous meningitis, trauma or closed head injury, multiple metabolic abnormalities (hypoglycemia, ketoacidosis, electrolyte imbalance, uremia, toxic exposure), seizure, and brain tumor. Even meningismus does not exclude alternative diagnoses such as subarachnoid hemorrhage, intracranial hemorrhage, and epidural abscess. In order to prevent morbidity and mortality from missed diagnoses, it is important to keep a high index of suspicion for acute bacterial meningitis and err on the side of starting treatment early and potentially unnecessarily (06).

Streptococcus pneumoniae and Neisseria meningitidis are the most common etiologic agents of bacterial meningitis after 1 year of age. In children less than 1 year of age, Group B streptococci and gram-negative enteric bacilli, particularly Escherichia coli, are the leading etiologic agents, presumably because of exposure to these agents during birth. Due to passive transfer of maternal antibodies, these neonates do not typically develop Haemophilus influenzae or pneumococcal or meningococcal meningitis (69).

In the setting of a preceding sinusitis, otitis media, head trauma, neurosurgical procedure, or cerebrospinal fluid leak, S pneumoniae and nontypeable H influenzae are both common etiologic agents of recurrent meningitis (47; 01) because both are a common part of “normal” skin and nasopharyngeal colonization. One study provides good evidence that surgical repair of CSF leaks effectively prevents recurrent bacterial meningitis (91).

In patients over 50 years of age, the most common causes of bacterial meningitis include S pneumoniae, Listeria monocytogenes, and gram-negative bacilli (80; 50). H influenzae is included in the gram-negative group, along with E coli, Enterobacter, and Pseudomonas. S pneumoniae is more likely in association with pneumonia, Pseudomonas in association with chronic lung disease, E coli, or Enterobacter in the setting of urinary tract infection, and S pneumoniae or H influenzae in the setting of sinusitis, otitis media, head trauma, or a neurosurgical procedure. Listeria monocytogenes can also be seen, especially in the immunosuppressed elderly, and Staphylococcus aureus is seen with dural disruption.

Diagnostic workup

Bacterial meningitis, including that caused by Streptococcus pneumoniae, should be considered and promptly treated in any patient with a compatible presentation, keeping in mind that the presentation may be atypical in some patients, especially young children and the elderly. Neutrophilic CSF pleocytosis and a decreased CSF glucose concentration are strongly suggestive of bacterial meningitis (06). Empiric therapy is initiated with a third- or fourth-generation cephalosporin and vancomycin along with dexamethasone (21).

If imaging is performed before CSF examination, images should be evaluated for contraindications for lumbar puncture. In such a scenario, empirical treatment should be initiated before the patient is sent for neuroimaging (21).

A minority of patients with bacterial meningitis, particularly with pneumococcal meningitis, have normal CSF leukocyte counts. This finding is more common in individuals who are immunocompromised and is associated with a poor outcome (100).

No tests are currently available that are rapid enough to confirm S pneumoniae as the causative organism in time to base initial treatment on that finding.

With S pneumoniae meningitis, as well as most other etiologic agents, both blood and CSF cultures will usually be positive. However, again, treatment should be initiated without delay, even prior to obtaining culture samples. Multiplex PCR platforms for simultaneous detection of multiple bacterial pathogens causing meningitis are now commercially available. A probe to detect unique S pneumoniae RNA sequences is also available. Methods for detecting antibody to capsular polysaccharide have proven neither sensitive nor specific enough to be clinically useful.

Historically, pneumococci have been identified in culture by the following characteristics: alpha-hemolysis of blood agar, catalase negativity, susceptibility to optochin, and solubility in bile salts (64). With the modern techniques of molecular biology, reliance on these bacterial properties may be less important, but cultures remain vital to determine antibiotic sensitivity.

Management

When bacterial meningitis is suspected, emergent antibiotic treatment must be initiated without waiting for speciation (06). In persons older than the neonatal period, empiric treatment is directed primarily against S pneumoniae and N meningitidis. Current recommendations for treatment of community-acquired meningitis for ages 3 months to 50 years is vancomycin 15 mg/kg IV every 8 to 12 hours and up to 2 g/day (maintain serum trough concentration of 15 to 20 ug/ml) plus either cefotaxime 50 mg/kg IV every 4 to 6 hours (maximum 2 g IV every 4 hours) or ceftriaxone 50 to 100 mg/kg IV every 12 hours (maximum 2 g IV every 12 hours). For patients over age 50 years, addition of ampicillin 2 g IV every 4 hours is recommended (96; 59; 98).

In adults, dexamethasone should ideally be added to empirical therapy before or at the same time as the first antibiotic dose, and it should be continued for 4 days in those with proven pneumococcal meningitis (105). The dose is 0.15 mg/kg every 6 hours (26; 96; 99). The European Society of Clinical Microbiology and Infectious Diseases guidelines suggest that corticosteroids can still be started up to 4 hours after commencement of antibiotic treatment (98). The UK guidelines recommend that for adults, dexamethasone can be given up to 12 hours after antibiotics are started (59). There has been some concern that dexamethasone could decrease the penetration of vancomycin into the CSF. However, a study demonstrated that appropriate levels of vancomycin are reached in the CSF of patients with pneumococcal meningitis receiving dexamethasone as long as proper serum levels of vancomycin are maintained (82). Dexamethasone 0.15 mg/kg every 6 hours, initiated prior to the first dose of antibiotic and continued for the first 2 to 4 days of treatment, decreases the risk of neurologic sequelae from community-acquired bacterial meningitis in children (53; 66; 93).

In addition to medication therapies, it is imperative that appropriate supportive care be instituted. Advancements in intensive care techniques offer significant benefit for patients with bacterial meningitis, including S pneumoniae meningitis.

Outcomes

Pneumococcal meningitis has always been associated with a high possibility of mortality and morbidity in adults. Authors from the Netherlands analyzed data between October 1998 and April 2002 and between January 2006 and July 2018. The outcome of pneumococcal meningitis in adults was better in the latter era of administration of conjugate vaccines and adjunctive dexamethasone compared to the former era, when these two interventions were used less. Nonetheless, in a cohort of 1783 patients (1816 pneumococcal meningitis episodes), there were 363 episodes of in-hospital death (20%). Unfavorable outcomes were recorded in 772 episodes (43%). Delayed cerebral venous thrombosis occurred in 29 patients (2%), and more half of these died (49). Neurofunctional disability in adults is frequent 1 year after community-acquired bacterial meningitis, particularly following pneumococcal meningitis. In a cohort of 281 patients at 12 months, 84 (30%) patients had neuro-functional disability and six patients died. Dexamethasone did not impact the likelihood of neurofunctional disability (02).

Special considerations

Pregnancy

Vaccination of pregnant women against Streptococcus pneumoniae is not recommended because it is not known whether these vaccines can cause fetal harm (04; 52; 79). A Cochrane Systematic Review also does not suggest that administration of pneumococcal vaccine during pregnancy is efficacious in reducing infant infections during pregnancy (19). Although the safety of the pneumococcal polysaccharide vaccine during pregnancy has not been evaluated, no adverse consequences have been reported among newborns whose mothers were inadvertently vaccinated during pregnancy (18).