The pathogenesis of encephalitis associated with the respiratory pathogen Mycoplasma
pneumoniae is not well understood. A direct infection of the central nervous system
(CNS) and an immune-mediated process have been discussed [1]. Recent observations
suggest that intrathecally detectable antibodies against the bacterium, which can
serve to establish the etiology of encephalitis, may indeed mediate the disease.
Mycoplasma pneumoniae is a major cause of upper and lower respiratory tract infections
in humans worldwide, particularly in children [2], [3]. Up to 40% of community-acquired
pneumonia in children admitted to the hospital are attributed to M. pneumoniae infection
[4]–[7]. Although the infection is rarely fatal, patients of every age can develop
severe and fulminant disease. Apart from the respiratory tract infection, M. pneumoniae
can cause extrapulmonary manifestations. They occur in up to 25% of manifest M. pneumoniae
infections and may affect almost every organ, including the skin as well as the hematologic,
cardiovascular, musculoskeletal, and nervous system [8]. Encephalitis is one of the
most common and severe complications [1]. M. pneumoniae infection is established in
5%–10% of pediatric encephalitis patients [9], [10], and up to 60% of them show neurologic
sequelae [10], [11].
It is important to establish the cause of encephalitis at an early stage in order
to specifically treat what can be treated and to avoid unnecessary treatment. The
diagnosis of M. pneumoniae encephalitis is challenging. The current diagnostic algorithm
of the “Consensus Statement of the International Encephalitis Consortium” [12] recommends
for the diagnosis of M. pneumoniae infection in children with encephalitis (1) serology
and polymerase chain reaction (PCR) from throat samples (routine studies), and if
positive test results and/or respiratory symptoms are present, then (2) additionally
PCR in cerebrospinal fluid (CSF) (conditional studies).
However, M. pneumoniae serology and PCR in the respiratory tract cannot discern between
colonization and infection in a clinically relevant time frame [13]. The main reason
for this is the relatively high prevalence of M. pneumoniae in the upper respiratory
tract of healthy children (up to 56%) [13], [14]. The demonstrated positive serological
results in such asymptomatic PCR-positive children (positive immunoglobulin (Ig) M
in 17%, IgG in 24%, and IgA in 6% of 66 cases) [13] may simply reflect one or more
previous encounters with M. pneumoniae and are not necessarily related to the presence
of M. pneumoniae in the respiratory tract. It is clear that this may give rise to
an overestimation of the M. pneumoniae-related disease burden. A more reliable diagnosis
of M. pneumoniae infection may be achieved by using paired patient sera in order to
detect seroconversion and/or a 4-fold increase in antibody titers in addition to PCR
(Table 1; table references: [13], [15]–[24]). However, such procedures are time-consuming
and are therefore neither practicable nor useful in an acutely ill patient.
10.1371/journal.ppat.1003983.t001
Table 1
Overview of diagnostic tests for M. pneumoniae.
Method
Test
Target/Antigen
Antibodies
Specimen
Performance1
Value
Comments
Direct identification of M. pneumoniae
PCR
Different target genes (e.g., P1 gene, 16S rDNA, 16S rRNA, RepMP elements, etc.)
-
Respiratory specimen (nasopharyngeal secretion, pharyngeal swab, sputum, bronchoalveolar
lavage), CSF, and other bodily fluids or tissues
High sensitivity, high specificity
RD2
NAATs provide fast results (in less than a day) and may be earlier than serology (because
antibody production requires several days); validation and standardization required
for routine diagnostic
Culture
-
-
Respiratory specimen (see above)
Low sensitivity, high specificity
AD
Special enriched broth or agar media; isolation takes up to 21 days
Nonspecific serological tests for M. pneumoniae
Cold-agglutinin test (“bedside test”)
Erythrocytes (I antigen)
Cold agglutinins (IgM)
Serum
Low sensitivity, low specificity
-3
Cold agglutinins target the I antigen of erythrocytes (alternative theory: cold agglutinins
target directly M. pneumoniae adhered to erythrocytes); positive in only about 50%
and in the first week of symptoms; less well studied in children; cross-reactivity
with other pathogens and noninfectious diseases
Specific serological tests for M. pneumoniae
CFT
Crude antigen extract with glycolipids and/or proteins
Igs (no discrimination between isotypes)
Serum
Sensitivity and specificity comparable to EIA
-3
Positive criteria: 4-fold titer increase between acute and convalescent sera or single
titer ≥1∶32; cross-reactivity with other pathogens and noninfectious diseases
PA
IgM and IgG simultaneously
-3
See above
EIA
Proteins (e.g., adhesion protein P1) and/or glycolipids
IgM, IgG,4
,
5 (IgA)6
Serum4, CSF5
,
7, other bodily fluids7
Moderate-high sensitivity, moderate-high specificity
RD
The sensitivity depends on the time point of the first serum and on the availability
of paired sera (for seroconversion and/or rise in titer); “gold standard”: 4-fold
titer increase as measured in paired sera
Immunoblotting
High sensitivity, high specificity8
AD
Confirmatory assay
IFA
Less sensitive and less specific than EIA
AD
Subjective interpretation
Abbreviations: AD, advanced diagnostic test; CFT, complement fixation test; CNS, central
nervous system; CSF, cerebrospinal fluid; EIA, enzyme immunoassay; IFA, immunofluorescent
assay; Ig, immunoglobulin; NAATs, nucleic acid amplification tests; PA, particle agglutination
assay; PCR, polymerase chain reaction; RD, routine diagnostic test; RepMP, repeated
M. pneumoniae DNA. References: [13], [15]–[24].
1
Qualitative statements included because of the wide range of test performances, which
depend on the assay, the patient cohort (children and/or adults), the reference standard
(PCR, culture, and/or serology), the respiratory specimen (for PCR), and the time
point of the sample collection after disease onset (for EIA)—e.g., sensitivities and
specificities for PCR [17], [18]: 79%–100% and 96%–99%; IgM EIA (in relation to PCR)
[19]: 35%–77% and 49%–100%; and for IgG EIA [17], [19]: 37%–100% (no indication on
specificity because of missing information on previous M. pneumoniae infections).
2
Epidemiological differentiation of clinical strains on the basis of differences in
the P1 gene by PCR or in the number of repetitive sequences at a given genomic locus
by multilocus variable-number tandem-repeat analysis (MLVA) [23].
3
Largely replaced by EIA.
4
Kinetics of antibody responses in blood.
IgM: onset: within 1 week after the onset of symptoms; peak: 3–6 weeks; persistence:
months (to years). IgG: onset and peak: 2 weeks after IgM; persistence: years (to
lifelong); reinfection in adults may lead directly to an IgG response in the absence
of an IgM response. IgA: onset, peak, and decrease earlier than IgM.
5
Antibody responses in the CNS differ from blood. There is no switch from an IgM to
an IgG response, the pattern of IgM, IgG, and IgA synthesis remains rather constant
and depends on the cause, and there is a long-lasting and slow decay of intrathecal
antibody synthesis [22]. In M. pneumoniae encephalitis, a dominant IgM response has
been observed [29].
6
The prevalence of serum IgA determined by EIA has been shown to be very low in PCR-positive
children with symptomatic respiratory tract infection (2.0%) [13].
7
To our knowledge, no validated test is available.
8
Immunoblotting with a combination of at least five specific M. pneumoniae proteins
showed sensitivities (in relation to PCR) of 83% (IgM), 51% (IgG), and 64% (IgA),
and specificities of 94%–100% (IgM), 98%–100% (IgG), and 93%–97% (IgA) [24].
The detection rate of M. pneumoniae by PCR in the CSF of M. pneumoniae encephalitis
patients is relatively low (0%–14%) [9], [10], [25], [26]. Moreover, various cases
with M. pneumoniae encephalitis in which bacterial DNA could not be detected in the
CSF had a more prolonged duration of respiratory symptoms before the onset of encephalitis
(>5–7 days) [10], [25], [27]. These cases indicate that M. pneumoniae encephalitis
may exemplify a postinfectious phenomenon that manifests after clearance of the bacteria
from the CNS or respiratory tract by the immune system. The immune response to M.
pneumoniae in the CNS or other sites may also contribute to the encephalitis (Figure
1; figure references: [1]).
10.1371/journal.ppat.1003983.g001
Figure 1
Proposed schematic pathomechanisms in M. pneumoniae encephalitis.
(Left) Respiratory tract infection. M. pneumoniae resides mostly extracellularly on
epithelial surfaces. Its close association allows the production of direct injury
by a variety of local cytotoxic effects. Furthermore, it can induce inflammatory responses,
elicited by both adhesion proteins and glycolipid epitopes that result in pneumonia.
(Right) Encephalitis. Extrapulmonary disease of the CNS is characterized by systemic
dissemination with resultant direct infection and local tissue injury (A) or immune-mediated
injury (B,C). The latter may occur as a result of cross-reactive antibodies against
myelin components, e.g., gangliosides and galactocerebroside C. These antibodies could
theoretically have originated from intrathecal synthesis (B) or from outside the CNS
(C). Figure adapted from [1]; see references in the text.
Interestingly, a promising diagnostic marker for M. pneumoniae encephalitis has recently
emerged from a few case studies. In one study, intrathecal synthesis of antibodies
to M. pneumoniae was reported in 14 cases of M. pneumoniae encephalitis (74%) [28].
The intrathecal production of antibodies is generally considered a highly specific
marker for infection of the CNS [22]. All cases that underwent PCR testing (93%) indeed
had a positive PCR targeting M. pneumoniae in the CSF [28] even though it has been
recently demonstrated that intrathecal antibody responses to M. pneumoniae but not
bacterial DNA can be present at the onset of clinical encephalitis [29]. In another
study, it was reported that intrathecal antibodies to M. pneumoniae were found to
cross-react with galactocerebroside C (GalC) in eight out of 21 (38%) of M. pneumoniae
encephalitis cases [30]. All eight cases showed a negative PCR targeting M. pneumoniae
in CSF. The cross-reactivity in these cases is likely induced by molecular mimicry
between bacterial glycolipids and host myelin glycolipids, including GalC and gangliosides
(Figure 2; figure references: [31]–[34]). Cross-reactive, anti-GalC antibodies have
previously been detected in patients with Guillain-Barré syndrome (GBS) who suffered
from a preceding M. pneumoniae infection [32], [35]–[38]. GBS is a typical postinfectious
immune-mediated peripheral neuropathy [39]. In GBS, cross-reactive antibodies cause
complement activation and formation of a membrane attack complex at the peripheral
nerves, resulting in neuromuscular paralysis. Anti-GalC antibodies have been associated
with demyelination in patients with GBS [35], [38]. Moreover, these anti-GalC antibodies
cause neuropathy in rabbits that are immunized with GalC [40]. Such antibodies may
also be involved in demyelination of central nerve cells in M. pneumoniae encephalitis,
as a significant correlation was found between the presence of anti-GalC antibodies
in the CSF and demyelination (p = 0.026) [30].
10.1371/journal.ppat.1003983.g002
Figure 2
Schematic structures responsible for molecular mimicry between M. pneumoniae and neuronal
cells.
(Left) M. pneumoniae adhesion proteins and glycolipids. The immunogenic and major
cytadherence proteins P1 and P30 are densely clustered at the tip structure. The P1
protein [31] and glycolipids, e.g., those forming a GalC-like structure [32], elicit
cross-reactive antibodies induced by molecular mimicry. (Right) Host myelin glycolipids,
to which antibodies were found in patients with M. pneumoniae encephalitis. Glycolipids
are organized in specialized functional microdomains called “lipid rafts” and play
a part in the maintenance of the cell membrane structure. Abbreviations: GalC, galactocerebroside
C; GQ1b, ganglioside quadrosialo 1b; GM1, ganglioside monosialo 1 (the numbers stand
for the order of migration on thin-layer chromatography, and the lower-case letters
stand for variations within basic structures); HMW, high-molecular-weight. Structures
of M. pneumoniae adhesion proteins and host glycolipids are adapted from [33] and
[34], respectively.
Anti-GalC antibodies have not only been detected in CSF but also in the serum of M.
pneumoniae encephalitis patients [30], [36], [41]–[43], including rates from 13% (2/15)
[30] to 100% (3/3) [41], respectively. It is possible that during inflammation the
blood-brain barrier (BBB) can become permeable, which would thereby enable antibodies
to cross the BBB and cause disease. As a consequence, the cross-reactive antibodies
in the CSF of M. pneumoniae encephalitis patients do not necessarily have to be produced
intrathecally (Figure 1).
M. pneumoniae infections may also be followed by the production of antibodies to gangliosides,
both in patients with GBS and in those with encephalitis. In M. pneumoniae encephalitis,
such antibodies were directed against GQ1b [44], [45] or GM1 [46] (Figure 2). Interestingly,
anti-GQ1b antibodies are associated with a distinct and severe encephalitis variant,
referred to as Bickerstaff brain stem encephalitis [47].
In conclusion, while PCR and serology may be of limited value in the diagnosis of
M. pneumoniae encephalitis, the detection of intrathecal antibodies to M. pneumoniae,
including cross-reactive antibodies against GalC and gangliosides, may be regarded
as a promising new diagnostic tool.
The routine diagnostic workup of M. pneumoniae encephalitis should therefore aim for
the detection of M. pneumoniae antibodies in both CSF and serum, in addition to M.
pneumoniae PCR in CSF. Intrathecal antibodies can be detected by widely accessible
enzyme immunoassays (EIAs) or immunoblotting (Table 1), while intrathecal antibody
synthesis can be established either by calculation of an antibody index [22] or through
parallel immunoblotting of simultaneously collected CSF and serum samples [48], [49].
Antiganglioside antibodies can be detected routinely by some specialized laboratories,
but their detection together with cross-reactive antibodies against GalC primarily
serve scientific purposes and may help to clarify M. pneumoniae antibodies' immune
target(s). Furthermore, their hypothesized role in the pathogenesis might provide
a basis for immunomodulatory treatment in M. pneumoniae encephalitis.