MAJOR ARTICLE

Acute Childhood Encephalitis

and Mycoplasma pneumoniae
Ari Bitnun,1 Elizabeth Lee Ford-Jones,1 Martin Petric,3 Daune MacGregor,2 Helen Heurter,1 Susan Nelson,4
Grant Johnson,3 and Susan Richardson3
1

Division of Infectious Diseases and 2Division of Neurology, Department of Pediatrics, and 3Division of Microbiology, Department of Pediatric
Laboratory Medicine, The Hospital for Sick Children, and 4Aventis-Pasteur, Ltd., Toronto

In a prospective 5-year study of children with acute encephalitis, evidence of Mycoplasma pneumoniae infection
was demonstrated in 50 (31%) of 159 children. In 11 (6.9%) of these patients, M. pneumoniae was determined
to be the probable cause of encephalitis on the basis of its detection in cerebrospinal fluid (CSF) by polymerase
chain reaction (PCR) or by positive results of serologic tests for M. pneumoniae and detection of the organism
in the throat by PCR. CSF PCR positivity correlated with a shorter prodromal illness (P p .015 ) and lack of
respiratory symptoms (P p .06 ). Long-term neurologic sequelae occurred in 64% of probable cases. Thirty
children (18.9%) who were seropositive for M. pneumoniae but did not have the organism detected by culture
or PCR had convincing evidence implicating other organisms as the cause of encephalitis, suggesting that
current serologic assays for M. pneumoniae are not sufficiently specific to establish a diagnosis of M. pneumoniae encephalitis.
Acute childhood encephalitis is a potentially devastating
illness with an incidence of 10.5 cases per 100,000
child-years in developed countries [1]. Among children
!1 year old, the incidence may be as high as 27.7 cases
per 100,000 child-years [2]. In the prevaccine era, measles, mumps, polio, and rubella were commonly implicated as etiologic agents of acute encephalitis [3, 4].
However, in more recent prospective studies, nonpolio
enteroviruses, respiratory viruses, herpesviruses, and
Mycoplasma pneumoniae have accounted for the majority of etiologically confirmed cases [1, 5, 6].
Most large series published to date have relied ex-

Received 5 May 2000; revised 5 October 2000; electronically published 21 May

2001.
Presented in part: 39th Interscience Conference on Antimicrobial Agents and
Chemotherapy, San Francisco, 2529 September 1999 (abstract 2095).
Grant support: The Meningitis D.R.E.A.M. (Douglas Russell Evely Annual
Memorial) Fund, The Hospital for Sick Children, Toronto.
Reprints or correspondence: Dr. Susan Richardson, Division of Microbiology,
Dept. of Pediatric Laboratory Medicine, The Hospital for Sick Children, 555
University Ave., Toronto, Ontario, M5G 1X8, Canada (susan.richardson@sickkids
.on.ca).
Clinical Infectious Diseases 2001; 32:167484
 2001 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2001/3212-0002$03.00

1674 CID 2001:32 (15 June) Bitnun et al.

clusively on serologic tests for the diagnosis of M. pneumoniae encephalitis [1, 3, 5, 713]. Unfortunately, the
glycolipid antigen extract of M. pneumoniae that is used
in such assays is not specific, and false-positive results
are a significant concern [1419]. Glycolipids that
cross-react serologically occur commonly in plants [16]
and in human brain tissue [17]. Fourfold or greater
rises in CF titer to M. pneumoniae have been demonstrated in 40% of adult patients with bacterial meningitis and in 10% of those with other bacteremic
infections [18]. False-positive M. pneumoniae IgM antibody responses have been detected in patients with
acute meningoencephalitis and acute pancreatitis [19].
Among patients who have acute encephalitis, the detection of M. pneumoniae in the CSF by means of culture or PCR provides strong evidence of causality. However, absence of the organism in the CSF does not rule
out M. pneumoniae as a cause, because CNS disease
may be immunologically mediated. In this respect, the
detection of M. pneumoniae in the respiratory tract of
a patient with acute encephalitis provides supportive
evidence of causality.
Since January 1994, The Hospital for Sick Children,
Toronto, has maintained a prospective encephalitis reg-

istry, and any child who is admitted to the hospital with acute
encephalitis undergoes an extensive neurologic and microbiological investigation [6]. In this report, we summarize data
compiled from January 1994 through December 1998 that pertain to the role of M. pneumoniae in acute childhood encephalitis.

PATIENTS AND METHODS

Enrollment criteria, data collection, and investigations.
Enrollment criteria, data collection, and investigations have
been described elsewhere [6]. In brief, patients were identified
by the registry nurse in a daily review of admissions. Patients
were excluded if they were 4 weeks old or 18 years old, if
they were known to have a neurologic disorder, or if they were
known to be immunodeficient. Children with postvaricella cerebellar ataxia were also excluded. In addition to the microbiological investigations detailed below, electroencephalographic
(EEG) analyses, cranial CT scans, MRI scans, and single-photon
emission CT were performed, as indicated.
Microbiology. Most of the microbiological methods have
been described elsewhere [6]. Acute-phase and convalescentphase serum samples were tested for M. pneumoniae by use of
the CF test and for M. pneumoniaespecific IgM by use of EIA
(GenBio). Serologic evidence of M. pneumoniae infection was
determined by seroconversion, which was defined by a 4-fold
increase in titer or a single titer of 1:128, as determined by
CF, or by the presence of M. pneumoniaespecific IgM. Antibody titers for herpes simplex virus (HSV), enteroviruses, adenovirus, and influenza A and B viruses were determined by
CF, and those for arboviruses (Powassan, St. Louis encephalitis,
and eastern and western equine encephalitis) were determined
by use of a hemagglutination inhibition assay. Serologic assays
for Epstein-Barr virus (EBV) included the test for heterophile
antibody (Oxoid) and EIAs for early antigen, viral capsid antigen, and Epstein-Barr nuclear antigen (Diasorin). Serologic
testing for human herpesvirus 6 (HHV-6; ABI), varicella-zoster
virus (VZV), and measles (Gull) was done by use of EIA,
whereas serologic testing for parvovirus B19 (Biotrin) was performed by use of indirect immunofluorescence microscopy until 1997 and, thereafter, by use of EIA. Serologic testing for
Bartonella henselae was performed by use of indirect immunofluorescence microscopy (Central Public Health Laboratory,
Ontario Ministry of Health, Toronto).
Detection of M. pneumoniae in throat and CSF specimens
was performed by means of culture before 1996 [6] and, thereafter, by PCR. The PCR assay was performed using the P11
and P13 primers for the P1 adhesin gene, which resulted in
a 209-bp amplicon [2022]. PCR reactions were performed in
a total volume of 50 mL with 2.0 U of AmpliTaq Gold (Perkin
Elmer), 200 mM of each dNTP, 2.0 mM of MgCl2, 25 pmol of

each primer, and 10 mL of sample DNA overlaid with mineral

oil. PCR involved a 2-step reaction that was performed on the
Robocycler 96 (Stratagene). The cycles were as follows: 1 cycle
of 10 min at 95C (for activation of AmpliTaq Gold), 40 cycles
of 1 min at 95C (for denaturation) and 1 min at 72C (for
annealing and extension), and 1 cycle of 3 min at 72C (for
final extension). The 209-bp product was then detected by gel
electrophoresis by means of ethidium bromide staining. An
extraction control (PBS) was run with each test to detect contamination. In addition, a reagent control and sensitivity control of 1 color-changing unit (CCU) were run. Each specimen
was also tested to detect inhibition of the PCR reaction (1 CCU
of M. pneumoniae was added to a duplicate PCR reaction). The
sensitivity of our PCR assay was determined to be 0.1 CCU by
use of a stock of M. pneumoniae with known concentration (as
determined by culture of serial dilutions to determine 1 CCU).
Virus cultures were performed with AGMK and RD cell lines,
for CSF and stool specimens, and with AGMK, MDCK, and
HEp-2 cell lines, for throat and nasopharyngeal specimens. CSF
was analyzed by reverse-transcriptase PCR and PCR for enteroviruses and all known herpesviruses, respectively [23]. Nasopharyngeal specimens were examined for the presence of respiratory syncytial virus, parainfluenza viruses (1, 2, and 3),
influenza A and B viruses, and adenovirus by means of direct
immunofluorescence microscopy and virus isolation in cell culture. Stool specimens were examined by use of electron microscopy for the presence of viral particles, and the specimens
were inoculated into cell culture as described elsewhere [6].
Definitions. Encephalopathy was defined as a depressed or
altered level of consciousness, including lethargy, extreme irritability, or a significant change in personality or behavior that
persisted for 24 h. Encephalitis was defined by the presence of
encephalopathy plus 2 of the following criteria: fever (temperature, 38.0C), seizure(s), focal neurologic findings, pleocytosis (WBC count, 15 cells/mL), EEG findings compatible with
encephalitis, or abnormal neuroimaging.
All children who fulfilled the study criteria for encephalitis
and in whom evidence of M. pneumoniae infection was detected
either by means of serologic testing or by culture or PCR of
throat or CSF specimens were included. Depending on the
strength of the evidence that implicated M. pneumoniae, cases
were classified as probable, possible, or indeterminate
with respect to M. pneumoniae being the etiologic agent. Probable M. pneumoniae encephalitis was defined by either of the
following 2 criteria: detection of M. pneumoniae in CSF specimens by means of PCR, culture, or both, with or without
confirmatory results of serologic tests for M. pneumoniae; or
detection of M. pneumoniae in throat specimens by means of
PCR, culture, or both, with confirmatory results of serologic
tests for M. pneumoniae. Possible M. pneumoniae encephalitis
was defined by either serologic evidence of M. pneumoniae
Encephalitis and M. pneumoniae CID 2001:32 (15 June) 1675

Table 1.

Microbiological data for 11 children with probable Mycoplasma pneumoniae encephalitis.

Results of serologic testing
for M. pneumoniae
Acute-phase
specimens

Convalescent-phase
specimens

Results of PCR
for detection of
M. pneumoniae

IgM EIA

CSF

Throat

Age,
years

IgM EIA

1:256

1:1024

None

10.5

1:128

1:512

None

12

1:256

1:512

Parainfluenza virus 3 NP by IF; HHV-6 IgM

and IgG EIA; Bartonella henselae serology
(1:128/1:128)

1:128

1:128

Enterovirus serology (1:4/1:16)

13

NSQ

NSQ

NSQ

None

1:64

NSQ

1:32

Parainfluenza virus 3 NP by IF; enterovirus

serology (1:16/!1:4)

13

NSQ

1:64

HSV-2 CSF PCR; HSV serology (1:16/1:16);

EBV Monospot; EBV EA, IgM VCA, IgG
VCA; EBNA

MMR vaccine 3 weeks before admission

Influenza A virus NP by IF; influenza A virus

serology (1:256/1:64)

10

1.5

!1:8

!1:8

HHV-6 IgM and IgG EIA

11

!1:8

!1:8

HSV-2 CSF PCR; HSV serology (!1:8/!1:8)

Patient

CF

!1:8

NSQ

NSQ

CF

!1:8

NSQ

Other potential etiologic agents

NOTE. The criteria for classification of cases of M. pneumoniae encephalitis as probable are described in the Patients and Methods section. EA, early
antigen; EBV, Epstein-Barr virus; EBNA, Epstein-Barr nuclear antigen; HHV-6, human herpesvirus 6; HSV, herpes simplex virus; HSV-2, herpes simplex virus type
2; IF, immunofluorescence testing; NP, nasopharyngeal swab; MMR, measles-mumps-rubella; NSQ, sample insufficient to perform assay; VCA, viral capsid antigen;
, positive finding; , negative finding.
a

Values in parentheses are acute-phase serologic titer/convalescent-phase serologic titer.

infection with negative results of culture and PCR of throat

and CSF specimens and the absence of convincing evidence for
other potential etiologic agents, or as positive results of culture
or PCR for M. pneumoniae in throat specimens but negative
results of serologic tests for M. pneumoniae. Indeterminate M.
pneumoniae encephalitis was defined as serologic evidence of
M. pneumoniae infection with negative results of culture and
PCR of throat and CSF specimens and convincing evidence
that implicated at least 1 other pathogen.
Convincing evidence for the presence of other pathogens was
determined by the presence of one of the following: (1) detection of the organism in the CSF by means of culture or PCR;
(2) detection of the organism in samples from a site other than
the CSF by means of culture, PCR, antigen detection techniques, or electron microscopy; (3) serologic evidence of acute
infection that was equally or more convincing than the evidence
for M. pneumoniae; (4) in cases of suspected varicella encephalitis, a clinical diagnosis of varicella made at the time of or
just before the development of encephalitis; or (5) a clinical
presentation consistent with tuberculous meningitis with an
appropriate exposure history and favorable response to antituberculous therapy.

1676 CID 2001:32 (15 June) Bitnun et al.

RESULTS
Evidence for M. pneumoniae infection was detected in 50 (31%)
of 159 children who were hospitalized with acute encephalitis
from January 1994 through December 1998. Eleven of these
patients (22%) had sufficient evidence of M. pneumoniae encephalitis for their cases to be classified as probable; among the
remaining 39 patients, 9 cases (18%) were categorized as possible, and 30 cases (60%) were classified as indeterminate. Patients with probable M. pneumoniae encephalitis included 6
boys and 5 girls, who had a mean age of 7 years (range, 113
years). Patients with possible M. pneumoniae encephalitis included 3 boys and 6 girls, who had a mean age of 9.5 years
(range, 117 years).
Microbiological findings. M. pneumoniae was detected by
PCR in CSF samples from 6 children and in throat specimens
obtained from an additional 5 children who were classified as
having probable M. pneumoniae encephalitis (table 1). No patient had M. pneumoniae detected at both sites. Three of the
6 children in whom M. pneumoniae was detected in CSF by
means of PCR lacked detectable antibody to this organism in
both acute-phase and convalescent-phase serum samples. All 3

Table 2.

Microbiological data for 9 children with possible Mycoplasma pneumoniae encephalitis.

Results of serologic testing for M. pneumoniae

Age,
years

Results of
PCR of throat
specimens for
M. pneumoniae

IgM EIA

12

13

17

Patient

14

1.2

Acute-phase
specimens
CF

Convalescent-phase
specimens
CF

1:8

1:32

None

1:128

NSQ

1:128

None

1:128

1:128

Parainfluenza virus 2 NP by culture;

parainfluenza virus 1 NP by IF

1:128

None

15

17

1:64

NSQ

16

12

1:2048

NSQ

17

1:128

18

1:128

19

20

15

Other potential etiologic agents

IgM EIA

1:4096

Bartonella henselae serology

(1:128/1:128)

NSQ

1:32

None

NSQ

1:128

None

1:128

NSQ

1:128

None

1:8

1:16

Influenza A virus NP by IF; influenza

A virus serology (!1:8/1:512)

NOTE. The criteria for classification of cases of M. pneumoniae encephalitis as possible are described in the Patients and Methods section. IF, immunofluorescence testing; NP, nasopharyngeal swab; NSQ, sample insufficient to perform assay; , positive finding; , negative finding.
a

Values in parentheses are acute-phase serologic titer/convalescent-phase serologic titer.

of these children were !2 years old, and only 1 of them had a

documented respiratory prodrome.
Seven of 11 patients who had probable M. pneumoniae encephalitis had evidence of coinfection with at least 1 other
organism (table 1). In 2 patients, coinfection of the CNS by
M. pneumoniae and HSV-2 was demonstrated by the detection
of both organisms in the CSF by PCR. Amplicons produced
by HSV-specific primers formed products characteristic for
HSV-2 when digested with restriction endonucleases [23].
When digested with the restriction endonuclease AluI, the 209bp amplicon produced by the M. pneumoniaespecific primers
yielded 2 base-pair fragments of 134 and 75 bp, which is characteristic of M. pneumoniae. Cross-reactivity between the 2 organisms was excluded by demonstrating that the M. pneumoniae DNA was not amplified by the HSV-2 primers and that
the HSV-2 DNA was not amplified by the M. pneumoniae
primers.
The microbiological evidence for the possible cases of M.
pneumoniae encephalitis is summarized in table 2. Viral respiratory coinfections were documented in 2 children. For 1
child, influenza A virus antigen was detected by immunofluorescence in a nasopharyngeal specimen, and a 14-fold rise in
titer to the influenza A virus was demonstrated between acutephase and convalescent-phase serum samples. The other child
had parainfluenza virus 2 isolated in culture of a specimen
from the nasopharynx. Results of serologic tests for B. henselae
in patient 16 were considered equivocal because of the lack of
change in titer between acute-phase and convalescent-phase
serum samples.

In all 30 of the children who were classified as having indeterminate M. pneumoniae encephalitis, the evidence that implicated other agents was equally or more compelling than that
for M. pneumoniae. The possible etiologic agents are summarized in table 3. Nineteen children had particularly strong evidence that implicated agents other than M. pneumoniae as the
cause of encephalitis. Enteroviruses were the likely etiologic
agent in 6 children. In 1 child, enterovirus was isolated in
culture of the CSF, and in another child it was detected by
means of reverse-transcriptase PCR of the CSF. The remaining
4 children had a 4-fold increase or decrease in the CF titer to
Table 3. Etiologic agents implicated in 30 children with
indeterminate Mycoplasma pneumoniae encephalitis.

Etiologic agent

No. of patients
(n p 30)a

Enteroviruses

Herpes simplex virus

Varicella-zoster virus

Epstein-Barr virus

Human herpesvirus 6

Influenza A virus

Adenovirus

Bartonella henselae

Mycobacterium tuberculosis

NOTE. The criteria for classification of cases of M. pneumoniae

encephalitis as indeterminate are described in the Patients and Methods
section.
a

More than 1 agent was implicated in 10 patients.

Encephalitis and M. pneumoniae CID 2001:32 (15 June) 1677

enterovirus. In 1 patient, HSV was detected in the CSF by PCR,

and a 4-fold rise in the CF antibody titer was demonstrated
between acute-phase and convalescent-phase serum samples.
For 3 children, detection of adenovirus in the nasopharynx or
stool was accompanied by positive results of serologic testing
for adenovirus. Two patients exhibited a 4-fold rise in CF antibody titer to influenza A virus. VZV was the likely cause of
encephalitis in 2 children who had active clinical varicella, 1
of whom also had VZV detected in the CSF by PCR. EBV was
implicated in 1 patient in whom heterophile antibody as well
as IgM and IgG to the EBV viral capsid antigen were detected
in acute-phase serum samples. Two children had a 4-fold rise
in antibody titer to B. henselae. Tuberculous meningitis was the
likely diagnosis for 2 children. Although neither case was confirmed microbiologically, the patients clinical presentation,
CSF findings (lymphocytic pleocytosis and elevated levels of
CSF protein), and response to antituberculous therapy strongly
implicated Mycobacterium tuberculosis as the cause of the encephalitic presentation. The remaining 11 patients had serologic evidence of infection with other agents, including human herpesvirus 6 (n p 2 ), EBV (n p 2), influenza A virus
(n p 1), enterovirus (n p 1), adenovirus (n p 2 ), and B. henselae (n p 3).
Clinical manifestations. Prodromal respiratory manifestations occurred in 7 of 11 patients with probable M. pneumoniae encephalitis and 6 of 9 patients with possible M. pneumoniae encephalitis (table 4). The duration of respiratory
manifestations before admission varied from several days to as
long as 4 weeks. Chest radiographs were performed for 6 patients with probable M. pneumoniae encephalitis and 3 patients
with possible M. pneumoniae encephalitis; focal infiltrates and
peribronchial thickening were detected in 4 and 2 patients,
respectively. In patients without preceding respiratory symptoms, prodromal symptoms included fever, headache, vomiting,
and diarrhea. One patient with probable M. pneumoniae encephalitis had no documented prodromal illness, and another
had a mild febrile illness 2 weeks before the development of
encephalitis, subsequent to receiving a routine measles-mumpsrubella vaccine.
The duration of prodromal illness was significantly shorter
among children with positive M. pneumoniae CSF PCR than
in children who had negative M. pneumoniae CSF PCR (P p
.015 by 2-tailed Fishers exact test). In 5 of 6 children who had
M. pneumoniae detected in the CSF but not in the throat, the
prodromal illness was 5 days in duration, whereas all 5 children in whom M. pneumoniae was detected in the throat but
not in the CSF had prodromal symptoms 7 days in duration.
Respiratory symptoms occurred in all children in whom M.
pneumoniae was detected in the throat but in only 2 of 6 children in whom M. pneumoniae was detected in the CSF (P p
.06 by 2-tailed Fishers exact test).
1678 CID 2001:32 (15 June) Bitnun et al.

Neurologic manifestations in patients with probable or possible M. pneumoniae encephalitis included seizures, focal motor
deficits, ataxia, and increased intracranial pressure (table 4). Of
the 13 patients with seizures, 8 had generalized tonic-clonic
seizures and 5 had focal motor seizures. Focal neurologic deficits, independent of seizures, included hemiparesis, dysarthria,
and aphasia. Encephalopathic manifestations included a decreased level of consciousness, disorientation, confusion, visual
hallucinations, and extreme irritability. Acute demyelinating
encephalomyelitis (ADEM) was diagnosed in 2 patients with
probable M. pneumoniae encephalitis, 1 of whom also had optic
neuritis. For both patients, PCR detected M. pneumoniae in
specimens from the throat but not the CSF.
Specific therapy directed against M. pneumoniae was administered to 4 children in whom M. pneumoniae encephalitis was
suspected (2 probable cases and 2 possible cases); 3 received
macrolides, and 1 received doxycycline. Ten children were
treated with acyclovir for presumed HSV encephalitis. Corticosteroids were administered to 1 patient with ADEM.
CSF findings. CSF specimens were available for all of the
patients with probable or possible M. pneumoniae encephalitis
(table 4). For the 12 patients with pleocytosis, the mean WBC
count was 47 10 9 cells/L (range, 6 10 9 to 98 10 9 cells/L)
in children with probable M. pneumoniae encephalitis and
35 10 9 cells/L (range, 7 10 9 to 110 10 9 cells/L) in children
with possible M. pneumoniae encephalitis. A predominance of
lymphocytes (87%100%) was documented in 9 of the 10 patients for whom a differential cell count was available. Among
patients with probable or possible M. pneumoniae encephalitis,
the mean CSF protein level was 42 mg/dL (range, 10119 mg/
dL) and 37 mg/dL (range, 1365 mg/dL), respectively. The CSF
glucose level was 2.86.0 mM.
EEG and neuroimaging findings. EEG and neuroimaging
(cranial CT, MRI, or both) were performed for all 20 patients
with probable and possible M. pneumoniae encephalitis; EEG
and neuroimaging abnormalities were demonstrated in 85%
and 35% of these patients, respectively (table 4). Neuroimaging
abnormalities included focal hemispheric lesions suggestive of
an ischemic injury in 2 patients, focal temporal enhancing and
edematous lesions in 1 patient in each category, and mild unilateral ventricular dilatation in 1 patient. Findings suggestive
of ADEM included diffuse demyelination in 1 patient and
edema of the basal ganglia, cerebral peduncles, and deep white
matter in another. Results of single-photon emission CT scanning were normal in both patients with ADEM. Auditory and
somatic evoked potentials were also normal in these 2 patients,
but visual evoked responses were abnormal in the patient with
concomitant optic neuritis.
Neurologic outcome. There were no deaths among patients
in any of the 3 categories of M. pneumoniae encephalitis. The
mean duration of follow-up for the 8 children with probable

Table 4. Most common clinical and laboratory manifestations of probable, possible, and
indeterminate Mycoplasma pneumoniae encephalitis (n p 50).
No. (%) of patients with specified
type of M. pneumoniae encephalitis
Probable
(n p 11)

Parameter

Possible
(n p 9)

Indeterminate
(n p 30)

Prodromal symptoms
None

4 (36)

3 (33)

3 (10)

NS

Cough/coryza/sore throat

7 (64)

6 (67)

11 (37)

NS

Fever

6 (55)

7 (78)

20 (67)

NS

Headache

2 (18)

5 (56)

13 (43)

NS

Vomiting

4 (36)

2 (22)

8 (27)

NS
NS

Neurologic manifestationsb
Seizures

7 (64)

5 (56)

13 (43)

Focal motor deficits

4 (36)

4 (44)

8 (27)

NS

Ataxia

4 (36)

1 (11)

1 (3)

.014

Increased intracranial pressure

1 (9)

NS

Leukocytosis (115 109 leukocytes/L)

5 (45)

3 (33)

8 (27)

NS

Anemia (!110 g/L)

1 (9)

1 (11)

NS

Thrombocytopenia (!150 10 platelets/L)

1 (9)

1 (3)

NS

Elevated ESR (25 mm/h)c

5 (63)

5 (63)

6 (40)

NS

6 (55)

7 (78)

27 (90)

.022

5 (45)

6 (67)

22 (76)

NS

4 (36)

4 (44)

12 (43)

NS

Abnormal

9 (82)

8 (89)

19 (90)

NS

Generalized slowing

7 (64)

4 (44)

13 (62)

NS

Epileptic focus

3 (27)

2 (22)

2 (10)

NS

PLED

2 (18)

NS

FIRDA/OIRDA

2 (18)

3 (33)

6 (29)

NS

Hematologic findingsc

CSF findings
Abnormal
d

Pleocytosis (15 WBCs/mL)

Elevated protein level (40 mg/dL)

EEG findingsf

Neuroimaging findings
Abnormal

5 (45)

2 (22)

12 (40)

NS

Focal edema or ischemia

3 (27)

1 (11)

4 (13)

NS

Focal enhancing lesions

1 (9)

2 (7)

NS

Demyelination

1 (9)

3 (10)

NS

Ventricular dilatation

1 (11)

2 (7)

NS

NOTE. The criteria for classification of cases of M. pneumoniae encephalitis as probable, possible, or
indeterminate are described in the Patients and Methods section. EEG, electroencephalography; ESR, erythrocyte sedimentation rate; FIRDA, frontal intermittent rhythmic delta activity; OIRDA, occipital intermittent
rhythmic delta activity; PLED, periodic lateralizing epileptiform discharge.
a

Comparison of probable and indeterminate cases by use of x2 statistic (2-tailed Fishers exact test if cell
no. !5).
b
By definition, encephalopathy was present in all patients; for details, see the Patients and Methods
section.
c
Available for 8 patients with probable M. pneumoniae encephalitis, 8 patients with possible M. pneumoniae encephalitis, and 15 patients with indeterminate M. pneumoniae encephalitis.
d
Available for 29 patients with indeterminate M. pneumoniae encephalitis.
e
Available for 28 patients with indeterminate M. pneumoniae encephalitis.
f
Available for 21 patients with indeterminate M. pneumoniae encephalitis.

M. pneumoniae encephalitis in whom neurologic deficits were

present at the time of discharge from the hospital was 1.8 years
(range, 04 years). At least 6 months of follow-up data were
available for 7 of 8 children with probable and 4 of 5 children
with possible M. pneumoniae encephalitis who had neurologic
deficits at the time of discharge. A seizure disorder that required
prolonged anticonvulsant therapy developed in 4 patients with
probable M. pneumoniae encephalitis and 1 patient with possible M. pneumoniae encephalitis. Mild cognitive impairments,
including difficulty with concentration, memory, reading, and
word finding, occurred in 4 of the 5 children who had seizure
disorders. Other persistent neurologic deficits that occurred in
patients with probable M. pneumoniae encephalitis included
hemiparesis (n p 1), expressive dysphasia (n p 1), dysarthria
(n p 1), and truncal ataxia (n p 1). The 2 children with
ADEM, 1 of whom was treated with corticosteroids, made full
recoveries over the course of several months. Three of the 4
children who were treated with macrolides or doxycycline experienced neurologic sequelae. Among children with possible
M. pneumoniae encephalitis, 1 patient experienced severe developmental delay and concomitant hemiplegia. Other abnormal findings in this group of children included a speech disorder (word-finding difficulty) in 2 patients and asymptomatic
ventriculomegaly and mild EEG abnormalities in 1.

DISCUSSION
M. pneumoniae was strongly implicated as the probable etiologic agent in 6.9% of 159 children with acute encephalitis.
This figure is consistent with data from previous studies suggesting that 1%10% of cases of acute childhood encephalitis
are caused by this organism [1, 711]. The criteria for diagnosis
of probable M. pneumoniae encephalitis included the detection
of M. pneumoniae by culture or PCR of specimens from the
throat, the CSF, or both to eliminate the potential impact of
false-positive serologic test results. The detection of M. pneumoniae by PCR in these children provides conclusive evidence
that all of them were infected, in the respiratory tract or CNS,
at the time of or just before the development of encephalitis.
By using more stringent diagnostic criteria than those of previous studies, we have strengthened the evidence implicating
M. pneumoniae as a cause of acute childhood encephalitis.
M. pneumoniae is typically considered to be a potential cause
of disease in children 5 years old who have respiratory symptoms. However, in our cohort, respiratory manifestations were
absent in 4 (36%) of 11 patients with probable M. pneumoniae
encephalitis and 3 (33%) of 9 patients with possible M. pneumoniae encephalitis. Among patients with probable M. pneumoniae encephalitis, respiratory symptoms occurred in all 5
children in whom M. pneumoniae was detected by PCR in the
throat but not in the CSF, but in only 2 of 6 children in whom
1680 CID 2001:32 (15 June) Bitnun et al.

M. pneumoniae was detected in the CSF but not in the throat.

The absence of respiratory manifestations has also been documented in several previous case reports of patients with encephalitis for whom PCR or culture detected M. pneumoniae
in the CSF [2427]. In a recently reported study of patients
with serologically confirmed systemic infections caused by M.
pneumoniae, M. pneumoniae was detected in serum by PCR in
only 1 of 25 patients with pneumonia but in 10 of 17 patients
without pneumonia (P ! .001) [28]. With respect to age, 4 of
11 children in our cohort who had probable M. pneumoniae
encephalitis and 2 of 9 children who had possible M. pneumoniae encephalitis were !5 years old (tables 1 and 2). Four
of these 6 children were !2 years old. Therefore, M. pneumoniae
infection should be considered in the differential diagnosis of
children with acute encephalitis, regardless of whether they have
signs or symptoms of respiratory illness and regardless of their
age.
The detection of M. pneumoniae in the brain tissue and CSF
of patients with encephalitis indicates that direct invasion of
the brain parenchyma is responsible for some cases of acute
encephalitis [2938]. We are aware of 2 cases in which M.
pneumoniae was detected in brain tissue; in one, the organism
was isolated in culture [29], and in the other, it was detected
by nucleic acid hybridization [30]. M. pneumoniae has also been
isolated from the CSF by culture in 7 patients [24, 3235] and
detected by PCR in an additional 26 patients, including those
in our cohort [2527, 31, 3638].
M. pneumoniae has been implicated as a cause of immunemediated neurologic syndromes, including ADEM, transversemyelitis, and Guillain-Barre syndrome [12, 36, 3946]. In our
cohort, ADEM was diagnosed in 2 children in whom M. pneumoniae was detected in the throat but not in the CSF by PCR.
The underlying pathogenesis of immune-mediated injury in
patients with M. pneumoniae infection is incompletely understood, but it likely stems from the presence of antigenic similarities between M. pneumoniae and human tissues. M. pneumoniae infection has been associated with the production of a
variety of anti-human antibodies, including IgM-class cold agglutinins, cold-reactive antilymphocyte antibodies, and antibodies to cardiolipin, smooth muscle, mitotic spindle apparatus, centriole, lung, liver, and brain [11, 17, 4749]. CF
antibodies to brain tissue antigens have been demonstrated in
patients with and without CNS manifestations [17, 47]. The
role of such antibodies in human disease has not been determined, although one report suggests that they are more common in subjects with CNS disease [50]. The deposition of immune complexes formed from such autoantibodies within small
venules in the CNS has been implicated as a possible mechanism of immune-mediated neurologic injury [43, 51].
In autoimmune-induced encephalitis, one would anticipate
a more prolonged duration of symptoms before the onset of

encephalitis and the presence of M. pneumoniae in the respiratory tract but not in the CSF. In our cohort, all 5 children
in whom M. pneumoniae was detected by PCR in the throat
but not in the CSF had prodromal symptoms of 7 days
duration. In contrast, 5 of the 6 children in whom M. pneumoniae was detected by PCR in the CSF but not in the throat
had prodromal symptoms of 5 days duration. These results
are consistent with those of Narita et al. [28, 36], who demonstrated a statistically significant association between the detection of M. pneumoniae in CSF samples and serum by PCR
and fever of 7 days duration before the onset of encephalitis.
This lends support to the contention that autoimmune mechanisms are likely responsible for some cases of M. pneumoniae
encephalitis, particularly those with prodromal symptoms of
157 days duration.
Cerebrovascular thromboembolic phenomena have also been
implicated as a potential cause of neurologic injury in patients
infected with M. pneumoniae [52, 53]. Whether the cerebral
infarcts that occurred in 2 of the children who we studied were
caused by thromboembolic events, an autoimmune vasculitis,
or some other pathologic process is uncertain. Neurotoxininduced neurologic disease occurs in mice infected with Mycoplasma neurolyticum and in turkeys infected with Mycoplasma
gallisepticum, but no such toxin has been demonstrated in humans infected with M. pneumoniae [52].
No immune response to M. pneumoniae could be demonstrated by either EIA or CF assays in the acute-phase or convalescent-phase serum samples of 3 of 6 children with probable
M. pneumoniae encephalitis for whom the results of CSF PCR
were positive (table 1). All 3 previously had been healthy, with
no evidence to suggest an immune deficiency. The absence of
a detectable serologic response could have been due to a lack
of sensitivity of the assays or a failure to develop specific antibody. False-negative results of serologic tests for M. pneumoniae have been described elsewhere [29, 5456]. In a study
of 3546 patients with pneumonia, 10% of culture-proven M.
pneumoniae infections were associated with acute-phase and
convalescent-phase CF titers of 1:16, and only 53% had a
4-fold rise in the CF titer [54]. In studies that used the microparticle agglutination test and indirect immunofluorescence
serologic assays, 15%25% of children with PCR-proven respiratory infections caused by M. pneumoniae had negative results of serologic testing for the microorganism [55, 56]. Falsenegative results of CF for M. pneumoniae have also been
documented in a fatal case of culture-proven infection of the
CNS [29] and in a child who had M. pneumoniae detected in
the CSF and the throat by PCR [25]. Thus, the absence of
detectable antibodies to M. pneumoniae does not eliminate it
as a possible cause of infection.
Strong evidence for coinfections, as determined by the direct
detection of an organism by use of culture, PCR, or antigen

detection techniques was demonstrated in 5 (45%) of the 11

patients who were classified as having probable M. pneumoniae
encephalitis (table 1). Respiratory viral infections can have cytotoxic effects on respiratory epithelium, and therefore it is conceivable that such infections could have facilitated bloodstream
invasion by M. pneumoniae. Dual CNS infection with HSV-2
and M. pneumoniae, as was demonstrated in 2 of the children
in our cohort, has not been previously reported. The presence
of detectable antibody to HSV in one of these children at the
onset of encephalitis and the lack of a 4-fold rise in HSV antibody titer in both children suggest that reactivation of latent
HSV infection, rather than primary HSV infection, occurred
in these 2 children. The pathogenetic role of other infectious
agents detected by means of serologic tests alone in patients
with probable M. pneumoniae encephalitis is questionable. M.
pneumoniae has been shown to induce non-antigen-specific T
celldependent antibody production in human B lymphocytes
in vivo [57, 58], and it is likely that the elevated levels of
antibody to these other infectious agents are the result of nonspecific immune activation by M. pneumoniae.
Twenty-eight (93%) of 30 children who had indeterminate
M. pneumoniae encephalitis had serologic evidence of infection
with 1 other agent. Thirteen (43%) had evidence of infection
with 2 agents, in addition to M. pneumoniae. A minority of
these cases might represent true dual viral mycoplasmal infections or mycoplasma-induced nonspecific immune activation.
However, in the majority of these cases, the evidence that implicated other agents as the cause of encephalitis was compelling, suggesting that false-positive results of serologic tests for
M. pneumoniae were likely. Such false-positive results could be
caused by cross-reactivity with human brain antigens [17, 19,
59] or by other inherent limitations in the specificity of serologic tests for M. pneumoniae [1415]. For all these reasons,
we believe that in the absence of corroborating culture or PCR
results, seropositivity is insufficient to establish a definitive diagnosis of M. pneumoniae encephalitis.
The clinical manifestations, CSF abnormalities, and EEG and
neuroimaging findings associated with M. pneumoniae encephalitis are indistinguishable from those of viral encephalitis [6].
Long-term neurologic sequelae have been documented in
48%54% of serologically confirmed cases of M. pneumoniae
encephalitis [7, 12]. In our cohort, 7 (64%) of 11 children with
probable M. pneumoniae encephalitis experienced neurologic
sequelae.
Antibiotic therapy has been temporally associated with clinical improvement in some cases of M. pneumoniae encephalitis
[12, 25, 31, 32, 34, 35, 37, 40, 42, 43] but not in others [11,
12, 26, 38, 4245, 59]. Complete neurologic recoveries without
antibiotic therapy have also been described [24, 36, 41, 42].
Unfortunately, it is not possible to draw any conclusion regarding the efficacy of antibiotic therapy from the results of
Encephalitis and M. pneumoniae CID 2001:32 (15 June) 1681

our study or from the data in the literature because of the small
number of children treated, the uncertainty regarding the natural history of untreated infection, and the failure to use an
antibiotic that penetrates the blood-brain barrier in many of
the reported cases. Nevertheless, until more-definitive data become available, it is reasonable to consider antibiotic therapy
for children who present with a short prodromal illness consistent with direct CNS infection. In theory, an antibiotic such
as chloramphenicol, doxycycline, or a quinolone should be
used, because of the antimycoplasmal activity of such antibiotics and their ability to traverse the blood-brain barrier.
The optimal management of this condition in children in
whom immune-mediated neurologic injury is likely remains uncertain. At present, no data support or refute the potential benefit
of antimicrobial therapy for such patients. Corticosteroids have
been temporally associated with clinical improvement in a small
number of patients with ADEM and serologically confirmed M.
pneumoniae infection [12, 4244, 60, 61]. The efficacy of corticosteroids in the treatment of M. pneumoniaeassociated transverse myelitis is less well established [45]. Plasmapheresis appeared to be effective in 1 patient with M. pneumoniae transverse
myelitis [62] and 1 patient with polyradiculitis [63]. No such
benefit was seen with iv immunoglobulin [40]. Despite the lack
of conclusive evidence, it is reasonable to consider the use of
these immune-modulating therapies in addition to antibiotics in
children with M. pneumoniae encephalitis who have severe disease.
Because of the difficulties associated with the serologic diagnosis and the prolonged incubation period required for culture, PCR is increasingly used for the diagnosis of M. pneumoniae infections [2022, 28, 36, 37, 55, 56, 6469]. In
preliminary experiments that involved 114 clinical respiratory
samples, 50 of which had culture results that were positive for
M. pneumoniae, our PCR assay had a sensitivity, specificity,
positive predictive value, and negative predictive value of 100%,
98.4%, 98%, and 100%, respectively, with culture serving as
the gold standard (unpublished data). The high sensitivity and
specificity of our assay are consistent with published data from
other centers that used the same PCR primers [2122].
Although the possibility of false-positive PCR results cannot
be absolutely excluded, such an event is unlikely, given the
design and execution of the assay. The detection of M. pneumoniae in the CSF of 6 children with encephalitis provides
strong evidence that this organism was responsible for their
illness, regardless of the results of serologic tests. The detection
of M. pneumoniae in the throat but not in the CSF (in 5 probable cases) provided less-conclusive evidence of causality and
therefore required the presence of supportive positive results
of serologic tests for M. pneumoniae.
In conclusion, M. pneumoniae is responsible for at least 6.9%
of cases of acute childhood encephalitis and is strongly asso1682 CID 2001:32 (15 June) Bitnun et al.

ciated with 12.5% of cases. Neurologic sequelae are common,

occurring in 48%64% of cases. Direct invasion of the CNS is
the probable pathogenetic mechanism in children with a brief
(5 days duration) prodromal illness. Immunologic phenomena, thromboembolic phenomena, or both may be the mechanisms responsible in children with more prolonged (7 days
duration) prodromal illness. Coinfections with respiratory viruses and herpesviruses are common. Currently available EIA
and CF tests for M. pneumoniae are not sufficiently specific to
reliably diagnose acute childhood encephalitis caused by M.
pneumoniae in the absence of corroborating evidence, such as
positive culture or PCR.

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