Aetiological diagnosis of brain abscesses and spinal infections: application of broad range bacterial polymerase chain reaction analysis.

Abstract

To evaluate the usefulness of the broad range bacterial rDNA polymerase chain reaction (PCR) method combined with DNA sequencing in the aetiological diagnosis of intracranial or spinal infections in neurosurgical patients.
In addition to conventional methods, the broad range bacterial PCR approach was applied to examine pus or tissue specimens from cerebral or spinal lesions in patients treated in a neurosurgical unit for a clinical or neuroradiological suspicion of bacterial brain abscess or spondylitis.
Among the 44 patients with intracranial or spinal lesions, the final diagnosis suggested bacterial disease in 25 patients, among whom the aetiological agent was identified in 17. A causative bacterial species was identified only by the rDNA PCR method in six cases, by both the PCR methodology and bacterial culture in six cases, and by bacterial culture alone in five. All samples in which a bacterial aetiology was identified only by the PCR approach were taken during antimicrobial treatment, and in three patients the method yielded the diagnosis even after >/= 12 days of parenteral treatment. One case also identified by the PCR approach alone involved a brain abscess caused by Mycoplasma hominis, which is not readily cultured by routine methods.
In patients with brain abscesses and spinal infections, the broad range bacterial rDNA PCR approach may be the only method to provide an aetiological diagnosis when the patient is receiving antimicrobial treatment, or when the causative agent is fastidious.

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PAPER
Aetiological diagnosis of brain abscesses and spinal
infections: application of broad range bacterial
polymerase chain reaction analysis
L Kupila, K Rantakokko-Jalava, J Jalava, S Nikkari, R Peltonen, O Meurman,
R J Marttila, E Kotilainen, P Kotilainen
.............................................................................................................................
J Neurol Neurosurg Psychiatry 2003;74:728–733
Objective: To evaluate the usefulness of the broad range bacterial rDNA polymerase chain reaction
(PCR) method combined with DNA sequencing in the aetiological diagnosis of intracranial or spinal
infections in neurosurgical patients.
Methods: In addition to conventional methods, the broad range bacterial PCR approach was applied
to examine pus or tissue specimens from cerebral or spinal lesions in patients treated in a neurosurgi-
cal unit for a clinical or neuroradiological suspicion of bacterial brain abscess or spondylitis.
Results: Among the 44 patients with intracranial or spinal lesions, the final diagnosis suggested bac-
terial disease in 25 patients, among whom the aetiological agent was identified in 17. A causative
bacterial species was identified only by the rDNA PCR method in six cases, by both the PCR method-
ology and bacterial culture in six cases, and by bacterial culture alone in five. All samples in which a
bacterial aetiology was identified only by the PCR approach were taken during antimicrobial
treatment, and in three patients the method yielded the diagnosis even after > 12 days of parenteral
treatment. One case also identified by the PCR approach alone involved a brain abscess caused by
Mycoplasma hominis, which is not readily cultured by routine methods.
Conclusions: In patients with brain abscesses and spinal infections, the broad range bacterial rDNA
PCR approach may be the only method to provide an aetiological diagnosis when the patient is receiv-
ing antimicrobial treatment, or when the causative agent is fastidious.
I
infections. In patients with suspected bacterial meningitis,
specific polymerase chain reaction (PCR) techniques have
been used for the identification of Neisseria meningitidis,1–3
Streptococcus pneumoniae,4 5and Listeria monocytogenes6or for the
simultaneous detection of Neisseria meningitidis, Haemophilus
influenzae, and streptococci in cerebrospinal fluid (CSF)
samples.7 8The aetiology of community acquired bacterial
meningitis has also been assessed using PCR with broad range
bacterial primers combined with DNA sequencing in CSF.9 10
These PCR primers are targeted at the most conserved bacte-
rial rDNA gene sequences, rendering possible the detection of
practically any bacterial species in the sample. The bacterial
speciescanthenbeidentifiedbycomparingtheDNAsequence
of the amplification product with previously published bacte-
rial sequence types.11
PCR techniques have also been applied in the aetiological
diagnostics of brain abscesses. Reports on the use of specific
PCR assays in cerebral specimens from patients with brain
abscesses have focused mainly on toxoplasma infections,12 13
but there are also papers describing the use of specific PCR
assays to identify, for example, Entamoeba histolytica in pus
from brain abscess14or Mycobacterium tuberculosis in tubercu-
lous brain lesions.15One case report has described identifica-
tion of Fusobacterium species in a brain abscess, and another,
Streptococcus pneumoniae in a subdural empyema, by the broad
range bacterial PCR approach alone.16 17
We have previously used broad range bacterial rDNA PCR to
analyse 536 clinical samples of various tissues from patients
admitted to hospital during the years 1994 to 1997. That work
also included samples obtained during neurosurgery, but the
patients involved were not further described nor the results
n recent years, molecular methods have increasingly been
used to detect microbes in various clinical samples, includ-
ing those from patients with central nervous system
specifically discussed.9Subsequently, the PCR method has
been applied in our hospital to analyse intracranial and spinal
specimens from neurosurgical patients, when the procedure
was considered clinically indicated by the attending clinicians.
We describe here our experience of the value of this technique
in diagnosing suspected bacterial intracranial or spinal infec-
tion at the neurosurgical department of a university hospital
in Finland.
METHODS
From the beginning of 1995 to the end of 2000, 44 pus or tis-
sue samples from neurosurgical patients treated at the
department of neurosurgery,Turku University Hospital,Turku,
Finland, were analysed by the PCR method, in addition to
conventional microbiological methods. These patients were
suspected of having bacterial infection in brain tissue or spinal
canal.
Patients and samples
The study collection included 24 intracranial and 20 spinal
samples. The intracranial pus or tissue samples were from
patients operated on for a clinical or neuroradiological suspi-
cion of brain abscess or subdural empyema, or when the
intraoperative nature of the lesion remained macroscopically
undefined. Eleven samples were obtained by trepanation and
puncture, and 13 by stereotactic or open biopsy (table 1). The
spinal samples were from patients who underwent neurosur-
gery for suspected spondylitis or spinal epidural abscess. The
samples included five pus and 15 tissue specimens, of which
18 were obtained by biopsy during laminectomy, one tissue
specimen was obtained by stereotactic biopsy, and one pus
specimen was obtained by puncture (table 2).
Data were collected on the final clinical diagnoses as well as
on the administration of antimicrobials and the timing of the
See end of article for
authors’ affiliations
.......................
Correspondence to:
Dr Laura Kupila,
Department of Neurology,
Turku University Central
Hospital, Kiinamyllynkatu
4-8, 20520 Turku, Finland;
laura.kupila@tyks.fi
Received 9 July 2002
In revised form
28 November 2002
Accepted
28 January 2003
.......................
728
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microbiological specimens with respect to antimicrobial treat-
ment. All positive PCR or culture results were reassessed to
determine the clinical importance of the findings.
DNA purification
DNA was extracted from the fresh tissue samples after protei-
nase K (0.1 mg/ml) digestion (56°C,two to 17 hours) with two
phenol-chloroform-isoamyl alcohol extractions followed by
one ether wash, as described earlier.18Pus samples were
concentrated by centrifugation19and DNA extraction was
done as described above.
PCR
The primers,reagents,and conditions used in the 23S and 16S
rDNA PCR have been described previously.10All samples were
initially screened for the presence of bacterial DNA by ampli-
fication of the 23S rRNA genes with oligonucleotide primers
MS 37 and MS 38.10On the basis of sequence analysis of the
23S rDNA, these primers cover several bacterial subdivisions,
as described previously.10
The bacterial DNA present in a 23S rDNA PCR positive
specimen was identified by sequencing the 23S or 16S rDNA,
or both. Amplification of the 23S rDNA was used in the initial
screening of the samples because of its higher sensitivity com-
pared with that of the previously described 16S rDNA PCR
method. The 16S rDNA PCR product was preferred for
sequencing because of the more abundant sequence data
presently available.
Special care was taken to avoid contamination of samples
with amplicons.20Strict measures were employed to separate
Table 1
PCR, DNA sequencing, bacterial culture, data on operative technique and preoperative antimicrobial treatment, and
final diagnosis
Results of analyses of specimens from 24 patients with suspected intracranial infection: broad range bacterial
No
Initial
diagnosis
Operative
technique
Intracranial
specimen
Bacterial culture
resultPCRSequencing result
Homology
(%)a
Sequence
length
(nt)a
Preoperative
antibioticsb
Final diagnosis
1Brain abscess PuncturePus
Str equinus+ Str intermedius98.1423 Yes (14
hours)
No
Brain abscess
2 Brain abscess
or metastasis
Brain abscess Puncture
Open
biopsy
Pus
Streptococcus (sp.
viridans group)
Str intermedius/
anginosus
Peptostreptococcus
sp
Propionibacterium
acnes
Bacteroides
gracilis,
Peptostreptococcus,
Str intermedius
–d
+Streptococcus (sp
milleri group)
Str intermedius/
anginosus
Peptostreptococcus
sp
Propionibacterium
acnes
Several speciesc
99.6458Brain abscess
3 Pus
+99.4349 Yes (5 days)Brain abscess
4 GlioblastomaOpen
biopsy
Puncture
Pus
+ 95.7 243 Yes (12
hours)
No
Brain abscess
5 Postoperative
brain abscess
Brain abscess Puncture
Pus
+ 99.7299 Postoperative
brain abscess
Brain abscess
6 Pus
+ No
7Postoperative
brain abscess
Post-traumatic
brain abscess
Postoperative
subdural
empyema
Postoperative
subdural
empyema
Brain abscess Puncture
Brain abscess Stereotactic
PuncturePus
+ Staph aureus98.6441 Yes (5 days)Postoperative
brain abscess
8 Puncture Pus–+
Mycoplasma
hominis
Failuree
99.8 499Yes (10 days) Post-traumatic
brain abscess
Postoperative
subdural
empyema
Postoperative
subdural
empyema
Brain abscess
9PuncturePus–+
No
10
Open
biopsy
Pus
Staph aureus– Yes (3 days)
11
12
Pus
Pus
Fusobacterium sp
–
–
–
No
Yes (60 days) Aspergillus sp,
biopsy brain abscess
Brain abscess
13Brain abscess PuncturePus–– Yesf(11
days)
Yesf(7 days)
No
14
15
Brain abscess Puncture
Brain tumour
or infection
CNS infection Stereotactic
Pus
Brain tissue
–
–
–
–
Brain abscess
Demyelination Stereotactic
biopsy
16
biopsy
Brain tissue Not done–NoUnspecific
gliosis
17CNS infection Stereotactic
biopsy
Stereotactic
biopsy
Cysta liquid –– Yes (13 days) Parvocellular
carcinoma
Glioblastoma
18 Unclear
progressive
brain disease
Unclear brain
lesion
Brain tumour
or abscess
Brain tumour
or abscess
Brain tumour
or abscess
Brain abscess Stereotactic
Brain tissue––Yes (one
dose)
19Puncture Brain tissue–– Yes (5 days)Glioblastoma
20Open
biopsy
Stereotactic
biopsy
Stereotactic
biopsy
Brain tissue Not done–Yes (one
dose)
No
Glioblastoma
21Brain tissue
Propionibacterium
sp
–
–Astrocytoma
22Brain tissue–NoLymphoma
23
biopsy
Open
biopsy
Brain tissue–– Yes (4 days)Lymphoma
24Tumour Mucous
liquid
––Yes (one
dose)
Mucocele
aThe per cent homology and the length of the overlapping sequence for the best match.
bAn effective antimicrobial treatment against the recognised microbe in the specimen; duration of preoperative antibiotic treatment is given in parentheses.
cThe sample was interpreted to contain several species if the electropherogram showed strong signals but multiple overlapping peaks in some locations.
dNegative result.
eSequencing failure was assumed if the sequencing signals reported by the instrument were weak.
fThe patient received ceftriaxone and metronidazole.
CNS, central nervous system; nt, nucleotides; PCR, polymerase chain reaction.
Diagnosis of brain abscesses and spinal infections729
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the pre-PCR facilities from the post-PCR areas. Gamma
irradiation and ultraviolet light irradiation were used to
destroy possible traces of environmental bacterial DNA in the
reagents.21 22
DNA sequencing
The sequencing reactions were done as described earlier,
either manually18or semiautomatedly,10by using a 373 A
Stretch DNA sequencer or an ABI Prism 310 genetic analyser
(Applied Biosystems, Foster City, California, USA).
Sequence analysis and databases
The 16S rDNA study sequences were compared with those in
a database made up of sequences obtained from GenBank,23
EMBL,24and the ribosomal database project25by using an
in-house algorithm.10For comparison of the 23S rDNA
sequences, the FastA program was used.26The interpretation
Table 2
bacterial PCR, DNA sequencing, bacterial culture, data on operative technique, preoperative antimicrobial treatment,
and blood cultures, and final diagnosis
Results of analyses of specimens from 20 patients with suspected spondylitis or epidural abscess: broad range
No
Initial
diagnosis
Operative
technique
Spinal
specimen
Bacterial
culture result PCR
Sequencing
result
Homology
(%)a
Sequence
length
(nt)a
Preoperative
antibioticsb
Blood culture
resultc
Final diagnosis
1 SpondylitisOpen
biopsy
Pus
Staph aureus+ Staphylococcus
sp, probably
several
speciesd
Staphylococcus
sp
Staph aureus
86.3 164Yes (8 days) Staph aureus
(–8 days)
Spondylitis and
epidural
abscess
2Epidural
abscess
Spondylitis
Open
biopsy
Open
biopsy
Open
biopsy
Open
biopsy
Pus–e
+ 94.7216 Yes (15
days)
Yes (12
days)
Yes (13
days)
Yes (4 days)
Staph aureus
(–15 days)
Staph aureus
(–14 days)
Not done
Epidural
abscess
Spondylitis
3 Bone and
disc
Pus
–+
98.6441
4Postoperative
spondylodiscitis
Vertebral
tumour
–+
Str intermedius 98.1423 Postoperative
spondylodiscitis
Spondylitis
5 Bone–+
Str
mitis/
gordonii/
oralis/peroris
Pseudomonas
sp
100418Not done
6Spondylitis PuncturePus–+97.4 268Yes (2 days)
Str mitis (–3
days)
Spondylitis and
infective
endocarditis
Spondylitis
7Spondylitis Stereotactic
biopsy
Open
biopsy
Bone
Staph aureus– NoStaph aureus
8Postoperative
epidural
abscess
Pus
Pseudomonas
aeruginosa
– NoPseudomonas
aeruginosa
Postoperative
spondylodiscitis
and epidural
abscess
Spondylitis
9 Vertebral
tumour
Spondylitis and
epidural
abscess
Epidural
abscess
Postoperative
spondylodiscitis
and epidural
abscess
Spondylitis
Open
biopsy
Open
biopsy
Bone–– Yesf(4 days) –
10
Bone–– No– Spondylitis
11 Open
biopsy
Open
biopsy
Bone––Yesg(10
days)
No
Not done Spondylitis
12
Disc–––Postoperative
spondylodiscitis
13Open
biopsy
Open
biopsy
Open
biopsy
Open
biopsy
Open
biopsy
Open
biopsy
Bone––Yesh
–Aseptic
spondylitisi
Aseptic
spondylitis
Aseptic
spondylitis
Lymphoma
14 Spondylitis Bone––NoNot done
15Spondylitis Bone–– No–
16 Spondylitis Bone–– Yes (one
dose)
Yes (8 days)
–
17SpondylitisBone–––Lymphoma
18Vertebral
tumour or
infection
Vertebral
tumour or
infection
Vertebral
tumour or
infection
Bone–+Failurej
Yes (one
dose)
Not doneLymphoma
19
Open
biopsy
Epidural
tumour
––Yes (one
dose)
Not doneLymphoma
20Open
biopsy
Epidural
tumour
–– Yes (one
dose)
Not done Plasma cell
granuloma
aThe per cent homology and the length of the overlapping sequence for the best match.
bAn effective antimicrobial treatment against the recognised microbe in the specimen; duration of preoperative antibiotic treatment is given in parentheses.
cThe number of days a positive blood culture result was obtained before the specimen for the PCR assay and culture was taken are given in parentheses.
Unless otherwise indicated, blood culture was taken on the same day as the PCR.
dThe sample was interpreted to contain several species if the electropherogram showed strong signals but multiple overlapping peaks in some locations.
eNegative result.
fThe patient received ofloxacin.
gThe patient received ceftriaxone and metronidazole.
hThe patient received trimethoprim prophylaxis for urinary tract infection.
iAseptic spondylitis was diagnosed as one component of an autoimmune disorder of unknown origin, as described in ref 29.
jSequencing failure was assumed if the sequencing signals reported by the instrument were weak.
nt, nucleotides; PCR, polymerase chain reaction.
730Kupila, Rantakokko-Jalava, Jalava, et al
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of the sequencing results was based on the guidelines given by
Stackebrandt and Goebel.27
Conventional microbiological methods
Samples referred for PCR analysis were at the same time also
sent to the clinical microbiology laboratory of the hospital for
bacterial cultures and Gram staining. For aerobic culture, the
specimens were inoculated on blood agar and chocolate agar
platesandincubatedforaminimumoftwodaysat35°CinCO2
atmosphere. For anaerobic culture, the specimens were inocu-
lated on fastidious anaerobe agar (LabM, Bury, Lancashire,
UK), kanamycin-vancomycin agar, and bacteroides-bile-
esculin agar plates and incubated for a minimum of four days
at 35°C in an MK3 anaerobic cabinet (Don Whitley Scientific,
West Yorkshire, UK). Before cultivation, biopsy specimens
were homogenised in brain-heart infusion broth (Gibco BRL,
Life Technologies, Paisley, Scotland). Plates were examined
daily for bacterial growth. Identification of isolated colonies
was based on routine microbiological methods, including the
VITEK system (bioMerieux, Marcy l’Etoile, France) and
different API test strips (bioMerieux).28Gram staining was
done on the specimen at the request of the attending clinician.
Blood cultures were taken if considered clinically indicated.
For blood cultures,the Bactec 9240 system (Becton Dickinson,
Sparks, Maryland, USA) was used.
RESULTS
Intracranial specimens
The final clinical diagnoses in the 24 patients with intracranial
lesions included brain abscess or subdural empyema in 14,
malignant tumour in seven, and demyelination, non-specific
gliosis, or mucocele in one. Bacterial 23S rDNA PCR was posi-
tive in nine of the 14 pus samples from patients with brain
abscesses or subdural empyemas. Eight of these 14 samples
were positive on bacterial culture. Results of the PCR tests,
DNA sequencing, and bacterial cultures of the intracranial
specimens are given in table 1,as are the data on the quality of
the specimens, the operative technique used, preoperative
antimicrobial treatment, and the final clinical diagnoses. The
specimens from patients 1, 2, 4, 5, 6, and 7 were included in
our previous study.9
From six patients with brain abscesses, bacteria were
detected in the specimens by both the PCR approach and bac-
terial culture. In five of these cases, the causative bacterial
species were identified by both sequencing and culture, at
least to the genus level: three cases were caused by
streptococci (patients 1–3); one case by Peptostreptococcus
species (patient 4); and one case by Propionibacterium acnes
(patient 5). In patient 6, the brain abscess was multibacterial
in aetiology—sequencing indicated the presence of several
bacterial species, and bacterial culture yielded Streptococcus
intermedius, Peptostreptococcus species, and Bacteroides gracilis.
Three patients with intracranial infections had specimens
that were positive by PCR but negative by culture:the first had
a postoperative brain abscess caused by Staphylococcus aureus,
based on the sequencing result (patient 7); the second had
post-traumatic brain abscesses, the specimen from which
yielded Mycoplasma hominis by sequencing (patient 8); in the
third case (patient 9) sequencing was not successful. In the
latter, the disease was classified as probably bacterial because
of the clinical features.The patient made an uneventful recov-
ery with antimicrobial treatment.
Three patients with brain lesions had PCR negative
samples, which were positive on culture. In patient 10, who
had undergone neurosurgery for a meningioma, Staphylococcus
aureus was identified from postoperative subdural empyema
only by culture.In patient 11,who was admitted with 15 brain
abscesses, bacterial culture was the only method to reveal
Fusobacterium species as the causative agent of the disease.The
patient was an alcoholic with dental caries and poor oral
hygiene, which was considered to be the source of the
infection. In patient 21, Propionibacterium species was grown
from the third PCR negative and culture positive cerebral tis-
sue specimen;in this patient,microbiological tests were taken
as the preoperative MRI finding was suggestive of either
tumour or brain abscess. On the basis of the clinical presenta-
tion and the histological finding of an astrocytoma in the tis-
sue specimen,the culture result was designated as contamina-
tion, and the patient was not given any antimicrobial
treatment.
In three patients with brain abscesses the specimens were
negative by both PCR and culture. In patient 12, Aspergillus
species was identified as the cause of the brain abscess at
necropsy. In patient 13, Gram staining of abscess material
revealed Gram positive cocci, suggesting a bacterial aetiology.
In patient 14, the disease was, on clinical grounds, most prob-
ably bacterial in origin.
PCR and bacterial cultures from brain tissue specimens
were negative in seven patients with non-infectious brain dis-
ease. In addition, two patients had PCR negative intracranial
samples which were not referred for bacterial culture because
of the high perioperative likelihood of non-infectious brain
disease.
Antimicrobial treatment and bacteriological findings
The specimens from the above intracranial bacterial infections
(definite or probable) were taken during antimicrobial
treatment in eight patients. The mean duration of the preop-
erative treatment was 5.3 days (range 12 hours to 11 days).
The causative bacteria were identified by both the PCR
approachandcultureinthreecases,byPCRaloneintwocases,
by culture alone in one case, and by neither method in two
cases.
Spinal specimens
The final clinical diagnoses of the 20 patients with spinal
lesions included spondylitis or epidural abscess in 12, aseptic
spondylitis in three, lymphoma in four, and plasma cell
granuloma in one. Among these samples, seven were positive
by PCR and three by bacterial culture.Results of the PCR tests,
DNA sequencing, and bacterial cultures of the spinal
specimens are given in table 2,as are the data on the quality of
the specimens and the operative technique used, preoperative
antimicrobial treatment, blood cultures, and final clinical
diagnoses of the respective patients. The specimens from
patients 1, 2, and 8 were included in our previous study.9
For patient 1 with spondylitis and epidural abscess,a spinal
pus sample obtained during laminectomy yielded Staphylococ-
cus aureus by culture and Staphylococcus species (probably
several species) by sequencing. Eight days earlier, Staphylococ-
cus aureus had been grown from the blood cultures of this
patient, further confirming that this organism was the cause
of her disease.
For four patients with spondylitis or epidural abscess,
causative bacteria were identified in pus samples in two cases
and in vertebral bone in two cases by the PCR approach,while
bacterial cultures remained negative. Staphylococcus species
and Staphylococcus aureus were identified by sequencing in the
samples of patients 2 and 3, respectively. These findings were
in agreement with the growth of Staphylococcus aureus from the
blood cultures of both patients two weeks earlier. Streptococcus
intermedius was identified by sequencing in the pus specimen
taken during reoperation from patient 4 with postoperative
spondylodiscitis; and Streptococcus species, in the specimen
from patient 5 with spondylitis.
The PCR assay was also positive in the pus aspirate from
patient 6, and sequencing of the PCR product yielded
Pseudomonas species. In this patient, spondylitis and spinal
abscess developed as a complication of infective endocarditis.
As Streptococcus mitis growing in the blood cultures of the
Diagnosis of brain abscesses and spinal infections 731
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patient three days before neurosurgery was the causative
agent of the endocarditis, Pseudomonas species recognised by
sequencing was designated as contamination.
Sequencing was not successful for the last PCR positive and
culture negative bone specimen (patient 18). Because of the
final diagnosis of lymphoma and the absence of any clinical
symptoms of infection, the positive PCR finding was
designated as contamination in this patient.
For two patients with spondylitis (patients 7 and 8), causa-
tive bacteria were identified from pus/bone specimens by cul-
ture while the PCR assays remained negative. One of these
specimens yielded Staphylococcus aureus, and the other Pseu-
domonas aeruginosa. The same bacterial species were grown
from the blood cultures taken simultaneously from the
respective patients, confirming the aetiological role of these
pathogens in their diseases.
The tissue samples from 11 patients were negative on both
PCR and culture. The final diagnosis was spondylitis/
spondylodiscitis of probable bacterial origin in four of these
patients (9–12). In three patients (13–15), the disease was
classified as aseptic spondylitis, as one component of an auto-
immune disorder of unknown origin.29In the five remaining
patients (16–20),spinal tumours were diagnosed by histology.
Antimicrobial treatment and bacteriological findings
The specimens from the above spinal bacterial (either definite
or probable) infections were taken during antimicrobial treat-
ment in eight patients. The mean duration of the preoperative
treatment was 8.5 days (range two to 15 days). The causative
bacteria were identified from pus or tissue specimens by the
PCR approach alone in four cases, by both PCR and culture in
one case, and by neither method in three cases.
DISCUSSION
This study was undertaken to evaluate the usefulness of the
broad range bacterial PCR and sequencing in the aetiological
diagnosis of infection in patients with clinically suspected
intracranial or spinal infection. We are not aware of any previ-
ous reports focusing on the systematic use of the PCR and
sequencing method to identify the aetiology of bacterial infec-
tion in neurosurgical patients. The results presented here are
in accordance with earlier reports on patients with other
infections showing that the broad range bacterial rDNA PCR
approach may be the only method to yield an aetiological
diagnosis in specific situations—that is, when the specimen is
taken during antimicrobial treatment or when the causative
agent is fastidious.9 10 18 30In fact, all our samples with which
the broad range bacterial PCR approach was the only success-
ful investigation were taken while the patient was receiving
antimicrobial treatment.Moreover, Mycoplasma hominis identi-
fied in one of these specimens is considered a fastidious
microbe.
Mycoplasma hominis is a rare pathogen which normally colo-
nises the genitourinary tract.31It is not as fastidious as other
mycoplasmas, and various
organism.32The few previously described cases of brain
abscesses caused by Mycoplasma hominis in adults were
diagnosed by bacterial culture.33–35This is the first time that the
organism has been identified from a brain abscess by a culture
independent method. In our patient, preoperative treatment
with clindamycin,which has recognised efficacy against Myco-
plasma hominis,36might also have contributed to the culture
negativity of the abscess material.Although clindamycin pen-
etrates poorly through the blood–brain barrier in healthy
individuals,36the situation may have been different in this
patient, who had a post-traumatic brain abscess following
brain contusion,leading to more effective penetration through
a disturbed blood–brain barrier.
One further specimen from a brain abscess revealing
Staphylococcus aureus was identified by the PCR method alone.
mediahaveyieldedthis
In five additional patients with brain abscesses, the aetiologi-
cal agent was identified by both the PCR approach and bacte-
rial culture. Except for the patients described here, data on
identification of the causative bacteria by 16S rDNA PCR and
sequencing directly from tissue or pus specimens from brain
abscess have been reported in anecdotal cases only.16 17Also,
16S rRNA sequencing has been applied in pure cultures from
brain abscesses to identify bacteria that may be difficult to
classify by their phenotypic properties. Such cases include
identification of Nocardia otitidiscaviarum from a brain abscess
in a renal transplantation patient37and Abiotrophia adiacens
from a brain abscess which had developed in a patient after
neurosurgery.38
Among our patients with bacterial spondylitis or epidural
abscess, the causative agent was identified in pus or vertebral
bone specimens by the PCR and sequencing method alone in
four patients, two of whom also had positive blood cultures.
PCR was the only method to identify the aetiological agent in
the two remaining cases from whom blood cultures were not
taken. It is notable that in patients with spondylitis, the PCR
method could identify the microbe in the specimen even after
a long course of parenteral antimicrobial treatment—among
the five patients whose specimens were successfully analysed
by the PCR approach, one had received parenteral anti-
microbial treatment for as long as 15 days, two for at least 12
days, and one for eight days. On the other hand, five of the six
specimens taken during antimicrobial treatment from spinal
lesions in patients with bacterial spondylitis were negative by
culture, possibly indicating good penetration of antimicrobial
agents into the spine. In our patients, blood culture proved
useful as a tool to reveal the aetiology of spondylitis. In all six
patients with aetiologically identified spinal infection in
whom blood cultures were done, the cultures yielded
corresponding pathogens.
A disadvantage associated with the application of the PCR
methodology in the clinical setting is the potential for false
positive results owing to cross contamination of target DNA
between samples, or contamination of reagents and speci-
mens with PCR amplicons. The use of the broad range bacte-
rial PCR approach involves an additional risk of introducing
bacterial DNA from the vessels and reagents used in various
phases of sample processing and amplification. Thus rigorous
measures were taken throughout the study period to avoid
amplicon or sample to sample contamination and to reduce
theamountof“background”
reaction.20–22Even so, false positive amplification results may
occur despite the most stringent precautions. In the present
study, two positive PCR assays were considered to have been
the result of contamination.
bacterial DNA inthe
Conclusions
We found the broad range bacterial rDNA PCR approach to be
useful in the aetiological diagnosis of infection in patients
with brain abscesses or spinal infections.The results presented
here show that in neurosurgical patients the PCR approach
may be the only method to provide the aetiological diagnosis
when the causative agent is fastidious, or when the patient is
receiving antimicrobial treatment. In patients with bacterial
spondylitis, this method may be successful even after > 12
days of parenteral antimicrobial treatment.
ACKNOWLEDGEMENTS
We thank Mrs Tiina Haarala,Tarja Laine,Merja Mikkola,Anne Peippo,
and Kirsi Sundholm for their excellent technical work.
.....................
Authors’ affiliations
L Kupila, R J Marttila, Department of Neurology, Turku University
Central Hospital, Turku, Finland
K Rantakokko-Jalava, Department of Medical Microbiology, Turku
University
732Kupila, Rantakokko-Jalava, Jalava, et al
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Page 6

J Jalava, Antimicrobial Research Laboratory, National Public Health
Institute, Turku
S Nikkari, O Meurman, Clinical Microbiology, Turku University Central
Hospital
P Kotilainen, R Peltonen, Department of Medicine, Turku University
Central Hospital
E Kotilainen, Department of Surgery, Division of Neurosurgery, Turku
University Central Hospital
Competing interests: none declared
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