Professional Documents
Culture Documents
Comparison of Multiplex PCR, Gram Stain, and Culture For Diagnosis of Acute Bacterial Meningitis
Comparison of Multiplex PCR, Gram Stain, and Culture For Diagnosis of Acute Bacterial Meningitis
Academic Sciences
Original Article
COMPARISON OF MULTIPLEX PCR, GRAM STAIN, AND CULTURE FOR DIAGNOSIS OF ACUTE
BACTERIAL MENINGITIS
MOHAMMAD AMMAR YAHIA, OMAR BALACH
Department of Laboratory Medicine, Section of Microbiology, Faculty of Medicine, Aleppo University, Syria.
Email: dr.ammar82@hotmail.com
Received: 01 May 2014 Revised and Accepted: 30 May 2014
ABSTRACT
Objectives: to compare characteristics of the multiplex PCR and gram stain with bacterial culture as reference method for detection of five
pathogens which multiplex PCR can detect: Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitides, Group B
streptococcus, and Listeria monocytogenes in cerebrospinal fluid (CSF) samples of patients were suspected of acute bacterial meningitis.
Methods: 110 CSF samples were obtained from 110 patients were suspected of acute bacterial meningitis as defined by WHO. Gram stain, bacterial
culture, and multiplex PCR tests were done for all samples.
Results: Gram stain for any bacteria was positive in 32 cases (29.1%), including the five pathogens in 11 cases (10%). Bacterial culture was positive
in 38 cases (34.5%), including the five pathogens in 8 cases (7.2%). Multiplex PCR was positive in 60 cases (54.5%) and the most bacteria detected
was Streptococcus pneumoniae in39/60 cases(65%), followed by Neisseria meningitides in 8/60 cases (13.3%). The sensitivity of multiplex PCR
was 100%. 50 cases of acute bacterial meningitis were diagnosed by multiplex PCR, while both gram stain and bacterial culture were negative in it.
Conclusions: The PCR method is a rapid, sensitive, and specific diagnostic test for acute bacterial meningitis. PCR is particularly useful for analyzing
CSF of patients who have been treated with antibiotics before lumbar puncture.
Keywords: Comparison, Bacterial meningitis, Multiplex PCR, Five pathogens.
INTRODUCTION
Bacterial meningitis is a medical emergency, and immediate steps
must be taken to establish the specific cause and initiate effective
therapy. The mortality rate of untreated disease approaches 100
percent and, even with optimal therapy, there is a high failure rate
[1,2].
To the best of our knowledge, this research is done for the first time
in Syrian Arab Republic.
MATERIALS AND METHODS
Yahia MA et al.
Multiplex PCR: A CSF samples (minimum 0.5 mL) were kept at 20C
until transportation to the Research Laboratory-Faculty of Medicine
(Aleppo University, Aleppo, Syria) for PCR analysis.
DNA extraction: CSF specimens were centrifuged at 15,000 rpm for
5 min. DNA was extracted from the cell pellet by using a nucleic acid
purification column according to the manufacturers instructions
(Qiagen Inc., Valencia, CA, USA). The DNA extraction was stored at 20 C for DNA amplification.
DNA amplification: We used Seeplex Meningitis ACE Detection kit
(Seeplex Meningitis-B, Korea) which detects five pathogens:
Streptococcus pneumoniae (SP), Haemophilus influenzae type b
(Hib), Neisseria meningitides (NM), Group B streptococcus (GBS),
and Listeria monocytogenes (LM). [Table 1]
Seeplex
Meningitis-B
Organism
Internal control
SP
Hib
NM
GBS
LM
Target gene
Target protein
GyrB
P6
ctrA
Cfb
Hly
Temperature
94
94
63
72
72
The amplified PCR products were electrophoresed in 2% (w/v) agarose gels and were stained with ethidium bromide.
Duration
15 min
0.5 min
1.5 min
1.5 min
10 min
426
Yahia MA et al.
All statistical analysis was performed with SPSS version 11.5 (SPSS
Inc, Chicago, IL, USA) [25].
RESULTS
A total of 110 CSF samples were collected from 110 patients who
were diagnosed with acute bacterial meningitis, based on clinical
symptoms and CSF ndings as described above.
The demographic and laboratory characteristics of patients were
included in this study are shown in table 3.
Gram stain for the five pathogens, which multiplex PCR can detect,
was positive in 11/110 cases (10%), of which 10 (90.9%) was S.
pneumoniae and 1 (9.1%) was N. meningitidis.
Male (%)
Mean CSF leukocyte count, cells/mm3 (range)
Neutrophils (%)
Mean CSF Glucose, mg/dl (range)
Mean CSF Protein, mg/dl (range)
Receipt of antibiotics before lumbar puncture
(%)
Patients (n =110)
4.2 (3 days-52
years)
60.9
2500 (25-25000)
62.7
30.9 (4-239)
234.6 (26-492)
65.5
Bacterial culture for the five pathogens, which multiplex PCR can
detect, was positive in 8/110 cases (7.2%) and all these cases were
S. pneumoniae.
Multiplex PCR test was positive in 60/110 cases (54.5%). Among the
multiplex PCR positive cases, S. pneumoniae was detected at the
highest incidence of 39 cases (65%), followed by N. meningitidis in 8
cases (13.3%), Listeria monocytogenes in 7 cases (11.7%), Group B
streptococcus in 4 cases (6.7%), and Hib in 2 cases (3.3%) [table 4].
Multiplex PCR was negative in all 30 CSF specimens that with culture
isolation of bacteria other than S. pneumoniae, N. meningitidis,
Listeria monocytogenes, Group B streptococcus, or Hib.
Table 4: Shows the results of bacterial culture, gram stain and multiplex PCR tests
laboratory test
Gram stain
for any bacteria
SP
NM
Gram negative bacilli
Gram positive cocci
Bacterial culture
for any bacteria
SP
Pseudomonas aeruginosa
Acinetobacter spp.
E. coli
Enterobacter aerogenes
Citrobacter freundii
Serratia marcescens
staphylococcus spp.
Burkholderia cepacia
Multiplex PCR
Any positive
SP
NM
Hib
LM
GBS
(a) *one patient showed simultaneous infection with 2 bacterial (Acinetobacter spp. and Citrobacterfreundii) isolated from two ventricular shunts,
right and left.
The results of the three tests (gram stain, bacterial culture, and multiplex PCR) for the five pathogens, which multiplex PCR can detect, are
summarized in the table 5.
Table 5: shows the results of the three tests (gram stain, bacterial culture, and multiplex PCR) for the five bacteria studied by multiplex PCR
Bacterial culture
+
+
+
+
-
Gram stain
+
+
+
+
-
multiplex PCR
+
+
+
+
-
N= 110
8
1
0
50
0
2
0
49
427
Yahia MA et al.
All other culture and gram stain results, including the absence of
bacteria or the presence of other bacterial species (in the absence of
S. pneumoniae, N. meningitidis, Listeria monocytogenes, Group B
streptococcus, and H. influenzae), were considered a negative test
result.
For the evaluation of performance characteristics of the gram stain
and multiplex PCR, results were compared with CSF cultures as
current gold standard [table 6].
Table 6: Shows performance characteristics of the gram stain and multiplex PCR
Gram stain
Multiplex PCR
Positive
Negative
Positive
Negative
Bacterial culture
Positive (n = 8)
8
0
8
0
Negative (n = 102)
3
99
52
50
Total
(n = 110)
11
99
60
50
However, this specicity of the multiplex PCR does not reect the
true percentage, because in many cases with a negative bacterial
culture an antibiotic had been prescribed before the bacterial
cultivation of the CSF. [Table 7]
Table 7: Shows performance characteristics of multiplex PCR among untreated patient before lumber puncture.
Multiplex PCR
Total
Positive
Negative
In the present study, there were few Hib influenza cases (3.3%) were
detected by multiplex PCR; this can be due to routine Hib vaccination for
Bacterial culture
Positive
6
0
6
negative
2
30
32
Total
8
30
38
In Japan, Chiba et al. [8] found that H. inuenzae was detected at the
highest incidence (45.2%), followed by S. pneumoniae (21.4%).
Yahia MA et al.
In this study, the gram stain was positive in 29.1%, which is higher
than that was found by Saravolatz et al. [15] (14.9%), and
Schuurman et al [19] (9.3%), but is lower than that was detected by
Favaro et al. [30] (75%).
Sensitivity and specificity of gram stain are 100%, 97.1%
respectively,which are higher than that for multiplex PCR (100%,
49% respectively), although gram stain was positive only in 1 case,
while multiplex PCR was positive in 50 cases.
However, the above information denotes that the gram stain and
bacterial culture cannot be a considered as a method for early and
exact diagnosis of meningitis
In conclusion, The PCR has high sensitivity and specificity for the
detection of bacterial pathogens such as S. pneumoniae in the CSF.
Further refinements in this technique may make it useful for the
diagnosis of bacterial meningitis, especially when results of CSF
gram stain and bacterial culture are negative and when patients had
received antibiotics before the lumbar puncture was done.
429