Bali Medical Journal (Bali Med J) 2016, Volume 4, Number 1: 44-47
P-ISSN.2089-1180, E-ISSN.2302-2914

The Role of Multiplex Polymerase Chain Reaction in Detecting Etiological Causes of Bacterial
Prostatitis Associated Benign Prostatic Hyperplasia
Bramastha A Rosadi1, Tjokorda GB Mahadewa2, Gede Wirya K Duarsa3
Department of Surgery Udayana University, Sanglah General Hospital Denpasar Bali
2
Department of Neuro Surgery Udayana University, Sanglah General Hospital Denpasar Bali
3
Department of Urology Udayana University, Sanglah General Hospital Denpasar Bali
Correspondence: Tel (+62) 361227 911. E-mail: rosadie.bramastha@gmail.com
1

Background: Benign Prostatic Hyperplasia (BPH) has been correlated with chronic prostatitis
according recent study. Chronic pelvic pain is the chief complain of BPH followed by prostatitis. The
gold standard of the etiological diagnosis is urine culture, but the negativity rate is still high. Multiplex
polymerase chain reaction (PCR) as a diagnostic tool in search of etiological causes could identify
microorganism on DNA level. This research aims to find out the role of multiplex polymerase chain
reaction as diagnostic tools on prostatitis patients. Material and Method: A total of 12 samples
collected during the TURP procedure in Sanglah General Hospital Denpasar – Bali from February until
May 2015. All of the samples has been diagnosed prostatitis clinically and perform urine culture test.
The prostate specimen taken was sent to the Pathological anatomy for histopathology diagnostic and
underwent multiplex PCR for etiologic diagnostic. Result: 12 samples have been declared as prostatitis
based on histopathology examination, and then were analyzed using multiplex PCR. 10 samples were
positive (6 were E. coli, 2 were C. trachomatis, the rest were N. gonorrhea and P. aeruginosa). The
urine culture revealed 9 positive, within the result 6 were E. coli, and the others were P. aeruginosa, M.
morganii and A. haemolyticus. Conclusion: In prostatitis patient, the etiological diagnostic was
important. Multiplex PCR as diagnostic tools could detect the microorganism on a negative urine
culture. The combination of the urine culture test and multiplex PCR revealed a better result on etiologic
diagnosis which leads to a better management of the disease.
Keywords: Prostatitis, Urine Culture, Multiplex Polymerase Chain Reaction
DOI: 10.15562/bmj.v4i1.188
Cite This Article: Rosadi B, Mahadewa T, Duarsa GWK. 2015. The Role of Multiplex Polymerase
Chain Reaction in Detecting Etiological Causes of Bacterial Prostatitis Associated Benign Prostatic
Hyperplasia. Bali Medical Journal 4 (1), 44-47. DOI: 10.15562/bmj.v4i1.188
INTRODUCTION
Benign Prostatic Hyperplasia (BPH) regard
as the most common of the degenerative diseases on
male and followed by lower urinary tract symptoms.
The symptoms although not life threatening but
troublesome, this symptom significantly affect the
quality of life of the patients. Histologically on
group of 41 – 50 years old patient, 20% has been
diagnosed as BPH, at the age of 51 – 60 years old
the prevalence was higher up until 50% and the
highest incidence on the age of 80’s within 90%. In
the world, the incidence in between 0,5 – 1,5 /
100.000 but the mortality is very low1.
The risk factor of BPH were unclear, newest
research leads to inflammatory diseases, genetic
predisposition and races. Approximately 50 % male
under the age of 50’s with BPH has a congenital
factor with the probability of autosomal dominance,
and the risk factor are 4 times fold1,2. The AUA

guidelines state that 90% of male on BPH within age
45 – 80 years old has lower urinary tract symptoms3.
These symptoms were the chief complaint of the
patients and commonly followed by chronic
prostatitis. 45 – 98 % prostate specimen post
operative were histopathology diagnosed as chronic
prostatitis 4.
The prostatitis was combination of infection,
chronic
pelvic
pain,
and
asymptomatic
inflammation.
Prostatitis
commonly
under
diagnosed, and accidentally revealed on post
operative histopathology examination 5. Prevalence
prostatitis in the world were vary, from 2% until 13
%5,6,7,8,9, In USA, approximately 5 – 12 million
peoples were diagnosed as prostatitis10. The cost to
treat prostatitis were abundant, there were 8.021.396
prostatitis cases and costing USD 3017 – 6534 per
patient with the sum of USD 84 million6,10,11. The
etiological microorganism of prostatitis was E. Coli,

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Bali Medical Journal (Bali Med J) 2016, Volume 4, Number 1: 44-47
P-ISSN.2089-1180, E-ISSN.2302-2914

Pseudomonas Spp., Chlamydia trachomatis,
Neisseria gonorrhea, Ureaplasma urealyiticum and
Mycoplasma genitalium12. There was outcome
controversy of the operative procedure of BPH
patient with prostatitis. Jonas et al state that BPH
patient with prostatitis still had a LUTS after the
surgery9,10, on the contrary Yalcinkaya et al state
that there were no significant differences of the
cases4.
Diagnostic procedure of prostatitis was based
on 4 tube examination and the etiological diagnostic
tools were urine culture5. The urine culture has many
limitation and revealed only P. aeruginosa and E.
coli. If the culture failed to detect the
microorganism, the diagnosis was straight to type III
prostatitis and treated by broad spectrum
antibiotics5. The invention of DNA hybridization
and nucleic acid amplification could detect N.
gonorrhea, the recent discovery of polymerase chain
reaction (PCR) was a bright light on detecting
etiological diagnosis of prostatitis12,13.
PCR as a diagnostic tool was a one step
examination, using one tube sample that
simultaneously detect vary of microorganism
including viral infection. PCR method were more
sensitive and cost effective compared to the others
traditional method13. This research aims to find out
the role of multiplex polymerase chain reaction
(mPCR) as diagnostic tools on prostatitis patients.
MATERIAL AND METHODS
Pure cultures of submitted strains from E.
coli, C. trachomatis, N. gonorrhea, U. urealyiticum,
M. genitalium, Corynebacterium spp. and P.
aeruginosa were maintained on Luria agar slants at
4°C for working purposes, while stock cultures were
stored in 15% glycerol at -80°C. Briefly, 10 ml
aliquots of cells were centrifuged at 12,000×g for 3
min at 4°C and subjected to DNA extraction. The
DNA was then purified and recovered in 200 μl of
elution buffer followed by dilution in 1 ml of
phosphate-buffered saline (PBS) containing 1
mg/ml chenodeoxycholate and stored at -80°C until
further use. Upon thawing, the specimens were
divided into aliquots for assessment and comparison
to cloned markers and a 3.0 kb pGEM-T easy vector
to ensure the DNA had originated from the correct
bacterial species.
A total of 12 consecutive samples from
February to May 2015 that has been diagnosed
prostatitis clinically collected, midstream urine was
taken. The urine specimens were collected into
sterile 20 ml conical tubes on-site and taken to the
microbiology division and plant to blood sheep agar
media for culture. Positive culture regard as
bacterial growth more than 100.000 colony forming
unit.
All 12 samples underwent operative
procedure, during the TURP procedure in Sanglah
General Hospital Denpasar – Bali the prostate

specimen taken from the first resection and sent to
the pathological anatomy for histopathology
diagnostic and underwent multiplex PCR for
etiologic diagnostic. The prostate specimen stores
on Eppendorf tube and sent directly to preservation
at the temperature of -80°C until further use.
The specimen undergo centrifugation at 3,000 rpm
for 20 minutes, the pellets were washed in 5 ml of
sterile water and then centrifuged again at 2,000 rpm
for 10 minutes. The DNA in the pellet was then
extracted by three rounds of resuspension in 30%
polyethylene glycol and 3 ml NaCl, followed by
incubation on ice for 30 min, and centrifugation at
room temperature for 5 min at 15,000 rpm. The final
pellet was frozen at -20°C. To confirm the
pathogenic bacterial infection, an initial 16S rDNA
consensus primer from the causative bacteria in all
specimens was investigated via PCR and automated
sequencing analysis. The amplified PCR products
were electrophoresed on 1.5% agarose gels
containing ethidium bromide in buffer and were
photographed with an Image Master.
RESULT
To establish the PCR conditions which
permit the amplification of sequences from E. Coli,
C. trachomatis, N. gonorrhea, U. urealyiticum, M.
genitalium, Corynebacterium spp. and P.
aeruginosa, the laboratory strains of these
organisms were cultured, followed by extracting
their DNA and performing PCR assays. The optimal
results were obtained at an annealing temperature of
54°C or 56°C. These temperatures allowed for the
amplification of the seven specific bands along with
reduced levels of non-specific amplification
products. These assays revealed that the 54°C
annealing temperature efficiently generated all
products.
The mean age of the sample was 68,8 ± 6,9
years old within a range of 60 until 78 years old. All
of the sample were married and has no history of
diabetes, Tb infection and another systemic
inflammation nor malignancy. The characteristic of
the sample was presented on table 1.
Table 1. Subject Characteristic
Subject Characteristic
n = 12
Age
Mean+ SD
68, 8 + 6,9
Min
60
Max
79
Educational Status
Elementary
6 (50,0)
Junior High
1 (8,3)
High
2 (16,7)
Post Graduate
3 (25,0)
Marital Status
Marriage
12 (100,0)
Occupation
State Government 2 (16,7)

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Bali Medical Journal (Bali Med J) 2016, Volume 4, Number 1: 44-47
P-ISSN.2089-1180, E-ISSN.2302-2914

Farmer
Private

7 (58,3)
3 (25,0)

All of the samples were analyzed using
multiplex PCR that has been establishing using the
previous state methods. 10 samples (83,3%) were
positive (6 were E. coli, 2 were C. trachomatis, the
rest were N. gonorrhea and P. aeruginosa). The
urine culture revealed 9 positive (70%), within the
result 6 were E. coli, and the others were P.
aeruginosa, M. morganii and A. haemolyticus.
There was interesting result from the examination,
we found concordance (agreement) of 58,3% from
the 2 examination and 41,7% disconcordance. Both
of the procedure successfully detect E. coli and P.
aeruginosa. The agreement between 2 examinations
presented on Table 2 and mapping of the result were
presented on Table 3.
Table 2. Agreement of the Procedure
PCR
Urine
Negative Positive
Negative 0 (0,0)
3
Disconcordance
(25,0) 41,7 % and
Concordance
Positive 2 (16,7)
7
(58,3) 58,3 %

Subject
1
2
3
4
5
6
7
8
9
10
11
12

Table 3. Result of Procedure
Urine
PCR
Microorganism
Result
Result
Positive
Positive
Escherichia
coli
Negative Positive
Chlamydia
trachomatis
Positive
Positive
Escherichia
coli
Positive
Positive
Pseudomonas
aeruginosa
Positive
Positive
Escherichia
coli
Positive
Positive
Escherichia
coli
Positive
Positive
Escherichia
coli
Positive
Negative Morganellamo
rganii
Positive
Positive
Escherichia
coli
Negative Positive
Chlamydia
trachomatis
Negative Positive
Neisseria
gonorrhea
Positive
Negative Acinetobacterh
aemolyticus

DISCUSSION
The diagnosis of prostatitis has traditionally
been largely dependent on methods such as culture,
enzyme immunoassay, DNA hybridization,

antibody staining as well as the examination of
rectal and prostatic fluids for the signs of
inflammation and infection5,6,12,13. The previous
methods did not profound as standard diagnostic
studies, mPCR described as being a more sensitive
detector of pathogenic bacteria compared to
traditional tests involved in the detection
process13,14.
This study describes a rapid one step method
for the detection of pathogenic bacteria in prostatitis
associated BPH patients by mPCR. We have tested
the 12 specimens by urine culture and found 58,3%
concordance with the mPCR results. Moreover,
mPCR had higher positivity rate (83,3%) compared
to urine culture (70%). The mPCR supremely detect
the STD microorganism that failed detected by urine
culture. The downside of the mPCR were mainly
based on primary DNA, consequently special
attention must be paid in the primer design13,14.
Moreover the amplification conditions must be
optimized since the reactions can be affected by
several factors, including temperature and duration
of DNA denaturation, primer annealing, extension
as well as the concentrations of the polymerase,
MgCl2 and primers13,15-17. This comes up to the
negative result like Morganella morganii and
Acinetobacter haemolyticus. We don’t have the
primary DNA of both of the microorganism. At
present, the universal features of mPCRs lead to
highly reliable, sensitive and specific multiple target
gene detection is not known. Nevertheless, our
sequencing data showed that our mPCR method
yields
highly
reliable,
pathogen-specific
information
CONCLUSION
In prostatitis associated benign prostate
hyperplasia patient, the etiological diagnostic was
important. Multiplex PCR as diagnostic tools could
detect the microorganism on a negative urine
culture. The combination of the urine culture test
and multiplex PCR revealed a better result on
etiologic diagnosis which leads to a better
management of the disease.
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