Viral implications in benign prostatic hypertrophy

…………, Mihaela Surcel2, Adriana Munteanu2, R. Huică2, C. Ursaciuc2

1. …………………..
2. Imunopathology laboratory, National Institute “Victor Babeș”, Bucharest, Romania

Abstract

Benign prostatic hyperplasia (BPH) can be determined by cell proliferation caused by persistent
local inflammatory processes. Viral infection associated with immune deficiencies constitutes a
possible factor in maintenance of chronic inflammation of the prostate.
To determine the possibility of involving the viral infection in the determinism of the processes
leading to hypertrophy of the prostate, a group of 50 patients with BPH without urinary tract
infection were tested for the presence of human papilloma virus (HPV), cytomegalovirus (CMV)
and Epstein - Barr virus (EBV). These viruses are the most common cause of inapparent viral
infections.

Specific DNA for HPV in prostate tissue was only found in 4% of patients, while 98% and 100%
patients were positive for serum anti-CMV IgG or anti-EBV, proving contact with viruses in the
priors. IgM anti-CMV were uncertain in 10% of the cases and weren’t positive for EBV, which
contradicted the acute infection.
The results showed that BPH may be associated with chronic inflammation post-viral infection
with CMV or EBV, or a result of the presence of these viruses in the prostate, while the
involvement of HPV in BPH has a low probability. The data suggest that viral investigation is a
possibility of enlarging screening in inflammatory or tumoral diseases of urinary tract.
Keywords: benign prostatic hypertrophy, human papilloma virus, cytomegalovirus, Epstein-Barr
virus.

INTRODUCTION
Benign prostatic hyperplasia (BPH) is the most common urological disease encountered in men
in second and third age. Although it is a public health problem, up to now it has not yet been
found a direct relationship between BPH and a certain causative agent, often the etiology of
hyperplasic process not being unique [1]. The main assumption of the appearance of
hypertrophic response of the prostate is enzyme mediated cell proliferation, generated through
steroid hormones and inflammatory response to local infectious factors [1, 2].
Persistent inflammation of the prostate may be due to viral infections occurred on the account of
immune disorders. Grafting of the infection in the prostate may be favored by some weaknesses
of antiviral immune response developed by cytotoxic T lymphocytes (T-CD8+) or antiviral
antibodies secreted by B lymphocytes [3]. Development of BPH as a result of a chain of events
that start from impaired immune response becomes a condition that can be associated with a viral
infection that can sometimes be asymptomatic. Given the frequency of inapparent viral
infections with human papilloma viruses (HPV), cytomegalovirus (CMV) and Epstein-Barr
(EBV) and also some literature data on their relation to prostate pathology [4,5,6], we believe it
is important a study on the presence of these infections in patients with BPH.
HPV infection has been incriminated in clinical history of 75% of men and women of
childbearing age and has been detected in malignant prostatic diseases [7, 8], rising suspicion of
involvement of this virus in the genesis of prostatic adenocarcinoma. The presence of HPV in
BPH has not been definitely proven, most of the information currently known come from
indirect analysis of the patients in witness group in prostatic adenocarcinoma studies [9, 10, 11,
12, and 13]. A large number of reports and articles from the literature raise the hypothesis of the
presence of HPV in the neoplasia of the esophagus, bladder, lung, and breast and also in prostate
diseases [14]. CMV infection was found in 50-85% of young adults [15, 16], but, in contrast to
HPV, the involvement of CMV in the benign or malignant prostate disease is controversial, the
data in literature being antagonistic [17]. Regarding EBV, few studies have researched its
presence in the prostate gland [18, 19].
This paper investigates the presence of HPV, CMV and EBV viruses in a group of patients with
BPH. We believe that such an investigation concerning viral participation in BPH can provide
some answers with therapeutic implications.
MATERIALS AND METHODS

1. Patients
 The study included a group of 50 patients with clinically and laboratory proven BPH,
admitted in 2013-2014 in the Urology Clinic of the Dr. Carol Davila Central Military
Emergency University Hospital. All patients underwent surgery: Transurethral resection
(TUR-P) - 37 patients, transvesical adenomectomy - 13 patients.
Patients with urinary tract infection and those who have received treatment with the 5-
alpha reductase inhibitor previously were excluded from the study to minimize the impact on
the appearance of prostate inflammation.
All patients signed an informed consent before study entry. In order to conduct the
study the consent of the local ethics committee of the hospital was obtained.

2. Detection of HPV
Fresh prostatic tissue fragments were collected from the patients from each prostatic
lobe. The fragments were frozen at -200 C until use.
Isolation and purification of genomic DNA (gDNA) of the prostate tissue samples was
performed with the kit "QIAmp DNA Mini Kit" (Qiagen, Hilden, Germany) according to the
manufacturer's instructions. The method involved proteolysis followed by adsorption on the
micro-formed membrane of silicate. Contaminants were removed by washing and centrifugation,
and elution of gDNA from the membrane was carried out using a saline buffer to finally obtain
200 µl gDNA.
The analysis of samples resulting from the extraction procedure was performed with the
spectrophotometers "Biospec-nano" (Shimadzu Biotech, Kyoto, Japan) and "Epoch-
multivolume" (Biotek, Winooski, VT, USA), determining the amount of DNA (ng / µl) and its
quality (ratio OD260 / OD280). A value of about twice the ratio OD260 / OD280 indicates a high
purity DNA.
Determination of the presence of the HPV consisted in HPV-specific DNA (HPV-DNA)
detection by polymerase chain reaction (PCR). The kit "HPV High Risk Screen Real-TM Quant"
(Sacace Biotechnologies, Como, Italy) was used, which is an amplification test in real time for
the detection of both the high risk HPV (types 16, 18, 31, 45) and the low-risk HPV (type 33, 35,
39, 51, 52, 56, 58, 59). Thus, the most common oncogenic HPV genotypes are detected. Each set
of samples contained 10 µl gDNA, 3 standards with known concentrations of HPV-DNA and a
negative control (NTC - No Template Control). Mix reaction tubes were processed in the device
 "RotorGene 6000" (Corbett Life Sciences, Qiagen, Hilden, Germany) according to the
manufacturer's program amplification kit (Table 1).

Stage Temperature (0C) Time No. cycle

1 95 15 min 1
95 5 sec
2 60 20 sec 5
72 15 sec
3 95 5 sec 40
Table 1 - The polymerization reaction amplification program

Signals obtained for NTC 3 standard and 4-channel fluorescence probes were analyzed:
FAM (green), JOE (Yellow) Rox (orange), Cy5 (red).
The results were automatically calculated using the "High Risk HPV Screen 4x Quant",
supplied by the manufacturer along with the kit. Test results are considered invalid in the absence
of a fluorescent signal, or negative if fluorescent signal is present only in FAM channel. The
results are considered positive for HPV types 16, 31, 33, 35, 52, 58 if there is a positive signal
(Ct ≤ 33) in the JOE channel; HPV type 18, 39, 45, 59 if there Rox positive signal channel; HPV
type 51, 56 if there is a positive signal in the Cy5 channel.
HPV-DNA load is calculated using the following formula:

HPV DNA copies per reaction

× 200000=log ⁡( HPV DNA ∈100000 cells )
genomic DNA copies per reaction
log ⁡¿

3. Detection of CMV
There were anti-CMV IgG and IgM antibodies detected in the patients' serum with the
Enzyme-Linked Immunosorbent Assay technique (Novalis Cytomegalvirus IgG / IgM ELISA
immunodiagnostic Novatec GMBH, Dietzembach, Germany) according to the manufacturer's
instructions. Samples of serum were added to a plastic 96-well plate coated with CMV antigen.
Each kit contains 3 controls (positive, negative, cut-off). After washing the wells, peroxidase-
coupled human anti-IgG / IgM antibodies were added. The formed complex was processed by
adding, after further washing, tetra-methyl-benzidine (TMB), generating a color reaction in
 proportion to the antibody level. The reaction was stopped with a solution of sulfuric acid and the
absorbance at 450/620 nm was read with the plate reader "Sunrise" ((Tecan, Männedorf,
Switzerland).
Cutoff value was determined by mediating the two absorbance obtained (cut off control
was assayed in duplicate). Samples are considered positive in an absorbance value greater than
the cut-off by over 10%. The samples are considered negative in an absorbance value less than
the cut-off value of more than 10%. Samples are considered "gray area" in an absorbance value
higher or lower than the cut-off less than 10%.
The results were expressed in Novatec units (NTU) according to the formula:

4. Detection of EBV
There were anti-EBV IgG and IgM antibodies detected in the patients' serum with the
Enzyme-Linked Immunosorbent Assay technique (Novalis Epstein-Barr virus IgG / IgM ELISA
immunodiagnostic Novatec GMBH, Dietzembach, Germany) according to the manufacturer's
instructions. r. Samples of serum were added to a plastic 96-well plate coated with EBV antigen.
After washing the wells, peroxidase-coupled human anti-IgG / IgM antibodies were added. The
formed complex was processed by adding, after further washing, tetra-methyl-benzidine (TMB),
generating a color reaction in proportion to the antibody level. The reaction was stopped with a
solution of sulfuric acid and the absorbance was read with the plate reader "Sunrise" ((Tecan,
Männedorf, Switzerland).
Cutoff value was determined by mediating the two absorbance obtained (cut off
control was assayed in duplicate). Samples are considered positive in an absorbance value
greater than the cut-off by over 10%. The samples are considered negative in an absorbance
value less than the cut-off value of more than 10%. Samples are considered "gray area" in an
absorbance value higher or lower than the cut-off less than 10%.
The results were expressed in Novatec units (NTU).

RESULTS
1. Determination of HPV DNA viral load
Table 2 presents the distribution of fluorescent signals (number of signals) on the 4 pursued
channels. NTC has not issued a positive fluorescent signal on the 4 channels, and the 3 standards
have shown positive signals depending on the concentration on all channels. Just one sample
(case 21, Fig.1) has issued a positive fluorescent signal (Ct ≤ 33) on Cy5 channel, which
corresponds to the presence in the prostatic tissue of HPV type 51 or 56. Another sample (case
48) which showed a weak positive result (Ct = 38.21), is also included in statistics.

Table 2 - Distribution of fluorescent signals on 4 channels

Fluorescent signals
Channel
NTC Standards Samples
FAM 0 3 0
JOE 0 3 0
Rox 0 3 0
Cy5 0 3 2

Given that this type of kit also evaluates the risk of cervical cancer induced by HPV, the result
has been inserted by the program in the non-significant risk group (Fig.1). Overall, the certain
presence of HPV in prostate occurred in 4% of the studied cases (Fig.2)
 Figure 1 - Automatic evaluation of the risk of cancer induced by HPV in 21st sample found
positive for HPV-DNA
2. Determination of anti-CMV antibodies
The results of determination of the presence of IgG and IgM anti-CMV antibodies is shown in
Table 3 and Figure 2. Of the 50 investigated patients, none revealed the presence of IgM anti-
CMV antibodies, while 49 (98%) patients showed positive for IgG anti-CMV antibodies. A total
of 5 cases showed an uncertain outcome (gray area) for IgM anti-CMV, which may suggest a
possible acute infection.
 Determination No. Positive results No. Uncertain results

IgM anti-CMV 0 5

IgG anti-CMV 49 1

Table 2 - CMV detection results from 50 patients with BPH

3. Determination of anti-EBV antibodies
The results of determination of the presence of EBV antibodies IgM and IgG-type is shown in
Table 4 and Figure 2. Of the 50 investigated patients, none revealed the presence of anti-EBV
IgM antibodies, whereas all patients showed positive for anti-EBV IgG antibodies. No patient
had an uncertain outcome (gray area) for IgM or IgG anti-EBV.

Determination No. Positive results No. Uncertain Results

IgM anti-EBV 0 0

IgG anti-EBV 50 0

Table 4 - EBV detection results from 50 patients with BPH

98 100
100

80

60

%
40

20

2
HPV CMV-IgG EBV-IgG
0

Figure 3 - Distribution of viral infections in tested case studies

Discussions
This paper aims to investigate the presence of viral infection in BPH to determine the possibility
of associating this type of infection in determinism of processes leading to prostate hypertrophy.
Thus, a group of 50 patients with BPH were tested for the presence of three types of viruses:
HPV, CMV, EBV. The choice was based both on literature studies concerning the presence of
infections in the adult population and also on the importance of the impact these viruses may
have on the immune system and on the inflammatory elements in the prostate. Moreover, this
study was combined with the determination of immunological features of this group of patients
[3] in order to establish possible correlations.
HPV virus usually causes inapparent infections, but the possibility of damaging some organs
must be taken into consideration. A number of inflammatory syndromes occurred without a
certain known determinism can be the result of infections with these viruses. The existence of
female genital cancer risk after inapparent HPV infection is a proof of this, bringing arguments
for an investigation in male patients. Since the inapparent HPV infection is frequent in males
(men being the virus reservoir that generates carcinogenic infections in women), intra-prostatic
search for the presence of HPV becomes an element of interest.
During the conducted investigation, the presence of HPV DNA was detected in two of the 50
investigated cases (4%). The investigation kit is intended to establish the degree of risk of
cervical cancer in women, the results obtained in men cannot be assigned to a certain degree of
risk. Considering that the use of this kit for analyzing the presence of HPV-DNA was performed
for the first time in our country for men and targeted a particular tissue, the results should be
interpreted only as intra-prostatic viral presence, without being able to determine whether such
presence gives or not a certain degree of risk of developing prostate cancer. The literature
provides conflicting information on a definite link between HPV infection and prostate cancer
[20,21].
The fact that it was possible to establish that there are cases of BPH with the presence of HPV
can be a starting point for introducing the investigation for the detection of HPV in the male
genital secretions or excised tissue after surgery of the genitals of the man. Both cases that were
positive for infection addressed to the RED channel, more precisely 51, 56 strains, that are
recently employed in high risk category. A slip from our point of view is the kit’s addressing to
the high risk strains only. So far we have not found in literature any association between these
virus strains and prostate adenoma.
We believe it is too early to establish a link between the presence of HPV and the development of
BPH, but we can do a number of correlations regarding the immune response of patients (data
not shown). The HPV-positive patient showed decrease of cytotoxic T-lymphocytes (T-CD8 +).
This is a general tendency of investigated patients [3], the CD8 + T-lymphocyte involvement in
cellular antiviral immune response suggesting a possible risk of infection in these patients.
Contact with the HPV does not necessarily mean that the virus entered into the prostate, the viral
infection being able to be maintained in genital secretions only. The existence of the virus in
prostate is proof that a possible HPV infection in the body and can be also found in this organ.
The investigations for the presence of CMV and EBV are discussed together because the results
are similar: the presence of anti-CMV IgG in 98% of cases and anti-EBV IgG in 100% of cases;
the uncertain presence of anti-CMV IgM in 10% of cases and the absence of anti-EBV IgM. This
emphasizes the rarity of acute viral infection cases or with a virus that replicates in the body,
although there is an anamnestic contact with the virus. Our results also suggest that even if the
virus could theoretically be stationed in the sleeping form in an organ, in most cases the
conditions for viral replication are not met . The presence of antiviral IgG in an overwhelming
majority of cases is somewhat surprising, and could conclude that most likely the infections were
inapparent. We have observed that most patients had decreases in lymphocytes B [3], which,
associated with the presence in their medical history of infection with EBV in 100% of cases, can
generate the assumption that these patients suffered effects of the lymphotropic action of EBV on
lymphocytes B.
In patients with BPH, the evolution towards adenoma can be the result of a chronic inflammation
post-viral infection or an effect of the presence of the virus, even if just transiently, in the
prostate. Under the circumstances of an inapparent acute infections, the prostatic cells reaction to
the presence of the virus can not be established or predicted, given the fact that the infection even
can not be diagnosed in the acute phase. From and anamnestic view, the existence of such an
infection can be evidenced by the presence of specific IgG antibodies. Patients with high levels
of anti-CMV IgG or anti-EBV IgG prove the existance of previous contact with the virus, but we
cannot determine precisely whether or not the virus had affected the prostate. The presence of
these antibodies in almost all patients with BPH may question whether CMV and EBV are
involved in chronic inflammatory processes that generate the cellular reaction inducing prostatic
hypertrophy.
The analysis of the casework and the conducted investigations do not provide sufficient data to
support viral infection influence on a possible malignant transformation in the prostate, even if
some of the patients had elevated PSA [3]. However, based on our results, we support the viral
investigation as a possibility of enlarging tumor screening in inflammatory diseases of urinary
tract.
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