Human papillomavirus and pterygium. Is the virus a risk
Nicolai Christian Sjo ¨, Christian von Buchwald, Jan Ulrik Prause, Bodil Norrild, Troels Vinding, Steffen
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Br J Ophthalmol 2007;91:1016–1018. doi: 10.1136/bjo.2006.108829
Background: Pterygium is a disease of unknown origin and
pathogenesis that might be vision threatening. It is charac-
terised by a wing-like conjunctival overgrowth of the cornea.
Several studies have investigated human papillomavirus (HPV)
as a risk factor for the development of pterygia, but the results
Aim: To investigate a large sample of pterygia for the presence
of HPV in order to clarify the putative association between
pterygia and HPV.
Methods: 100 specimens of pterygium from Danish patients
and 20 normal conjunctival biopsy specimens were investi-
gated for the presence of HPV with PCR technique using
b-globin primers to access the quality of the extracted DNA and
the HPV primers MY09/11 and GP5+/6+. HPV-positive
specimens underwent subsequent HPV typing with type-specific
HPV primers and further investigation with DNA in situ
Results: 90 of 100 investigated pterygia proved suitable for
HPV analysis by PCR. As b-globin could not be amplified, 10
specimens were excluded from the study. 4 of 90 pterygia
harboured HPV. HPV type 6 was identified in all four HPV-
positive pterygia. The 20 normal conjunctival biopsy specimens
were b-globin positive and HPV negative. All four pterygia that
were HPV type 6 positive were DNA ISH negative.
Conclusions: The low presence of HPV DNA in pterygia does
not support the hypothesis that HPV is involved in the
development of pterygia in Denmark.
cornea (fig 1).1The overgrowth consists of a stromal part
containing fibroblasts and blood vessels and is accompanied by
an inflammatory cell infiltrate and an abnormal accumulation
of extracellular matrix, which is composed of elastin and
collagen1(fig 1). The stromal part is covered by flattened
conjunctival epithelium (fig 1). Bowman’s layer beneath the
pterygium may undergo destruction by the advancing fibro-
vascular tissue, resulting in a corneal scar. Pterygium is a
common eye disease and has a worldwide distribution,
occurring at highest prevalence and most severely in tropical
regions. In Denmark, the prevalence of pterygium has been
estimated to be 0.7%,2whereas the prevalence was found to be
9.9% in Singapore3and 23.4% among the coloured population
of Barbados.4Originally believed to be a degenerative condition
of the conjunctiva, recent theories are considering pterygium as
a growth disorder.5
There is compelling epidemiological evidence that damage
mediated by ultraviolet (UV) light acts as a trigger for the
pathogenesis of pterygium.3 4 6–9In accordance with this, a
terygium is a disease of unknown origin and pathogenesis
that might be vision threatening. The disease is charac-
terised by a wing-like conjunctival overgrowth of the
‘‘two-hit’’ theory has been suggested.10Detorakis et al10suggest
that pterygium is caused due to a combination of an oncogenic
agent and a pre-existing genetic damage produced by UV light
(or inherited factors).
Human papillomavirus (HPV) is composed of a closed
circular double-stranded DNA genome of which at least 100
www.stdgen.lanl.gov). The different HPV subtypes are asso-
ciated with primarily benign or malignant epithelial tumours—
for example, HPV 6 and 11 are associated with benign neoplasia
and HPV 16 and 18 are closely linked with malignancy of the
uterine cervical squamous epithelium.11HPV is also associated
with epithelial tumours of the conjunctiva, including conjunc-
tival papilloma12and conjunctival carcinoma.13Several studies
have investigated HPV as a risk factor for the development of
pterygium, but the outcome seems inconclusive as the
proportion of HPV-positive pterygia ranges from 0% to
100%14–17and the materials investigated were limited.
The purpose of the present study was to investigate a large
material of pterygium for the presence of HPV in order to clarify
the putative association between pterygium and HPV.
MATERIALS AND METHODS
In all, 100 specimens of primary pterygium from Danish
patients registered during the period 1994–2003 were collected
from the files of the Eye Pathology Institute, University of
Copenhagen, Copenhagen, Denmark. Gender and age of
patients were registered, and all material were reviewed
Furthermore, 20 normal conjunctival biopsy specimens excised
during the years 2000–1 were included as controls.
of the diagnosis.
Polymerase chain reaction
Three sections from each paraffin wax block were cut. The
sections were placed in 1.5 ml tubes and 50 ml digestion buffer
(10 mM Tris pH 7.0, 1 mM EDTA and K 200 mg/ml proteinase)
was added. The specimens were digested at 65˚C for 3 h,
spinned and were transferred to a new tube for the aqueous
phase. Boiling for 10 min inactivated the proteinase K. The
samples, together with appropriate positive and negative
controls, were amplified with primers targeting a 288 bp
fragment of the single copy b-globin gene in order to ensure
the integrity of the DNA as described by Sebbelov et al.18
b-Globin-negative pterygia were excluded from further analysis.
HPV PCR was performed with the generally accepted
consensus primers GP5+/GP6+19and MY 09/1120; these primers
amplify the majority of the known mucosa-tropic HPV types.
The PCR product, 15 ml, was run in a 1.5% submerged agarose
gel in Tris-acetate-EDTA buffer, stained with ethidium bromide
and viewedinUV transillumination.The samplewas
Abbreviations: HPV, human papillomavirus; ISH, in situ hybridisation;
considered HPV positive when a band of a predicted size
(,150 bp for the GP5+/GP6+ primers or 450 bp for the MY09/
11 primers) was identified on the gel. HPV-positive specimens
underwent subsequent HPV typing with type-specific HPV
primers 6, 11, 16, 18, 31 and 33.21
DNA in situ hybridisation
HPV PCR-positive pterygia and two HPV PCR-negative con-
junctival biopsy specimens were investigated with DNA in situ
hybridisation (ISH). Sections of 5 mm thickness were cut and
then immersed successively in toluene, 100%, 90%, 60% and
30% ethanol, and distilled water. For unmasking of nucleic
acids, the specimens were treated with 50 mg/ml of proteinase K
for 20 min at 37˚C. Denaturation of target DNA and probe was
achieved by heat treatment at 75˚C for 10 min and 95˚C for
5 min, respectively. Specimens were incubated with 50 ng/ml
digoxigenin-labelled HPV DNA probe 6, 11 or 16 at 37˚C
overnight. Sections were then subjected to two post-hybridisa-
tion washes in standard saline citrate buffer and incubated in
phosphate-buffered saline containing 5% (weight/volume)
bovine serum albumin. Thereafter, sections were incubated
for 1 h with alkaline phosphatase conjugated anti-digoxigenin
1/2000, followed by precipitation with nitroblue tetrazolium
chloride and bromochloroindoylphosphate. Finally, sections
were counterstained for 10 s in Mayers haematoxylin. Positive
control sections were included in the experiment.
The 100 patients consisted of 57 men and 43 women. Age range
was 37–85 years, with a mean age of 63.4 years. All pterygia
were primary lesions.
In all, 90 of 100 investigated pterygia proved suitable for HPV
analysis by PCR. As b-globin could not be amplified in 10
specimens these were excluded from the study. In all, 4 of 90
pterygia harboured HPV. HPV type 6 was identified in all four
HPV-positive pterygia (fig 1). All 20 normal conjunctival biopsy
specimens were b-globin positive and HPV negative.
DNA ISH results
The four pterygia that were HPV type 6 positive had been
analysed using PCR investigations, and all were DNA ISH
UV light is associated with the development of pterygium,8
which is observed only in humans, and the lesion has never
been described in animals. Therefore, risk factors other than UV
radiation must be significant in the pathogenesis of pterygium.
Our investigation of 100 pterygia questions the possibility that
HPV is involved in the pathogenesis of pterygium. The low-risk
HPV type 6 was identified in only four specimens using the PCR
technique. However, subsequent DNA ISH investigations were
unable to confirm the presence of HPV. It is well known that
the PCR technique is superior in sensitivity to the ISH
technique.18 22Therefore, the four HPV PCR-positive but HPV
DNA ISH-negative specimens could be due to contamination of
the investigated specimens or a non-significant, incidental
occurrence of an actual infection with a low number of virions.
Nonetheless, owing to the small number of HPV-positive
pterygia in our material, it seems unlikely that HPV is an
essential factor in the development of pterygia in Denmark.
Other investigations of pterygium have shown great geo-
graphical variance in the proportion of HPV-positive ptery-
gium.14–16 23 24Using PCR, HPV was not identified in 65 pterygia
from Taiwan14and, likewise, Dushku et al24were unable to
detect HPV in an investigation of 13 pterygia in a study from
the USA. On the other hand, 30% of pterygium from Greek
patients were HPV positive23; in a British study,15the HPV
prevalence was 50% and Piras et al16found HPV in 100% of 17
investigated Italian pterygia. However, none of the previous
investigations haveused immunohistochemistry
to show the localisation of HPV in their HPV-positive
pterygia.15 16 23 24
A mechanism that might cause HPV to become undetectable
by standard PCR assays is the integration of HPV DNA in the
host cell genome in HPV-positive tissue. Integration of HPV
results in the interruption of HPV DNA in the early region of the
genome.25Nevertheless, previous studies performed on cervical
carcinoma in The Netherlands indicate that this mechanism
plays only a minor role.26The pathogenesis of HPV infections
might also support that HPV is not associated with pterygium.
HPV infections are confined to the keratinocytes of the skin and
mucosal surfaces.27Therefore, the virus is highly adapted to this
cell type and productive HPV infection is unlikely to occur in
overgrowth of the cornea. (B) The basophilic appearance of elastotic
degeneration is a characteristic feature of the stromal layer of pterygium.
(H&E,6100). (C) Agarose gel with PCR products viewed in ultraviolet-light
transillumination. Lane M contains the DNA band marker that is used to
estimate the size of the amplificated products. A circle marks the human
papillomavirus (HPV) type 6 positive control in lane 1. Lanes 2 and 5 are
negative controls, whereas lanes 3 and 4 are HPV-negative specimens.
Lanes 6 and 7 are considered HPV type 6 positive due to the presence of a
DNA band of the appropriate size.
(A) Pterygium characterised by a wing-like conjunctival
Human papillomavirus and pterygium 1017
any other tissue types.27Additionally, epidemiological data of
pterygium prevalence and HPV infection contradict. The
prevalence of pterygium increases linearly with age,3 4whereas
HPV infection is predominantly seen among younger people in
their 20s and 30s.28However, in recent years, new types of skin
HPV have been identified and are more common than
considered previously. These observations indicate that there
is a reservoir of superficially located virus on the skin.29Perhaps
the different HPV frequencies in geographically distinct
populations reflect that pterygium is a multifactorial disease
resulting from a non-specific response to various different
insults. A number of proinflammatory cytokines, angiogenic
and fibrogenic growth factors and their receptors have been
reported in pterygium, which parallels their involvement in the
corneal wound-healing cascade.30 31In particular, basic-fibro-
endothelial growth factor and heparin-binding epidermal
growth factor are over-expressed in pterygium and, further-
more, directly or indirectly, induced by UV light.5 31It has
recently been shown that key proinflammatory cytokines and
growth factors are modulated by exposure to UV light through
intracellular pathways, which supports the role of cumulative
UV damage in pterygium formation.31
causative agents of this process include herpes virus23and
factors that cause repeated insults to the interpalpebral
corneoscleral junction (eg, dust and wind exposure).1
In conclusion, our finding of the very limited presence of HPV
DNA in pterygium does not support the hypothesis that HPV is
involved in the development of pterygium in Denmark. The
geographical variance in the proportion of HPV-positive
pterygium may reflect that pterygium is a multifactorial
Landsforeningen Værn om Synet; The John and Birte Meyer
Foundation; Betzy, Dagny og Caja Bojesens Mindefond; Synoptik
Fonden; The Danish Eye Research Foundation and Michaelsen Fonden
supported this study.
Nicolai Christian Sjo ¨, Jan Ulrik Prause, Steffen Heegaard, Eye Pathology
Institute, University of Copenhagen, Copenhagen, Denmark
Christian von Buchwald, Department of Oto-rhino-laryngology,
Rigshospitalet, Copenhagen, Denmark
Bodil Norrild, Institute of Molecular Pathology, University of Copenhagen,
Competing interests: None declared.
Correspondence to: S Heegaard, Eye Pathology Institute, University of
Copenhagen, Frederik V’s Vej 11, DK-2100 Copenhagen, Denmark;
Accepted 8 December 2006
Published Online First 19 December 2006
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1018 Sjo ¨, von Buchwald, Prause