Human papillomavirus in normal conjunctival tissue and in
conjunctival papilloma: types and frequencies in a large series
Nicolai Christian Sjo ¨, Christian von Buchwald, Patricia Cassonnet, Bodil Norrild, Jan Ulrik Prause,
Troels Vinding, Steffen Heegaard
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Br J Ophthalmol 2007;91:1014–1015. doi: 10.1136/bjo.2006.108811
Aim: To examine conjunctival papilloma and normal conjunc-
tival tissue for the presence of human papillomavirus (HPV).
Methods: Archival paraffin wax-embedded tissue from 165
conjunctival papillomas and from 20 histological normal con-
junctival biopsy specimens was analysed for the presence of HPV
by PCR. Specimens considered HPV positive using consensus
primers, but with a negative or uncertain PCR result using type-
specific HPV probes, were analysed with DNA sequencing.
Results: HPV was present in 86 of 106 (81%) b-globin-positive
papillomas. HPV type 6 was positive in 80 cases, HPV type 11
was identified in 5 cases and HPV type 45 was present in a
single papilloma. All the 20 normal conjunctival biopsy
specimens were b-globin positive and HPV negative.
Conclusion: There is a strong association between HPV and
conjunctival papilloma. The study presents the largest material
of conjunctival papilloma investigated for HPV and the first
investigation of HPV in normal conjunctival tissue. HPV types 6
and 11 are the most common HPV types in conjunctival
papilloma. This also is the first report of HPV type 45 in
and have different oncogenic potentials.2So far, low-risk HPV
types 6 and 11 and high-risk HPV types 16 and 33 have been
identified in conjunctival papilloma.1 3–6To our knowledge,
similar studies on normal conjunctiva have not been reported
to date. Therefore, it is still unclear whether HPV plays a crucial
role in the development of conjunctival papilloma.
The purpose of this study was to analyse the largest material
of conjunctival papilloma to date (fig 1) and normal con-
junctival tissue for the presence of various HPV types. The aim
was also to investigate for HPV types not described previously
in conjunctival papilloma, and to determine whether HPV is a
common pathogen on the normal ocular surface.
uman papillomavirus (HPV) is associated with the
occurrence of conjunctival papilloma.1The individual
HPVtypesaretrophic forcutaneousor mucosal epithelium
MATERIALS AND METHODS
We studied 165 cases of conjunctival papilloma from the files of
the Eye Pathology Institute, Copenhagen, Denmark. The
formalin-fixed, paraffin wax-embedded specimens were regis-
tered during the period 1988–97. A histological review of the
diagnosis was made in each case. The material did not include
our previous series of 55 conjunctival papillomas.1
A total of 20 normal conjunctival biopsy specimens from
patients undergoing strabismus surgery served as controls.
Before excision biopsy, the conjunctiva underwent slit-lamp
evaluation to exclude patients with clinical signs of ocular
surface disease. All biopsy specimens were taken from the nasal
and inferior part of the conjunctiva, which is known to be the
most frequent location of papilloma.7The investigation was
approved by the Danish National Committee on Biomedical
Research Ethics, Denmark.
PCR and DNA sequencing
PCR was carried out as described earlier.1 8
considered HPV positive using consensus primers, but with a
negative or uncertain PCR result using type-specific probes,
were analysed with DNA sequencing as described previously9
using GP5+/6+ primers.
Abbreviation: HPV, human papillomavirus
exophytic, papillary growth. (B) The epithelium of the conjunctival
papilloma is acanthotic (asterisk) and wrapped over the stems of
fibrovascular cores (arrow) (H&E, magnification 612.5). (C) PCR
investigation of conjunctival papilloma. Agarose gel with MY09/11 PCR
products viewed in UV-light transillumination. Lane M contains the DNA
band marker and lane 1 contains the positive control. Lanes 2–6 are
considered HPV positive because of the presence of a DNA band of the
appropriate size. Lane 7 contains the negative control.
(A) Conjunctival papilloma (arrow) showing the characteristic
Tissue control Download full-text
From each block from which sections were used for PCR
analysis, an extra final cut was made for histological examina-
tion to ensure the presence of sufficient tissue.
The papillomas were excised from patients aged 17–82 years
(mean age 37 years) and the normal controls originated from
patients aged 18–65 years (mean age 27 years).
The b-globin fragment could not be amplified in 59 cases
(36%) of the papilloma series and these specimens were
excluded from further analysis.
HPV was detected in 86 of the remaining 106 papillomas
(81%) using MY09/11 and GP5+/6+ consensus primers (fig 1).
Using type-specific primers and DNA sequencing, HPV type 6
was positive in 80 cases, HPV type 11 was identified in 5 cases
and HPV type 45 in a single papilloma (table 1).
All the 20 normal conjunctival biopsy specimens were b-
globin positive and HPV negative.
This investigation confirms that most conjunctival papillomas
harbour HPV (81%). In accordance with the benign nature of
conjunctival papilloma, the low-risk HPV types 6 and 11 were
found to be predominant among HPV-positive papillomas. Only
in one case was an HPV type other than 6 or 11 identified—that
is, HPV type 45. This is the first report of HPV type 45 in
conjunctival papilloma. HPV type 45 is a high-risk HPV type
primarily associated with low-grade cervical intraepithelial
neoplasia of the uterus.12Histopathological examination of
the HPV 45-positive conjunctival papilloma in this study did
not disclose any signs of dysplasia. The HPV 45-positive
papilloma recurred twice. However, it is known that 11% of
the papillomas recur one or more times.7
In our material, 36% of the specimens could not be used for
PCR investigations because amplifiable DNA could not be
demonstrated. Formalin and paraffin wax contain large
amounts of PCR inhibitors.13Furthermore, it is known that
the formalin-induced DNA degradation begins after a few
hours of formalin fixation.13At the Eye Pathology Institute,
specimens are received by mail and thus formalin fixation time
is never ,24 h and in some cases is .3 days. This could explain
why DNA extraction was unsuccessful in 36% of our cases.
This study is the first investigation of normal conjunctival
tissue for the presence of HPV, and we found that all normal
conjunctival biopsy specimens were HPV negative. This
indicates that HPV is associated with conjunctival papilloma.
Furthermore, our study shows that the conjunctiva is not an
HPV reservoir, in contrast with the genital mucosa.14This is in
accordance with the outcome of similar studies on the
neighbouring nasal and oral cavity, where HPV is likewise not
present in normal mucosa.15 16
In conclusion, our study presents the largest material of
conjunctival papilloma investigated for HPV and the first
investigation of a putative HPV presence in normal conjunctival
tissue. Our investigation shows that HPV is associated with
We thank Michaelsen Fonden, Landsforeningen Værn om Synet, The
John and Birte Meyer Foundation, Betzy, Dagny og Caja Bojesens
Mindefond, Synoptik Fonden and the Danish Eye Research Foundation
for supporting 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
Patricia Cassonnet, Unite ´ des Papillomavirus, Institut Pasteur, Paris, France
Bodil Norrild, Protein Laboratory, University of Copenhagen,
Troels Vinding, Department of Ophthalmology, Rigshospitalet,
Competing interests: None declared.
Correspondence to: Dr S Heegaard, Eye Pathology Institute, University of
Copenhagen, Frederik V’s Vej 11, DK-2100 Copenhagen Ø, Denmark;
Accepted 8 December 2006
Published Online First 13 December 2006
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for the presence of human papillomavirus using the PCR
Previous investigations of conjunctival papilloma
StudyCases HPV types HPV frequency (%)
Saegusa et al (1995)
Sjo ¨ et al (2001)
Eng et al (2002)
6, 11, 45
HPV, human papillomavirus.
Case reports are not included.
HPV in normal conjunctival tissue and in conjunctival papilloma1015