JCM Accepts, published online ahead of print on 9 April 2014 J. Clin. Microbiol. doi:10.1128/JCM.00286-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Improving oral HPV detection using toothbrush sampling in HIV positive
2
men who have sex with men
3 4
Jason J. Onga#, Tim Readb,c, Marcus Chenb,c, Sandra Walkerb, Matthew Lawd,
5
Catriona Bradshawb,c, Suzanne M. Garlande,f,g, Sepehr N. Tabrizie,f,g, Alyssa
6
Cornallf , Andrew Grulichd, Jane Hockinga, Christopher K. Fairleyb,c
7 8
Melbourne School of Population and Global Health, University of Melbourne,
9
Australiaa; Melbourne Sexual Health Centre, Australiab; Central Clinical School,
10
Monash Universityc; Kirby Institue, University of New South Wales, Australiad;
11
Department of Obstetrics and Gynaecology, University of Melbourne, Australiae;
12
Regional HPV Lab Net Reference laboratory, Department of Microbiology and
13
Infectious Diseases, The Royal Women’s Hospital, Australiaf; Murdoch Childrens
14
Research Institute, Parkville, Australiag
15 16
Running title: Improving oral HPV detection using toothbrush sampling
17 18
# address correspondence to Jason Ong, [email protected]
19
1
20
Abstract
21
Pre- and post-abrasion oral rinse samples(ORS) and toothbrush sample detected
22
human papillomavirus(HPV) DNA in at least one sample among 45 (26%) of 173
23
HIV-positive men who have sex with men. There was moderate agreement
24
between HPV genotype detection pre-abrasion and post-abrasion ORS(κ=0.49,
25
95% CI:0.37-0.61). There was good agreement between post-abrasion ORS and
26
toothbrush(κ=0.70, 95% CI:0.60-0.80). Sensitivities for HPV genotypes detected
27
were 80% (95% CI: 69-88) for pre-abrasion ORS, 65%(95% CI:54-76) for post-
28
abrasion ORS and 75%(95% CI:63-84) for toothbrush.
29
Word count: 78
30
2
31
Human papillomavirus (HPV) causes some forms of oropharyngeal cancer,
32
most commonly in the lingual and palatine tonsils or base of tongue(1, 2). It is
33
estimated that more than half have high risk (hr-)HPV detected, the most
34
common genotype being HPV-16(3, 4). Incidence of oropharyngeal cancer has
35
increased in younger individuals and the proportion of these tumours associated
36
with HPV continues to rise(5-8).
37
Currently there is no universally agreed method of oral sampling for detection of
38
HPV DNA. The most common sampling method is the oral rinse swirl or gargle
39
(ORS)(9-12). Alternatives include using a flocked Nylon swab(13), biopsy(14),
40
cytobrush(11, 15) or oral mucosal scraping(16). An untested sampling method
41
is using ORS immediately after brushing one’s teeth and gums. A study by our
42
group found the likelihood of detecting oral HPV fell in a linear fashion by about
43
14% with each additional hour after brushing teeth, suggesting that abrasion of
44
oral mucosa may improve collection of infected cells in an oral rinse(13). This
45
finding suggests that current sampling techniques may be improved by prior
46
epithelial abrasion similar to that used for anogenital HPV detection in men(17).
47
The detection of HPV seems to vary widely depending on the sampling method
48
as well as detection methods with prevalence rates ranging from 7% to 45%
49
even within similar populations(18). It remains unclear whether one sampling
50
method is superior to another for detection of oral HPV.
51
Men who have sex with men (MSM) attending the Melbourne Sexual
52
Health Centre (MSHC) HIV clinic from December 2012 to August 2013, were
53
recruited. In the single clinic visit, participants provided 3 samples for HPV
54
detection and genotyping. The first sample was a pre-abrasion ORS that
3
55
involved swishing and gargling 20 mls of sterile saline in the oral cavity for 20-
56
30 seconds and then spitting this into a sterile specimen cup. The post-abrasion
57
ORS sample was collected directly after participants brushed their teeth with a
58
new toothbrush (Dentitex, medium grade bristles), but otherwise identically to
59
the pre-abrasion ORS. The toothbrush was then placed in 10 mls of phosphate
60
buffered saline (PBS) and rotated.
61
The resuspended cells from the toothbrush and ORS samples were centrifuged
62
for 10 minutes at 14,000 xg and subsequently resuspended in 400μl of PBS. An
63
aliquot of 200μl was extracted by the automated MagNA Pure 96 isolation and
64
purification system (Roche Molecular Systems) using DNA and Viral NA Small
65
Volume isolation kit. Following nucleic acid isolation, all samples were initially
66
assessed for DNA adequacy with a quantitative PCR for a 260bp fragment of the
67
human beta-globin gene using 10pmol each of β-globin primers GH20 [5’-
68
GAAGAGCCAAGGACAGGTAC-3’] and PCO4 [5’-CAACTTCATCCACGTTCACC-3’]
69
and 2pmol of adapted probe PCO3 [5’-6-FAM-ACACAACTGTGTTCACTAGC-TAM-
70
3’](19). Samples which were HPV positive by PCR-ELISA(20) were subsequently
71
genotyped by LINEAR ARRAY®(LA)HPV Genotyping Test (Roche
72
Diagnostics)(21, 22), with modification as described previously(23, 24).
73
Samples which were HPV positive by PCR-ELISA but negative by LA were
74
amplified using the more sensitive HPV SPF10-LiPA 25 assay version 1(25).
75
Those defined as hr-HPV genotypes are according to recent International Agency
76
for Research on Cancer nomenclature(26).
77
The PCR ELISA utilized the well established Ll consensus primers PGMY09/(27,
78
28) combined with sensitive detection using HPV Ll generic probe and captured
4
79
on streptavidin-coated plates(Roche Biochemicals) with the bound hybrid
80
detected by an anti-digoxigenin peroxidase conjugate by use of the colorimetic
81
substrate ABTS(20). The established sensitivity of this assay in the laboratory is
82
10 copies per reaction for the mucosal types detected.
83
Statistical analyses were performed using STATA (version 13.1). 95%
84
confidence intervals for sensitivities was calculated using an exact binomial
85
confidence interval. We used kappa-values to quantify agreement of HPV
86
detection comparing each sampling method. This research was approved by the
87
Alfred Health Human Ethics Committee (Project 384/12).
88
Two hundred and ten HIV-positive MSM attending Melbourne Sexual
89
Health Centre were approached with 173 (69%) men consenting to participate.
90
The mean age of participants was 52 years (range 23-87). They had an average
91
duration of HIV of 11.8 years (range 2-31), with mean current CD4 count of 679
92
cells/μl and 91% had a suppressed viral load (
1
Improving oral HPV detection using toothbrush sampling in HIV positive
2
men who have sex with men
3 4
Jason J. Onga#, Tim Readb,c, Marcus Chenb,c, Sandra Walkerb, Matthew Lawd,
5
Catriona Bradshawb,c, Suzanne M. Garlande,f,g, Sepehr N. Tabrizie,f,g, Alyssa
6
Cornallf , Andrew Grulichd, Jane Hockinga, Christopher K. Fairleyb,c
7 8
Melbourne School of Population and Global Health, University of Melbourne,
9
Australiaa; Melbourne Sexual Health Centre, Australiab; Central Clinical School,
10
Monash Universityc; Kirby Institue, University of New South Wales, Australiad;
11
Department of Obstetrics and Gynaecology, University of Melbourne, Australiae;
12
Regional HPV Lab Net Reference laboratory, Department of Microbiology and
13
Infectious Diseases, The Royal Women’s Hospital, Australiaf; Murdoch Childrens
14
Research Institute, Parkville, Australiag
15 16
Running title: Improving oral HPV detection using toothbrush sampling
17 18
# address correspondence to Jason Ong, [email protected]
19
1
20
Abstract
21
Pre- and post-abrasion oral rinse samples(ORS) and toothbrush sample detected
22
human papillomavirus(HPV) DNA in at least one sample among 45 (26%) of 173
23
HIV-positive men who have sex with men. There was moderate agreement
24
between HPV genotype detection pre-abrasion and post-abrasion ORS(κ=0.49,
25
95% CI:0.37-0.61). There was good agreement between post-abrasion ORS and
26
toothbrush(κ=0.70, 95% CI:0.60-0.80). Sensitivities for HPV genotypes detected
27
were 80% (95% CI: 69-88) for pre-abrasion ORS, 65%(95% CI:54-76) for post-
28
abrasion ORS and 75%(95% CI:63-84) for toothbrush.
29
Word count: 78
30
2
31
Human papillomavirus (HPV) causes some forms of oropharyngeal cancer,
32
most commonly in the lingual and palatine tonsils or base of tongue(1, 2). It is
33
estimated that more than half have high risk (hr-)HPV detected, the most
34
common genotype being HPV-16(3, 4). Incidence of oropharyngeal cancer has
35
increased in younger individuals and the proportion of these tumours associated
36
with HPV continues to rise(5-8).
37
Currently there is no universally agreed method of oral sampling for detection of
38
HPV DNA. The most common sampling method is the oral rinse swirl or gargle
39
(ORS)(9-12). Alternatives include using a flocked Nylon swab(13), biopsy(14),
40
cytobrush(11, 15) or oral mucosal scraping(16). An untested sampling method
41
is using ORS immediately after brushing one’s teeth and gums. A study by our
42
group found the likelihood of detecting oral HPV fell in a linear fashion by about
43
14% with each additional hour after brushing teeth, suggesting that abrasion of
44
oral mucosa may improve collection of infected cells in an oral rinse(13). This
45
finding suggests that current sampling techniques may be improved by prior
46
epithelial abrasion similar to that used for anogenital HPV detection in men(17).
47
The detection of HPV seems to vary widely depending on the sampling method
48
as well as detection methods with prevalence rates ranging from 7% to 45%
49
even within similar populations(18). It remains unclear whether one sampling
50
method is superior to another for detection of oral HPV.
51
Men who have sex with men (MSM) attending the Melbourne Sexual
52
Health Centre (MSHC) HIV clinic from December 2012 to August 2013, were
53
recruited. In the single clinic visit, participants provided 3 samples for HPV
54
detection and genotyping. The first sample was a pre-abrasion ORS that
3
55
involved swishing and gargling 20 mls of sterile saline in the oral cavity for 20-
56
30 seconds and then spitting this into a sterile specimen cup. The post-abrasion
57
ORS sample was collected directly after participants brushed their teeth with a
58
new toothbrush (Dentitex, medium grade bristles), but otherwise identically to
59
the pre-abrasion ORS. The toothbrush was then placed in 10 mls of phosphate
60
buffered saline (PBS) and rotated.
61
The resuspended cells from the toothbrush and ORS samples were centrifuged
62
for 10 minutes at 14,000 xg and subsequently resuspended in 400μl of PBS. An
63
aliquot of 200μl was extracted by the automated MagNA Pure 96 isolation and
64
purification system (Roche Molecular Systems) using DNA and Viral NA Small
65
Volume isolation kit. Following nucleic acid isolation, all samples were initially
66
assessed for DNA adequacy with a quantitative PCR for a 260bp fragment of the
67
human beta-globin gene using 10pmol each of β-globin primers GH20 [5’-
68
GAAGAGCCAAGGACAGGTAC-3’] and PCO4 [5’-CAACTTCATCCACGTTCACC-3’]
69
and 2pmol of adapted probe PCO3 [5’-6-FAM-ACACAACTGTGTTCACTAGC-TAM-
70
3’](19). Samples which were HPV positive by PCR-ELISA(20) were subsequently
71
genotyped by LINEAR ARRAY®(LA)HPV Genotyping Test (Roche
72
Diagnostics)(21, 22), with modification as described previously(23, 24).
73
Samples which were HPV positive by PCR-ELISA but negative by LA were
74
amplified using the more sensitive HPV SPF10-LiPA 25 assay version 1(25).
75
Those defined as hr-HPV genotypes are according to recent International Agency
76
for Research on Cancer nomenclature(26).
77
The PCR ELISA utilized the well established Ll consensus primers PGMY09/(27,
78
28) combined with sensitive detection using HPV Ll generic probe and captured
4
79
on streptavidin-coated plates(Roche Biochemicals) with the bound hybrid
80
detected by an anti-digoxigenin peroxidase conjugate by use of the colorimetic
81
substrate ABTS(20). The established sensitivity of this assay in the laboratory is
82
10 copies per reaction for the mucosal types detected.
83
Statistical analyses were performed using STATA (version 13.1). 95%
84
confidence intervals for sensitivities was calculated using an exact binomial
85
confidence interval. We used kappa-values to quantify agreement of HPV
86
detection comparing each sampling method. This research was approved by the
87
Alfred Health Human Ethics Committee (Project 384/12).
88
Two hundred and ten HIV-positive MSM attending Melbourne Sexual
89
Health Centre were approached with 173 (69%) men consenting to participate.
90
The mean age of participants was 52 years (range 23-87). They had an average
91
duration of HIV of 11.8 years (range 2-31), with mean current CD4 count of 679
92
cells/μl and 91% had a suppressed viral load (
