1. Prevalence of Human Papillomavirus (HPV) Genotypes in HIV-1 Infected Women in Seattle, WA and Nairobi, Kenya Results from the Women HIV Interdisciplinary Network (WHIN)
HPV is considered the most common known sexually transmitted agent worldwide. At a any given point in time 10% of women are positive for cervical HPV DNA this represents approximately 291 million women globally. Cervical cancer is the number one cancer cause of year of life lost in the developing world.
African women have the highest prevalence of HPV.
The currently available HPV vaccine effectively protect against the most common oncogenic types of HPV ( 16 and 18).
Given the type specific immunity of these vaccines and the potential for geographic variation on prevalence of HPV types it is important to understand the distribution of HPV genotypes in particular among HIV infected women
Certain HPV types are associated with squamous intraepithelial lesions. Specifically HPV 16, 18, 45 and 31 cause over half of the low grade lesions and about 65% of the high grade lesions.
Amneris E. Luque*, Jane Hitti, Christina Mwachari, Christopher Lane, Susan Messing, Susan E. Cohn, Robert Rose and Robert Coombs

2. Background-HPV Prevalence
HPV is the most common known sexually transmitted agent worldwide
At any given point in time 10% of women are positive for cervical HPV DNA ( 291 million women)
Certain HPV types are associated with squamous intraepithelial lesions. HPV 16 and 18 cause about 65% of the high grade lesions

3. Cervical Cancer in the Developing World
83% of cases in developing world
#1 cancer cause of YLL in developing world
Source: NEJM 2005;353:2101-4

4. Background-HIV and HPV
When compared with HIV-uninfected women, HIV Infected women have:
higher rates of HPV infection
higher prevalence of infection with multiple types
of HPV
higher prevalence of oncogenic (high-risk) HPV
higher prevalence of cervical dysplasia
A different distribution of HPV genotypes
There appears to be a geographic variation in the distribution of HPV genotypes, but this has not been widely studied in HIV-infected women.
HIV infection has a significant impact on HPV infection and the progression of HPV-related cervical disease.
HIV infected women not only have a high prevalence of HPV infection but also are more likely to be infected with multiple HPV genotypes concurrently. They are also more likely to be infected with HPV genotypes that are considered high risk for progressing to cervical cancer.

5. Study Aim
To assess the prevalence of HPV genotypes in two distinct populations of HIV-1 infected women
To assess the prevalence of HPV genotypes in two geographically distinct populations, we compared the HPV genotype distribution between HIV-1 infected women from Seattle and Nairobi

6. Methods
HIV-1 infected women were enrolled in Seattle, WA and had evaluations at set intervals, over a 3 year period ( )
HIV-1 infected women with CD4+ cell count > 350 cells/mm3 were enrolled from Nairobi, Kenya and had similar evaluations as the Seattle participants at a single point in time

7. Methods (cont 1.) Informed consent
Standardized history and physical examination
Questionnaire
Sample collection
Cervical Cytology
Cervico-vaginal lavage samples (CVL)

8. Methods (cont 2.)
Total DNA extraction in CVL specimens was followed by PCR amplification and genotype analysis by reverse line blot
Reverse line blot used a set of 27 immobilized oligonucleotide probes, each corresponding to an individual HPV genotype.

9. Reverse Line Blot Assay
Type Specific Probe Application
Rotate 90°
The reverse line blot assay util;izes an immobilized probe array in which oligonucleotide probes are deposited in a linear array

10. Reverse Line Blot Assay
Biotinylated PCR Product Application
The PCR product is generated using biotinylated primers. The presence of biotinylated primers bound to specific probe is detected using streavidin-conjugated with alkaline phosphatase and a chromogenic soluble substrate to produce a color line at the position of the positive probe
Detect using StrepAvidin-conjugated with Alkaline Phosphatase

11. On the right the different 27specific probes are listed with the Beta globin controls at the bottom. The numbers on the top of the columns represent consecutive patients 1,2,3,4,5,6,7,8,9,10 etc… ( only a number every 5 is written to prevent crowding.

12. Statistical Analyses
Demographics and cross sectional data is presented by site.
A logistic regression analysis was used to assess the relationship between grouped HPV types and cytological results.
All statistical tests are two-sided with alpha <.05

13. Baseline demographic information, stratified by site
Seattle
n=38
Nairobi
n= 50
Age (years) mean + sd
Parity
Race/ethnicity: n (%)
Caucasian
12 (32) --
African American
20 (53)
Asian/Pacific islander
2 (5)
Hispanic
Native American
1 (3)
Other
Kikuyo
24 (48)
Luo
8 (16)
Luhya
6 (12)
Kamba
3 (6)
Other/unknown
9 (18)

14. Baseline demographic information, stratified by site
Seattle
n=38
Nairobi
n= 50
HIV Risk Factor: n (%)
IVDU
9 (24)
Heterosexual
25 (66)
50 (100)
Blood transfusion
2 (5)
Other/unknown
Lifetime sexual partners
Median ( IQR)
11(5-20)
3 (2-5)*
Oral contraceptive use: n (%)
Ever
29 (76)
30 (60)
Current
2 (4)
On ARVT: n (%)
20 (53)
0 (0)
CD4 cell count: cells/mm3
346
538 **
HIV RNA: copies/ml
1,036
13,942
*P = Wilcoxon rank-sum test
**P = .008

15. Results-Prevalence of HPV risk types and number of lifetime sexual partners
No. lifetime sexual partners ( entire group)
No HPV
Only low risk HPV
HPV 16/18
Other high-risk HPV
<5
29 (85%)
1 (3%)
3 (9%) ≥5
17 (68%)
2 (8%)
1 (4%)
5 (20%)

16. Results-Prevalence of HPV risk types and number of lifetime sexual partners in the Nairobi group
No. lifetime sexual partners
No HPV
High or Low Risk HPV
<5
27 (84%)
5 (16%) ≥5
6 (50%)
6(50%)
P =0.04 Fisher’s exact test

17. Results-HPV genotypes by site
Cohort
# of CVL samples
Samples Positive for beta globin
Samples Positive for HPV DNA
HPV Infections
HPV types
(number of occurences)
Seattle
(n=37) 63
38 (60%)
15 (40%)
Triple:1
Dual:5
Single:5
Non-typeable:4
56 (5), 66 (4), 6 (3), MM8(2), 16 (1), 33 (1), 53 (1), MM7 (1)
Nairobi
(n=50) 49
37 (76%)
11 (30%)
Dual:1
Single: 9
53 (3), 6 (3), 33 (2), 58 (2) 16 (1), 18 (1), 62 (1), MM8 (1)
Numbers in bold denote high-risk HPV types

18. Results-Cervical Cytology by Site
Seattle
n=64
Nairobi
n=50
Total
Normal 48 39 87
Abnormal 15 7 22
ASCUS 8 2 10
ASCUS-D
LGSIL 4 6
HGSIL 1 3
Other
Inadequate
Missing

19. HPV detected in CVL specimen
Results-Cervical cytology, according to HPV genotypes detected in CVL specimens
Cytological results
HPV detected in CVL specimen
MM other
ASCUS 2
LGSIL 1
HGSIL
Inadequate
Other
WNL 4 3
Total 5 6

20. Phylogenetic Tree of 118 HPV Types
Virology, 324, (2004)

21. Conclusions
Participants from Nairobi had significantly fewer sexual partners and had more commonly a single HPV type detected in CVL than those from Seattle.
HIV-1 infected women in Seattle and Nairobi had geographically distinct set of HPV genotypes
Patients in this study had a higher prevalence of high-risk HPV types other than 16 and 18.

22. Conclusions
Women with more than one HPV type detected in CVL were 6.25 more likely to have an abnormal Pap smear than those with only one or no HPV detected
Women were 7.52 times more likely to have an abnormal pap smear when they had high risk HPV present vs. none.
Currently available HPV vaccines may not protect against other prevalent HPV types in these women.

23. Acknowledgements
Kenya Research Institute
Christina Mwachari
University of Washington in Seattle
Bob Coombs
Jane Hitti
Katie Paul
University of Rochester
Richard C Reichman
Robert Rose
Susan Messing
Chris Lane
Susan Cohn
Women in Nairobi and Seattle who participated in the study