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DETECTION AND CONCENTRATION OF PLASMA AFLATOXIN IS ASSOCIATED WITH DETECTION OF
ONCOGENIC HUMAN PAPILLOMAVIRUS IN KENYAN WOMEN
Jianjun Zhang1,2, Omenge Orang’o1,3, Philip Tonui3, Yan Tong4, Titus Maina5, Stephen Kiptoo6,
Katpen Muthoka6, John Groopman7, Joshua Smith7,
Erin Madeen7, Aaron Ermel8, Patrick Loehrer8, Darron R. Brown8,9
1These two authors contributed equally to this study and manuscript.
2Department of Epidemiology, Fairbanks School of Public Health, Indiana University,
Indianapolis, IN, USA, 46204
3Department of Reproductive Health, Moi University, Eldoret, Kenya
4Department of Biostatistics, Indiana University School of Medicine and Fairbanks School of
Public Health, Indianapolis, IN, USA, 46204
5Department of Molecular Biology, Maseno University, Kenya
6Academic Model Providing Access to Healthcare (AMPATH), Eldoret, Kenya
7Department of Environmental Health and Engineering, Johns Hopkins Bloomberg School of
Public Health, Baltimore, MD USA, 21205
8Department of Medicine, 9Department of Microbiology and Immunology, Indiana University
School of Medicine, Indianapolis, IN, USA, 46204
© The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of
America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-
NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits
non-commercial reproduction and distribution of the work, in any medium, provided the original work is
not altered or transformed in any way, and that the work is properly cited. For commercial re-use,
please contact journals.permissions@oup.com
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FOOTNOTES PAGE
Potential Conflict of Interest: Dr. Brown currently receives research funding and has received
consulting fees in the past from Merck and Co., Inc.
Funding statement: This work was funded by the National Cancer Institute 1U54CA190151-01,
AMPATH-Oncology Institute: HPV and Cervical Cancer in Kenyan Women with HIV/AIDS
(Loehrer, Omenge, Brown) and by the internal pilot grant mechanism of the Indiana University
Simon Cancer Center (Jianjun Zhang).
Meeting where the information has been presented: This work will be presented in Abstract
form at the ASCO 2019 Meeting in Chicago, IL, in June of 2019.
Corresponding author contact information:
Darron R. Brown, MD, MPH
Department of Medicine
Indiana University School of Medicine
Indianapolis, IN 46202
Email: darbrow@iu.edu
Phone: (317) 274-8115
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ABSTRACT
BACKGROUND. Cervical cancer is common in Kenyan women. Cofactors in addition to infection
with oncogenic human papillomavirus (HPV) are likely to be important in causing cervical
cancer, as only a small percentage of HPV-infected women will develop this malignancy.
Kenyan women are exposed to dietary aflatoxin, a potent carcinogen and immunosuppressive
agent, which may be such a co-factor.
METHODS. Demographics, behavioral data, plasma, and cervical swabs were collected from 88
HIV-uninfected Kenyan women without cervical dysplasia. HPV detection was compared
between women with or without plasma AFB1-lys and evaluated in relation to AFB1-lys
concentration.
RESULTS. Valid HPV testing results were available for 86 women (mean age 34.0 years); 49
women (57.0%) had AFB1-lys detected and 37 (43.0%) had none. AFB1-lys detection was not
associated with age, being married, having more than secondary school education, home
ownership, living at a walking distance to health care ≥60 minutes, number of lifetime sex
partners, or age of first sex. AFB1-lys detection and plasma concentrations were associated
with detection of oncogenic HPV types.
CONCLUSIONS. AFB1-lys-positivity and higher plasma AFB1-lys concentrations were associated
with higher risk of oncogenic HPV detection in cervical samples from Kenya women. Further
studies are needed to determine if aflatoxin interacts with HPV in a synergistic manner to
increase the risk of cervical cancer.
Abstract word count: 199
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INTRODUCTION
Oncogenic types of human papillomavirus (“high-risk”, or HR-HPV) are the causative agents of
cervical cancer [1-3]. Cervical cancer is one of the most common malignancies in women living
in sub-Saharan African countries, including Kenya [4]. The incidence rate (15 per 100,000
women per year) and mortality rate (12 per 100,000 women per year) of cervical cancer in
Kenya far exceed rates for women living in the United States (4 and 1 per 100,000 women per
year, respectively) [5]. The reasons why some, but not all women develop malignant
consequences of HR-HPV infection are poorly understood. Co-factors are likely to be important:
women who are infected with human immunodeficiency virus (HIV) have a higher prevalence of
HR-HPV infection and a higher incidence of HPV-associated malignancies compared to HIV-
uninfected women [6-9]. However, while HIV may account for much of the high incidence of
cervical cancer in Kenya, additional co-factors are likely to play a role, and HIV-uninfected
Kenyan women continue to suffer from a high burden of cervical cancer.
Another potentially important co-factor for cervical cancer is chronic ingestion of aflatoxin.
Contamination of corn crops by aflatoxin, a mycotoxin produced by Aspergillus species, is a
food safety and security issue, particularly for people living in developing countries with
temperate and tropical climates [10]. Chronic aflatoxin exposure is associated with a high
incidence of hepatocellular carcinoma, one of the major cancers in men living in sub-Saharan
Africa, Southeast Asia, China, and South Korea [11-14]. Little is known about the effects of
aflatoxin on other human cancers.
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Aflatoxins are also immunosuppressive agents. It is possible the immunosuppression caused by
aflatoxin ingestion could lead to poor immune control of oncogenic HPV infections, and
therefore persistence and increased detection in cervical samples. A study was therefore
conducted to determine if aflatoxin exposure in Kenyan women was associated with increased
detection of oncogenic HPV in cervical samples.
METHODS
Study Population
Women were enrolled from September 2015 to October 2016 at the Academic Model Providing
Access to Healthcare (AMPATH) Cervical Cancer Screening Program (CCSP) at Moi Teaching and
Referral Hospital in Eldoret in a prospective cohort study conducted to investigate biological,
behavioral, and environmental factors that contribute to the risk of oncogenic HPV detection
among women during four years of follow-up. Women aged 18 to 45 years living within 30 km
of Eldoret presenting for screening at the CCSP were asked to participate if they had a normal
visual inspection with acetic acid, or VIA that day. A total of 285 women were approached for
participation in the overall project on which the current analysis was based; 223 gave consent
and were enrolled. The HIV status was not available for one woman, and cervical samples from
two women were inadequate based on negative β-globin control results. These three women
were therefore excluded from the study, leaving 220 women in the cohort, including 115 HIV-
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infected and 105 HIV-uninfected women. Plasma obtained at enrollment was available for 88 of
105 HIV-uninfected women. No blood sample was available for the remaining 17 women. Two
of these 88 women had inadequate swab samples for HPV testing (based on beta-globin
testing), leaving the final number of 86 women included in this analysis.
Interview and questionnaire
Structured face-to-face interviews of enrollees by trained researchers were conducted at
enrollment to capture social, behavioral, and biological information, including age, marital
status, educational level, home ownership, walking distance to the local clinic, number of
lifetime sexual partners, age of first sex, percentage of condom-protected coital events,
number of lifetime pregnancies, and history of cervical cancer screening.
Sample collection
At enrollment, a nurse or physician collected a cervical swab for HPV testing as part of the
pelvic examination and inspection of the cervix. Swabs were placed in standard transport media
and then frozen at -80°C in the AMPATH Reference Laboratory. Plasma was collected and
frozen at -20°C at the same laboratory.
HPV testing
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Specimens were transported on dry ice to the Kenya Medical Research Institute-University of
Massachusetts Medical School (KEMRI-UMMS) laboratory for processing and subsequent
genotyping. The Roche Linear Array was used to determine HPV types (Roche Molecular
Systems, Inc., Branchburg, NJ USA) as previously described [15]. HPV 16-positive, HPV 16-
negative, and human β-globin (used to assess specimen adequacy) controls provided by the
manufacturer were tested with each batch of samples.
Individual HPV types were determined, and in addition, HPV types were grouped into “high-risk”
(HR-HPV) and “low-risk” (LR-HPV) based on the designation in the Roche Linear Array
instructions, or HR-HPV types as designated by the International Agency for the Research on
Cancer (IARC) [16]. HPV types were further grouped into A9 and A7 types [17]. The specific HPV
types included in each of these groups are detailed in the Results.
Aflatoxin-albumin adduct (AFB1-lys) detection in plasma samples
Plasma aflatoxin B1-lysine (AFB1-lys) was measured at the Department of Environmental Health
and Engineering of the Johns Hopkins Bloomberg School of Public Health, using a minor
variation of the method reported by McCoy and colleagues [18]. Serum (150 μL) was spiked
with an internal standard (0.5 ng AFB1-d4-lysine in 100 μL), combined with Pronase (EMD
Millipore, Billerica MA, USA) protease solution (3.25 mg in 0.5 mL phosphate-buffered saline),
and incubated for 18 h at 37°C. Solid-phase extraction–processed samples (Oasis MAX columns;
Waters, Milford, MA, USA) were analyzed with ultra-high pressure liquid chromatography
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(UHPLC)-isotope dilution mass spectrometry on a ThermoFisher Scientific (San Jose, CA, USA)
system composed of a Vanquish UHPLC and a TSQ Quantis triple quadrupole mass
spectrometer in positive electrospray ionization mode [19, 20]. The limit of quantification (<20%
CV) was 14 pg AFB1-lys/mL serum.
Statistical analysis
Demographic and behavioral characteristics of participants were compared between women
with and without detectable AFB1-lys in plasma using t-test, Wilcoxon rank test, or Chi-square
test as appropriate. Logistic regression models were fit to examine 1) associations between HPV
detections and plasma AFB1-lys detection (yes vs. no), and 2) associations between HPV
detections and plasma AFB1-lys concentrations. Demographic and behavioral characteristics
including age, being married, having more than secondary school education, home ownership,
living at a walking distance to health care ≥60 minutes, number of lifetime sex partners, or age
of first sex (vaginal intercourse) were included in all logistic regression models as potential
confounders. All analyses were performed using SAS Version 9.4 (Cary, NC).
Ethics considerations
Study approval was granted from the local review board at Moi Teaching and Referral Hospital
(MTRH) and Moi University, Eldoret, Kenya, the Kenya Medical Research Institute’s Scientific
and Ethics Review Unit (KEMRI-SERU) and the Institutional Review Board of Indiana University.
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RESULTS
Overall characteristics of participants and aflatoxin detection
Of 86 women with an available plasma sample and valid HPV testing results, 49 (57.0%) had
detectable AFB1-lys in plasma and 37 (43.0%) had no detection of AFB1-lys (Table 1). Detection
of AFB1-lys in plasma samples was not associated with age, being married, having more than
secondary school education, home ownership, living at a walking distance to health care ≥60
minutes, number of lifetime sex partners, or age of first sex (Table 1).
Associations of AFB1-lys detection and plasma AFB1-lys concentration with HPV detections
Substantial variation existed in plasma AFB1-lys concentrations among the 49 women with
detected aflatoxin in plasma, ranging from 0.015 to 0.209 pg/µL (data not shown). The counts
and percentages of HPV detections in women with and without plasma AFB1-lys detection, and
N, Median (IQR) of plasma AFB1-lys concentration (pg/uL) for women with and without HPV
detections are shown in Supplementary Table 1.
As shown in Table 2, logistic regression analysis indicated that detection of AFB1-lys in plasma
was associated with detection of A9 HPV types (HPV 16, 31, 33, 35, 52, and 58) as a group in
cervical swabs (OR=15.66, 95%CI=2.03 - 120.87, P=0.008) after adjustment for age, being
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married, having more than secondary school education, home ownership, living at a walking
distance to health care ≥60 minutes, number of lifetime sex partners, or age of first sex .
Detection of AFB1-lys in plasma was also associated with non-HPV 16 A9 detection (OR=39.05,
95%CI=2.34 - 650.82, P=0.011, Supplementary Table 2) but not with other groups of HPV types
(Table 2 and Supplementary Table 2).
Plasma AFB1-lys concentrations were associated with detection of A9 HPV types as a group
(ORper 0.1 pg/uL increase=8.19, 95%CI=1.61 - 41.66, P=0.011) after adjustment for age, being
married, having more than secondary school education, home ownership, living at a walking
distance to health care ≥60 minutes, number of lifetime sex partners, or age of first sex (Table
2). In addition, plasma AFB1-lys concentrations were associated with detection of all HR-HPV
types (ORper 0.1 pg/uL increase=2.88, 95%CI=1.01 - 8.24, P=0.048) and non-HPV 16 A9 types
(ORper 0.1 pg/uL increase=11.21, 95%CI=1.77 - 70.98, P=0.010) after adjustment for age, being
married, having more than secondary school education, home ownership, living at a walking
distance to health care ≥60 minutes, number of lifetime sex partners, or age of first sex
(Supplementary Table 3), but were not associated with other groups of HPV types (Table 2 and
Supplementary Table 3). In addition to plasma AFB1-lys detection or plasma AFB1-lys
concentration, certain demographic and behavioral characteristics significantly associated with
HPV detection were identified from regression models (Table 2 and Supplementary Tables 2
and 3).
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All individual A9 types except HPV 16 were detected more often in women with plasma AFB1-lys
detection than in women without AFB1-lys detection, although these differences were not
statistically significant (Figure 1). The number of detections for any individual A9 HPV type were
small. For example, there was one HPV 16 case in either of the plasma AFB1-lys detected or
undetected women (Figure 1). The AFB1-lys plasma concentration was significantly associated
with HPV 52 detection. The median AFB1-lys plasma concentration for 4 women with HPV 52
detected was 0.088 pg/uL (IQR 0.073 – 0.099). The median AFB1-lys plasma concentration for
82 women with no HPV 52 detected was 0.030 pg/uL (IQR 0.000 – 0.080), P=0.042. Plasma
AFB1-lys concentration was not significantly associated with detection of other individual A9 or
other HPV types (data not shown).
DISCUSSION
Aflatoxins are produced by Aspergillus species during growth or after harvesting of crops such
as corn and ground nuts. More than 4 billion people are exposed to aflatoxins in their foods,
mainly corn and ground nuts [21-23]. In many sub-Saharan countries, corn is the major source
of calories for most people. The poorest families are the most likely to be exposed: Leroy et al.,
showed that higher serum aflatoxin levels in plasma from adult women from Eastern Province
in Kenya were associated with lower household socio-economic status [24].
The incidence of cervical cancer is extremely high in women living in sub-Saharan Africa, where
screening programs as well as vaccination against HPV are available to very few [25, 26].
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Although HIV infection greatly accelerates the natural history of cervical cancer, other cofactors
are likely to be important, because only a small percentage of women infected with oncogenic
HPV will ever develop cervical cancer, whether HIV-infected or not. What factors in addition to
HIV are likely to be important?
In the current study of HIV-uninfected adult Kenyan women, more than half had detectable
aflatoxin in plasma. This is consistent with other studies in sub-Saharan Africa demonstrating a
high percentage of children and adults with aflatoxin detected in blood samples [27-29]. This is
in stark contrast to the situation in the United States where less than 1% of adults have
detectable aflatoxin in blood [30]. In the current study, aflatoxin detection was associated with
an increased risk of detection of oncogenic HPV types in cervical swabs from these women.
Aflatoxin plasma concentrations were also associated with an increased risk of oncogenic HPV
detection, and although median differences between plasma aflatoxin concentrations are
seemingly small, other studies indicate that such differences may have an impact in cancer risk.
There have been several studies in humans to reveal the relationship between exposure to
aflatoxin and the formation of the aflatoxin albumin adduct measured in the study. Based upon
the work of Skipper and Tannenbaum [31] the accumulation of aflatoxin adduct in albumin
would be 30 times the single daily exposure given the 28 to 30 day lifetime of the protein.
When human volunteers were exposed to low levels of C-14 radiolabeled aflatoxin in the diet a
measurement of aflatoxin albumin adduct formation was made by accelerator mass
spectrometry [32]. These findings when combined to the work in Gambia of Wild et al., also
showed a relationship between exposure and albumin addict formation [33]. Collectively, these
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data indicate that at the limit of detection found in this study the daily exposure to aflatoxin
was approximately 100 ng per day. Our available epidemiologic studies from a number of
different populations at risk for liver cancer indicate that exposures up between 2 and 10 µg
per day equate to substantial risk of liver cancer [34].
In addition to its oncogenic properties, aflatoxin is a potent immunosuppressive compound [35,
36]. HPV infections occur more frequently in immunosuppressed people, including those
infected with HIV and other conditions than in otherwise healthy people, as do HPV-associated
cancers [1, 37, 38]. A possible mechanism for increased A9 HPV detection in women with
detectable plasma aflatoxin may be aflatoxin-induced immunosuppression, leading to
suboptimal immunological control of HR-HPV, the causative agents of cervical cancer. In vitro
studies indicate a potent effect of aflatoxin on markers of immunity, even at very low doses
[39]. Documented effects include reduced phagocytosis of monocytes against Candida albicans,
and decreased secretion of interleukins and tumor necrosis factor [39]. Aflatoxin exposure is
associated with reduced salivary IgA and a higher prevalence of malarial parasitemia [39]. In
addition, studies of HIV-infected individuals in Ghana show an association of aflatoxin detection
with higher HIV viral loads [40].
Aflatoxins are potent carcinogens that contribute heavily to the worldwide burden of
hepatocellular carcinoma [41, 42]. For viral pathogens such as the hepatitis viruses B and C
viruses, a synergistic effect with aflatoxin on development of hepatocellular carcinoma is well
established [43, 44]. Although the relationship between aflatoxin exposure and other cancers is
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not well established, it is possible that oncogenic HPV types (such as the A9 HPVs) and dietary
aflatoxin act synergistically in increasing the risk of cervical cancer in Kenyan women. In
addition, aflatoxins have been detected in cervical tissue and could potentially act directly on
cervical cells in the carcinogenic process [45].
If aflatoxin is involved in cervical cancer development, what can be done? Several specific
strategies have been proposed and utilized to reduce aflatoxin exposure [1, 23, 46-49]. Some
of these strategies can be applied before harvest and some after harvest. Some of these
measures include biocontrol using atoxicigenic Aspegillis species, enhancement of host plant
resistance by genetic manipulation, and integrated management systems at the level of the
farm itself.
Some limitations of the present study need to be considered. Our reported results were based
on a modest sample size that may give us suboptimal power for the data analysis. However, we
have obtained statistically significant associations between circulating levels of aflatoxin and
the risk of cervical detection of oncogenic HPV. Another limitation is that dietary factors were
not adjusted as a potential confounder in our data analysis, which might have distorted the
findings of the present study to some extent. This confounding could arise because animal and
human studies have revealed that malnourished subjects exhibit suppressed immunity and
thereby may be susceptible to aflatoxin exposure, aflatoxin adduct formation, and persistent
HPV infection [22, 50]. In addition, the results of our study may be subject to multiple
comparisons due to a relatively large number of the models presented. However, this problem
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is unlikely to occur as all exposure and outcome variables included in the constructed models
were carefully selected in terms of the findings of previous studies and biological relevance.
In summary, AFB1-lys was detected in plasma samples from 57% of HIV-uninfected Kenyan
women without cervical dysplasia. AFB1-lys plasma detection and concentration of aflatoxin
were associated with increased detection of the A9 group of oncogenic HPV types in cervical
samples from HIV-uninfected Kenyan women who had normal VIA examinations. Further
studies are needed to determine if exposure to aflatoxin interacts with HPV infection (and
possibly HIV co-infection) to modulate the risk of cervical cancer in women in Kenya and other
developing countries in which aflatoxin exposure is frequent.
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Table 1. Characteristics of women with or without plasma AFB1-lys detection
Characteristics
Plasma AFB1-lys detection
No
N=37
Yes
N=49
P-value
Median age (IQR)
34.0 (29.0, 38.0)
34.0 (30.0, 38.0)
.8181
Married
24 (65%)
36 (73%)
.3902
More than secondary school education
4 (11%)
9 (18%)
.3332
Home ownership
9 (24%)
15 (31%)
.5202
Walking distance to health care ≥60 mins
2 (5%)
6 (12%)
.4573
Median number of lifetime sex partners (IQR)
2.0 (1.0, 4.0)
3.0 (1.0, 4.0)
.6604
Median age of first sex (IQR)
18.0 (17.0, 20.0)
17.0 (16.0, 20.0)
.5061
1P-value from t-test
2P-value from Chi-square test
3P-value from Fisher’s exact test
4P-value from Wilcoxon rank sum test
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Table 2. Logistic regression analysis of associations of IARC HR-HPV, A9 HPV, A7 HPV and ≥2 HR-HPV types detection with plasma AFB1-lys
detection, plasma AFB1-lys concentration and characteristics of women
Variables
IARC HR-HPV1
A9 HPV2
A7 HPV3
≥2 HR-HPV types4
OR (95%CI)
P-
value
OR (95%CI)
P-
value
OR (95%CI)
P-
value
OR (95%CI)
P-
value
In models with plasma AFB1-lys detection
Plasma AFB1-lys detection
1.47 (0.48-4.52)
0.505
15.66 (2.03-120.87)
0.008
0.23 (0.03-1.86)
0.170
1.76 (0.11-28.89)
0.693
Age
1.00 (0.90-1.12)
0.934
1.06 (0.91-1.23)
0.441
0.90 (0.74-1.09)
0.266
0.83 (0.65-1.07)
0.158
Married
0.46 (0.14-1.59)
0.220
0.20 (0.04-1.10)
0.064
7.84 (0.46-134.09)
0.155
0.09 (0.005-1.82)
0.117
More than secondary school education
1.79 (0.40-7.96)
0.446
4.64 (0.60-36.13)
0.143
1.33 (0.09-19.04)
0.834
9.77 (0.61-157.25)
0.108
Home ownership
0.45 (0.10-2.08)
0.309
0.04 (0.002-0.82)
0.037
1.52 (0.18-12.33)
0.696
0.48 (0-2.90)
0.531
Walking distance to health care ≥60 mins
1.39 (0.25-7.73)
0.707
0.93 (0.09-9.51)
0.954
2.17 (0.09-51.93)
0.633
1.86 (0-13.14)
0.999
Number of lifetime sex partners
1.26 (0.98-1.62)
0.071
1.06 (0.73-1.52)
0.774
1.67 (1.08-2.58)
0.021
1.00 (0.57-1.76)
0.990
Age of first sex
1.14 (0.96-1.36)
0.135
1.23 (0.98-1.54)
0.074
1.31 (0.97-1.77)
0.078
0.96 (0.62-1.49)
0.858
In models with plasma AFB1-lys
concentration
Plasma AFB1-lys concentration (pg/uL)5
1.76 (0.57-5.45)
0.331
8.19 (1.61-41.66)
0.011
0.26 (0.02-2.86)
0.268
1.12 (0.04-30.43)
0.947
Age
1.00 (0.90-1.12)
0.935
1.05 (0.92-1.21)
0.471
0.90 (0.74-1.09)
0.268
0.78 (0.59-1.03)
0.082
Married
0.45 (0.13-1.54)
0.202
0.21 (0.04-1.16)
0.074
7.19 (0.41-125.35)
0.176
0.05 (0.001-1.71)
0.096
More than secondary school education
1.92 (0.43-8.56)
0.393
5.05 (0.70-36.50)
0.108
0.88 (0.07-11.57)
0.924
23.22 (0.92-585.46)
0.056
Home ownership
0.47 (0.10-2.15)
0.334
0.08 (0.006-1.15)
0.063
1.37 (0.17-11.26)
0.771
0.48 (0-2.63)
0.515
Walking distance to health care ≥60 mins
1.24 (0.21-7.23)
0.809
0.60 (0.05-7.12)
0.689
2.02 (0.08-54.48)
0.676
13.97 (0-265.48)
0.999
Number of lifetime sex partners
1.28 (0.99-1.64)
0.056
1.16 (0.81-1.65)
0.415
1.61 (1.05-2.46)
0.028
0.86 (0.43-1.74)
0.678
Age of first sex
1.15 (0.96-1.38)
0.122
1.23 (0.98-1.53)
0.070
1.31 (0.98-1.76)
0.076
1.01 (0.65-1.55)
0.982
1IARC HR-HPV: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66
2A9 HPV: HPV 16, 31, 33, 35, 52, 58
3A7 HPV: HPV 18, 39, 45, 59, 68
4HR-HPV (High-Risk HPV): HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, IS39
5OR (95% CI) per 0.1 pg/uL increase of plasma AFB1-lys concentration
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