Association of Plasma Aflatoxin With Persistent Detection of Oncogenic Human Papillomaviruses in Cervical Samples From Kenyan Women Enrolled in a Longitudinal Study

Background Cervical cancer is common among Kenyan women and is caused by oncogenic human papillomaviruses (HR-HPV). Identification of factors that increase HR-HPV persistence is critically important. Kenyan women exposed to aflatoxin have an increased risk of cervical HR-HPV detection. This analysis was performed to examine associations between aflatoxin and HR-HPV persistence. Methods Kenyan women were enrolled in a prospective study. The analytical cohort for this analysis included 67 HIV-uninfected women (mean age 34 years) who completed at least two of three annual study visits and had an available blood sample. Plasma aflatoxin was detected using ultra-high pressure liquid chromatography (UHPLC)-isotope dilution mass spectrometry. Annual cervical swabs were tested for HPV (Roche Linear Array). Ordinal logistic regression models were tted to examine associations of aflatoxin and HPV persistence. Results Aflatoxin was detected in 59.7% of women and was associated with higher risk of persistent detection of any HPV type (OR = 3.03, 95%CI = 1.08–8.55, P = 0.036), HR-HPV types (OR = 3.63, 95%CI = 1.30–10.13, P = 0.014), and HR-HPV types not included in the 9-valent HPV vaccine (OR = 4.46, 95%CI = 1.13–17.58, P = 0.032). Conclusions Aflatoxin detection was associated with increased risk of HR-HPV persistence in Kenyan women. Further studies are needed to determine if aflatoxin synergistically interacts with HR-HPV to increase cervical cancer risk.


Introduction
Cervical cancer is a common malignancy among Kenyan women [1][2][3]. The incidence rate of cervical cancer in Kenya is 31.3 per 100,000 women per year and the mortality rate is 25 per 100,000 women per year, gures considerably higher than those in wealthy countries [4,5]. Oncogenic types of human papillomaviruses ("high-risk", or HR-HPV) are the causative agents of cervical cancer. However, only a small percentage of women infected with HR-HPV will develop cancer, indicating the importance of cofactors associated with HR-HPV persistence that contribute to the occurrence of cervical cancer [6]. Women with persistent infection with oncogenic HPV are at signi cantly higher risk of cervical cancer [7].

-lys) detection in plasma samples
Plasma a atoxin B1-lysine (AFB 1 -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 [15,20]. Brie y, plasma (150 µL) was spiked with an internal standard (0.5 ng AFB 1 -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 hours at 37°C.
Solid-phase extraction-processed samples (Oasis MAX columns; Waters, Milford, MA, USA) were analyzed with ultra-high pressure liquid chromatography (UHPLC)-isotope dilution mass spectrometry on a ThermoFisher Scienti c (San Jose, CA, USA) system composed of a Vanquish UHPLC and a TSQ Quantis triple quadrupole mass spectrometer in positive electrospray ionization mode [21,22].

Persistent HPV detection
Type-speci c HPV testing results obtained from the enrollment, 12-month and 24-month cervical samples were combined to determine the detection category of each speci c HPV type for each woman. Three categories of HPV detection were determined: No detection, Incident Detection, and Persistent Detection. To be included, two or three of a participant's cervical samples (Enrollment, 12-month, or 24-month) had to be available; one of the three samples could be missing. The type-speci c HPV detection categories were de ned as follows: I. No detection: No detection for the speci c HPV type at any of the three timepoints; II. Incident detection: One sample positive for detection of a speci c HPV type, but other samples were negative for that type; III. Persistent detection: Two samples taken one year apart, or two years apart were positive for detection of a speci c HPV type. The third sample could be negative for that type (or missing).
At the level of study participants, a woman's HPV detection status was de ned as the highest level of HPV detection category in the descending order of "Persistent detection," "Incident detection," and "No detection" among her type-speci c HPV detection episodes within a combined group of speci c HPV types. For example, a woman is classi ed as at the status of persistent detection in HR-HPV if any typespeci c "persistence detection" episode is identi ed among the HR-HPV types. The subsequent analysis of HPV detection was conducted at the level of participant.

Statistical analysis
Demographic and behavioral characteristics of participants at enrollment (age, marital status, educational level, home ownership, walking distance to health care of ⩾60 min, number of lifetime sex partners, and age of rst sex) were summarized by descriptive statistics and compared between women with and without detectable plasma AFB 1 -lys using t-tests, chi-square tests, or Wilcoxon rank sum tests.
Frequencies and percentages of HPV detections ("No detection", "Incident detection" and "Persistent detection") in women were compared between those with and without detectable plasma AFB 1 -lys using chi-square tests or Fisher's exact tests. Plasma AFB1-lys concentration (pg/uL) were summarized in mean, standard deviation (std), median and interquartile range (IQR) and compared among women with different HPV detection status using Wilcoxon rank sum tests. In addition, ordinal logistic regression models were tted to examine associations of HPV detection (persistent detection vs. incident detection vs. no detection) with plasma a atoxin detection, controlling for demographic and behavioral characteristics of the women as confounders. The proportional odds assumption was examined for each tted ordinal logistic regression model to ensure validity of the model. All analyses were performed using SAS Version 9.4 (Cary, NC).

Results
Overall characteristics of participants and a atoxin (AFB 1lys) detection  A total of 87 women in the original cohort had plasma samples tested for AFB 1 -lys, including 67 women consisting of the analytical cohort and 20 women who did not complete at least two study visits.
Comparisons between the 67 women in this analytical cohort and the 20 women who did not complete at least two visits were conducted with respect to the demographics/behavioral characteristics and plasma AFB1-lys detection/concentration. No signi cant differences in these variables were found between the two groups of women (data not shown).

Association of plasma AFB 1 -lys detection with persistent HPV detection
Frequencies and percentages of HPV detections ("no detection", "incident detection" and "persistent detection") in women with and without plasma AFB 1 -lys detection and mean (STD) and median (IQR) of plasma AFB 1 -lys concentration (pg/uL) among women with different HPV detections are shown in Table   2. There was a trend of signi cantly increasing plasma AFB 1 -lys concentrations among women who had no detection, incident detection, or persistent detection for any HPV type (p = 0.036), HR-HPV types (p = 0.020), or vaccine-unprotected HR-HPV types (p = 0.017) ( Table 2). Similar trends in increasing plasma AFB 1 -lys concentrations were observed for some other groups of HPV types, however, these were not signi cant (Table 2).  29.6% for incident detection, p = 0.038). Similar patterns of HPV detections between women with and without detectable plasma AFB 1 -lys were found for all other HPV combination types except for LR-HPV types. However these were not statistically signi cant, possibly due to small sample sizes.
A total of 13 episodes of type-speci c persistent HPV detections occurred in 12 women (Table 3). Among these episodes, 12 were HR-HPV types, including 10 episodes that occurred in 9 of 40 women with detectable plasma AFB 1 -lys, and 2 episodes occurring in 2 of 27 women without detectable plasma AFB 1lys (Table 3). HPV 18 was the most frequently detected persistent type (3 episodes), all occurring in women with detectable plasma AFB 1 -lys (Table 3).  Table 4). The proportional odds assumption was validated for each ordinal logistic regression model. There was no statistically signi cant association of detectable plasma AFB 1 -lys with persistent detection of sub-groups of HR-HPV types, for HR-HPV types protected by the 9-valent HPV vaccine (Vaccine-protected HR-HPV types), or for low-risk (LR) HPV types (Supplemental Table 1).

Discussion
In this longitudinal study, women with detectable a atoxin biomarkers in plasma had a higher risk of persistent detection of oncogenic cervical HPV. Although only a small percentage of HPV-infected women will eventually develop cervical cancer, women with persistent detection of HR-HPV are at the highest risk for this malignancy [23,24]. A atoxins are mycotoxins produced by certain Aspergillus species during growth or after harvesting of corn and several other crops [25]. These compounds are classi ed by the International Agency for Research on Cancer (IARC) as class I carcinogens [26]. In addition, a atoxins are potent immunosuppressive agents [27][28][29][30]. Exposure to a atoxins contributes heavily to the worldwide burden of hepatocellular carcinoma, but the contribution of a atoxin exposure to other cancers is unknown [31,32]. This study revealed an association between a atoxin exposure and persistent HR-HPV detection, the major risk factor for cervical cancer.
A previous cross-sectional study showed signi cant associations between plasma a atoxin biomarkers and detection of A9 HPV types in cervical samples among HIV-uninfected Kenyan women [15]. The current analysis employed a subset of the original cohort with the longitudinal follow-up data on HPV testing, disclosing the relationship of a atoxins with persistent detections of HR-HPV, and raising the possibility that a atoxin could be a contributing factor to cervical cancer. We are not aware of other studies describing an association of a atoxin with HPV persistence, cervical dysplasia, or cancer.
It is possible that HR-HPV types and dietary a atoxin act synergistically in increasing the risk of cervical cancer in Kenyan women. A atoxins have been detected in cervical tissue and could potentially act directly on cervical cells in the carcinogenic process, but this hypothesis has not been studied [33]. It is also possible the immunosuppression caused by a atoxin could lead to poor immune control of oncogenic HPV infections, leading to persistence. These hypotheses need to be further investigated. In addition, it has a tremendous public health impact to investigate the role of the interaction between a atoxin exposure and persistent HPV infection in the etiology, pathogenesis, and prevention of cervical cancer and its precursor lesions in large epidemiological studies especially in developing countries.
A atoxin exposure is widespread in many sub-Saharan African countries, largely due to consumption of contaminated corn, the major source of daily calories for many people, especially for poor families [34][35][36]. Leroy et al., showed higher serum a atoxin levels from adult Kenyan women associated with lower household socio-economic status [37]. Women with the lowest socio-economic status also have the lowest rates of cervical cancer screening, and therefore bear the highest burden of cervical cancer [38,39]. A atoxin, as a potential environmental risk factor of cervical cancer, demands more recognition for public health emphasis.
Some limitations of the present study include a modest sample size, as not all women initially analyzed for the association of a atoxin detection and HR-HPV detection continued in the longitudinal study.
However, our analysis showed that there were no signi cant differences in demographic/behavioral characteristics and plasma AFB 1 -lys detection/concentration between the women who remained in the original study and included in this analysis compared to those who did not continue in the original study. Another potential limitation is that dietary factors that modulate immune functions were not included as potential confounders in our data analysis, which could possibly distort the ndings of the present study. For example, malnourishment may contribute to suppressed immunity and render such women more susceptible to the toxic effects of a atoxin, and thus, more prone to persistent HPV infection [34,40]. 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 is unlikely because all exposure and outcome variables included in the constructed models were carefully selected in terms of the ndings of previous studies and biological relevance.
In summary, detection of plasma a atoxin biomarkers was associated with increased persistence of oncogenic HPV types, in cervical samples from HIV- written informed consent, either in Swahili or English, for participation in the study and for use of clinical specimens. All study procedures were performed in accordance with relevant guidelines and regulations outlined by the Ethics Review Boards indicated above.

Consent for publication
The Authors give the Publisher the permission to publish this work.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author (Dr. Brown and the additional authors) upon reasonable request and with permission of AMPATH.

Competing interests
Dr. Brown currently receives research funding and has received royalties and consulting fees in the past from Merck and Co., Inc. Dr. Brown serves on the Scienti c Advisory Board for PDS, Inc. The other Authors do not possess any potential con icts of interest.
Funding National Cancer Institute, United States, 1U54CA190151-01, and National Cancer Institute, United States, P30 CA082709 Authors' contributions YT contributed to conceptualization, methodology, data curation, formal analysis, validation, writing (original, review and editing).
PT contributed to study supervision, investigation, writing (review and editing).
OO contributed to funding acquisition, project administration, and writing (review and editing).
JZ contributed to funding acquisition, writing (review and editing).
TM contributed to investigation (performance of laboratory tests).
KM contributed to study supervision and writing (review and editing).
JG contributed to methodology, formal analysis, resources, and writing (review and editing).
JS contributed to investigation (performance of laboratory tests).
EM contributed to investigation (performance of laboratory tests).
AE contributed to study supervision, and writing (review and editing).
PL contributed to funding acquisition, project administration, and writing (review and editing).
DB contributed to funding acquisition, project administration, conceptualization, methodology, study supervision, writing (original draft, review and editing).