Methods
Study population
The Ludwig-McGill study cohort study of HPV natural history recruited
women at maternal health clinics in São Paulo, Brazil, between 1993 and
1997. Details of the study design have been published
elsewhere.20 Eligible women had to be between 18-60
years old, permanent residents of São Paulo, not currently pregnant,
with an intact uterus, and with no history of treatment for cervical
disease. During the first year, women were followed-up every 4 months;
at each visit they provided cervical specimens for HPV genotyping and
underwent an interview administered by study nurses collecting data on
sociodemographic information, reproductive health, sexual behaviors,
smoking, and diet. Women provided signed informed consent to study
participation. The study was approved by the ethical review boards of
all participating institutions: McGill University, Montreal, Canada;
University of Toronto, Ontario, Canada; and the Ludwig Institute for
Cancer Research and the Hospital Maternidade Vila Nova Cachoeirinha,
both in São Paulo, Brazil.
The current analysis is restricted to a random subset of 505 women
sampled from the full cohort who had a follow-up visit within 10 days of
the 1-year follow-up date, had valid samples (either β-globin or
HPV-positive samples) at the baseline and 1-year visits, and who had
complete questionnaire and HPV genotyping data. Our previous publication
details the selection process, baseline characteristics, and the
betapapillomavirus prevalence at baseline (1st visit)
and 1-year (4th visit) in this subset of
women.19 For the current study, we extended genotyping
to the 4 month (2nd visit) and 8-month
(3rd visit) samples collected from these women in
order to measure betapapillomavirus incidence rates over the first year
of follow-up and increase the number of observations for analyses.
HPV genotyping
Exfoliated cervical cells were digested with 100 µg/ml proteinase K for
3-18 hours at 55°C, and DNA was obtained by spin column chromatography.
Samples were tested for alphapapillomaviruses by polymerase chain
reaction (PCR) amplification using MY09/11 or PGMY09/11 generic primers,
and genotyped using hybridization with HPV type-specific oligonucleotide
probes or restriction fragment length polymorphism analysis. This
methodology allowed the identification of potentially more than 40
genital alphapapillomavirus genus types from the following species: α-1:
HPVs 32, 42; α-3: HPVs 61, 62, 72, 81, 83, 84, 89; α-4: HPV57; α-5: HPVs
26, 51, 69, 82; α-6: HPVs 53, 56, 66; α-7: HPVs 18, 39, 45, 59, 68, 70;
α-8: HPV40; α-9: HPVs 16, 31, 33, 35, 52, 58, 67; α-10: HPVs 6, 11, 44;
α-11: HPVs 34, 73; α-13: HPV54; and α-14: HPV71.21
The presence of betapapillomaviruses was assessed by a type-specific,
multiplex genotyping PCR assay followed by genotyping via a bead-based
Luminex technology.22 This assay distinguishes 43
betapapillomavirus genus types from the following species: β-1: HPVs 5,
8, 12, 14, 19, 20, 21, 24, 25, 36, 47, 93, 98, 99, 105, 118 124, 143;
β-2: HPVs 9, 15, 17, 22, 23, 37, 38, 80, 100, 104, 107, 110, 111, 113,
120, 122, 145, 151; β-3: HPVs 49, 75, 76, 115; β-4: HPV92; and β5: HPVs
96, 150. In this assay, results are expressed as the median fluorescence
intensity (MFI) of at least 100 beads per bead set. For each probe, the
MFI values obtained when no PCR product was added to the hybridization
mixture were considered the background values.
Different bead preparations
impact MFI values; because the samples from visits 2 & 3 were tested
using a different preparation of beads than the samples from visits 1 &
4, a different MFI cutoff value was used for these visits. Based on MFI
values from positive and negative controls reads, cutoffs were computed
by adding 20 MFI to 1.1X the median background values in samples
obtained in visits 1 and 4;20 and by adding 50 MFI to
1.1X the median background values for samples obtained in visits 2 and
3, with the exception of HPV100 for which the cutoff was computed by
adding 100 MFI to 1.1X the median background values for visits 2 and 3.
Statistical analysis
All analyses were type-specific, with HPV type as the unit of
observation. The prevalences of betapapillomaviruses were summed across
the four visits to derive a time-averaged measure of prevalence over the
first year of the study. The incidence rates of betapapillomaviruses
were calculated in women who were negative for that specific
betapapillomavirus type at the previous visit, with the denominator
being the time between visits. We pooled results by summing observations
over all HPV types.
To assess whether betapapillomaviruses and alphapapillomaviruses occur
more frequently together than expected, we used permutation tests.
Permutation tests work by performing rearrangements of the observed data
without replacement (permutations).23 The permutations
allow deriving an expected distribution of betapapillomaviruses and
alphapapillomaviruses under the assumption that infections are
independently distributed across individuals. Because there was evidence
of within-genus clustering, we implemented two versions of the
permutation test (Figure 1). The first test assumed full independence of
all HPV types; in this case, the results for each individual HPV type
are permutated across participants, with each HPV type resampled
separately. This first test will give the expected distribution of
type-specific detections assuming all HPV types are fully independent,
both across and within genera. The second test assumed independence
between papillomavirus genera but not within genus; in this case, the
set of results from all HPV types within a genus from the same person
are permutated across participants, with each genus resampled
separately. This second test will give the expected distribution of
type-specific detections assuming that alphapapillomaviruses are
independent from betapapillomaviruses and vice-versa, but accounting for
the tendency of HPV types from the same genus to cluster together. We
performed 2500 permutation resamples for both tests. We divided the
observed number of co-detections by the mean number of co-detections in
the permutation resamples to obtain observed over expected values. The
95% confidence intervals were obtained using the 2.5-97.5 percentiles
of the permutation resample distribution.
To assess the cross-sectional association between alphapapillomavirus
and betapapillomavirus prevalence, we calculated odds ratios (OR) using
random effects logistic regression models, with a woman-level random
intercept. To assess the prospective association between
alphapapillomavirus and betapapillomavirus incidence, we calculated
hazard ratios (HR) using Cox proportional hazard regression models,
using the robust sandwich estimate of the covariance to account for
multiple HPV types per woman.24 All models were fitted
separately for alphapapillomavirus and betapapillomavirus types and used
the HPV type as the unit of analysis. To assess interactions between
genera, the models included as predictors whether the woman was positive
for any HPV type of a different genus at either the same visit sample
(logistic regressions) or the previous visit sample (Cox regressions).
Multivariable models were adjusted for age and sexual behaviors reported
since the previous visit. Sexual behavior variables were included as a
priori predictors because our previous publication had found that
lifetime number of sex partners might be inversely associated with
betapapillomavirus prevalence.19