INTRODUCTION
Squamous cervical cancer is the second most common cancer in women worldwide.1 2-4
HIV-1 and HPV are both transmitted sexually and share common risk factors. It is therefore not surprising that HIV-1-infected women have a higher prevalence of cervical HPV infections than HIV-negative women,5,6 7,8 9 10-12 13 14
Most epidemiological studies of HPV infection have been performed in non-HIV-infected women,14-16 14,16 15-19 20 13 21 22 22-25
It is widely accepted that a specific diagnosis of HPV infection requires detection of HPV nucleic acids in clinical samples. At present, routine diagnosis of HPV infection in Spanish hospitals involves 2 steps: first, qualitative HPV detection is performed by second-generation hybrid capture (HC-2) and only those samples that are positive are subsequently assayed with multiplex polymerase chain reaction (mPCR). However, conflicting results are available on the agreement between these techniques in routine clinical practice.26,27
The objective of this study was to provide epidemiological data regarding prevalence, persistence, clearance, and incidence of different HPV genotypes in cervical samples obtained from HIV-1-infected women without previous cervical pathology background and normal Papanicolau smears. A secondary objective was to compare the performance of HC-2 and mPCR as diagnostic tools in this setting.
PATIENTS AND METHODS
Study Design
This was a single-center retrospective analysis of a prospective cohort of HIV-1-infected women studied for HPV infection using HC-2. The hospital's ethics committee approved the prospective protocol.
Study Population
The study cohort included HIV-1-infected women who were naive for highly active antiretroviral therapy (HAART), who had CD4 counts below 500 cells per cubic millimeter, and who had consented to participate. At the time of recruitment, women had to have 2 consecutive normal Papanicolaou smear tests and no prior history of cervical dysplasia or cervical cancer in their gynecological records. The cohort was recruited between March 1999 and January 2003 from all HIV-1-infected women who attended our center. These women have been prospectively followed to date. Of 160 women screened, 18 presented cytological abnormalities at baseline and 3 refused to participate in the study.
To be included in the present analysis, women had to have at least 2 frozen cervical secretion samples available for testing taken at least 24 weeks apart (baseline and follow-up). If more than 1 follow-up sample was available, the most recent one was analyzed.
Date of birth, dates of cervical sampling, date of HIV diagnosis, previous pregnancies, current or previous intravenous drug use, and current or previous smoking habit were auto recorded. Most recent (±1 week from the date of sample) and nadir CD4 counts were determined by flow cytometry. HIV-1 RNA load (HIV_VL) (±1 week from the date of sample) was determined by Nuclisens (detection limit 80 copies/mL; bioMerieux, Inc, Durham, NC).
Detection of HPV Infection
Cervical secretions were analyzed for HPV DNA [Digene Cervical Sampler system (Digene Corporation, Gaithersburg, MD), which includes cervical brush and preserving solution]. Samples were frozen at −20°C until analysis by HC-2. The remainder was frozen until analysis by mPCR.
Second-Generation Hybrid Capture
The HC-2 consists of molecular hybridization by means of an RNA probe. Reaction is detected by chemoluminescence, with qualitative detection of the presence of the different DNA of high intermediate oncogenic risk (HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and low oncogenic risk (HPV genotypes 6, 11, 42, 43, and 44). The HC-2 was performed according to the supplier's instructions (Digene Corporation). Results were quantified by a luminometer and expressed as the ratio between the light units emitted by the sample being tested and the mean of those emitted by 3 positive controls. Samples with a ratio ≥1 (1 pg/mL of HPV DNA) were considered positive for HPV.
Multiplex PCR
The mPCR was subsequently performed on the same sample used for HC-2, as follows: DNA was extracted from cell suspensions using the QiAMP Viral RNA kit (QIAGEN, Hilden, Germany). HPV detection and typing was performed using the F-HPV typing (Molgentix, Barcelona, Spain) following the manufacturer's instructions. Briefly, extracted DNA was amplified using a multiplex F-HPV polymerase chain reaction (PCR) with a set of 16 fluorescently labeled primers recognizing HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 within the E6 and E7 regions of the HPV genome, the regions most likely to be retained after viral integration. A human short tandem repeat included in the same mPCR kit was used as an internal control to check for DNA integrity and absence of PCR inhibitors. The F-HPV amplification was performed in a final PCR volume of 25 μL containing 20 μL of ready-to-use reaction mixture and 5 μL of total extracted DNA for 35 cycles lasting 30 seconds at 95°C, 30 seconds at 64°C, and 30 seconds at 95°C and a final extension of 10 minutes at 60°C. Positive and negative controls were included in each run.
Fragment analysis was carried out by electrophoresis of 2 μL of PCR product in 20 μL highly deionized formamide (Applied Biosystems, Foster City, CA) and 0.3 μL GeneScan-500 LIZ through a 36 cm capillary on a 3130 Genetic Analyzer (Applied Biosystems). Automated HPV genotypes were generated using GeneMapper 3.7 software recognizing fluorescent peaks with a specific color (FAM, VIC, NED, and PET dyes) and size (amplicons between 156 and 489 base pairs). Samples showing at least 1 HPV type-specific peak were scored as positive. Samples showing a single peak on the electrophoretogram were interpreted as consistent with a single infection; the presence of 2 or more peaks reflected simultaneous infection with multiple HPV types. The absence of HPV-specific peaks was only interpreted as negative result in presence of detectable products for the human short tandem repeat marker.
Cytological Diagnosis
Cervical cytology was assessed by the Papanicolaou test, a standard diagnostic technique used routinely in gynecologic examinations. The cytological samples were obtained at the baseline and the last follow-up visit from the exocervix with an Ayre spatula and from the endocervix with a cotton swab. The cytological changes were classified according to the Bethesda System28
Statistical Analysis
This cohort study was planned as an exploratory study; thus, no formal calculation of the sample size to confirm a prespecified hypothesis was performed. The complete sample, defined as all patients with at least 1 mPCR and HC-2 result available at baseline and follow-up, was analyzed.
Definitions
Persistence was defined as detection of HPV infection in both baseline and follow-up samples. Clearance was defined as detection of HPV infection at baseline and absence of detection in the follow-up sample. Incidence was defined as absence of HPV detection at baseline but detection of HPV infection in the follow-up sample. Type-specific prevalence, persistence, clearance, and incidence were also assessed by means of mPCR.
Statistical Comparisons
In this study, we compared the prevalence, persistence, clearance, and incidence obtained by mPCR and HC-2. Differences between diagnostic techniques were evaluated by the χ2 test for qualitative variables. The 95% confidence intervals (CIs) were calculated. Time to persistence, clearance, and incidence was calculated in months (95% CI) as estimated with the Kaplan-Meier method. The degree of agreement between HC-2 and mPCR was analyzed using the kappa index (K). Kappa is a measurement of the degree of nonrandom agreement between observers or between measurements of the same variable. Kappa values lie between 0 and 1, with 0 indicating lack of agreement and 1 indicating complete concordance.
Clearance and incidence of HPV infection were also analyzed with Kaplan-Meier curves and differences were assessed with the Mantel-Haenzel log-rank test. Bivariate analysis and multivariate proportional hazards regression (Cox regression) were performed to determine which of the following factors was associated with clearance and incidence: age, time since HIV diagnosis, baseline CD4, baseline HIV-1 RNA load, and baseline number of genotypes. The association between HPV infection status and cytologic lesion grade (ASCUS, LSIL, HSIL, and CA) was analyzed by the χ2 test. The cytologic lesion grade was grouped into 2 categories according to the severity of lesion (ASCUS/LSIL and HSIL/CA) to improve the statistical power of the model. Odds ratios and hazard ratios and their corresponding 95% CIs were estimated. A P value ≤0.05 was considered statistically significant.
Data analysis was performed using the statistical software programs SAS version 9.1 (SAS Institute Inc, Cary, NC).
RESULTS
Patient Characteristics
From the original cohort of 139 HIV-1-infected women, 93 individuals met the inclusion criteria and were included in this study (Fig. 1 ). Patients were excluded for the following reasons: frozen samples were not available in 21 subjects, 12 had only 1 sample available, and in 3 subjects, the follow-up sample available was collected less than 24 weeks after the baseline sample. In addition, 10 patients were lost to follow-up (2 deaths).
FIGURE 1: Flow chart according to the results of both diagnostic procedures.
The median age was 35 years [mean (SD), 35 (7) years; range, 20-64 years]. The median baseline CD4 count was 274 cells per cubic millimeter [mean (SD), 283 (157) cells/mm3 ; range, 14-499 cells/mm3 ]. Thirty-four percent had nadir CD4 counts below 200 cells per cubic millimeter. The median HIV viral load was 10,000 copies per milliliter [mean (SD), 55,055 (146,710] copies/mL, range, 80-999,999 copies/mL]. All patients started HAART soon after the baseline sample was taken and almost all follow-up samples were while patients were receiving HAART.
Forty-nine percent (46/93) of the patients had prior history of intravenous drug abuse; 39% (36/93) had become pregnant at least once; only 10% (9/93) had no previous history of smoking; and 5% (5/93) of the patients had no information on risky behavior. Table 1 summarizes the baseline characteristics of the study population with stratification according to HPV status (HPV positive or HPV nondetectable by means of HC-2 results).
TABLE 1: Baseline Characteristics Stratifying the Study Population in HPV Positive or HPV Nondetectable by Means of Hybrid Capture Results
Detection of HPV Infection
Multiplex PCR
The mPCR yielded a result (either positive or negative) for HPV infection in all patients (100% applicability). HPV infection (all HPV genotypes) was detected in 59 patients [mean (SD) age, 34 (8) years; range, 20-64 years]. Therefore, the baseline prevalence of HPV infection was 63% (95% CI, 53% to 73%). Twenty patients (22%) were infected with only 1 genotype and 39 (42%) with multiple genotypes. The prevalence at follow-up (mean, 20 months; range, 6-44 months after baseline) was 61% (57/93 patients; 95% CI, 51% to 71%), and 29 of 93 subjects (31%) were infected with 1 genotype, whereas 28 of 93 women (30%) were infected with multiple genotypes. HPV-16 genotype (28%) was the most predominant, followed by HPV-33 (18%), HPV-52 (12%), HPV-58 (11%), and HPV-39 (11%).
Table 2 summarizes the prevalence (at baseline and follow-up), persistence, clearance, and incidence of the different HPV genotypes according to the mPCR method. The actuarial probability of clearance at 36 months was 16% and the incidence was 45% (Fig. 2 ). No variable was associated with clearance or incidence in the bivariate and multivariate analyses.
TABLE 2: Prevalence (Baseline and Follow-up), Persistence, Clearance, and Incidence of the Different HPV Genotypes
FIGURE 2: Kaplan-Meier plots of time to HPV infection clearance (upper plots) and incidence (lower plots), as assessed by mPCR (left) and HC-2 (right). The 95% CIs are indicated by dashed lines. Circles indicate censored data.
Second-Generation Hybrid Capture
HPV infection was detected in 38 patients by HC-2 [mean age, 34 (6) years; range, 23-55 years] irrespective of HPV type, a figure which corresponds to a baseline prevalence of 41% (95% CI, 31% to 51%). The prevalence of high-, low-, and both high- and low-risk HPV type infection was 23%, 3%, and 15%, respectively. Overall prevalence of HPV infection at follow-up was 47% (44/93 patients; 95% CI, 37% to 57%), with 25 (27%), 5 (5%), and 14 (15%) patients being infected with high-, low-, and both high-risk and low-risk HPV types, respectively. The actuarial probability of clearance at 36 months was 34%, and the incidence was 62% (Fig. 2 ). No variable was associated with clearance or incidence in bivariate and multivariate analysis.
mPCR Versus HC-2
Table 3 shows the comparison between mPCR (positive to any HPV genotype versus nondetectable) and HC-2 (positive to any HPV genotype versus nondetectable) for prevalence, persistence, clearance, and incidence estimations of HPV infection. mPCR detected 22% more HPV infections at baseline and 14% more HPV infections at follow-up. Table 4 displays the agreement between mPCR and HC-2 in the diagnosis of HPV infection.
TABLE 3: Prevalence, Persistence, Clearance, and Incidence Considering Positive or Nondetectable Hybrid Capture or mPCR, Both HPV Diagnostic Techniques
TABLE 4: Agreement Between HC-2 and mPCR in HPV Infection (n = 93)
Cytological Diagnosis
The cervical cytological follow-up sample was not available in 2 patients. According to the cytological evaluation, 64% (58/91) of women remained free of cervical abnormalities, 10% (9/91) developed an ASCUS, 16% (15/91) LSIL, 8% (7/91) HSIL, and 2% (2/91) CA.
There was a clear association between persistent HPV-positive status and development of cervical abnormalities (P < 0.02). For women with normal baseline cervical cytology and persistent HPV infection (positive for HPV infection at baseline and follow-up) determined by mPCR, 48% (24/50) had normal cytology, 36% (18/50) had ASCUS/LSIL, and 16% (8/50) had HSIL/CA at follow-up. This distribution according to HC-2 was 30% (8/27) with normal cytology, 44% (12/27) with ASCUS/LSIL, and 26% (7/27) with HSIL/CA. In the group of 23 women with persistent HPV infection determined by mPCR but not by HC-2, the cervical cytology at follow-up was 65% (15/23) normal cytology, 31% (7/23) ASCUS/LSIL, and 4% (1/23) HSIL/CA. The cytological data are detailed in Table 5 . Although both techniques presented similar results in the prediction of high-grade lesions (Table 5 ), the HC-2 would fail to detect 35% of carriers who progressed to abnormal cytology as HPV positive. This figure could be an underestimate, relaxing a more rigorous gynecological control.
TABLE 5: Distribution of Cervical Cytology in HIV-Infected Women Comparing the HR-HPV Status During the Follow-Up Period Detected by Either mPCR or HC-2 Techniques
None of our patients had to be treated during the study period but 4 required surgical resection soon after the follow-up sample was taken. Histology confirmed diagnosis of CIN-3/CA. Two of the treated women had a relapse and required retreatment.
DISCUSSION
This study provides detailed epidemiological data on the different HPV genotypes isolated from cervical specimens of a Spanish cohort of HIV-positive women. The results point to an alarmingly high prevalence and persistence of HPV infection among HIV-1-infected women without preexisting cervical pathology and normal Papanicolau smear cytology. Moreover, the presence of HR-HPV genotypes other than genotypes 16 or 18 should be taken into consideration when evaluating the effectiveness of future vaccination strategies in the HIV population. It is also important to choose an appropriate diagnostic tool because HPV infection could be underdiagnosed using HC-2 methods compared with mPCR.
It is well established that HPV infection may progress to cervical cancer after several asymptomatic years. Early identification of HIV-1-positive women infected with HR-HPV genotypes is essential for proper follow-up and treatment before invasive cervical cancer develops. This study indicates that HPV infection is highly prevalent (63%) in HIV-1-positive women with normal Papanicolau smears. Moreover, most HPV infection corresponds to genotypes associated with high oncogenic risk and, in agreement with previous studies,10-12,29 30,31
Limited data are available regarding the epidemiology of cervical HPV infection in HIV-1-infected women. Our study included women with normal cervical cytology, this could cause a selection bias for patients included given that genotypes HPV-16 and HPV-18 are more likely to be associated with abnormal cervical cytology. Likewise, HPV-16-positive and HPV-18-positive women with normal cervical cytology may be protected for some reason (possibly genetic) and not progress. Nevertheless, the results of our study show that the HPV-16 was the most predominant genotype, followed by HPV-33, HPV-58, HPV-52, HPV-39, and HPV-18 (Table 2 ). Geographic differences may partly explain the different prevalence of HPV genotypes observed in other studies.23,24
Persistent infection with HR-HPV genotypes is considered as a risk factor for developing cervical precancerous lesions and cancer.32-36 37-40 41,42
Early diagnosis and treatment may modify the natural history of HPV infection. Therefore, sensitive and reliable diagnostic techniques are needed to assess HPV infection in HIV-1-infected women, particularly in the presence of normal cervical cytology.43
None of the potential baseline risk factors assessed was associated with higher or lower risk of clearance, persistence, or incidence of HPV infection in HIV-1-infected women. This may be because of the baseline characteristics of our cohort (women with <500 CD4+ counts/mm3 , 23% with nadir CD4 counts <200 cells/mm3 ) or the small magnitude of changes in the epidemiological end points during the follow-up. Another explanation is that, although HPV and HIV-1 infections have overlapping risk factors, HIV-1 infection itself does not exercise a large influence on the natural history of HPV infection, except when severe immune deficiency occurs.44
In conclusion, cervical infection with oncogenic HPV genotypes is highly prevalent and persistent among HIV-1-infected women with normal current or previous cervical pathology and normal Papanicolau smears. Our results reinforce the need for close monitoring of HPV infection in all HIV-infected women, particularly in those with CD4 counts below 500 cells per cubic millimeter.
ACKNOWLEDGMENTS
We thank Dr. Raquel Jerez and Dr. Jesús Villoria (medicest, external biostatistical Clinical Research Organization, Madrid) for their advice on methodology. Special thanks also go to the female patients of our HIV unit. HIV-HPV Study Group-University Hospital Germans Trias i Pujol, Badalona (Barcelona), Autonomous University of Barcelona: Department of Gynecology: Dr. N. Grane; HIV Clinical Unit and Internal Medicine Department: Dr. C. Rey-Joly, Dr. A. Bonjoch, Dr. M. Jabaloyas, Dr. T. Jou, Dr. J. Molto, Dr. E. Negredo, Dr. J. Romeu, and Dr. C. Tural; Nurses of HIV Clinical Unit: Ms. Carmen Alcalde (head nurse), Ms. Rosa Guerola, and Ms. Anna Salas; General Lab, Barcelona: I. Castilla (laboratory technician). Contributors: G.S., S.V., and B.C. designed the study protocol; S.V., L.D., R.P., and B.C. wrote the article; L.D., M.P.C., and M.B. performed HPV detection and genotyping with mPCR; E.C. and M.L. analyzed cervical cytology samples and performed HPV detection with HC-2; A.T. collected gynecological specimens and was responsible for the gynecological clinical follow-up; statistical analysis was performed by S.V. and L.D. All authors read and approved the final article.
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