Bree Weaver

Human papillomavirus detection and typing in thin prep cervical cytologic specimens comparing the Digene Hybrid Capture II Assay, the Roche Linear Array HPV Genotyping Assay, and the Kurabo GeneSquare Microarray Assay

Three methods for the detection of HPV DNA were compared in cervical cytologic specimens: the Digene Hybrid Capture II Assay (HC), the Roche Linear Array HPV Genotyping Assay (LA) and the Kurabo GeneSquare Microarray (GS). The main goals of the study were to correlate cytology with HPV detection and to determine agreement between assay pairs for HPV detection. Thin-prep Pap smears were performed and supernates were tested by HC, LA, and GS. For specimens reacting with the HPV 52/33/35/58 probe in the LA assay, type-specific PCR was performed for HPV types 52, 33, 35, or 58. Binomial proportions and kappa coefficients were calculated for agreement between assays. Cytology results and supernatant were available for 202 subjects. HPV detection increased with worsening cytologic abnormality in all three assays. For all cytologic groups, LA and GS detected more HPV (all and oncogenic) than HC. However, for detection of oncogenic HPV types represented in all three assays, differences between assays were less pronounced. The highest agreement was between LA and GS. In four of 12 specimens reacting with the HPV 52/33/35/58 probe in the LA assay but deemed HPV 52-LA-negative using an algorithm provided by the manufacturer, the presence of HPV 52 was confirmed using type-specific HPV 52 PCR. All four of these specimens were also GS-positive for HPV 52.

Association of Chlamydia trachomatis Infection With Redetection of Human Papillomavirus After Apparent Clearance

BACKGROUND Persistent infection with oncogenic human papillomavirus (HPV) is associated with an increased risk of cervical malignancy. Redetection of type-specific HPV after a period of nondetection may be caused by reactivation of a low-level persistent infection. Little is known about factors associated with type-specific HPV redetection. METHODS For a longitudinal cohort of adolescent women with frequent behavioral and sexually transmitted infection (STI) information (every 3 months), Cox proportional hazard models were used to assess the influence of sexual behaviors and STIs on the redetection of oncogenic or high-risk HPV infections. RESULTS A total of 210 type-specific high-risk HPV detection episode periods were identified in this longitudinal cohort; 71 (33.8%) were characterized by a period of nondetection followed by redetection. Chlamydia trachomatis (hazard ratio [HR], 3.14; 95% confidence interval [CI], 1.44-6.86) was associated with redetection; redetection was >2 times more likely with each additional self-reported sex partner in the past 3 months (HR, 2.26; 95% CI, 1.35-3.78). CONCLUSIONS This study demonstrates the role of C. trachomatis and number of recent sexual partners in type-specific HPV redetection. Given that persistent oncogenic HPV infections are associated with cancer-related outcomes, understanding the potential role of such factors in the pathogenesis of HPV-related outcomes is important.

High Frequency of Human Papillomavirus Detection in the Vagina Before First Vaginal Intercourse Among Females Enrolled in a Longitudinal Cohort Study

BACKGROUND Genital human papillomavirus (HPV) infection is believed to be primarily sexually transmitted. Few studies have documented the detection of HPV in the vagina before first vaginal intercourse. METHODS We used a longitudinally followed cohort of adolescent females without prior vaginal intercourse to examine the frequency of detection of vaginal HPV and the association between first reported HPV detection and noncoital sexual behaviors. RESULTS HPV was detected in 45.5% of subjects (10 of 22) before first vaginal sex. Seven of these 10 subjects reported noncoital behaviors that, in part, might have explained genital transmission. CONCLUSIONS HPV can be detected in the vagina before first sexual intercourse, highlighting the need for early vaccination.

Natural History of Multiple Human Papillomavirus Infections in Female Adolescents With Prolonged Follow-up

PURPOSE The aim of the study was to better characterize the natural history of human papillomavirus (HPV) infections in female adolescents. METHODS Female adolescents were enrolled in a longitudinal study. Self-vaginal samples were obtained every 3 months and tested for HPV. No participants received HPV vaccination. The findings for 40 female adolescents with the longest follow-up are reported in this study. RESULTS Average age at the time of enrollment was 15.2 years (range: 14-17; SD: .97). Mean duration of follow-up was 6.7 years (range: 4.4-9.2; SD: 1.2). In all, 32 participants (80%) reported being involved in sexual activity before their enrollment in the study; all reported being involved in sexual activity before enrollment; all reported being involved in sexual activity during follow-up. Baseline and cumulative prevalence of HPV among participants was 55% and 100%, respectively. During the study, each participant tested positive for a mean of 14 HPV types. Cumulatively, HPV 16 was detected in 29 of 40 participants (72.5%). Mean duration of high- and low-risk infections was 655.9 (median: 433) and 524.1 days (median: 334), respectively. CONCLUSION With prolonged follow-up, HPV infections with multiple types were found in all participants. Most had infection with HPV-16 or HPV-18, the oncogenic types represented in current vaccines, as well as infection with other oncogenic types. These data reinforce the importance of vaccine and non-vaccine strategies for prevention of HPV infections.

Low-level persistence of human papillomavirus 16 DNA in a cohort of closely followed adolescent women†

Most human papillomavirus (HPV) infections in young women become undetectable by standard assays after a few months. It is possible that many HPV infections do not actually clear, but persist at very low levels for years, becoming detected again later in life. The purpose of this study is to describe HPV 16 clearance, reappearance, and low‐level persistence in a cohort of adolescent women. Adolescent women (N = 66), not vaccinated against HPV, were recruited from 1998 to 2008 into a longitudinal study. Self‐collected vaginal samples were obtained quarterly and tested for HPV by Linear Array HPV Genotyping Test (LA‐HPV). To explore low‐level persistence, a type‐specific nested PCR for HPV 16 (TSN‐PCR‐16) was developed. Women with HPV 16 detected by LA‐HPV had their negative swabs retested with TSN‐PCR‐16. Forty‐two participants with HPV 16, followed for a mean of 6.3 years, were analyzed. Using LA‐HPV, the median duration of HPV 16 detection was 428 days (SD 852.5 days). TSN‐PCR‐16 detected HPV 16 during periods of LA‐HPV non‐detection in samples from many women. Using a combination of LA‐HPV and TSN‐PCR‐16 results, the median duration of HPV 16 detection was 1,022.5 days (SD 943.7 days). The durations of detection differed significantly between the two methods (P = 0.0042) with a mean difference of 434.5 days. In adolescent females, duration of HPV 16 detection was significantly longer when TSN‐PCR‐16 was combined with LA‐HPV. Some apparently cleared HPV 16 could be shown to persist at low levels using nested PCR. J. Med. Virol. 83:1362–1369, 2011.

The natural history of incident gonococcal infection in adolescent women

Background: The natural history of Neisseria gonorrhoeae (GC) infections is largely unknown. The objective of the current study was to use sequential weekly vaginal samples and molecular techniques to describe the natural history of incident gonorrhea infections in adolescent women. Methods: A cohort of 387 adolescent women aged 14 to 17 were enrolled from urban, primary care clinics and followed longitudinally for a period of up to 8 years. Weekly vaginal swabs and daily diaries were provided during 12-week periods biannually, beginning and ending with a clinic visit, where all identified infections were treated. For this study, specimens and data from 16 women who became infected with GC during a weekly sampling period were analyzed. Results: GC organism load was highly variable between subjects. The number of organisms did not significantly differ across the first 6 weeks of infection (P = 0.59). Organism load did not differ among women with a previously documented GC infection at week 1 (P = 0.43) or across the first 6 weeks of infection (P = 0.67). The association of concurrent chlamydial infection on gonorrhea organism load was borderline significant over the first 6 weeks of infection (P = 0.06). Conclusions: Individual shedding patterns varied widely, and GC organism load did not decline in women for at least several weeks and were not associated with genitourinary symptoms. Chlamydia coinfection is associated with higher GC organism loads, potentially increasing chances of transmission. This study utilized a standardized quantification technique to assess GC organism load.

DNA detection and seroprevalence of human papillomavirus in a cohort of adolescent women.

Objectives Human papillomavirus (HPV) infections are common in adolescent women, while the rare cancerous sequelae of HPV infections do not generally occur until the 4th or 5th decades of life. This prospective study of a cohort of adolescent women was performed to further our knowledge of the natural history of incident and prevalent HPV infections. Methods Self-vaginal swabs collected from high-risk, unvaccinated adolescent women in a longitudinal study were analysed for HPV DNA. Sera were collected at enrolment and later tested for HPV antibodies. Statistical analysis was performed to determine the HPV genotype distribution and duration of detection, and to determine rates of seropositivity and seroconversion for HPV types represented in the assays. Results 146 subjects (mean enrolment age=15.4 years; mean duration of follow-up=5.8 years) had samples adequate for analysis of HPV detection, and 95 of these subjects had paired sera available. The cumulative prevalence for high-risk and low-risk HPV types was 95.9% and 91.1%, respectively. HPV types 6, 11, 16 and 18 (HPV types represented in the quadrivalent vaccine) were found at some point in 40.4%, 6.2%, 48% and 24% of participants, respectively. Serological data confirmed exposure to these vaccine-covered types, as well as to other high-risk HPV types. Conclusions In this cohort of adolescent women, high- and low-risk HPV types were frequently detected, and serological data confirmed exposure in most subjects. The high-prevalence HPV types represented in the quadrivalent HPV vaccine further support vaccination of women at an age well before sexual debut.

Human Papillomavirus in Older Women: New Infection or Reactivation?

(See the major article by Gravitt et al, on pages 272–80.) Human papillomavirus (HPV) can be detected in exfoliated cervical cells or vaginal swab samples from approximately 25%–50% of young, sexually active women, according to cross-sectional studies, and from a higher percentage, according to longitudinal studies. In up to 90% of cases, the infection “clears” within 1 or 2 years, meaning that specific HPV types cannot be detected by polymerase chain reaction (PCR) assays of cervical or vaginal swab samples [1]. “Clearance” implies that the individual is no longer infected and does not need to worry about possible long-term sequelae of the infection. Proving that HPV is absolutely gone is, of course, impossible. An alternative hypothesis is that HPV can exist in a low-level persistent state and can reactivate later in life and cause disease. Determining that an HPV infection has cleared should not be based on 1 or 2 negative test results, as nearly all studies have done [2–5]. Several studies involving younger women indicate that type-specific HPV can be detected again after a long period of apparent clearance, but it has not been established whether type-specific HPV redetection is due to reactivation of a low-level persistent infection or the result of a new infection [6–9]. The questions of why and how low-level persistence happens are not understood. A small focus of infected cells may simply be inadequately sampled, or the HPV load may drop to only a few copies per cell at the time of HPV integration into the host genome, making detection unlikely. The resulting low viral copy number may be below the lower limit of detection of standard HPV PCR assays, resulting in falsely negative HPV testing results. This small focus of cells could persist under immunologic control until waning control later in life allows lesion expansion and subsequent HPV redetection. Although our understanding of HPV is incomplete, relatively more is known about early events (at the time of initial infection) and late events (the malignancies associated with oncogenic HPV), compared with the long period between initial infection and the diagnosis of cervical cancer. The prevalence of HPV infection peaks in the early 20s, and after a gradual decline, a second peak in HPV prevalence occurs in the fifth or sixth decades of life in North American, European, and Central/South American women [10]. Cervical cancer, essentially all of which is caused by infection with oncogenic HPV types, also peaks around the fifth or sixth decades of life. Many studies have demonstrated that persistent oncogenic HPV detection is associated with cervical cancer. “Persistence” in these studies was generally defined as 2–4 semiannually collected cervical swabs positive for the same HPV type, just prior to the diagnosis of the high-grade cervical lesion. The question remains of when this infection initially occurred: is it the same HPV isolate acquired in the womans teens or early 20s, or does it involve a new infection acquired later in life (during ages 45–60 years), in the years immediately prior to the diagnosis of cancer? A study by Gravitt et al in this issue of the Journal was performed to address these and other questions about HPV detection and possible reactivation of a preexisting or “prevalent” HPV infection in older women [11]. This study involved cohort analysis, a method used to identify birth cohorts at increased risk for specific outcomes (such as detection of oncogenic HPV) and risk factors for those outcomes. Cohort effects are variations in the risk of a health outcome according to birth year (or years) that are related to differences in the exposure of the cohort to risk factors for that particular outcome [12]. The authors enrolled a cohort of 843 women aged 35–60 years and stratified these women into 2 groups: those with <5 lifetime sex partners (and, thus, at a lower risk of oncogenic HPV acquisition), and those with ≥5 lifetime sex partners (and, thus, at a higher risk of oncogenic HPV infection). The age-specific HPV prevalence was estimated in these 2 groups of women. The age-specific prevalence of oncogenic HPV declined among women with <5 lifetime sex partners but not among those with ≥5 lifetime sex partners. Additionally, the population attributable risk for oncogenic HPV infection due to ≥5 lifetime sex partners was higher among older women (87.2%), compared with younger woman (28.0%). In contrast, the population attributable risk associated with a new sex partner was 28% among younger women, compared with 7.7% among older women. The authors concluded that there might be an interaction of age and lifetime number of sex partners on oncogenic HPV infection. The authors also concluded that this interaction of age and lifetime number of sex partners on oncogenic HPV infection suggested that older women might be at risk for HPV “reactivation.” Thus, the older women in the study, who were likely infected with oncogenic HPV during the interval spanning the late 1960s through the 1970s—the period of the US sexual revolution—had a lower overall risk of HPV infection, because they reported a lower overall number of lifetime number of sex partners. However, oncogenic HPV prevalence declined with age only among older women with <5 lifetime sex partners. One can conclude from this study that the risk of oncogenic HPV reactivation may increase after the age of 50 years and that reactivation contributes to a large fraction of HPV detection at older ages, compared with the fraction resulting from new HPV infections. What is the importance of HPV reactivation? What is the cause of reactivation? Among immunosuppressed individuals, oncogenic HPV present for many years at very low levels may be responsible for the high rate of HPV-related disease. The high rate of disease among these individuals may result from reactivation of low-level persistent HPV as immunity wanes [13]. What about the phenomenon known as immunosenescence, which involves a reduction in many aspects of immune system function and naturally occurs during the aging process? Immunosenescence leading to reactivation of HPV has been hypothesized as an explanation for higher prevalence proportions among older women [14]. In summary, although we now have safe and effective vaccines to prevent infection and disease with the 2 most important oncogenic HPV types (HPV 16 and HPV 18) in younger women, it will be decades before reductions in cervical cancer will be seen. Women >30 years old who are unvaccinated today are at continued risk of cervical cancer for the next 20–30 years. The questions asked by Gravitt et al have great importance from epidemiologic, behavioral, and clinical perspectives. Older women should not be told that detection of HPV always indicates a new infection, but rather that detection of HPV could result from an infection acquired many years ago. Further research is needed to help better understand the natural history of HPV infection in older women and to understand the importance of HPV persistence and reactivation in all women.

Necrotizing retinitis due to syphilis in a patient with AIDS

The ocular manifestations of syphilis are varied. Ocular syphilis can occur during any stage of infection and involve virtually any part of the eye. In immunocompetent individuals, the most common etiologies include syphilitic uveitis. Although the clinical presentation of ocular syphilis in HIV-infected patients is also widespread, posterior segment involvement has been more commonly described particularly in patients with AIDS. The diagnosis of syphilitic retinitis is challenging since its clinical presentation mimics retinitis caused by other viral etiologies. In addition, HIV-infected individuals with syphilis are more likely to develop aberrant serologic responses. Recognition of syphilitic retinitis and prompt initiation of penicillin therapy is of critical importance since syphilitic retinitis generally responds well to treatment and loss of vision is reversible. In this report, we describe a 39-year-old female with advanced stages of AIDS who developed necrotizing retinitis due to syphilis. Prompt initiation of intravenous penicillin led to excellent visual outcome for this patient despite significantly decreased visual acuity on presentation.

P3.061 Mycoplasma Genitalium DNA Detected from Adolescent Males in a Longitudinal Cohort

Background Mycoplasma genitalium (MG) causes non-gonococcal urethritis as well as asymptomatic infections although most data on the incidence and natural history of MG is from adults. Methods Participants were 14–17 year old men in a longitudinal study of STI and the urethral microbiome. Urine samples were collected monthly and batch tested retrospectively for MG DNA using PCR. Urine samples were tested in real-time for chlamydia, gonorrhoea, trichomonas and white blood cells (WBC): infections by these organisms were treated. White blood cell count (WBC) was measured by automated cell count of fresh urine. Dysuria and urethral discharge were self-reported on cell phone diaries. Results Among 75 participants (mean age 16.0 at enrollment), 6 (8.0%) men have at least one MG positive sample, with a total of 14 MG positive monthly urine samples. The prevalence of Chlamydia or gonorrhoea infection was 19/75 (25.3%) and 1/75 (1.3%), respectively. All but one participant was positive for at least two consecutive months, and one participant was positive for 4 consecutive months. One participant was positive only once, was co-infected with chlamydia, but treatment could not be confirmed. No other MG positive visits occurred simultaneously with other STI. None of the participants reported symptoms or sexual behaviours within a 15 day window of the positive visit. Average urine WBC was 21.8 WBC/ml urine although only 3/14 MG positive samples were associated with urine WBC > 28.5/ml (commonly used as a diagnostic threshold for pyuria). Conclusions MG in adolescent men is more common than gonorrhoea, persistent without treatment for up to 120 days, and is typically not associated with symptoms or pyuria. These data add to emerging understanding of the prevalence and natural history of sexually transmitted MG and support the importance of more detailed understanding of sexual and reproductive health morbidity associated with these infections.