Development of a SimpleProbe real-Time PCR Assay for rapid detection and identification of the US novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC).

Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_NmUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade.

Redetection of human papillomavirus type 16 infections of the cervix in mid-adult life.

PURPOSE: To assess whether HPV 16 originally detected in adolescent women can be redetected in adulthood. METHODS: A convenience sample of 27 adult women with known HPV 16 detection during adolescence was assessed for HPV 16 redetection. A comparison of the long control region (LCR) DNA sequences was performed on some of the original and redetected HPV 16 isolates. RESULTS: Median age at reenrollment was 27.5 years (interquartile range of 26.7-29.6). Reenrollment occurred six years on average after the original HPV 16 detection. Eleven of 27 women had HPV 16 redetected. Some of these HPV 16 infections had apparently cleared during adolescence. LCR sequencing was successful in paired isolates from 6 women; in 5 of 6 cases the redetected HPV 16 isolates were identical to those detected during adolescence, CONCLUSIONS: HPV 16 may be episodically detected in young women, even over long time periods. HPV 16 redetection with identical LCR sequences suggests low-level persistent infection rather than true clearance, although newly acquired infection with an identical HPV 16 isolate cannot be excluded. However, this study suggests that a new HPV 16-positive test in a clinical setting may not indicate a new infection.

Invasive cervical cancers in the United States, Botswana and Kenya: HPV type distribution and health policy implications.

BACKGROUND: More deaths occur in African women from invasive cervical cancer (ICC) than from any other malignancy. ICC is caused by infection with oncogenic types of human papillomavirus (HPV). Co-infection with the human immunodeficiency virus (HIV) accelerates the natural history of ICC, and may influence the HPV type distribution. Because HPV vaccines are available, this malignancy is theoretically preventable, but the vaccines are largely type-specific in protection against infection. Data on specific HPV types causing ICC in African women is limited, and many studies utilized swab samples rather than actual cancer tissue. A previous study using archived, ICC tissue from women in Botswana identified an unusual HPV type distribution. A similar study was therefore performed in a second sub-Saharan country to provide additional information on the HPV type distribution in ICC. METHODS: Archived, formalin-fixed, paraffin-embedded ICCs were acquired from women in the United States, Kenya, or Botswana. DNA was extracted and HPV genotyping performed by Roche Linear Array. HIV sequences were identified in ICCs by PCR. RESULTS: HPV types 16 or 18 (HPV 16/18) were identified in 93.5 % of HPV-positive ICCs from the U.S., 93.8 % from Kenya, and 61.8 % from Botswana (p < 0.0001). Non-HPV 16/18 types were detected in 10.9 % of HPV-positive cancers from the U.S., 17.2 % from Kenya, and 47.8 % from Botswana (p < 0.0001). HIV was detected in 2.2, 31.5, and 32.4 % from ICCs from the U.S., Kenya, or Botswana, respectively (p = 0.0002). The distribution of HPV types was not significantly different between HIVinfected or HIV-uninfected women. The percentages of ICCs theoretically covered by the bivalent/quadrivalent HPV vaccines were 93.5, 93.9, and 61.8 % from the U.S., Kenya and Botswana, respectively, and increased to 100, 98, and 77.8 % for the nanovalent vaccine. CONCLUSIONS: HPV 16/18 caused most ICCs from the U.S. and western Kenya. Fewer ICCs contained HPV 16/18 in Botswana. HIV co-infection did not influence the HPV type distribution in ICCs from African women from the two countries. Available HPV vaccines should provide protection against most ICCs in the U.S. and Kenya. The recently developed nanovalent vaccine may be more suitable for countries where non-HPV 16/18 types are frequently detected in ICC.

Episodic detection of human papillomavirus within a longitudinal cohort of young women.

Redetection of a type-specific human papillomavirus (HPV) infection may represent reinfection. However, a growing body of literature suggests that reactivation of HPV is common and that episodic detection of a HPV infection may represent reactivation of a persistent virus. A cohort of prospectively followed adolescent women (N = 150), ages 14-17, was observed on average 6.4 years. The authors describe the redetection of 37 HPV types and associated factors of redetection of high-risk (HR) and low-risk (LR) types using Cox proportional hazard models. Of 1,248 HPV type-specific infections, 286 (22.9%) were associated with redetection after apparent clearance. Chlamydia infections (HR = 1.99 [95%CI, 1.15-3.49]) and non-condom use (HR = 1.1 [95%CI, 1.04-1.99]) were associated with increased redetection of HR-HPV infections. Oral contraceptive pills (HR = 2.73 [95%CI, 1.52-4.90]) and number of sexual partners (HR = 1.44 [95%CI, 1.04-1.99]) were associated with increased redetection of LR-HPV infections. Episodic detection of HPV is common for HR- and LR-HPV types. This finding and identified factors or redetection have clinical implications and enhances the understanding of HPV natural history.

Invasive cervical cancers from women living in the United States or Botswana: differences in human papillomavirus type distribution.

BACKGROUND: Cervical cancer is the primary cause of cancer-related deaths in women living in Botswana. METHODS: Paraffin-embedded blocks of formalin-fixed invasive cervical cancer specimens were identified from women living in the U.S. (n = 50) or Botswana (n = 171) from which DNA was extracted. Thin-section PCR was performed on each sample for HPV types and HIV. Comparisons were made between HPV types and groups of types identified in cancers. RESULTS: HPV DNA was identified in 92.0% of specimens from the U.S. containing amplifiable human DNA, and 79.5% of specimens from Botswana. HPV 16 was detected in 40 of 46 HPV-positive specimens (87.0%) from the U.S. vs. 58 of 136 (42.7%) from Botswana (p < 0.001). In contrast, non-HPV 16/18 types, all A9 species (HPV16, 31, 33, 35, 52, and 58), non-HPV 16 A9 (HPV 31, 33, 35, 52, and 58), HPV 18, all A7 types (18, 39, 45, 59, and 68) types were detected significantly more often in specimens from Botswana. The prevalence of non-HPV 18 A7 types did not differ significantly between the two groups. For specimens from Botswana, 31.6% contained PCR-amplifiable HIV sequences, compared to 3.9% in U.S. specimens. Stratifying the samples from Botswana by HIV status, HPV 31 was detected significantly more often in HIV-positive specimens. Other HPV types and groups of types were not significantly different between HIV-positive and HIV-negative specimens from Botswana. CONCLUSION: This study demonstrates that there may be important HPV type differences in invasive cervical cancers occurring in women living in the United States or Botswana. Factors in addition to HIV may be driving these differences.

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.

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.

Association of condom use, sexual behaviors, and sexually transmitted infections with the duration of genital human papillomavirus infection among adolescent women.

OBJECTIVE: To examine the association of potentially modifiable factors such as condom use, sexual behaviors, and concurrent sexually transmitted infections with duration of genital human papillomavirus (HPV) infections among adolescent women. DESIGN: Longitudinal observational study. SETTING: Study conducted at 3 inner-city clinics in Indianapolis, Ind. PARTICIPANTS: Forty-nine HPV-positive adolescents were tested frequently for HPV infection and provided sexual behavior diaries. Main Exposures Condom use, sexual behaviors, number of partners, and concurrent infections with Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis. MAIN OUTCOME MEASURES: Time from onset to clearance of type-specific HPV infections was analyzed with proportional hazard models. Adjusted hazard ratios (AHRs) were used to assess the effects of risk factors on the duration of HPV infection. Because viral clearance is a preferred outcome, a variable with an AHR less than 1 was considered a risk factor (ie, associated with reduced chance of viral clearance and prolonged infection). RESULTS: Prolonged HPV infection was associated with oncogenic HPV types (AHR, 0.58 [95% confidence interval (CI), 0.39-0.84]) less than median level of condom use during an HPV infection (AHR, 0.53 [95% CI, 0.33-0.84]) and coinfection with C trachomatis (AHR, 0.58 [95% CI, 0.31-0.89]) or T vaginalis (AHR, 0.32 [95% CI, 0.16-0.64]). Not having multiple sexual partners during an HPV infection was associated with early HPV clearance (AHR, 5.52 [95% CI, 3.28-9.30]). CONCLUSIONS: These findings support public health messages of reducing the number of sexual partners, promoting routine condom use, and frequent sexually transmitted infection screening that may be beneficial with HPV infections.

Detection of specific human papillomavirus types in paraffin-embedded sections of cervical carcinomas.

Human papillomaviruses (HPV) are the causative agents of most cervical carcinomas. A complete understanding of the HPV types that cause cervical carcinoma is needed as vaccines are designed. Fresh tissues are not always available for such studies. We therefore sought to determine the feasibility of HPV studies using formalin-fixed, paraffin-embedded sections of 56 cervical carcinomas, correlating typing information with the pathology and physical state of the HPV sequences within cells. Sections from each specimen were used to extract and purify DNA. Specific HPV types were identified using a PCR/reverse blot strip assay. Tyramide signal-amplified, fluorescent DNA in situ hybridization (FISH) was used to localize HPV within cells. Human beta-globin sequences were amplified in DNA from all specimens. HPV sequences from oncogenic types were identified in 52 of 56 (92.9%) by PCR/reverse blot strip assay, and in one additional case using an HPV 16 multiplex PCR assay. HPV 16 was the most commonly detected type, present in most cases as a solitary isolate. Thirty- five of 42 HPV 16 or HPV 18 PCR-positive specimens were also positive in the FISH assay, in most cases in a pattern consistent with viral integration. We conclude that HPV typing from formalin-fixed, paraffin-embedded sections of cervical carcinomas is possible, with a sensitivity that is similar to that found in studies using fresh tissue.

The human papillomavirus type 11 E1--E4 protein is a transglutaminase 3 substrate and induces abnormalities of the cornified cell envelope.

The human papillomavirus (HPV) E1--E4 protein is detected in the cytoplasm of differentiated keratinocytes, near the cornified cell envelope. HPV does not induce lysis of the infected keratinocyte, and the normally durable cornified cell envelope that forms during keratinocyte differentiation would seemingly inhibit viral egress. HPV infection induces abnormalities of the cornified cell envelope, but the exact mechanisms involved are not well understood. We tested whether the HPV 11 E1--E4 protein, which co-localizes the cell envelope and co-purifies with cell envelope fragments, could serve as an in vitro substrate for transglutaminases. We found evidence of E1--E4 cross-linking by endogenous transglutaminases in an in situ assay using frozen sections of human foreskin, and in addition, E1--E4 protein was cross-linked by recombinant transglutaminase 3 (but not transglutaminase 1) in an in vitro cross-linking assay. We also tested whether expression of E1--E4 in differentiated keratinocytes would induce morphologic alterations of cornified cell envelopes. Differentiated keratinocytes expressing E1--E4 were disorganized and pleomorphic compared to control cells, and cell envelopes purified from E1--E4-expressing cells were small, fragmented, and rough bordered compared to the round, smooth bordered cell envelopes from control cells. We conclude from these in vitro experiments that the E1--E4 protein is cross-linked by transglutaminase 3, and that E1--E4 expression in differentiated keratinocytes induces morphologic abnormalities of the cornified cell envelope.

A longitudinal study of genital human papillomavirus infection in a cohort of closely followed adolescent women.

BACKGROUND: We performed a study to better characterize the natural history of genital human papillomavirus (HPV) infection in a cohort of closely followed adolescent women. METHODS: A cohort of 60 adolescent women was followed over a 2.2-year period, on average. A median of 41.5 self-collected vaginal and clinician-obtained cervical swabs were obtained from each subject. RESULTS: HPV was detected in 45.3% of all adequate specimens, by use of a polymerase chain reaction/reverse blot strip assay. Oncogenic--or high-risk (HR)--HPV types were detected in 38.6% of specimens, and nononcogenic--or low-risk (LR)--types were detected in 19.6% of specimens. During the entire study period, 49 of 60 subjects tested positive for HPV (cumulative prevalence, 81.7%). The most frequently detected HR types were HPV types 52, 16, and 59. Infections with multiple HPV types were common. The median duration of persistence of a specific HPV type was 168 days, and HR types were more persistent than LR types. Abnormal cervical cytological results occurred in 37% of the adolescent women and were significantly associated with HR HPV infection. CONCLUSIONS: The cumulative prevalence of HPV infection in sexually active adolescent women is extremely high, involves numerous HPV types, and frequently results in cervical dysplasia.

Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation.

Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

Distribution of human papillomavirus types in cervicovaginal washings from women evaluated in a sexually transmitted diseases clinic.

BACKGROUND: Women evaluated for sexually transmitted diseases (STDs) may be at increased risk for human papillomavirus (HPV) infection, a condition associated with cervical dysplasia. The distribution of HPV types in such a population is unknown. GOAL: The goal was to determine the prevalence of HPV infection, the distribution of HPV types, and the type distribution in relation to cervical dysplasia in women in an STD clinic. STUDY DESIGN: Cervicovaginal lavage and Papanicolaou smear specimens were obtained from 295 women. Lavage specimens were analyzed for HPV by polymerase chain reaction/reverse blot strip assay. RESULTS: Cervical cytologic findings were abnormal for 19.7% of women. HPV DNA was detected in 49.2% of women (high-risk HPV in 42.4%). HPV positivity correlated with the degree of cytologic abnormality. In women with dysplasia, HPV types 16, 66, 83, 56, 52, and 59 were commonly detected. Specimens containing abundant HPV DNA occurred most often in women with dysplasia. CONCLUSIONS: HPV infection was common in women attending an STD clinic. Numerous individual HPV types were associated with cervical dysplasia, including "low-risk" types.