Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

Background Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. Methods Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR. Results Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence's of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6 %, 12.8 %, 4.6 % and 8.3 % respectively. Conclusions Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4 + and CD8 + T cells from the cervix responded to the HPV-16 antigens and that interferon-γ (IFN-γ) production was HPV type-specific. Comparing HPV-specific T-cell IFN-γ responses at the cervix with those in the blood, we found that while CD4 + and CD8 + T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-γ responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4 + and CD8 + T-cell IFN-γ responses to E7 were of similar magnitude in both compartments and CD8 + responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.

Strategies for the prevention of cervical cancer by human papillomavirus vaccination

As cervical cancer is causally associated with 14 high-risk types of human papillomavirus (HPV), a successful HPV vaccine will have a major impact on this disease. Although some persistent HPV infections progress to cervical cancer, host immunity is generally able to clear most HPV infections. Both cell-mediated and antibody responses have been implicated in influencing the susceptibility, persistence or clearance of genital HPV infection. There have been two clinical trials that show that vaccines based on virus-like particles (VLPs) made from the major capsid protein, L1, are able to type specifically protect against cervical intra-epithelial neoplasia and infection. However, there is no evidence that even a mixed VLP vaccine will protect against types not included in the vaccine, and a major challenge that remains is how to engineer protection across a broader spectrum of viruses. Strategies for production of HPV vaccines using different vaccine vectors and different production systems are also reviewed. © 2005 Elsevier Ltd. All rights reserved.

Vaccination strategies for the prevention of cervical cancer

Infection with high-risk human papillomaviruses (HPVs) is an essential step in the multistep process leading to cervical cancer. There are approximately 120 different types of HPV identified: of these, 18 are high-risk types associated with cervical cancer, with HPV-16 being the dominant type in most parts of the world. The major capsid protein of papillomavirus, produced in a number of expression systems, self assembles to form virus-like particles. Virus-like particles are the basis of the first generation of HPV vaccines presently being tested in clinical trials. Virus-like particles are highly immunogenic and afford protection from infection both in animal models and in Phase IIb clinical trials. A number of Phase III trials are in progress to determine if the vaccine will protect against cervical disease and, in some cases, genital warts. However, it is predicted that these vaccines will be too expensive for the developing world, where they are desperately needed. Another problem is that they will be type specific. Novel approaches to the production of virus-like particles in plants, second-generation vaccine approaches including viral and bacterial vaccine vectors and DNA vaccines, as well as different routes of immunization, are also reviewed. © 2005 Future Drugs Ltd.

Factors influencing the impact of primary and secondary prevention strategies for cervical cancer among Queensland women

Since the introduction of the National Human Papillomavirus Vaccine Program (NHPVP) in 2007, few studies have assessed women's knowledge, beliefs and attitudes towards cervical screening and human papillomavirus (HPV) vaccination in Australia. It is imperative to ascertain this, as substantial changes are anticipated to the National Cervical Screening Program (NCSP) through a process called 'the Renewal', to ensure any changes that are introduced will be acceptable and well understood by women. The objectives of this study were to describe Queensland women's current knowledge of cervical cancer/screening and HPV, their beliefs and attitudes towards Pap smears and the HPV vaccine and seek their advice on effective methods for communicating changes to the NCSP in their communities. This research was a descriptive-exploratory study that incorporated a combination of qualitative and quantitative methods within the context of the Health Belief Model (HBM). A computer-assisted telephone interview (CATI) survey of 1002 Queensland women was conducted in Phase 1 of the study. During Phase 2 of the study, 23 focus groups were conducted throughout Queensland to gather in-depth information about women's knowledge, awareness and acceptance about cervical cancer prevention strategies. This study found high levels of awareness of HPV (over 60%) and the HPV vaccine (over 86%) amongst Queensland women. However, it also identified considerable uncertainty amongst participants about perceived susceptibility to cervical cancer, especially, the link between cervical cancer, HPV and sexual activity. Women also had limited understanding of the benefit of the Pap smear as a preventative strategy, with many women thinking the main purpose of the Pap smear was for the early detection of cancer. Despite high awareness of HPV, women participating in this study also had significant knowledge deficits about their susceptibility to HPV and the severity of HPV infection. Queensland women had high levels of awareness of the HPV vaccine, which was most commonly via the media. High acceptance of the HPV vaccine was found amongst participants although awareness of the full benefits of vaccination was not evident with little acknowledgement that the quadrivalent vaccine used in the NHPVP would also prevent genital warts. Extensive barriers to having Pap smears, including physical and psychological discomfort, were identified and the most common barriers to vaccination were concerns about side effects and a lack of information upon which to make a decision about consent. Women described enablers for screening participation, such as reminder systems and practitioner characteristics, and expressed positive views towards self collected testing as an enabler, particularly for women who did not attend screening. As this study was conducted with Queensland women it may therefore not be representative of women from other parts of Australia and as participants were more likely to report they were regular screeners than Queensland women overall, these results may not be representative of women least likely to participate in cervical screening. The use of self-reported cervical screening history may also have led to over-reporting of screening status and previous abnormalities by participants. This study reveals significant gaps in Queensland women's knowledge that require effective communication strategies to address. Recommendations from this study highlight the need for increased community education to raise awareness about primary and secondary cervical cancer prevention strategies, training of cervical screening providers in sensitive examination techniques, a reduction in costs associated with screening, the exploration of alternative service models and communication plans that incorporate methods women trust and recommend for disseminating information about changes to the NCSP. This study is the first large study to explore women's perceptions of the Pap smear and barriers to screening, their knowledge about HPV and their attitudes towards the HPV vaccine in Queensland, since the introduction of the NHPVP. It highlights considerable uncertainty about many aspects of cervical cancer and primary and secondary prevention strategies available in Australia and identified many barriers to cervical screening and concerns about HPV vaccination. These knowledge gaps and barriers need to be taken into account and addressed within the context of anticipated changes to the NCSP to ensure benefits are maximised for women in future primary and secondary cervical cancer prevention strategies in the Australian context.

Cervical auscultation in the diagnosis of oropharyngeal aspiration in children : a study protocol for a randomised controlled trial

Background Oropharyngeal aspiration (OPA) can lead to recurrent respiratory illnesses and chronic lung disease in children. Current clinical feeding evaluations performed by speech pathologists have poor reliability in detecting OPA when compared to radiological procedures such as the modified barium swallow (MBS). Improved ability to diagnose OPA accurately via clinical evaluation potentially reduces reliance on expensive, less readily available radiological procedures. Our study investigates the utility of adding cervical auscultation (CA), a technique of listening to swallowing sounds, in improving the diagnostic accuracy of a clinical evaluation for the detection of OPA. Methods We plan an open, unblinded, randomised controlled trial at a paediatric tertiary teaching hospital. Two hundred and sixteen children fulfilling the inclusion criteria will be randomised to one of the two clinical assessment techniques for the clinical detection of OPA: (1) clinical feeding evaluation only (CFE) group or (2) clinical feeding evaluation with cervical auscultation (CFE + CA) group. All children will then undergo an MBS to determine radiologically assessed OPA. The primary outcome is the presence or absence of OPA, as determined on MBS using the Penetration-Aspiration Scale. Our main objective is to determine the sensitivity, specificity, negative and positive predictive values of ‘CFE + CA’ versus ‘CFE’ only compared to MBS-identified OPA. Discussion Early detection and appropriate management of OPA is important to prevent chronic pulmonary disease and poor growth in children. As the reliability of CFE to detect OPA is low, a technique that can improve the diagnostic accuracy of the CFE will help minimise consequences to the paediatric respiratory system. Cervical auscultation is a technique that has previously been documented as a clinical adjunct to the CFE; however, no published RCTs addressing the reliability of this technique in children exist. Our study will be the first to establish the utility of CA in assessing and diagnosing OPA risk in young children.

The case for routine cervical mediastinoscopy prior to radical surgery for malignant pleural mesothelioma

Objectives: To assess whether cervical mediastinoscopy is necessary before radical resection of malignant pleural mesothelioma (MPM). Methods: Patients who underwent radical excision of MPM in a 48-month period were prospectively followed for evidence of disease recurrence and death. Histological evidence of extra pleural lymph node metastases was correlated with survival. Lymph node size at intraoperative lymphadenectomy was correlated with the presence of metastatic tumour. Results: The 55 patients who underwent radical resection (51 extra pleural pneumonectomies and 4 radical pleurectomies) comprised 50 men and 5 women with a median age of 58 years, range 41-70. Histological examination revealed 50 epithelioid, four biphasic and one sarcomatoid histology. Postoperative IMIG T stage was stage I 4, II 11, III 30 and IV 10. Postoperatively the 17 patients with metastases to the extra pleural lymph nodes had significantly shorter survival (median 4.4 months, 95% CI 3.2-5.4) than those without (median survival 16.3 months, 95% CI 11.6-21.0) P=0.012 Kaplan-Meier analysis. Seventy-seven extra pleural lymph nodes without metastases were measured with a mean long axis diameter of 16.9 mm (range 4-55) ; 22 positive nodes had a mean long axis diameter of 15.2 mm (range 6-30). In 15 of the 17 patients with positive extra pleural nodes, the nodes could have been biopsied at cervical mediastinoscopy. Conclusions: This study confirms that extra pleural nodal metastases are related to poor survival. Pathological nodal involvement cannot be predicted from nodal dimensions. These data suggest that all patients being considered for radical resection of MPM should preferentially undergo preoperative cervical mediastinoscopy irrespective of radiological findings. © 2003 Elsevier B.V. All rights reserved.

Mouse estrous cycle regulation of vaginal versus uterine cytokines, chemokines, α-/β-defensins and TLRs

This study investigates the cyclic changes in innate immunity in the female reproductive tract (FRT) of mice during the estrous cycle. By examining uterine and vaginal tissues and secretions we show that innate immunity varies with the stage of the estrous cycle and site in the FRT. Secretions from the uterine lumen contained cytokines and chemokines that were significantly higher at proestrus and estrus relative to that measured at diestrus. In contrast, analysis of vaginal secretions indicated that only IL-1β and CXCL1/mouse KC changed during the cycle, with highest levels measured at diestrus and estrus. In contrast, vaginal α-defensin 2 and β-defensins 1-4 mRNA levels peaked at proestrus and estrus and are expressed 1-4 logs greater than that seen in the uterus. These studies further indicate that TLR5 and TLR12 in the uterus, and TLR1, TLR2, TLR5 and TLR13 in the vagina varies with stage of the estrous cycle, with some peaking at proestrus/estrus and others at diestrus. Overall, these studies indicate that innate immune parameters in the uterus and vagina are separate and discrete, and regulated precisely during the estrous cycle.

Alterations in cervical muscle activity in functional and stressful tasks in female office workers with neck pain

This study determined differences between computer workers with varying levels of neck pain in terms of work stressors, employee strain, electromyography (EMG) amplitude and heart rate response to various tasks. Participants included 85 workers (33, no pain; 38, mild pain; 14, moderate pain) and 22 non-working controls. Work stressors evaluated were job demands, decision authority, and social support. Heart rate was recorded during three tasks: copy-typing, typing with superimposed stress and a colour word task. Measures included electromyography signals from the sternocleidomastoid (SCM), anterior scalene (AS), cervical extensor (CE) and upper trapezius (UT) muscles bilaterally. Results showed no difference between groups in work stressors or employee strain measures. Workers with and without pain had higher measured levels of EMG amplitude in SCM, AS and CE muscles during the tasks than controls (all P < 0.02). In workers with neck pain, the UT had difficulty in switching off on completion of tasks compared with controls and workers without pain. There was an increase in heart rate, perceived tension and pain and decrease in accuracy for all groups during the stressful tasks with symptomatic workers producing more typing errors than controls and workers without pain. These findings suggest an altered muscle recruitment pattern in the neck flexor and extensor muscles. Whether this is a consequence or source of the musculoskeletal disorder cannot be determined from this study. It is possible that workers currently without symptoms may be at risk of developing a musculoskeletal disorder.

High level of MT-MMP expression is associated with invasiveness of cervical cancer cells

MMP-2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane-associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF-β, but its physiological regulation is still largely unresolved. MT-MMP was recently discovered and described as a potential gelatinase-A activator. In the present study, we investigated the expression of MT-MMP (membrane-type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro-transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT-MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT-MMP expression in invasive cervical carcinoma and lymphnode metastases compared to its expression in non-invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT-MMP. Taken together, our results clearly correlated high level MT-MMP expression with invasiveness, and thus suggested that MT-MMP might play a crucial role in cervical tumor invasion.

Vimentin expression in cervical carcinomas : association with invasive and migratory potential

Vimentin is an intermediate filament protein normally expressed in mesenchymal cells, but evidence is accumulating in the literature which suggests that the aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastatic potential. Vimentin expression has previously been associated with invasive properties in an in vitro model consisting of a set of HPV-33-transformed cervical keratinocyte cell lines. In the present study, in order to emphasize those in vitro findings, the expression of vimentin has been investigated in cervical neoplasms of different grades, using immunohistochemistry. A clear association is reported between vimentin expression and metastatic progression, since vimentin was detected in all invasive carcinomas and lymph node metastases, but not in CIN III lesions. These in vivo results are compared with present and previous data obtained in vitro on cervical keratinocyte cell lines, where vimentin expression also correlated with in vitro invasiveness.

Performance measures for Australian laboratories reporting cervical cytology : a decade of data 1998–2008

Aim Performance measures for Australian laboratories reporting cervical cytology are a set of quantifiable measures relating to the profile and accuracy of reporting. This study reviews aggregate data collected over the ten years in which participation in the performance measures has been mandatory. Methods Laboratories submit annual data on performance measures relating to the profile of reporting, including reporting rates for technically unsatisfactory specimens, high grade or possible high grade abnormalities and abnormal reports. Cytology-histology correlation data and review findings of negative smears reported from women with histological high grade disease are also collected. Suggested acceptable standards are set for each measure. This study reviews the aggregate data submitted by all laboratories for the years 1998-2008 and examines trends in reporting and the performance of laboratories against the suggested standards. Results The performance of Australian laboratories has shown continued improvement over the study period. There has been a fall in the proportion of laboratories with data outside the acceptable standard range in all performance measures. Laboratories are reporting a greater proportion of specimens as definite or possible high grade abnormality. This is partly attributable to an increase in the proportion of abnormal results classified as high grade or possible high grade abnormality. Despite this, the positive predictive value for high grade and possible high grade abnormalities has continued to rise. Conclusion Performance measures for cervical cytology have provided a valuable addition to external quality assurance procedures in Australia. They have documented continued improvements in the aggregate performance, as well as providing benchmarking data and goals for acceptable performance for individual laboratories.

Flight-training effect on the cervical muscle isometric strength of trainee pilots

External stimulus/loading initiates adaptations within skeletal muscle. It has been previously found that the cervical area has the highest loading while performing flying maneuvers under +Gz. The first purpose of this study was to examine the neck muscle response to the physical environment associated with flight training, incorporating limited exposure to +Gz force, in a Pilatus PC-9 aircraft. The second purpose was to examine the short-term range of movement (ROM) response to flight training. Isometric cervical muscle strength and ROM was monitored in 9 RAAF pilots completing an 8-mo flight-training course at Pearce Airbase in Western Australia, and in 10 controls matched for gender, age, height, and weight. Isometric cervical muscle strength and ROM were measured at baseline and at 8 mo using the multi-cervical rehabilitation unit (Hanoun Medical, Downsview, Ontario, Canada). Results indicated that an increase in pilot neck strength was limited to flexion while in a neutral position. No strength changes were recorded in any other site in the pilots or for the controls. These findings suggest that short-term exposure to the physical environment associated with flight training had a limited significant effect on increasing isometric cervical muscle strength. No significant changes were observed in pilot ROM, indicating that short-term exposure to flight does not effect ROM.