HPV testing in cervical cancer screening has been proposed as an alternative or complementary to cytology in women older than 30 years. However, adequate clinical sensitivity and specificity are crucial for a new test to be implemented. Hybrid Capture 2 (HC2) has proved good clinical performance in selecting women at risk for high-grade intraepithelial lesions with a high sensitivity and specificity. cobas HPV Test has been recently launched and its performance in different clinical settings needs to be determined.The aim of this study was to evaluate the cobas HPV Test for the detection of cervical HPV infection in a population of women in Catalonia (Spain) using HC2 as a reference.Cervical liquid cytology samples from 958 women have been studied. Sensitivity was analyzed in 60 samples from patients with a high-grade intraepithelial lesion (≥CIN2) on histology and specificity was determined in 898 samples from women with no ≥CIN2. All cases had HC2 and cobas HPV Test performed. Statistical analyses of sensitivity, specificity and comparison between HC2 and cobas HPV Test by a non-inferiority test were applied.Sensitivity of HC2 and cobas HPV Test for detecting ≥CIN2 proved identical (98.3%) while specificity was 85.3% and 86.2% respectively. The non-inferiority test demonstrated that cobas HPV Test surpassed 90% sensitivity and 98% specificity of HC2.The cobas HPV Test results fulfilled sensitivity and specificity requirements for HPV based cervical cancer screening and for the triage of minor cytological abnormalities, allowing its introduction in clinical settings. High-risk human papillomavirus (HR-HPV) have been demonstrated as the causal agents of cervical cancer www.fda.gov). Some studies have shown that the risk for CIN2 or worse (≥CIN2) at 3–5 years after a negative HPV test is 40–50% lower than after a negative cytology due to its high negative predictive value Hybrid capture 2 , the most widespread HPV test, is considered to have an adequate clinical sensitivity with an acceptable specificity Recently other HPV tests have been developed with limited information on clinical performance. Foreseeing the situation, guidelines were published by a group of experts to clinically validate HPV tests against the gold standard HPV test HC2, mainly focusing on the detection of ≥CIN2 in women ≥30 years old. A new candidate test should detect ≥CIN2 with sensitivity not less than 90% and specificity at least of 98% compared to HC2 The main objective of our study was to evaluate the cobas HPV Test from Roche Molecular System  in the detection of ≥CIN2 in comparison to the results of HC2 in opportunistic screening for cervical cancer in Catalonia, Spain.The project was approved by the PSMar Ethics Committee (CEIC-PSMAR 2010/4009/I). Samples were obtained from clinical diagnosis material. After diagnosis, part of the material was anonymized and collected for future research projects. The CEIC-PSMAR approved the use of these samples for the project (waived the need of specific informed consent).The Pathology department of the Hospital del Mar  is the reference laboratory for cervical cytology in an area with a population of approximately 150.000 women, of which 105.000 are 30 years or older. From a total of 2700 consecutive samples from September 2009 until October 2010 that had HC2 performed, 958 that fulfilled the eligibility criteria were included in the study. One group, consisting of 898 cases was used for the evaluation of HPV test specificity. These cases corresponded to women with the following inclusion criteria: ≥30 years, no abnormal cytology in Hospital del Mar in the previous 5 years, minimum of 10 ml of PreservCyt cell suspension. A second group, consisting of 60 samples, was used to determine the sensitivity of the HPV test for detecting intraepithelial high-grade lesions. All 60 cases corresponded to histologically confirmed ≥CIN2 selected, including 22 CIN 2 and 38 CIN 3. The only inclusion criteria for these cases was a minimum amount of 10 ml of PreservCyt cell suspension, with no limits on age or past history of abnormal cytology.The samples were collected in a ThinPrep vial with 30 ml of PreservCyt  and a slide for cytologic study was produced first. This procedure is based on a system that aspirates the cell suspension sample through a filter and stops aspiration when an adequate number of epithelial cells are obtained, therefore depending on the amount of cellularity the sample that remains is variable. Experienced cytotechnologists performed the screening with assistance of the automated system Imager (Hologic) and the results were reported according to the Bethesda system Intralaboratory reproducibility was assessed in 546 samples and consisted of performance by a different technician. The microbiology laboratory in Hospital de Valme, Sevilla, Spain was responsible for the testing of 445 samples to evaluate interlaboratory reproducibility, using as a reference the results from the first test at Hospital del Mar. The smaller sample size in this part of the study was due to consumption of some samples.HC2 is based on hybridization with RNA probes to detect a total of 13 HR-HPV  and signal amplification. First, PreservCyt samples must be converted to be suitable for HC2. In short, 6ml are centrifuged with a conversion buffer and then the pellet is resuspended in denaturing reagent and incubated for 45 minutes at 65°C. Following denaturation, hybridization is performed on a microtiter plate mixing 75 µl of the sample and 25 µl of a cocktail of RNA probes for 60 minutes at 65°C. Then the samples and controls are transferred to another microtiter plate with anti-hybrid antibodies and incubated for 60 minutes. Signal amplification is based on peroxidase-labelled anti-hybrid antibodies. The final step consists on adding a substrate that gives a chemiluminiscent signal that is measured by a luminometer. The analytical sensitivity of the test is set at 1 pg of HPV DNA/ml by the positive controls. The result is reported as positive when the semi-quantitative value (RLU: Relative Light Units), that is the ratio between each sample RLU, and the mean RLU of 3 positive controls, is ≥1.The cobas HPV Test, recently approved by the FDA, is a fully automated test based on a simultaneous extraction of the HPV and cellular DNA, followed by a real-time PCR designed to detect 14 high risk HPVs using LightCycler®480 technology. Analytical sensitivity measured with different amounts of SiHa and HeLa cells in PreservCyt solution, according to the kit insert, is 100 copies/ml for HPV 16 and 40 copies/ml for HPV 18. In short, 400 µl of each sample in Preservcyt are directly processed from the original vial with a minimum amount of 3 ml for DNA extraction with the X 480 system. Denaturation is achieved with high temperature and chaotropic agents. DNA is purified by magnetic beads system and a microtiter plate with the samples is generated. The plate is then introduced in the z 480 real-time PCR system. HPV 16 and 18 are detected separately and other 12 HR-HPV types  are detected as a pool by a cocktail of probes with 3 different fluorochromes. In the same reaction the human beta-globin gene is amplified and detected by a different fluorochrome as a control for the entire process. A positive and a negative control are also included. The results are expressed as negative or positive for each HPV 16, HPV 18 or other HR-HPVs. When neither beta-globin nor HPV is detected the result is invalid. The value of cycle threshold (Ct) in quantitative PCR inversely correlates with the amount of target DNA present in the sample, in this case HPV DNA. Ct of each sample is not displayed in the results sheet but it is easily accessible in the system.To estimate the clinical sensitivity and specificity conventional contingency tables were used, calculating percentages and their 95% confidence intervals. Cobas HPV Test sensitivity and specificity for ≥CIN2 were compared to HC2 by a non-inferiority score test, according to the suggested relative sensitivity for ≥CIN2 of at least 90% and relative specificity of ≥98% A Mann-Whitney U test was performed to compare RLU and Ct positive values between discordant vs. concordant results and diagnostic categories. Intraclass correlation coefficient (ICC) was applied to assess reproducibility of Cts in the intralaboratory study. In all analysis a p value less than 0.05 was considered statistically significant. Analyses were performed with SPSS 15.0 .The mean age of all the women included in the study was 46.78 years (range 23–89). Patients with ≥CIN2 had a mean age of 34.93 (range 23–63).A valid cobas HPV Test assay was obtained in all samples tested except for one case that was excluded, showing both negative beta-globin and HPV results. HC2 and cobas HPV Test sensitivities were identical : 95.1–100), missing a different ≥CIN2 each. Among <CIN2 cases there were 54 cases HPV positive by one method only, of these 31 were HPV positive by HC2 and 23 by cobas HPV Test. Specificity was 85.3% (95% C.I.: 83–87.6) for HC2 and 86.2% (95% C.I.: 83.9–88.4) for cobas HPV Test. Agreement between both tests including all samples was 94.2% with a kappa value of 0.814 .P = 0.009 and relative specificity of 98%, T = 3.045, P = 0.001).The analysis of sensitivity and specificity for ≥CIN2 for cobas HPV Test compared to the gold standard HC2 by a non-inferiority test showed that the candidate test was not inferior , in 2 CIN 1 and in 8 negative cytology samples. Other HR-HPV were detected in 34≥CIN2 (26 cases without HPV 16 or 18), 29 LSIL, 3 ASC-US and 71 cytology negative samples.Regarding intralaboratory reproducibility for the HPV negative/positive results, kappa was 0.957. Only 9 out of 546 samples showed discordant results . Type-spInterlaboratory reproducibility showed a kappa value of 0.971 for an HPV positive/negative result, with only 5 discordant results . HPV typAs a measure for viral load reproducibility, intraclass correlation coefficient (ICC) between Cts of the intralaboratory study depicted high values: 0.995 for HPV 16, 0.947 for HPV 18 and 0.984 for HR-HPVs. ICCs were higher for Cts below 35 than for Cts over 35.The comparison of semi-quantitative viral load values of HC2 and cobas HPV Test from women with or without ≥CIN2 showed statistically significant differences in HC2 RLU and cobas HPV Test Ct values. That is to say that ≥CIN2 samples with an HPV positive result, showed higher mean viral loads by both methods, with the exception of samples harbouring HPV 16 or HPV 18, where no differences according to cytological diagnosis were found .Discordant results accounted for 32 HC2 positive/cobas HPV Test negative and 24 cobas HPV Test positive/HC2 negative cases. Cases with concordant HPV positive results showed statistically significant higher viral loads than cases with discordant results .The introduction of HPV testing in cervical cancer screening based on a better sensitivity compared to cytology has the risk of an excessive number of women without relevant lesions being referred for colposcopy. It is important to remark that the key issue is to detect HPV infections that may reveal ≥CIN2 on biopsy, avoiding the clinically irrelevant infections. The high negative predictive value of HC2 has been proved useful in long term studies not only in triaging women with minor cytological abnormalities but also in screening women over the age of 30, whose risk of an HPV infection being persistent is higher than in younger women In this series of consecutive cases, the cobas HPV test system showed excellent results regarding both sensitivity and specificity. Non-inferiority test analysis consolidates it as being at least as good as HC2, the present gold standard in HPV testing in cervical samples. Both intralaboratory and interlaboratory reproducibility kappa values were in the category considered almost perfect, overcoming the minimal requirements for a clinical test. Recently, other groups have published similar results in other settings, one of them being the ATHENA trial including 1578 women with ASC-US that was used for the FDA approval of cobas HPV Test HPV genotyping by cobas HPV Test showed more HPV 16 among ≥CIN2 than in other diagnostic categories, supporting the idea that it is a more aggressive type Regarding the semi-quantitative results given by both tests, RLU by HC2 and PCR cycle of detection (Ct) by cobas HPV Test, although higher viral loads were found statistically significant related to worse diagnosis, it does not seem very useful from a clinical point of view, due to a large distribution of values in each diagnostic category. However it is interesting that statistically significant differences were not found for HPV 16 or HPV 18 Ct values.Beta-globin amplification is important in PCR based technology and our results showed that most cases had good quality DNA, only one being rejected. This finding confirms that liquid based cytology, in particular ThinPrep, is a good method for collecting and preserving cervical cells and DNA for molecular analyses.Cases with concordant positive HC2 and cobas HPV Test results showed statistically significant higher viral loads (high HC2 RLUs/low Cts in cobas HPV Test) than cases with discordant results, explaining that low viral loads near cut off values account for a lower concordance between tests. A more detailed analysis of discordant results using other techniques may give more information on the small differences between tests, probably irrelevant from the clinical point of view since both tests shared a sensitivity >95% and a specificity >85%. Viral load by cobas HPV test was reproducible when comparing results of intralaboratory results, especially when Cts were under 35, however its clinical applicability cannot be drawn from this study.In our setting, cobas HPV Test has proved to be a user-friendly system with high reproducibility and easy interpretation of results. We found some advantages in cobas compared to HC2. First of all there is no need for conversion when using PreservCyt samples. This procedure must be manually performed in badges of no more that 20 samples and takes approximately 2 hours. Moreover it is a possible cause of contamination and increases the risk of pipetting errors. The separate information on HPV types 16 and 18 may have clinical relevance regarding risk of persistence and progression, however our study was not designed to evaluate this objective and prospective studies would be more informative. We consider very valuable the DNA amplification of cellular beta-globin to assure good quality of the sample in the cobas HPV test. Actually, a negative result from a HC2 test could be due to the absence or low amount of cells on a sample while a cobas HPV test would yield an invalid result.We feel confident that our results confirm a reliable clinical performance of cobas HPV Test allowing its introduction in cervical cancer screening protocols.


A group of approximately 13 human papillomaviruses (HPVs) are responsible for virtually all cervical cancers as well as the majority of vulvar, vaginal anal and penile cancers, as well as at least half of oropharyngeal cancers. Currently two prophylactic vaccines are highly efficacious in preventing HPV 16 and 18 associated disease, and the quadrivalent vaccine also prevents HPV 6 and 11 warts. Currently therapeutic vaccines targeting the viral E6 and E7 oncogenes are being developed. One topic that will be discussed is whether the prophylactic vaccine can have utility in any setting where individuals are already infected. Secondly, this review will discuss the basis of long-term immunity and whether it is afforded by both vaccination and natural infection. Finally, strategies to provide more broad based coverage to other HPV types will be discussed.


To examine LMP10 expression and its possible impact on clinical outcome in human papillomavirus (HPV) positive and HPV-negative tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC).Outcome is better in HPV-positive TSCC and BOTSCC compared to matching HPV-negative tumours, with roughly 80% vs. 40% 5-year disease free survival (DFS) with less aggressive treatment than today’s chemoradiotherapy. Since current treatment often results in harmful side effects, less intensive therapy, with sustained patient survival would be an attractive alternative. However, other markers together with HPV status are necessary to select patients and for this purpose LMP10 expression is investigated here in parallel to HPV status and clinical outcome.From 385 patients diagnosed between 2000 and 2007 at the Karolinska University Hospital, 278 formalin fixed paraffin embedded TSCC and BOTSCC biopsies, with known HPV DNA status, were tested for LMP10 nuclear and cytoplasmic expression (fraction of positive cells and staining intensity). The data was then correlated to clinical outcome.An absent/low compared to a moderate/high LMP10 nuclear fraction of positive cells was correlated to a better 3-year DFS in the HPV-positive group of patients (log-rank p = 0.005), but not in the HPV-negative group. In the HPV-negative group of patients, in contrast to the HPV-positive group, moderate/high LMP10 cytoplasmic fraction and weak/moderate/high LMP10 cytoplasmic intensity correlated to a better 3-year DFS (p = 0.003 and p = 0.001) and 3-year overall survival (p = 0.001 and 0.009).LMP10 nuclear expression in the HPV-positive group and LMP10 cytoplasmic expression in the HPV-negative group of patients correlated to better clinical outcome. In 2007, the International Agency for Cancer Research (IARC) acknowledged human papillomavirus (HPV) as a risk factor for oropharyngeal squamous cell carcinoma (OPSCC) based on data by others and us . Defects in different APM components have been found in many tumours and have shown correlation with clinical outcome of patients LMP10 (low molecular weight protein 10) is a beta subunit of the 20S proteasome, identified as a third IFN-g inducible unit by Nandi et al 2006 In this study, we therefore examined for LMP10 expression in HPV-positive and HPV-negative TSCC and BOTSCC, in 278 patients diagnosed between 2000–2007 at the Karolinska University Hospital, and any correlation of LMP10 expression to survival.The study was performed according to ethical permissions 2005/431-31/4, 2005/1330-32 and 2009/1278-31/4 from the Regional Ethical Committee at Karolinska Institutet, Stockholm, Sweden. Written informed consent was obtained from all participants in the study.During 2000–2007, 385 patients diagnosed with TSCC and BOTSCC  at the Karolinska University Hospital, were identified through the Swedish Cancer Registry. Of these, 278 patients, with diagnostic pre-treatment biopsies, previously tested for HPV DNA and with sufficient material, were included in this study HPV DNA of 27 HPV types and betaglobin was assayed by a bead-based multiplex assay on a Magpix instrument (Luminex Corporation) as previously described In brief, formalin fixed paraffin embedded (FFPE) tumour biopsy slides (4 µm) were de-paraffinized in xylene and rehydrated in ethanol of decreasing concentrations. Afterwards they were heated in citrate buffer (pH 6.0) for antigen retrieval. Unspecific sites were blocked with 1.5% horse serum diluted in phosphate buffered saline (PBS). Paraffin sections were incubated overnight at +8°C with primary antibody, MECL-1 C-2 (anti-LMP10 antibody from Santa Cruz Biotechnology), in a moist chamber followed by secondary antibody application and a standard ABC-HRP kit for antigen detection. The chromogen-3′-diaminobenzydine (DAB) was used for visualization. Haematoxylin was applied for counterstaining followed by dehydration in ethanol of increasing concentrations and xylene.LMP10 cytoplasmic and nuclear staining, including negative and positive controls , was evaBoth patient characteristics and categorical data were analysed using the Chi square-test or, for comparison of mean values, the student T-test. Two-tailed p-values (5% significance level) were reported for all analyses. Survival was measured in days of diagnosis until an event occurred, or until 3 years after diagnosis when patients were censored. Death due to any cause (event) was included in the definition of overall survival (OS), while only disease recurrence (TSCC or BOTSCC) was used for calculation of disease free survival (DFS). Notably, death of patients without any prior recurrence was censored at day 0 when calculating DFS. Kaplan-Meier estimator was used for DFS and OS, and differences in the survival of patients were tested using the log-rank test. The Cox proportional hazards model was used for the calculation of the adjusted and unadjusted hazard ratios (HRs). All calculations and analyses were performed using IBM Corp., SPSS Statistics, version 21.0.The characteristics of the 278 patients, 195 (70%) with TSCC and 83 (30%) with BOTSCC and their tumours are shown in LMP10 nuclear and cytoplasmic expression was evaluated for all 278 patients and is exemplified in LMP10 nuclear expression was evaluated both for the fraction of positive cells in the nucleus, and for the intensity of nuclear staining, and was correlated to survival of the 258 patients treated with curative intent (illustrated as Kaplan-Meier curves in An absent/low (≤25%), as compared to a moderate/high (26–100%), fraction of LMP10 nuclear positive cells correlated to a better 3-year DFS in the whole group of patients, and for patients with HPV-positive tumours, but not among patients with HPV-negative tumours  . There wAn absent, as compared to weak/moderate/strong (present), LMP10 nuclear intensity staining was correlated with a better 3-year DFS for the whole group (p = 0.01) and the HPV-positive group (p = 0.037), but not for the HPV-negative group (p = 0.473) . For 3-yThe possible influence of LMP10 nuclear expression as well as LMP10 nuclear intensity staining was pursued separately for the HPV-positive and HPV-negative groups.A multivariate Cox-regression analysis was performed for patients with HPV-positive and HPV-negative tumours separately, including the fraction of LMP10 nuclear positive cells or LMP10 nuclear intensity, age and tumour stage, with regard to 3-year DFS and 3-year OS. For the HPV-positive group an absent/low fraction as compared to a moderate/high fraction of LMP10 positive cells was still a determinant of a better 3-year DFS (p = 0.027) , but notLMP10 cytoplasmic expression was evaluated both for the fraction of positive cells and intensity of staining and was correlated to survival for the 258 patients treated with curative intent  LMP10 cytoplasmic intensity staining as compared to absent LMP10 staining was correlated to a better 3-year DFS for patients with HPV-negative tumours, but not for the whole group or for patients with HPV-positive tumours  . For 3-yThe possible influence of LMP10 cytoplasmic expression was pursued separately for the HPV-positive and HPV-negative groups . For thaThe corresponding analysis for the HPV-positive group suggested that cytoplasmic LMP10 expression had no impact on clinical outcome (3-year DFS and 3-year OS) .In recent studies we have demonstrated that absent or low HLA class I expression was correlated to better survival in patients with HPV-positive TSCC and BOTSCC, while the opposite was the case for patients with HPV-negative TSCC and BOTSCC In the first analysis, HPV-positive TSCC and BOTSCC were examined and the analysis included the fraction of LMP10 nuclear positive cells, age, stage and HLA class I expression assayed as intensity of HCA2-staining. As presented in In the second analysis, HPV-negative TSCC and BOTSCC were studied and the analysis contained the fraction of LMP10 cytoplasmic positive cells, age, stage and the fraction of HLA class I positive cells assayed by HCA2-staining. As presented in The present study investigated LMP10 expression both in the nuclear and cytoplasmic compartments, in relation to HPV status and clinical outcome in 195 TSCC and 83 BOTSCC cases. LMP10’s nuclear and cytoplasmic expression, including both the fraction of positive cells and the intensity of staining was similar in HPV-positive and HPV-negative tumours, except for the higher fraction of LMP10 cytoplasmic positive cells in the HPV-positive group.Furthermore, an absent/low fraction of LMP10 positive cells in the nucleus, as well as absent nuclear staining intensity correlated to a better 3-year DFS for patients with HPV-positive tumours, but not for those with HPV-negative tumours. The reverse was found for cytoplasmic LMP10 staining, where a moderate/high fraction of positive cells and weak/moderate/high intensity was correlated to better 3-year DFS and OS in the HPV-negative, but not in the HPV-positive group.The reason for the difference with regards to nuclear and cytoplasmic LMP10 staining and survival for the HPV-positive and HPV-negative groups is not obvious. In earlier studies LMP10 cytoplasmic expression has been evaluated in bladder and oesophageal cancer for both the fraction of positive cells and the intensity of staining, but was not shown to have any prognostic impact et al. demonstrated a correlation with survival in HNSCC for both LMP2 and LMP7 To our knowledge there are no prior studies on LMP10 expression in relation to survival in HNSCC. Meissner The finding that a low nuclear LMP10 expression correlated to a better 3-year DFS in the HPV-positive group, while a corresponding high cytoplasmic LMP10 expression correlated to a better 3-year DFS in the HPV-negative group suggests several implications. First of all, our study suggests that LMP10 could be used as a prognostic marker for patients with either HPV-positive or HPV-negative tumours. We have previously been able to show that a biomarker, in the same pathway, HLA class I expression, can be used in a differential way to predict clinical outcome in HPV-positive and HPV-negative tumours. Thus, an absent/low HLA class I expression is a positive prognostic factor for patients with HPV-positive tumours, while the opposite is true for patients with HPV-negative tumours, where a high HLA class I expression is of benefit It is not known if the presence of HPV can influence LMP10 expression directly, and even if this is the case, the mechanism behind this regulation is not known. It has been shown that HPV E5 has the potential to down-regulate the expression of HLA class I There are some limitations in our study. Primarily the study is retrospective, but this may not affect the results. In addition, there were fewer HPV-negative cases, which is a direct representation of the high HPV prevalence in OSCC in patients from Sweden today.In conclusion, this study shows that LMP10 expression in TSCC and BOTSCC is correlated to disease free survival for both patients with HPV-positive and HPV-negative tumours and has the potential to be used in a clinical setting. However, since nuclear LMP10 expression predicts survival for patients with HPV-positive tumours, while cytoplasmic LMP10 expression correlates to survival for HPV-negative tumours, LMP10 expression would need to be analysed in relation to tumour HPV status.


Amniotic fluid embolism (AFE) is a rare but severe complication of pregnancy. A recent systematic review highlighted apparent differences in the incidence, with studies estimating the incidence of AFE to be more than three times higher in North America than Europe. The aim of this study was to examine population-based regional or national data from five high-resource countries in order to investigate incidence, risk factors and outcomes of AFE and to investigate whether any variation identified could be ascribed to methodological differences between the studies.We reviewed available data sources on the incidence of AFE in Australia, Canada, the Netherlands, the United Kingdom and the USA. Where information was available, the risk factors and outcomes of AFE were examined.The reported incidence of AFE ranged from 1.9 cases per 100 000 maternities (UK) to 6.1 per 100 000 maternities . There was a clear distinction between rates estimated using different methodologies. The lowest estimated incidence rates were obtained through validated case identification (range 1.9-2.5 cases per 100 000 maternities); rates obtained from retrospective analysis of population discharge databases were significantly higher (range 5.5-6.1 per 100 000 admissions with delivery diagnosis). Older maternal age and induction of labour were consistently associated with AFE.Recommendation 1: Comparisons of AFE incidence estimates should be restricted to studies using similar methodology. The recommended approaches would be either population-based database studies using additional criteria to exclude false positive cases, or tailored data collection using existing specific population-based systems.Recommendation 2: Comparisons of AFE incidence between and within countries would be facilitated by development of an agreed case definition and an agreed set of criteria to minimise inclusion of false positive cases for database studies.Recommendation 3: Groups conducting detailed population-based studies on AFE should develop an agreed strategy to allow combined analysis of data obtained using consistent methodologies in order to identify potentially modifiable risk factors.Recommendation 4: Future specific studies on AFE should aim to collect information on management and longer-term outcomes for both mothers and infants in order to guide best practice, counselling and service planning. Amniotic fluid embolism (AFE) is a rare but severe complication of pregnancy. The rarity of the condition and the fact that AFE is a diagnosis of exclusion, make it particularly challenging to study and therefore difficult to obtain reliable information about incidence, risk factors, management and outcomes. Centre-based studies, because of the small population they cover, or because of the long historical period which has to be studied in order to identify a sufficient number of cases, rarely generate robust and reproducible results which can be generalised to today's obstetric populations. As AFE is infrequent, the most robust studies of the condition are population-based studies, ideally incorporating large numbers of pregnant women in order to have sufficient statistical power to generate stable incidence estimates and examine a range of risk factors to assess the independent risk associated with each. Multinational studies can enhance further the robustness, timeliness and hence utility of study results.A recent systematic review highlighted apparent differences in the incidence of AFE in different studies , with esWe reviewed available data sources on the incidence of AFE in Australia, Canada, the Netherlands, the United Kingdom and the USA. Where information was available, data on the risk factors and outcomes of AFE were also reviewed. Because of variations in definitions of AFE used between countries and between data sources, we did not restrict our analysis to AFE defined using any one particular classification. The definitions and codes used to identify cases in each country are shown in table Maternal Deaths in Australia reports [Fatal cases were identified through four published triennial  reports -10. The  reports  an adminIn New South Wales (NSW) data on incident cases  were obtained from the Admitted Patient Data Collection (APDC), a census of all hospital discharges from public and private hospitals, and linked to the Midwives Data Collection (MDC), a legislated surveillance system of all births in NSW completed by attending midwives and doctors . DiagnosData for Victoria were derived from the Victorian Perinatal Data Collection (VPDC) which is completed by the birth attendant  for each birth . DiagnosAs previously reported , informaFatal cases were identified from the Dutch Confidential enquiries into the causes of maternal mortality between 1983-1992 and 1993-2005 . IncidenFatal cases were identified through the United Kingdom (UK) confidential enquiries into maternal deaths between 2003 and 2008 ,18.UK data on incident cases of AFE  were obtained through the UK Obstetric Surveillance System (UKOSS) between February 2005 and January 2010 . In eachFatal cases were identified through the Pregnancy Mortality Surveillance System (PMSS) in the Division of Reproductive Health at the Centers for Disease Control and Prevention (CDC) . Since 1Non-fatal and fatal incident cases were obtained from the Nationwide Inpatient Sample (NIS) for 1999-2008. The NIS is part of the Agency for Healthcare Research and Quality's Healthcare Cost and Utilization Project. The NIS .Incidence rates for AFE with 95% confidence intervals were calculated using as a denominator the number of maternities, deliveries or live births recorded regionally or nationally during each study period. Putative risk factors, identified from factors previously reported in the literature  -4,22, weThe UK AFE study was approved by the London Multi-centre Research Ethics Committee (ref 04/MRE02/46) and the linkage and use of New South Wales data was approved by the NSW Population and Health Services Research Ethics Committee (2006-06-011). The Dutch LEMMoN study was centrally approved by the medical ethics committee of the Leiden University Medical Centre (ref P04-020). No other permissions were required for use of the data presented.The reported incidence of AFE ranged from 1.9 cases per 100 000 maternities (UK) to 6.1 per 100 000 maternities  Table . There wMortality ratios ranged from 0.4 per 100 000 live births in the Netherlands from 1993-2005 to 1.3 per 100 000 live births in the United States in 1997-2001 and 1.1 in Australia excluding Victoria in 1994-2005. Case-fatality rates ranged from 11 to 43%. There were no clear differences in estimates of maternal mortality due to AFE or case fatality rates due to AFE using any different methodology, noting the limited power to detect differences due to small numbers of fatal cases  born to mothers with AFE in the Netherlands were stillborn or died in the neonatal period; the figure was 5 of 75 infants (7%) in the UK and 6 of 19 (32%) in NSW.This analysis demonstrates differences in the reported incidence of AFE in high-resource countries; the reported incidences vary according to the study methodology. Incidence estimates generated from analysis of population databases without additional criteria to exclude false positive cases produce more than double the estimates generated from analyses using specific validated case identification; an estimate from a population database analysis with additional criteria to exclude false positive cases was compatible with the estimates from the validated case identification studies. Comparison of the AFE incidence using a population database with and without criteria to exclude false positive cases also found this pattern, with the estimated incidence from the unselected population database double that when the additional criteria were used. This suggests that the noted European-North American differences in incidence  are alsoIt is difficult to determine which of these estimates is likely to represent a value closest to the "true" incidence of AFE. The apparently lower case fatality amongst cases identified through database studies supports the argument that these studies include a number of false positive cases. The observed association with conditions which may form part of the differential diagnosis of amniotic fluid embolism, for example eclampsia, also supports this hypothesis. Conversely, the higher case fatality observed in studies with case validation may simply indicate that these studies identify more severe cases. Database studies without case validation may thus have high sensitivity but conversely low positive predictive value due to the inclusion of a number of false positive cases, whereas studies with case validation may have lower sensitivity, due to lower case ascertainment and false negative cases, but higher positive predictive value. Perhaps the gold standard would be a database study with subsequent case validation through examination of the medical records. Such an approach, however, in the context of a rare condition such as AFE is difficult due to the large number of individual centres at which medical records would need to be accessed. All studies are complicated by the additional fact that AFE is a diagnosis of exclusion; internationally accepted diagnostic criteria for non-fatal cases do not exist, and there is a place for development of such criteria, using as a model, for example, the criteria used within the UK . DiffereThus the optimal approach to investigating AFE may be dependent on the purpose of the study, since each of the practical approaches has both advantages and disadvantages. Population-based database studies, where suitable sources of information exist, are likely to be the most economical option for studying the condition; however, there is likely to be a degree of inclusion of false positive cases, over-estimation of incidence and thus non-uniformity of the cases examined. The use of additional criteria to exclude false positive cases within database studies, the approach taken in New South Wales , may oveRecommendation 1: Comparisons of AFE incidence estimates should be restricted to studies using similar methodology. Depending on the available resources and research questions, the recommended approaches would be either population-based database studies using additional criteria to exclude false positive cases, or tailored data collection using existing population-based systems specifically designed to facilitate study of rare pregnancy conditions.Recommendation 2: Comparisons of AFE incidence between and within countries would be facilitated by development of an agreed case definition, particularly for non-fatal cases, and an agreed set of criteria to minimise inclusion of false positive cases for studies conducted using population-based databases.We identified very little consistency among countries in the factors associated with occurrence of AFE and no factors consistently associated with fatality from AFE. Maternal age appeared to be associated with the occurrence of AFE in all populations examined, and this may therefore represent a true association, although the mechanisms by which older maternal age predisposes to AFE remain hypothetical and may include disruption of the uterine vasculature or minor degrees of abnormal placental invasion. The available information on placental abnormalities suggests that placenta praevia and placental abruption substantially increase (3-10 times) the risk of AFE. Given the differing methodologies and therefore differing biases due to potential inclusion of false positive cases or selection of more severe cases, other reported associations are very difficult to interpret. One of the most widely reported associations, with induction of labour, was noted to be statistically significant in some studies , with only certain induction methods  or the association was not statistically significant, although the estimated odds ratio was compatible with those reported in most other studies (US) . This ilWith other factors, such as mode of delivery, issues concerning the timing of the event make the data difficult to interpret. The data we examined were often limited, frequently because the timing of the AFE in relation to the delivery was not available, and hence we were unable to identify cases in which a caesarean or operative vaginal delivery was a cause and not a consequence of the AFE. In the studies where timing was available, small numbers of women in certain subgroups, for example the number of cases undergoing operative vaginal delivery, limited statistical power. It is important to note, however, in the context of rising caesarean delivery rates worldwide, that there was a significant association with caesarean delivery in the one country (UK) in which we were able to investigate specifically cases where the AFE occurred after delivery. Analysis of pooled international data, obtained using consistent methodologies with agreed definitions and case validation, would provide more reliable information on these associated factors and hence provide the potential to develop appropriate preventive strategies which would otherwise be limited.Recommendation 3: Groups conducting detailed population-based studies on AFE should develop an agreed strategy to allow combined analysis of data obtained using consistent methodologies in order to identify potentially modifiable risk factors.There were no clear differences in maternal mortality ratios due to AFE between countries or by study methodology. Information on other outcomes for women was very limited, although data on cerebral injury  suggest Recommendation 4: Future specific studies on AFE should aim to collect information on management and longer-term outcomes for both mothers and infants in order to guide best practice, counselling and service planning.This analysis used data from five high resource countries, and the results are thus generalizable only to countries with similar resource settings. As discussed above, data on AFE were obtained using different study methodologies, and therefore this limits the comparability of some of the results. AFE is a rare condition, and all studies have limited power to detect true associations as statistically significant. We hope that this may be addressed in the future by further international collaborative studies, an approach that we would advocate for research into all rare conditions in pregnancy.This analysis has highlighted the benefits of detailed comparison of AFE incidence and ascertainment methods from different population-based studies and has identified a number of difficulties with making direct international comparisons. The study methodology impacts on estimates of disease incidence and case fatality, and may also account for inconsistencies in reported risk factors. There is a need for consistent study methodologies, including agreed case definition and case validation criteria. The use of such unified methodologies will allow for valid international comparisons of incidence in the future, and may permit pooling of international data to provide more reliable information on associated factors, management and outcomes, thus allowing for development of preventive and treatment strategies to improve outcomes of this rare but serious condition.The authors declare that they have no competing interests.MKn conceived the study, participated in the workshop, presented data, contributed to discussions and drafted the manuscript/paper. CB participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. PB participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. MKr participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. GL participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. JO participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. CR participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. CS participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. ES participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. JvR participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. JZ participated in the workshop, presented data, contributed to discussions and participated in revising the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/12/7/prepub


HPV typing using formalin fixed paraffin embedded (FFPE) cervical tissue is used to evaluate HPV vaccine impact, but DNA yield and quality in FFPE specimens can negatively affect test results. This study aimed to evaluate 2 commercial assays for HPV detection and typing using FFPE cervical specimens.Four large North Carolina pathology laboratories provided FFPE specimens from 299 women ages18 and older diagnosed with cervical disease from 2001 to 2006. For each woman, one diagnostic block was selected and unstained serial sections were prepared for DNA typing. Extracts from samples with residual lesion were used to detect and type HPV using parallel and serial testing algorithms with the Linear Array and LiPA HPV genotyping assays.LA and LiPA concordance was 0.61 for detecting any high-risk (HR) and 0.20 for detecting any low-risk (LR) types, with significant differences in marginal proportions for HPV16, 51, 52, and any HR types. Discordant results were most often LiPA-positive, LA-negative. The parallel algorithm yielded the highest prevalence of any HPV type (95.7%). HR type prevalence was similar using parallel (93.1%) and serial (92.1%) approaches. HPV16, 33, and 52 prevalence was slightly lower using the serial algorithm, but the median number of HR types per woman (1) did not differ by algorithm. Using the serial algorithm, HPV DNA was detected in >85% of invasive and >95% of pre-invasive lesions. The most common type was HPV16, followed by 52, 18, 31, 33, and 35; HPV16/18 was detected in 56.5% of specimens. Multiple HPV types were more common in lower grade lesions.We developed an efficient algorithm for testing and reporting results of two commercial assays for HPV detection and typing in FFPE specimens, and describe HPV type distribution in pre-invasive and invasive cervical lesions in a state-based sample prior to HPV vaccine introduction. Reducing the burden of cervical cancer is the key benefit of HPV prophylactic vaccines. In the U.S., state-based cancer registries will serve as the basis for evaluating vaccine impact on invasive HPV-associated cancers, including cervical cancers, as these lesions are reportable In addition to monitoring pre-invasive and invasive cervical endpoints, determining HPV type-distribution in these lesions is important to ensure vaccine-associated types are decreasing as vaccination coverage increases. Linking state cancer registries and sentinel systems to diagnostic pathology laboratories makes it possible to retrieve representative tissue for HPV detection and typing. Standardized laboratory methods adapted for optimal HPV testing in these archived formalin-fixed paraffin-embedded (FFPE) tissues are needed to assure that time trends in data reflect reliable measurement The objectives of this study were to 1) develop an efficient algorithm for testing and reporting results of two commercial assays for HPV detection and typing in archived FFPE tissue specimens (LA and LiPA), and 2) describe HPV type distribution in CIN2/3, AIS, squamous cell carcinoma (SCC), and adenocarcinoma (ADC) in a state-based sample prior to HPV vaccine introduction.This study was approved by the University of North Carolina at Chapel Hill Institutional Review Board. Written consent from the patients was waived by the ethics board because the data were obtained and analyzed anonymously.Four pathology laboratories in North Carolina participated in this study: Carolinas Medical Center, Duke University Health System, Laboratory Corporation of America® Holdings, and the University of North Carolina Health Care. Combined, these laboratories serve a large majority of the population in North Carolina and cover a wide geographical distribution within the state. Each lab searched their pathology database to generate a complete list of CIN2, CIN3, AIS, and invasive cervical cancer cases diagnosed in women ages 18 years and older from January 1, 2001 to June 1, 2006. After stratifying cases by diagnosis , a random sample of 25 cases was selected within each stratum . One block representative of the histology from each of the selected cases was retrieved for type-specific HPV DNA testing. Demographic information retrieved from the pathology records that was linked to the de-identified case number included histologic diagnosis , and other data when available, including age at diagnosis, race, and insurance coverage.Histopathology review: Serial sections were cut from each block using precautions to prevent PCR contamination between cases, including single-use disposable microtome blades, cleaning the microtome between cases, and direct transfer of sections for PCR from microtome to sterile tubes using new single-use applicators (no contact with waterbath). The first and last sections were stained with Hematoxylin and Eosin (H&E). Intervening sections were transferred into 2 mL conical screw cap tubes with tether caps, one 10-micron section or two 5-micron sections per tube . A study pathologist (ERU) reviewed the H&E stained sections to confirm that histology was representative of the diagnosis. Samples that did not have representative material were not processed.DNA isolation: DNA was extracted and purified by a modified protocol using the DNeasy kit  as described elsewhere HPV Genotyping Tests: All DNA extracts were tested with LA and LiPA HPV typing assays, both based on L1 consensus PCR and type-specific hybridization. The LA  uses biotinylated PGMY09/11 and β-globin primer sets and detects 37 individual HPV types , 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, 89, IS39) along with β-globin as an internal control. The manufacturer's protocol was followed with the exceptions of using 10 µL extract in the 100-µL PCR reaction and automated hybridization and washing of the reverse line blot with the Beeblot instrument . Samples positive for the XR(52) probe on the LA HPV strip that were also positive for HPV33, 35 and 58 were further evaluated to confirm or exclude the presence of HPV 52 using an HPV 52 quantitative PCR assay with a threshold of 50 viral copies LiPA  uses SPF10 primers and detects 28 HPV types  as well as a generic probe to detect other HPV types (HPV X) and a genomic control probe. The PCR and amplicon hybridization to the HPV genotyping strip  followed the manufacturer's protocols. The manufacturer's algorithm for interpretation of typing result based on pattern of positive probes was followed. This algorithm includes reporting unequivocal detection of types as well as “possible types”. The ambiguity results from certain hybridization patterns and affects only types 39, 52, 54, 68, 69, and 71. Samples negative for both the genomic control probe and HPV were considered inadequate for evaluation.Concordance between LA and LiPA: For specimens with adequate LA and LiPA results, we calculated kappa to assess congruence of the type-specific results and performed McNemar's test to identify differences in the marginal frequencies by typing assay. Because of the large number of comparisons, we used a threshold of p<.01 to assess significance of the McNemar's test. We performed these analyses for each HPV type present in both tests. We defined HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, as high risk (HR) HPV types, HPV 26, 53, 67, 69, 70, 73, 82, 85, and IS39 as “Possible HR”, and HPV 6, 11, 32, 40, 42, 43, 44, 54, 55, 61, 62, 64, 71, 72, 74, 81, 83, 84, 87, 89 as low risk (LR) HPV parallel algorithm, in which results of both tests were considered for all samples (positive result = positive for either test) and 2) serial algorithm, in which the LiPA assay results were only considered for those samples that were inadequate or negative for any HPV type by LA. We calculated the type-specific prevalence and overall HR or LR prevalence by each algorithm. We also calculated the mean and median number of HPV types detected per woman for each algorithm.We evaluated two algorithms for combining the LA and LiPA results. These were 1) HPV Type Distribution: We examined HPV type distribution stratified by grade of cervical diagnosis. For evaluating vaccine-type HPV prevalence, we examined cases positive for HPV16 or 18 within each diagnosis stratum. We used Pearson's χ2 test for independence to evaluate bivariate associations between vaccine-type HPV prevalence and race, stratified by diagnosis category. The Cochran-Armitage test was used to examine age trends of HPV 16/18 positivity within each diagnosis grade for 3 age groups . We examined bivariate associations between single and multiple infections and 1) disease grade, and 2) age group using Pearson's χ2 test. Two-sided statistical tests were considered significant at the alpha level of 0.05. Data were analyzed using SAS .p<.01). One sample was only tested with LA due to insufficient residual volume for LiPA assay. The remaining 255 samples with valid results in LA and LiPA were used for comparison of the methods. When comparing LA to unequivocal LiPA results for 25 types common to both assays, individual type concordance ranged from 0.33 to 1.00 (median 0.76) . Of the 282 samples processed for HPV testing, 15 (5.3%) were inadequate by LA only, 7 (2.5%) by LiPA, and 4 (1.4%) by both methods (an 0.76) . ConcordAs expected, the parallel algorithm yielded the highest prevalence of any HPV type (95.7%) as well as HR HPV (93.1%) . ApplyinOf the 278 specimens with typing results, 32.7% (n = 91) were CIN2, 31.3% (n = 89) were CIN3, and 30.9% (n = 86) were SCC. AIS comprised <1% of specimens (n = 2), and ADC accounted for only 2.5% (n = 7). The remaining 5 specimens were from subjects who were diagnosed with cervical cancer other than SCC and ADC (classified as ‘other invasive’), for a total of 98 (35.3%) invasive lesions. Proportionally higher CIN2 lesions (14.3%) were typed by LiPA than CIN3/AIS (12.4%) or invasive lesions (6.1%), but the difference was not statistically significant (p = .17).Among women with race information (n = 204) in all diagnosis grades, about half were non-Hispanic white, a third were non-Hispanic black, and 10% were Hispanic. Median age increased with increasing severity of squamous lesions, from 27 years in CIN2 to 33 in CIN3 to 40 in ICC (p<.0001). Median age for AIS was higher at 41 years and highest for ADC (48 years).Vaccine type HPV 16 or 18 was detected in 59.2% of invasive cancer specimens. As shown in Overall, HPV 16 or 18 was detected in 56.5% of specimens. The proportion of HPV 16/18-related lesions appeared to increase with disease severity, although this trend was not statistically significant . We founPrevalence and type distribution of HPV in cervical precancers and invasive cancers is not well-described in the United States Several studies have reported high concordance between the LA and LiPA assays for detecting HPV DNA in cervical cells As expected, HPV16 was the most commonly detected HPV type in all diagnosis categories, followed by HPV 52, 18, 31, 33, and 45. These findings are similar to that found in non-Hispanic white and Hispanic women with CIN3/AIS and invasive cervical cancer in the U.S. Our study has several limitations. First, our population was restricted to adult female residents of North Carolina and may not be generalizable to other U.S. populations. However, we obtained specimens from 4 large pathology laboratories that serve a large majority of residents of North Carolina to minimize selection bias. Second, because the study was cross-sectional in nature, it was neither possible to investigate the effects of aging on HPV persistence or type distribution, nor to examine the influence of HPV types on disease progression. Third, we had limited statistical power to investigate HPV coinfection patterns by disease grade. Fourth, we did not perform additional testing to validate typing results discrepant between LA and LiPA. We assumed that results positive by either method were correct, but some might actually be falsely positive. Concordance between LA and LiPA may differ depending on method of sample collection, extraction method, or disease prevalence.In summary, we used specimens available from a pilot study to develop an efficient approach to HPV testing of archived FFPE specimens. Currently, the serial algorithm is being applied to a variety of other population-based studies of HPV type distribution in cancer and precancer specimens


Endemic malaria and helminth infections in sub-Saharan Africa can act as immunological modulators and impact responses to standard immunizations. We conducted a cohort study to measure the influence of malaria and helminth infections on the immunogenicity of the bivalent HPV-16/18 vaccine. We evaluated the association between malaria and helminth infections, and HPV-16/18 antibody responses among 298 Tanzanian females aged 10–25 years enrolled in a randomized controlled trial of the HPV-16/18 vaccine. Malaria parasitaemia was diagnosed by examination of blood smears, and helminth infections were diagnosed by examination of urine and stool samples, respectively. Geometric mean antibody titres (GMT) against HPV-16/18 antibodies were measured by enzyme-linked immunosorbent assay. Parasitic infections were common; one-third (30.4%) of participants had a helminth infection and 10.2% had malaria parasitaemia. Overall, the vaccine induced high HPV-16/18 GMTs, and there was no evidence of a reduction in HPV-16 or HPV-18 GMT at Month 7 or Month 12 follow-up visits among participants with helminths or malaria. There was some evidence that participants with malaria had increased GMTs compared to those without malaria. The data show high HPV immunogenicity regardless of the presence of malaria and helminth infections. The mechanism and significance for the increase in GMT in those with malaria is unknown. GSK Biologicals was the funding source for the studies. Both studies were approved by the ethics committees of the National Institute for Medical Research (NIMR), Tanzania and the London School of Hygiene & Tropical Medicine (LSHTM), United Kingdom. The helminth/malaria study was registered under ControlledTrials.com (ISRCTN90378590).The HPV 021 trial  to receive either three doses of HPV-16/18 AS04-adjuvanted vaccine (vaccine group) or Al(OH)3 (placebo group) at 0,1 and 6 months. After enrolment (Month 0), participants returned to the clinic at Months 1, 2, 4, 6, 7, 8, 10 and 12 for follow-up visit procedures. Participants were tested for malaria and helminth infections at the Month 7 visit, one month after the scheduled vaccine dose 3. In addition, participants could attend government health services for investigation and management of any illnesses between booked study visits. A record was kept of investigations and treatments given through these other health services.The primary objective of this analysis was to evaluate the association of malaria parasitaemia and helminth infection with antibody responses against HPV-16 and HPV-18 one month (Month 7) and six months (Month 12) after the last scheduled vaccine dose in African females aged 10–25 years.Potential participants were recruited from schools, colleges and family planning clinics in Mwanza, and invited to attend a screening visit for eligibility approximately one month prior to enrolment. Prior to screening, informed consent was obtained from participants aged 18–25 years. For participants aged 10–17 years, we sought consent from a parent or legally authorized representative, as well as assent from the participant. Participants were eligible for enrolment if they were aged 10–25 years at the time of first vaccination, HIV negative, not pregnant, had not had more than six lifetime sexual partners, were free of obvious health problems as established by medical history and examination, had no history of neurologic disorders and were willing to use contraception or to abstain from sex if sexually active for 30 days prior to vaccination and for two months after completion of vaccination. The enrolment was age-stratified, with one-third of participants in the 10–14 years age-stratum and the remainder in the 15–25 years age-stratum.Schistosoma haematobium and (iii) three separate stool samples (during the week following the Month 7 visit) for the diagnosis of Schistosoma mansoni, Ancylostoma duodenale (hookworm), Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiura and Taenia spp. Participants who tested positive for malaria or helminth infections were provided with treatment by study clinicians at a subsequent study visit.Study procedures for the HPV 021 trial have been described in detail elsewhere 2.12.1.1Pairs of thick and thin peripheral blood films from each patient were stained with Giemsa stain in Mwanza, and examined by light microscopy at NIMR in Mwanza, and confirmed at LSHTM. Each thick film was scanned under oil immersion for at least 5 min and the presence of asexual malaria parasites or sexual gametocytes was recorded. Where parasites were seen, the number per 200 white blood cells (WBC) on the thick film was counted and multiplied by 40 to give number of parasites per microliter  2.1.2S. haematobium.A 10 mL aliquot from each urine sample was filtered through 25 mm, 12 μm Millipore filters on Swinnex filter holders. After filtration, the filter was placed onto a glass slide using blunt forceps adding a drop of saline and a glass coverslip. The filter was then examined at the NIMR laboratory under light microscopy for the eggs of S. mansoni, hookworm, S stercoralis, A. lumbricoides, T. trichiura and Taenia spp. using the Kato-Katz method Stool samples were examined at the NIMR laboratory for quantitative egg counts for S. stercoralis in a subset of samples. Approximately 50 mg of unfixed fresh faeces were mixed with distilled water in a 20 mL universal tube In addition, charcoal culture was used to confirm 2.1.3As part of the HPV 021 trial, serological assays for immunogenicity were performed at a GSK laboratory in Belgium. ELISA was used to determine antibodies to HPV-16 and HPV-18 as described previously 2.2®  and analysed using STATA11.0 . Sociodemographic characteristics of participants attending the Month 7 visit were tabulated by infection status and overall. The prevalence of malaria parasitaemia and each helminth infection at Month 7 was tabulated by age group and overall. Helminth infection intensities were classified into light, moderate and heavy, according to WHO guidelines S. mansoni; 2000 and 4000 EPG for hookworm; 1000 and 10,000 EPG for T. trichiura and 5000 and 50,000 EPG for A. lumbricoides, respectively. For S. haematobium, egg counts from urine were classified into two categories only, light (<50 eggs/10 mL of urine) and heavy (≥50 eggs/10 mL of urine or visible haematuria). There were too few participants in the vaccine-arm who were co-infected with both malaria and helminth infections (n = 8), or multiple helminth infections (n = 6) to examine the relationship between co-infection and HPV immunogenicity.Data were double entered and verified in DMSys10 (IgG concentration). Geometric mean titres  and 95% confidence intervals (CI) were calculated.Because the anti-HPV-16 and HPV-18 IgG antibody concentrations showed skewed distributions, HPV antibody results were transformed as logn = 195) or the Month 12 visit (n = 196) and had immunogenicity results. Box plots were used to graphically examine the distribution of raw antibody responses by malaria and helminth infection status. Linear regression was used to compare mean log-transformed IgG antibody between participants with and without any helminth infection, and with and without malaria. Regression coefficients and confidence limits were back-transformed to express results as ratios of geometric means (GMR). These analyses controlled for potential confounding by age of participants, and number of vaccine doses received. Analyses of malaria and HPV vaccine antibody response controlled for presence of any helminth infection. Similarly, the analyses of helminth infection and HPV vaccine antibody response controlled for malaria parasitaemia. There were insufficient data to examine associations with specific helminth infections.The analysis of HPV vaccine antibody response, and malaria and helminth infection was restricted to participants in the vaccine-arm who attended the Month 7 visit  participants attended the Month 7 visit  and 308 (92%) attended the Month 12 visit . The most common reason for discontinuation was withdrawal of consent (4%). The majority (96%) of participants received all three vaccine or placebo doses ; number All participants were of African origin and were HIV-seronegative at baseline. The median age of participants was 18 years (IQR = 13–19). More than three-quarters of participants (82%) were currently students. Most (89%) participants were single. Approximately one-third (37%) of participants lived in houses constructed from cement blocks, and 40% lived in homes constructed from mud bricks . As prev3.2S. mansoni was the most commonly detected helminth, found in one-quarter of participants (24.0%), followed by hookworm (5.7%). S. haematobium was rare; only two (0.7%) participants tested positive. The prevalence of malaria parasitaemia was somewhat higher in younger participants (p = 0.24).At Month 7, approximately one-third (38.1%) of participants tested positive for either malaria parasitaemia or helminth infection. The prevalence of malaria parasitaemia in the entire cohort was 10.2%  and in ticipants , althougS. mansoni infections were light infections, 17.6% were moderate and 4.4% were heavy. Of the two S. haematobium infections, one was light and one was heavy. All (100%) of the hookworm, A. lumbricoides, T. trichiura and Taenia spp. infections were categorized as light infections.Three quarters (77.9%) of 3.3As previously reported, all initially seronegative participants in the vaccinated cohort seroconverted for anti-HPV-16 and -18 antibodies, and remained seropositive up to Month 7. At Month 12, all initially seronegative participants in the vaccine group remained seropositive for anti-HPV-16, and all except one (13-year-old girl) remained seropositive for anti-HPV-18 n = 195), the GMT HPV-16 antibody response at Month 7 was 10,786 EU/mL , and the GMT HPV-18 antibody response was 3701 EU/mL (95% CI 3156–4340) (P < 0.001).HPV immunogenicity was high at Month 7 and Month 12. Among the vaccinated cohort who attended the Month 7 visit and had antibody results (56–4340) . As prevN = 40) was 18,248 EU/mL , and for 15–25-year-olds (N = 67) was 6493 EU/mL (95% CI 4606–9153). Similarly, the GMT HPV-18 antibody response among helminth and malaria uninfected 10–14-year-olds at Month 7 was 5255 EU/mL (95% CI 4109–6720), and for 15–25-year-olds was 2479 EU/mL (95% CI 1807–3399).Antibody responses to HPV-16/18 among 107 vaccine-arm participants without helminths or malaria parasitaemia were high. The GMT HPV-16 antibody response among helminth and malaria uninfected 10–14-year-olds at Month 7 (P = 0.05). Participants with malaria parasites had a 1.2 fold higher GMT HPV-18 antibody response at Month 7 compared to participants without malaria .There was some evidence that participants with malaria parasitaemia at Month 7 had a higher GMT HPV-16 and HPV-18 antibody response ; Fig. 1.P = 0.16) (P = 0.79)  . There w = 0.79) .P > 0.99), after controlling for age, number of vaccine doses received and malaria parasitaemia (P = 0.64). Similar results were seen at Month 12. Although mean antibody response was highest in participants with higher intensity helminth infections, there was no evidence of a signficant difference (At Month 7 and Month 12, GMT antibody responses were similar in participants with and without helminth infections . The GMRsitaemia ; Fig. 1.fference .4This is the first study to examine the effect of malaria and helminth infections on HPV vaccine antibody responses. The incidence of cervical cancer is extremely high in many countries in sub-Saharan Africa which are considering the implementation of HPV vaccination as a cervical cancer control strategy but which also have a high prevalence of endemic malaria and helminth infections. These infections can impact immune responses to vaccinations We observed some evidence of an association between malaria parasitaemia and a higher antibody response to the HPV-16/18 vaccine, which persisted adjusting for age. This association appeared weaker at Month 12 than Month 7 perhaps because there was a longer interval between the timing of the malaria and helminth tests and the antibody data. There was no observed effect of helminth infection, or intensity of helminth infection, on HPV-16/18 antibody response. The mechanism and significance of the increase in HPV-16/18 GMTs among malaria infected individuals is unclear. It is possible that malaria may induce a broader spectrum antibody response than helminths, which may potentiate the immune response to the HPV vaccine. We were unable to assess whether this observation was sustained beyond 12 months of follow-up.As in all observational studies, these findings may be distorted by unmeasured confounders. We attempted to control for potential confounding by age and number of vaccine doses received, which produced little change in the effect estimates. This study also had a small sample size, and a relatively small number of participants with helminth and malaria infections. Results should therefore be interpreted with caution. Sensitivity of the Kato-Katz method in diagnosing helminth infections is relatively low, although we attempted to increase the sensitivity by collecting 3 stool samples from each participant Both animal and human studies indicate that parasitic infections can impair long-term responses to vaccination In summary, we found high HPV immunogenicity regardless of the presence of malaria and helminth infections among young girls and women in Tanzania. There was some evidence of enhanced antibody titres to HPV vaccine genotypes in participants with malaria parasitaemia. Additional research on the impact of parasitic infection on the long-term duration of protection from HPV vaccines is warranted.


Our study aimed to assess adult women’s knowledge of human papillomavirus (HPV) and cervical cancer, and characterize their attitudes towards potential screening and prevention strategies.Women were participants of an HIV-discordant couples cohort in Nairobi, Kenya. An interviewer-administered questionnaire was used to obtain information on sociodemographic status, and sexual and medical history at baseline and on knowledge and attitudes towards Pap smears, self-sampling, and HPV vaccination at study exit.Only 14% of the 409 women  had ever had a Pap smear prior to study enrollment and very few women had ever heard of HPV (18%). Although most women knew that Pap smears detect cervical cancer (69%), very few knew that routine Pap screening is the main way to prevent ICC (18%). Most women reported a high level of cultural acceptability for Pap smear screening and a low level of physical discomfort during Pap smear collection. In addition, over 80% of women reported that they would feel comfortable using a self-sampling device (82%) and would prefer at-home sample collection (84%). Nearly all women (94%) reported willingness to be vaccinated to prevent cervical cancer if offered at no or low cost.These findings highlight the need to educate women on routine use of Pap smears in the prevention of cervical cancer and demonstrate that vaccination and self-sampling would be acceptable modalities for cervical cancer prevention and screening. Cervical cancer is a preventable disease, yet the number of cases globally is expected to almost double by the year 2025 Traditionally, Pap smear, combined with treatment of cervical precancer and early stage cancer, has been successful in preventing up to 80% of invasive cervical cancer cases in developed countries Uptake of self-sampling has been shown to be successful and HPV testing from self-collected samples is highly sensitive for detection of cervical intraepithelial neoplasia grade 2/3 in both clinic and home settings With the increase in technology and opportunity to prevent cervical cancer worldwide, it is important to understand attitudes and barriers of screening among women at high-risk of invasive cervical cancer in low-resource settings. Therefore, our study aimed to assess women’s knowledge of HPV and cervical cancer, and their attitudes towards potential screening strategies, including routine Pap smears, self-sampling for HPV DNA testing, and HPV vaccination. These data will be critical for successful implementation and high uptake of community-level cervical cancer screening programs.Women were recruited into a cohort of HIV-1-discordant couples identified in voluntary counseling and testing centers in Nairobi, Kenya, from May 2007 to October 2009. Couples were eligible to enroll in the study if they reported ≥3 sex acts in the previous 3 months, planned to stay in Nairobi in their current relationship for at least 2 years, and if one member of the couple was HIV-1-infected and the other HIV-1 susceptible. Women who were pregnant and participants using antiretroviral therapy at the time of enrollment were excluded. At enrollment and at quarterly follow-up visits, the HIV status of the uninfected partner was determined using the Determine® HIV–1/2 Rapid Test  or Bioline Recombigen HIV Test , with confirmation by the Vironostika® HIV Uni-form II Ag/Ab ELISA kit . In the HIV-1-infected partner, CD4+ T-cell counts were taken at enrollment and every six months using a FACSCaliber flow cytometer .At enrollment, clinical staff administered a questionnaire to obtain information on sociodemographic characteristics, sexual history and behavior, history of Pap screening, and medical history. Questionnaires were presented in English or Kiswahili, depending on participant preference, and were administered individually to ensure confidentiality. A medical examination was also conducted during biannual study visits, which included collection of a cervical Pap smear. All participants with abnormal cervical cytology were followed up with a repeat Pap test or colposcopy and biopsy as recommended.At the final study visit, up to two years after study enrollment, clinical staff administered an extended questionnaire to obtain additional information on male circumcision, pregnancy history, domestic violence, ARV medication history, and cervical cancer and screening. Relevant questions fell into three categories to assess women’s: 1) knowledge of HPV and the causes and prevention of cervical cancer, 2) attitudes towards Pap smear screening after having had biannual screening in the study, and 3) attitudes towards routine Pap smear screening, self-sampling and HPV vaccination. Specifically, participants were asked if they had ever heard of HPV and if they knew the cause of cervical cancer or how to prevent it. Participants gave detailed histories of Pap smear screening prior to the study or the reasons why they had never had any screening, and were also asked about future intentions for Pap smear screening. Women rated their level of pain during study-conducted Pap smears, beliefs of cultural acceptability, and feelings of necessity of Pap smear screening. Finally, women were asked if they would feel comfortable collecting their own vaginal samples at home and if they would consider HPV vaccination if it were offered at no or low cost. No intervention or education on cervical cancer and screening was provided during the study unless medically indicated, and all responses were based on women’s one-time self-report.Written informed consent was obtained from all participants. The study was conducted according to procedures approved by the University of Washington Institutional Review Board and the Kenyatta National Hospital Ethics and Research Committee.A descriptive summary of knowledge and acceptability overall and stratified by HIV-status is presented. The numbers and percentages of each response are presented. Measures of Pap smear acceptability, necessity and pain-level are based on a scale of 0 to 100 and women’s responses are summarized by the mean, median and interquartile range. Exact logistic regression was used to calculate unadjusted odds ratios and 95% confidence intervals to describe the associations between baseline socioeconomic indicators and medical and sexual history and: 1) having ever had a Pap smear prior to study enrollment, 2) having ever heard of human papillomavirus, 3) knowing that Pap smear screening is conducted to prevent invasive cervical cancer, and 4) feeling that Pap smears are completely acceptable (rating of 100) versus less than completely acceptable. All analyses were conducted using SAS version 9.2 .We interviewed 409 women from HIV-1-discordant couples, 268 (65%) of whom were HIV-positive at baseline with a median CD4+ T-cell count of 463  reported having had a Pap smear prior to the study. Of those who reported having a Pap smear prior to enrollment in the study, most had been done as part of routine care (42%) or as part of a prior research study (19%) and the remaining were conducted for unknown reasons (23%). Those who had never had a Pap smear reported that they did not get screened because they did not know what a Pap smear was or why they needed one (77%). After having at least two Pap smears as part of the study protocol, nearly all women (93%) said that they would seek out Pap screening in the future, with the hospital being the most commonly cited place at which they can be screened. Three-quarters of women said they would be willing to pay up to 400 Kenyan shillings for a Pap smear (approximately 5 US dollars).When asked about methods of screening and prevention, including self-sampling for HPV testing and HPV vaccination, the majority of the women (82%) reported that they would be comfortable using an at-home cervico-vaginal self-sampling device. In fact, 84% of all women said they would prefer this method over having a sample collected in a clinic. Despite their willingness to consider self-sampling, the majority of women (91%) had concerns regarding self-sampling, with the ability to properly collect the sample being the most commonly cited concern (67%). HIV-negative women were slightly more likely to report no concerns (16%) as compared to HIV-positive women (5%). Nearly all HIV-negative (94%) and HIV-positive women (94%) said they would get a vaccine to prevent cervical cancer if offered to them in the future at no or low cost.When asked about the level of acceptability, pain, and necessity that they felt towards Pap smear screening, nearly all women (95%) gave Pap smears the highest possible rating for necessity . About hWomen age 30 years and older , with at least a secondary education , who had ever heard of HPV  or knew that Pap smears were used to prevent invasive cervical cancer  were more likely to have ever had a Pap smear prior to enrolling in the study . Women wWomen with at least a secondary education compared to less than a secondary education , those who had ever had a Pap smear prior to enrollment in the study compared to never previously screened , and those who knew compared with those who did not know that Pap smears were conducted to as part of preventing cervical cancer  were more likely to have heard of HPV. Similarly, women with at least a secondary education compared to less than a secondary education  and those who had ever versus never heard of HPV  were more likely to know that Pap smears can identify cervical abnormalities before cancer develops in order to prevent the development of invasive cancer. A woman’s history of Pap smear screening , knowledge of HPV  and knowledge that Pap smears are a tool to prevent invasive cervical cancer  were associated with giving routine Pap smear screening the highest possible rating for acceptability. Although education was associated with a history of Pap smear screening, knowledge of HPV and knowledge that Pap smears are used in the prevention of cervical cancer, education was not associated with acceptability of Pap smears .The majority of women knew that Pap smears are used to detect cervical cancer (69%), but very few knew that routine Pap screening is the main way to prevent cervical cancer (18%). Most women did not know the cause of cervical cancer (78%) and only 18% had ever heard of HPV. However, it was reassuring to find that most women held positive attitudes towards future Pap smear screening, self-sampling, and HPV vaccination. Our findings highlight the need to educate and reinforce to women that routine Pap smear screening is a key part of preventing invasive cervical cancer so that the fear of being diagnosed with cancer is not a barrier to screening.Despite the fact that knowledge of HPV was very low in our cohort, almost all women reported that they would be willing to be vaccinated against HPV if the vaccine was available at no or low cost. This finding is similar to other studies Consistent with previous studies that document low uptake of Pap smear screening in sub-Saharan Africa, previous Pap smear screening was low in our cohort, with most women (86%) reporting never having had a Pap smear prior to joining the study Without an intervention or educational campaign, it is likely that the strongest predictor of future screening is having been screened in the past. Identifying the characteristics of women who have and have not had previous screening can help to target screening and outreach efforts. In this cohort, women with less education, who were HIV-positive, or didn’t have knowledge of HPV or Pap smears, were less likely to have had previous Pap smear screening. Given the cross-sectional study design, it is unclear whether women learned of HPV during Pap smear screening or whether they learned about HPV from other sources and thus sought out cervical cancer screening. However, in a setting such as Nairobi, where Pap smear screening is available, although access and campaigns are limited, and where HPV testing is now being advertised, it is likely that women who had previous Pap smears for a research study, routine care or diagnostic purposes learned of HPV during or in response to screening. In addition, these findings are consistent with a previous study of cervical cancer patients and non-patients in Nairobi, which found a lower level of education and lack of knowledge of cervical cancer to be associated with a decreased likelihood of having had previous pap screening Women’s knowledge and attitudes were self-reported when they exited the study so there was no follow-up to confirm their feelings or intentions. Pap smear screening conducted during the study may have influenced women’s perception towards Pap smear acceptability, as compared to women who have never been screened. However, after having at least one Pap smear during the study, women reported little physical discomfort associated with Pap smear screening and most responded positively toward future use of a self-sampling device and HPV vaccination. All women in our cohort were in stable, HIV-discordant partnerships and so their responses may not be generalizable to other HIV-negative and HIV-positive women in the general population. However, this is a very relevant population of women since they are of screening age, HIV-positive or at risk for HIV, and many are mothers with daughters potentially eligible for HPV prophylactic vaccination. Furthermore, very little data exist on women’s knowledge and attitudes towards cervical cancer and prevention, especially from East Africa where the incidence and mortality rate of cervical cancer are one of the highest worldwide Despite low levels of knowledge of HPV and that the fact that Pap smears are a tool to prevent, not just detect, cervical cancer, women reported a high-level of acceptability for cervical cancer prevention measures, including Pap screening, HPV vaccination, and self-sampling. Our findings highlight the need for education regarding the cause and prevention of cervical cancer. Currently over 100 sites in Kenya offer regular screening, however, awareness is low and cervical cancer screening coverage for all women age 18–69 years is only 3.2%


Well-documented cases of untreated cervical intra-epithelial dysplasia resulting in fatal progression of invasive cervical cancer are scarce because of a long pre-invasive state, the availability of cervical cytology screening programs, and the efficacy of the treatment of both pre-invasive and early-stage invasive lesions.We present a well-documented case of a 29-year-old Caucasian woman who was found, through routine conventional cervical cytology screening, to have pathologic Papanicolaou (Pap) grade III D lesions . She subsequently died as a result of human papillomavirus type 18-associated cervical cancer after she refused all recommended curative therapeutic procedures over a period of 13 years.This case clearly demonstrates a caveat against the promotion and use of complementary alternative medicine as pseudo-immunologic approaches outside evidence-based medicine paths. It also demonstrates the impact of the individualized demands in diagnosis, treatment and palliative care of patients with advanced cancer express their will to refuse evidence-based treatment recommendations. Cases of intra-epithelial disease of the cervix are almost entirely attributable to human papillomavirus (HPV) infection. A minority of women exposed to HPV develop a persistent infection that affects the squamocolumnar junction where the ectocervix and endocervix meet. Within that junction, dynamic changes of the epithelium occur due to puberty, pregnancy, menopause and hormonal stimulation. The epithelium is vulnerable to noxae associated with smoking, contraceptive use and infection with other sexually transmitted diseases. Alterations of the epithelium are assessed by conventional cervical cytology screening and are scored according to either the Bethesda or the Papanicolaou system. The occurrence of reactive changes and/or cell abnormalities triggers either repetitions of the cytology screening to exclude temporary alterations or a cervical biopsy for histological diagnosis of cervical intra-epithelial neoplasia and cervical cancer. With the advent of HPV vaccination  and HPV Invasive cervical cancer has a long pre-invasive state, and cervical cytology screening programs are available. Moreover, HPV vaccination has been shown to be a successful tool of primary prevention , and treHerein we report a rather rare, yet well-documented case of a 29-year-old woman who, during the course of her disease, accepted multiple diagnostic procedures but refused any curative treatment beginning with the first assessment of cervical dysplasia and early-stage invasive cancer 10 years later. She finally refused to accept any interventional medical strategies, except for palliative care, at the stage of locally progressed and metastasized cervical cancer.A 29-year-old Caucasian woman was seen for her routine annual gynecologic examination, and conventional cytological screening of her cervix uteri revealed a pathologic finding scored as grade IIID under the Papanicolaou system. Repeat screening performed one year later revealed a grade IV pathologic finding, suggesting a high-grade squamous intra-epithelial lesion. Our patient refused the recommended diagnostic and therapeutic procedure of conization, and she was placed on a non-specified homeopathic therapy consisting of a vitamin C-containing regimen and subcutaneous administration of mistletoe lectins.At the time of the first pathologic Papanicolaou test, our patient reported a normal menstrual cycle, no pregnancies, no use of oral contraceptives, no presence of any previous diseases or any surgery, no allergies, no smoking, and no use of illicit drugs. There was no evident lack in body hygiene. Except for her father's stomach cancer, her family and cancer-specific anamneses were unremarkable. On the basis of her grade V in Papanicoleau test, she was sent to our hospital's out-patient department.A gynecologic examination at that time revealed obvious tumor growth confined to her cervix with no signs of extension to her vagina. A cervical biopsy showed moderately differentiated (tumor grade II) large-cell non-keratinizing squamous cell carcinoma of the cervix uteri. Neither lymphatic nor venous vascular space involvement was reported, but dense inflammatory cell infiltration of the tumor stroma was noted. Clinical staging was completed by cystoscopy, proctoscopy, and chest radiography , which revealed stage IB2 cancer. Additional information was obtained by extended staging procedures, including computed tomographic (CT) and laparoscopic sampling of her para-aortic lymph nodes .However, our patient refused to undergo any further diagnostic procedures and instead underwent complementary medical treatment. This included regional hyperthermia, which led to her self-admission to a local hospital. She presented there with reduced physical status, large edema of the legs, and moist rales in her lungs. She also reported lower abdominal pain. Her clinical work-up revealed significant progression of her disease, which now included bilateral parametrial involvement, broad involvement of her dorsal bladder wall, infiltration of her outer rectum wall, pericardial and pleural effusion, bilateral hydronephrosis with laboratory signs of uremia , and tumor anemia with hemoglobin at 71 g/L. Two courses of hemodialysis were performed initially, followed by a right-sided nephrostomy after the failure of ureteral stenting due to tumor extension to her bladder. It was decided to commence hemodialysis on the basis of the patient's request for consequent evidence-based, palliative medical care after restoration of her renal function.Restaging was performed, which indicated involvement of her bladder wall and adhesions to the ileocecal area Figure . All thrChelidonium majus), all of which are purported to have antitumoral activity.Our patient was repeatedly admitted to both Brixen and Innsbruck hospitals for erythrocyte transfusions because of spontaneous uterine hemorrhage and further local tumor progression Figure . Acute lSeven months later our patient was admitted to the hospital with clinical signs of chronic large bowel obstruction, and laparotomy and side-to-side ileoascendostomy became necessary, during which her left ureter Figure , descendFor HPV testing, we isolated DNA from the paraffin-embedded tumor shown in Figure We have presented the case of a woman who had a pathologic cervical cytology screening at the age of 29 years, and died as a result of cervical cancer at the age of 42 after she had denied all recommended curative therapeutic procedures for 13 years. Except for the noted HPV type 18 infection, our patient's demographic characteristics included the typical socioeconomic and epidemiologic risk factors known for cervical cancer in that she was Caucasian, had a high socioeconomic status, reported no cigarette smoking, was nulliparous and reported no history of apparent promiscuity. It appears to be a rather rare case in terms of, on the one hand, accepting far-reaching diagnostic procedures such as laparoscopic lymph node sampling, and on the other, consistently refusing to accept all proposed evidence-based treatment recommendations over a total period of 13 years, with no psychological disorder being apparent.Documented cases of untreated cervical dysplasia are rare, and ours appears to be only the second report published during the past 10 years that is retrievable in the currently available medical literature databases. The other case, reported in 2002, described a very short interval of one year between the diagnosis of cervical dysplasia and metastases in the bone, liver, and orbit . This shOf note, we want to stress that the apparent inefficacy of the complementary alternative medical treatments practiced worldwide, which should have exerted a stimulatory effect on the immune system and hence an antineoplastic influence. Effects in preventing high-grade cervical lesions to date have been noted only for bivalent and quadrivalent vaccines against HPV type 16 or 18 and HPV types 6, 11, 16, and18, with vaccine efficacies well above 90% [HPV type 16 or 18 infections are responsible for approximately 60-80% of all invasive cancers, varying according to the patient's socioeconomic status . Pre-invThis ten-year period of uninfluenced tumor growth also allowed for systemic spread and a pattern of distant metastasis that, to the best of our knowledge, has not thus far been reported in the literature, but suggests a much more complex homing pattern of disseminated tumor cells. Overall, cervical cancer has a low propensity for distant hematogenous metastatic spread. The first clinical sign of metastasis to para-aortic lymph nodes, that is, beyond the true pelvis, was assessed after 10 years on the basis of a CT scan. Furthermore, the most common sites of distant metastasis are the lung, liver, bone and, rarely, the peritoneum. Single reports would add the orbit  and boneIn summary, we have presented an unusual case of untreated, presumably HPV type 18-induced cervical dysplasia with progression to invasive and finally metastatic cervical cancer that demonstrated a ten-year lead time between the diagnosis of dysplasia and invasive cancer. This case serves as a caveat against the promotion and use of complementary alternative medicine as pseudo-immunologic approaches outside evidence-based medicine paths. It also highlights the individualized demands in diagnosis, treatment and palliative care of advanced cancer patients who express their will to refuse evidence-based treatment recommendations.Written informed consent for publication could not be obtained despite all reasonable attempts to trace the patient's family. Every effort was made to protect the identity of our patient, and there is no reason to believe that any of her relatives would object to publication.The authors declare that they have no competing interests.IS, PW, SF, AS and CM cared for the patient during her time in the hospital (LKH Innsbruck and LKH Brixen) and assisted in data collection and the preparation of the manuscript. SB, DR and AGZ were the major contributors in writing the manuscript. EMH performed the histological examination of the tumor tissues. UW performed the HPV testing. All authors read and approved the final manuscript.