Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences.

UNLABELLED: Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. IMPORTANCE: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.

Type-specific HPV prevalence in invasive cervical cancer in the UK prior to national HPV immunisation programme: baseline for monitoring the effects of immunisation.

AIMS: To establish the human papillomavirus (HPV) type-specific prevalence in cervical cancer and high-grade cervical lesions in the UK prior to the introduction of national HPV vaccination. METHODS: Specimens of cervical cancer (n=1235) and cervical intraepithelial neoplasia (CIN)3 (n=2268) were tested for HPV genotypes in England, Scotland, Wales and Northern Ireland. Data were pooled and weighted estimates presented. RESULTS: Among cervical cancer cases, 95.8% were positive for at least one high-risk (HR) HPV type. Restricting to those with HR HPV, the proportion positive for HPV16 and/or HPV18 was similar across countries (weighted overall prevalence 83.0%). This proportion decreased with increasing age at diagnosis (p=0.0005). HPV31, HPV33, HPV45, HPV52 and/or HPV58 were detected in 16.1% of HR HPV-positive cervical cancers and there was no significant association with age for these types. For HR HPV-positive CIN3 cases, there was a similar age-specific pattern with the highest positivity of HPV16 and/or HPV18 in the youngest age group (77.2%). The proportion of HR HPV CIN3 cases positive for HPV31, HPV33, HPV45, HPV52 and/or HPV58 was 36.3% in those aged <30 years at diagnosis. CONCLUSIONS: The prevalence of HPV 16 and/or 18 was high in all UK countries and highest in those diagnosed at a younger age. The UK is well placed to monitor the impact of HPV vaccination on type-specific HPV prevalence in cervical disease.

Activity, safety, and feasibility of cidofovir and imiquimod for treatment of vulval intraepithelial neoplasia (RT³VIN): a multicentre, open-label, randomised, phase 2 trial.

BACKGROUND: Vulval intraepithelial neoplasia is a skin disorder affecting the vulva that, if left untreated, can become cancerous. Currently, the standard treatment for patients with vulval intraepithelial neoplasia is surgery, but this approach does not guarantee cure and can be disfiguring, causing physical and psychological problems, particularly in women of reproductive age. We aimed to assess the activity, safety, and feasibility of two topical treatments--cidofovir and imiquimod--as an alternative to surgery in female patients with vulval intraepithelial neoplasia. METHODS: We recruited female patients (age 16 years or older) from 32 centres to an open-label, randomised, phase 2 trial. Eligibility criteria were biopsy-proven vulval intraepithelial neoplasia grade 3 and at least one lesion that could be measured accurately. We randomly allocated patients to topical treatment with either 1% cidofovir (supplied as a gel in a 10 g tube, to last 6 weeks) or 5% imiquimod (one 250 mg sachet for every application), to be self-applied three times a week for a maximum of 24 weeks. Randomisation (1:1) was done by stratified minimisation via a central computerised system, with stratification by hospital, disease focality, and presentation stage. The primary endpoint was a histologically confirmed complete response at the post-treatment assessment visit 6 weeks after the end of treatment (a maximum of 30 weeks after treatment started). Analysis of the primary endpoint was by intention to treat. Secondary outcomes were toxic effects (to assess safety) and adherence to treatment (to assess feasibility). We present results after all patients had reached the primary endpoint assessment point at 6 weeks; 2-year follow-up of complete responders continues. This trial is registered with Current Controlled Trials, ISRCTN 34420460. FINDINGS: Between Oct 21, 2009, and Jan 11, 2013, 180 participants were enrolled to the study; 89 patients were randomly allocated cidofovir and 91 were assigned imiquimod. At the post-treatment assessment visit, a complete response had been achieved by 41 (46%; 90% CI 37·0-55·3) patients allocated cidofovir and by 42 (46%; 37·2-55·3) patients assigned imiquimod. After 6 weeks of treatment, 156 (87%) patients (78 in each group) had adhered to the treatment regimen. Five patients in the cidofovir group and seven in the imiquimod group either withdrew or were lost to follow-up before the first 6-week safety assessment. Adverse events of grade 3 or higher were reported in 31 (37%) of 84 patients allocated cidofovir and 39 (46%) of 84 patients assigned imiquimod; the most frequent grade 3 and 4 events were pain in the vulva, pruritus, fatigue, and headache. INTERPRETATION: Cidofovir and imiquimod were active, safe, and feasible for treatment of vulval intraepithelial neoplasia and warrant further investigation in a phase 3 setting. Both drugs are effective alternatives to surgery for female patients with vulval intraepithelial neoplasia after exclusion of occult invasive disease. FUNDING: Cancer Research UK.

HPV integration detection in CaSki and SiHa using detection of integrated papillomavirus sequences and restriction-site PCR.

Human Papillomavirus (HPV) infection is the primary cause of cervical neoplasia. HPV DNA is integrated into the human genome in the majority of cervical cancers. The nature of integration may differ with integration incorporating a single copy of HPV or occurring in concatenated form. Our understanding of HPV tumorigenesis is largely based on studies using characterised cell lines with defined integration sites; these cell lines provide an invaluable standard for validation of diagnostic assays. Cell lines also further understanding of integration mechanisms in clinical samples. The objective of this study was to explore integration assays and to investigate integration events in cell lines where HPV is integrated in concatenated form. Restriction site PCR and detection of integrated papillomavirus sequences were performed on DNA from SiHa and CaSki. A novel integration site on Xq27.3 and HPV genome rearrangements were detected in CaSki DNA. However, where integration was previously detected by FISH in CaSki, and reported to be integrated in concatenated form, integration was not detected by DIPS or RS-PCR. The data presented illustrate that HPV copy number can hinder integration detection; this needs consideration when interpreting results from tests applied to clinical samples.

Global genomic diversity of human papillomavirus 6 based on 724 isolates and 190 complete genome sequences.

UNLABELLED: Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Human papillomavirus infection is rare in nonmalignant tonsil tissue in the UK: implications for tonsil cancer precursor lesions.

The incidence of human papillomavirus (HPV)-associated tonsil cancer is increasing but the prevalence of HPV, and of premalignant precursors, in tonsil tissue is unknown. We aimed to assess prevalence of HPV infection in nonmalignant tonsillar crypt epithelia and to histopathologically characterise positive samples. Formalin-fixed paraffin-embedded (FFPE) tonsil tissue specimens were obtained from an age- and sex-stratified random sample of patients aged 0-69 years whose paired tonsils were archived following elective tonsillectomy at hospitals throughout England and Southern Scotland from 2004 to 2008. Homogenised fresh-frozen tonsil tissue was also obtained from archive for two random subsets of males aged 25-34 and over 44. HPV status was assessed in all samples for 20 mucosal HPV types by GP5+/6+ polymerase chain reaction (PCR) enzyme immunoassay and by HPV16 type-specific PCR targeting the E6 gene. In the homogenised material, HPV status was also assessed for 44 HPV types by SPF10-PCR enzyme immunoassay. Of 4,095 randomly sampled FFPE specimens, amplifiable DNA was extracted from 3,377 (82.5%) and from 511 of 524 (97.5%) homogenised tonsils. HPV DNA was identified in 0 of 3,377 (0%, 95% CI 0-0.089%) fixed samples and 0 of 511 (0%, 95% CI 0-0.58%) homogenised samples. This suggests HPV infection may be rare in tonsil reticulated crypt epithelia. Furthermore, we found no evidence of HPV-associated premalignant neoplasia. These data suggest that if HPV-associated premalignant lesions do occur, they are likely to be rare and may have a high risk of progression to carcinoma.

Does the HPV vaccination programme have implications for cervical screening programmes in the UK?

In the UK, a national HPV immunisation programme was implemented in 2008 for girls aged 12-13 years. In addition a catch-up programme was implemented for older girls up to 18 years of age from 2009 to 2011, with an uptake rate of 49.4%. Information about future uptake of cervical screening according to vaccination statistics is important in order to understand the impact of the vaccination programme and implications for a national cervical screening programme. We analysed data on a cohort of women who had been offered the HPV vaccine in the catch-up programme and were invited for cervical screening between 2010 and 2012 in Wales (n=30,882), in a record-linked database study, to describe the cervical screening uptake and clinical outcome according to HPV vaccination status. In our cohort, 48.5% (n=14,966) women had had HPV vaccination and 45.9% (n=14,164) women attended for cervical screening. Women who were unvaccinated were less likely to attend cervical screening (adjusted OR 0.58; 95% CI (0.55, 0.61)). Of those who attended for screening, 13.9% of vaccinated women had abnormal cytology reported compared to 16.7% of women who were unvaccinated. Women who lived in areas with high levels of social deprivation were less likely to be vaccinated (Quintile 5 OR 0.48 95% CI (0.45, 0.52)) or attend cervical screening (Quintile 5 OR 0.70; 95% CI (0.65, 0.75)) compared to those who lived in the least deprived areas. These data highlight the need for new strategies to address inequalities in cervical screening uptake and can inform further mathematical modelling work to clarify the impact of the HPV vaccination programme on future cervical cancer incidence.

UK population based study to predict impact of HPV vaccination.

BACKGROUND: In 2008 a human papillomavirus (HPV) vaccination programme for cervical cancer prevention was implemented in the UK. Surveillance of vaccine uptake, impact on prevalence of HPV infection and cervical cancer incidence were identified as key measures to evaluate the intervention. OBJECTIVE: To determine baseline HPV prevalence in unvaccinated women and predict impact of HPV vaccination on high-grade cervical disease (CIN2+). STUDY DESIGN: A pseudo-anonymous prospective cohort was sampled on entry to the routine cervical screening programme between March 2009 and November 2010. In total, 13,306 eligible females were identified and high-risk (hrHPV) type specific status determined. Potential impact of prophylactic vaccination on CIN2+ was calculated by applying HPV vaccine clinical trial data to the baseline HPV type-specific data. RESULTS: Of 13,306 samples tested, 3545 (26.6%) were confirmed positive for at least one hrHPV type and 1325 (10%) were positive for low risk HPV. HPV16 was the predominant type detected in cases positive with either single or multiple hrHPV infection(s) (5.2% and 4.7%, respectively). Based on hrHPV type-specific data, Gardasil would have prevented 33.2% HPV16/18 unrelated CIN2+ compared to 47.1% for Cervarix. This difference was not statistically significant. CONCLUSION: Prior to the introduction of the HPV vaccine, approximately one-quarter of young women were positive for hrHPV and one-tenth positive for HPV16. Post-vaccination, we anticipate a substantial absolute risk reduction in high-grade cervical disease associated with both targeted and non-targeted hrHPV types. There is no significant difference between the two commercially available vaccines in terms of clinical impact.

Cervical cancers associated with human papillomavirus types 16, 18 and 45 are diagnosed in younger women than cancers associated with other types: a cross-sectional observational study in Wales and Scotland (UK).

BACKGROUND: Most cervical cancers are attributable to infection with one of fourteen types of human papillomavirus (HPV), but HPV types differ in oncogenic potential. Characterisation of cancers associated with specific HPV types is required to predict the likely impact of current prophylactic vaccines and the potential benefits of vaccine formulations including additional HPV types. OBJECTIVE: The study aimed to correlate HPV type with histology and age at diagnosis, in Invasive Cervical Cancers (ICCs) from two of the devolved countries of the UK (Wales and Scotland). STUDY DESIGN: Centralised histopathology review and rigorously standardised HPV-DNA typing were applied to 592 ICC diagnosed 2001-2006. HPV status was analysed in relation to histology and age at diagnosis. RESULTS: HPV infection was confirmed in 535/592 cases. Among the 497 tumours infected with single HPV types, the three most common types were HPV16 (62% 95%CI: 57.6-66.1), HPV18 (18.9% 95%CI: 15.7-22.6) and HPV45 (5.4% 95%CI: 3.7-7.8). HPV16 or 18 were present in 80.9% of HPV positive cases. Women with tumours associated with HPV types 16, 18 and 45 were on average 10.5 years younger at diagnosis than women with tumours associated with other HPV types. CONCLUSIONS: Prophylactic vaccines targeting HPV16 and 18 could potentially prevent up to 80.9% of ICC in the populations investigated. Cancers associated with HPV16, 18 and 45 were diagnosed at younger ages, supporting the hypothesis of faster progression than for tumours caused by other HPV types.

Knowledge and awareness of HPV and the HPV vaccine among young women in the first routinely vaccinated cohort in England.

A national school-based human papillomavirus (HPV) vaccination programme has been available for 12-13 year old females in the UK since 2008, offering protection against HPV types 16 and 18, which are responsible for the majority of cervical cancer. Little is known about HPV knowledge in girls who have been offered the vaccine. Girls offered the school-based vaccine in the first routine cohort (n=1033) were recruited from 13 schools in London three years post-vaccination. Participants completed a questionnaire about HPV awareness, knowledge about HPV and the vaccine, and demographic characteristics including vaccine status. About a fifth of the girls reported they were unaware of the HPV infection. Among those who reported being aware of HPV (n=759) knowledge was relatively low. Approximately half of the participants knew that HPV infection causes cervical cancer, condoms can reduce the risk of transmission and that cervical screening is needed regardless of vaccination status. These results are helpful in benchmarking HPV-related knowledge in vaccinated girls and could be used in the development of appropriate educational messages to accompany the first cervical screening invitation in this cohort in the future.

Human papillomavirus type distribution in vulval intraepithelial neoplasia determined using PapilloCheck DNA Microarray.

Vulval intraepithelial neoplasia is a precursor of vulval carcinoma, and is frequently associated with human papillomavirus (HPV) infection. Estimates of HPV prevalence in vulval intraepithelial neoplasia vary widely in the UK. The objective of this study was to assess HPV infection in a sample of women with vulval intraepithelial neoplasia, confirmed histologically, and determine the proportion of disease associated with HPV types targeted by prophylactic HPV vaccines. HPV infection was assessed in biopsies from 59 patients using the Greiner Bio-One PapilloCheck® DNA chip assay. Valid results were obtained for 54 cases. HPV infection was present in 43 of the 54 cases (79.6%: 95% CI 67.1-88.2%). The most common HPV types were HPV 16 (33/54: 61.1%), HPV 33 (8/54: 14.8%), HPV 6 (5/54: 9.3%), and HPV 42 (3/54: 5.6%). The mean age of HPV positive women was significantly less than the mean age of HPV negative women. This is the largest UK series of vulval intraepithelial neoplasia in which HPV type has been investigated, and 34/54 (63.0%, 95% CI: 49.6-78.6%) cases were associated with HPV 16/18, which are targeted by current prophylactic HPV vaccines.

The risk of cervical cancer associated with specific types of human papillomavirus: a case-control study in a UK population.

Mounting evidence supports incorporation of HPV testing into cervical screening; however, the optimal test format and target population have yet to be confirmed. Assessment of the potential benefits of type-specific testing requires estimation of the risk associated with infection with individual HPV types. However, the risk posed by individual HPV types may be population specific and influenced by cervical screening practice. The existing data on HPV type-specific risk is derived largely from unscreened populations. Our study addressed the lack of data on HPV type-specific risk in cytologically screened populations using a case-control study of 262 invasive cervical cancers diagnosed in Wales between 2000 and 2006, and 8,428 controls who attended for cytological screening in 2004. The analysis showed that the odds ratios (ORs) for infection with HPV 16 and 18 are considerable; 2770 (95% CI 1050-7320) and 950 (95% CI 330-2740), respectively, and that the OR for other oncogenic types are in general considerably less (ranging, where quantified, from 20.2 to 386 in the same population). The effect of age on OR associated with particular HPV types was also assessed; this indicated that infection with a high-risk HPV in women older than 40 years was associated with an approximately 30-fold increased risk of invasive cervical cancer relative to women younger than 40 years. These results indicate that there is significant prognostic information associated with knowledge of HPV type.

TA-CIN, a vaccine incorporating a recombinant HPV fusion protein (HPV16 L2E6E7) for the potential treatment of HPV16-associated genital diseases.

Commercially available prophylactic HPV vaccines for cervical cancer prevention have limited use in women with previous viral exposure. Therefore, a therapeutic HPV vaccine would benefit patients with HPV-associated genital diseases. Being developed by Cancer Research Technology Ltd, under license from Xenova Group plc, TA-CIN (Tissue Antigen - Cervical Intraepithelial Neoplasia) is a fusion protein vaccine comprising the HPV16 viral proteins L2, E6 and E7 for the treatment of HPV16-associated genital diseases. In mouse models, TA-CIN induced dose-dependent HPV16-specific CD4 and CD8 T-cell responses, which were enhanced when boosted with the vaccinia-based vector vaccine TA-HPV (Therapeutic Antigen - HPV). A phase I clinical trial of TA-CIN in healthy volunteers reported no serious adverse events and HPV16-specific cellular immune responses. Phase II trials in patients with anogenital and vulval intraepithelial neoplasia investigated heterologous prime/boost strategies with TA-CIN/TA-HPV and TA-HPV/TA-CIN, but neither of the regimens offered advantages over single-agent TA-HPV. A recent phase II trial investigating imiquimod/TA-CIN in patients with vulval intraepithelial neoplasia demonstrated significant infiltration of CD4 and CD8 T-cells in lesion responders and complete lesion regression in 63% of patients. More comprehensive case-controlled trials are needed to define responders to immunotherapy with TA-CIN and verify its prophylactic and therapeutic properties.

The potential impact of human papillomavirus vaccination in contemporary cytologically screened populations may be underestimated: an observational retrospective analysis of invasive cervical cancers.

The aim of this study was to determine the proportion of invasive cervical cancers attributable to human papillomavirus (HPV) types 16 and 18 in a contemporary, cytologically well-screened UK population. This was achieved in a retrospective observational analysis by HPV typing 453 archival invasive cervical cancers diagnosed between January 1, 2000 and September 1, 2006. Pathological material was collected from 9 hospitals across Wales (UK), and HPV typing and pathology review was conducted at a central laboratory. Genotyping for high-risk HPV DNA was performed by PCR-enzyme immunoassay using the GP5+/6+ primer set. DNA was successfully extracted from 297 cases. Two hundred and eighty cases were included in the final analysis. The proportion of cases which had only HPV 16 and/or 18 was 219 of 280 (78.2%, 95% CI = 73.0-82.7); the proportion of cases which had HPV 16 or 18 and another HPV type was 230 of 280 (82.1%, 95% CI = 77.2-86.2). The proportion of cervical cancers associated with infection with HPV types 16 and 18 has previously been estimated at around 70%. The appropriate figure for a cytologically well-screened UK population appears to be approximately 80%. Hence, the potential impact of the current vaccination programme may be underestimated.

The role of respiratory viruses in cystic fibrosis.

BACKGROUND: Previous studies have suggested a role played by respiratory viruses in the exacerbation of cystic fibrosis (CF). However, the impact of respiratory viruses could have been underestimated because of the low detection rate by conventional laboratory methods. METHODS: Children with CF had nasal swabs and sputum samples obtained on a routine basis and when they developed respiratory exacerbations. Nucleic Acid Sequence Based Amplification (NASBA) was used to detect respiratory viruses from nasal swabs. The definition of a respiratory exacerbation was when the symptom score totalled to 4 or more, or if the peak expiratory flow fell by more than 50 l/min from the child's usual best value, or if the parent subjectively felt that the child was developing a cold. RESULTS: 71 patients had 165 reported episodes of respiratory exacerbations. 138 exacerbation samples were obtained of which 63 (46%) were positive for respiratory viruses. In contrast, 23 of 136 asymptomatic nasal swabs (16.9%) were positive for respiratory viruses. There was significantly more viruses being detected during respiratory exacerbations, in particular influenza A, influenza B and rhinovirus (p<0.05). Upper respiratory symptoms significantly correlated with positive respiratory viral detection (p<0.05). This study also showed that viral respiratory exacerbations in CF could be independent from bacterial infections. CONCLUSIONS: Respiratory viruses are associated with exacerbations in CF and upper respiratory symptoms are strong predictors for their presence. 'Real-time' NASBA has a rapid turn-around time and has the potential to aid clinical decision making, such as the use of anti-virals and administration of antibiotics.

Is there a role for influenza vaccination in cystic fibrosis?

BACKGROUND: Influenza vaccination is generally recommended to patients with Cystic Fibrosis (CF). Previous studies have shown that influenza infections cause worsening lung functions, disease progression and increase propensity of bacterial infections in CF. However, the clinical evidence in the effectiveness of influenza vaccination in CF is lacking. AIMS: This study retrospectively reviewed the influenza vaccination status in the patients with CF and compared the influenza infection rates between the vaccinated and non-vaccinated groups. Finally the factors associated with non-adherence with vaccination were determined. METHODS: Nasal swabs were obtained from 63 patients with CF between the age of 6 months to 18 years routinely and during respiratory exacerbations between October 2003 to April 2004. Influenza A and Influenza B were detected using Nucleic Acid Sequence Based Amplification (NASBA). The influenza vaccination status of these patients was retrospectively reviewed. RESULTS: 41 patients (65%) were vaccinated against influenza. For the 22 patients (35%) who were not vaccinated, 7 of them were scared of needles leading to non-adherence. Influenza virus was detected on 5 occasions; 3 were Influenza A (60%) and 2 were Influenza B (40%). 1 virus in the vaccinated group and 4 in the non-vaccinated were detected during the study period (p-value=0.046). CONCLUSIONS: Although the current available evidence to support routine influenza vaccination is limited in CF, this study has shown that such practice may yet play a role in preventing its subsequent acquisition.

Development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza A.

The development and introduction of effective treatment for influenza A in the form of neuraminidase inhibitors have made the rapid diagnosis of infection important especially in high-risk populations. The aim of this study was to develop a real-time nucleic acid sequenced based amplification (NASBA) using a molecular beacon that could detect a wide range of influenza A subtypes and strains in a single reaction by targeting a conserved region of the influenza genome, and to evaluate its sensitivity and specificity against traditional laboratory techniques on a range of clinical samples usefulness during the 2003/2004 influenza season. The results demonstrated the assay to be highly sensitive and specific, detecting <0.1 TCID50 of virus stock. Three hundred eighty-nine clinical samples were tested in total from two patient groups. Overall, the real-time NASBA assay detected 64% (66/103) more influenza positive samples than cell culture and direct immunofluorescence (IF) and, therefore, was shown to be more sensitive in detecting influenza A in a wide range of respiratory samples than traditional methods. In conclusion, the real-time influenza A assay demonstrated clinical usefulness in both hospital and community populations.

Development and evaluation of NucliSens basic kit NASBA for diagnosis of parainfluenza virus infection with 'end-point' and 'real-time' detection.

New methods for the detection of human parainfluenza viruses (HPIVs) were developed. These were based on nucleic acid sequence-based amplification (NASBA) and utilised the NucliSens Basic Kit. Primers and probes were selected from the haemagglutinin neuraminidase (HN) gene of HPIV1, HPIV2 and HPIV3, and from the phosphoprotein (P) of HPIV4a and -4b. Synthetic RNA, titrated control virus stocks and respiratory specimens (n=44) were utilised to evaluate performance of the assays. Detection of NASBA products was by probe hybridisation and electrochemiluminescence (ECL) ('end-point' detection) or using molecular beacons ('real-time' detection). The assays using ECL detection proved to be both sensitive and specific. Typically, less than or equal to 100 RNA copies or one TCID(50) input was detectable with no cross-reaction between the specific HPIV assays and other respiratory viruses. Results for clinical samples were concordant with those obtained by 'conventional' procedures by classical viral diagnostic methods. 'Real-time' detection utilised probes specific for either HPIV1 or HPIV3 with similar performance characteristics to the assays with 'end-point' detection. The feasibility of multiplexing targets together was confirmed using a combined HPIV1 and HPIV3 assay with good results for ECL and molecular beacon detection on control material and clinical samples.