TP53 is one direct ZBTB11 transcriptional target. TP53 repression is dependent on ZBTB11 cys116, which is a functionally critical, metal ion-coordinating residue within a novel viral integrase-like zinc finger domain. To our knowledge, this is the first description of a function for this domain in a cellular protein. We demonstrate that the PU.1–ZBTB11–TP53 pathway is conserved from fish to mammals. Finally, Zbtb11 mutant rescue experiments point to a ZBTB11-regulated TP53 requirement in development of other organs.In response to infection and injury, the neutrophil population rapidly expands and then quickly re-establishes the basal state when inflammation resolves. The exact pathways governing neutrophil/macrophage lineage outputs from a common granulocyte-macrophage progenitor are still not completely understood. From a forward genetic screen in zebrafish, we identify the transcriptional repressor, ZBTB11, as critical for basal and emergency granulopoiesis. ZBTB11 sits in a pathway directly downstream of master myeloid regulators including PU.1, and  TP53.Neutrophils are increased in response to injury and infection but how they form from a common granulocyte-macrophage progenitor is unclear. Here, the authors identify a role for the transcriptional repressor ZBTB11 in zebrafish, which is regulated by master myeloid regulators and represses  Trillions of neutrophils are required every day for host defence. In response to threats like infection and injury, the neutrophil population must be rapidly expanded25TP53 has long been synonymous with its central role as a tumour suppressor and protector of genome integrity. Its functional sphere of influence extends beyond this role, however, and TP53 has a key role in HSC development. High levels of TP53 are associated with HSC quiescence and expression of TP53 must be downregulated in order for HSCs to exit quiescence and enter the cell cycle7Zbtb11  domain containing 11) is an enigmatic member of the ZBTB (BTB-ZF or POK—Pox virus and Krüppel-like zinc fingers) superfamily of ∼49 proteins characterized by the family defining protein–protein interacting BTB domain and varying numbers of zinc fingers101113111517TP53, pin-pointing key residues that are required for ZBTB11 function as determinants of a novel N-terminal HHCC  zinc finger and establish a Zbtb11–Tp53-dependent pathway that regulates neuronal cell death.Herein we define a biological function for Zbtb11 and provide the first functional evidence for its role in myeloid lineage development. We describe a new evolutionarily conserved genetic and biochemical pathway connecting the master myeloid regulator PU.1 and ZBTB11 in basal neutrophil development and emergency granulopoiesis, and show that early in haemopoiesis Zbtb11 is required for neutrophil but not macrophage development. We identify ZBTB11 as a new transcriptional repressor of marsanne (mne) as a temperature-sensitive mutant with defective myeloid development evidenced by a deficit of cells expressing the neutrophil markers myeloperoxidase (mpx) and lysozyme (lyz), and myelomonocytic markers nephrosin (nspn1) and l-plastin (lcp1) ) is preserved for the life of the embryo (5 days post fertilization (d.p.f.)), localizing the haemopoietic defect to the myeloid compartment  . A similp1)  Zbtb11 and independent non-complementing CRISPR/Cas9-generated indel zbtb11 alleles validated the positional cloning  of fluorescence-activated cell sorting (FACS)-sorted adult zebrafish kidney marrow confirms expression of Zbtb11 in adult haemopoietic cells, with highest levels in myeloid cells , normal (28 °C) and permissive (23 °C) temperatures. In all cases, the population sizes of macrophages were not significantly different between mne and WT, showing that at 48 h.p.f. Zbtb11 deficiency does not impair basal macrophage development  cells. The myeloid specification determinant Pu.1  expression is slightly elevated compared to WT at 48 h.p.f. , which could indicate Pu.1 modulation by a Zbtb11-mediated potential negative feedback loop, though this remains to be explored. Collectively, these data identify a new myeloid transcription factor-ZBTB11 axis that is evolutionarily conserved in fish and mammals.Zbtb11-dependent transcriptional networks and its upstream genetic regulators have not been defined. To determine where Zbtb11 is placed with regard to the known haemopoietic transcriptional hierarchy, 2.9 kb of the human u.1 ref.  positive did not , furthert manner . These f11 locus  in granumne RNA were prepared from both whole embryos and FACS-purified mpx-EGFP or lyz:dsRed-expressing cells, and subjected to global RNA expression profiling. Tp53 was elevated in mne compared to WT in both analyses : logFC=3.5; neutrophils (RNA sequencing (RNAseq)): logFC=2.91, FDR=0.0000001). Tp53 was of particular interest because of the known requirement for its downregulation during the maturation of haemopoietic lineages724tp53 transcripts in mne was accompanied by corresponding functional protein. The high levels of the Δ113Tp53 alternative transcript shown by whole-mount in situ hybridization (WISH) in mne indicate that the upregulation of tp53 transcripts in mne results in stabilized activated Tp53 protein capable of transactivating its target genes, which include Δ113Tp53  reporter, in which Annexin 5-mVenus expression serves as a marker for cell death, was crossed onto mne. Quantification of Annexin 5+ cells demonstrated a significant increase in global apoptosis in mne  reporter was again employed. It also served as an internal control to monitor efficacy of the tp53 translation blocking morpholino used to knock down Tp53 levels, which would be predicted to rescue any Tp53-dependent cell death phenotype. Indeed, the abnormally high global cell death in mne embryos was normalized to WT levels in mne tp53 morphants CX2C zinc finger motif  measured . Furthermeasured . Togethemne compared to WT at 48 h.p.f. and this requirement continues into definitive haemopoiesis where haemopoietic stem cell-dependent neutrophil expansion fails to occur in mne in response to immune challenge or cytokine stimulation. Thus Zbtb11 appears to be important for both basal and emergency granulopoiesis. That macrophage number is indistinguishable between WT and mne at 48 h.p.f. and that mne macrophages can reconstitute after ablation indicate that intact Zbtb11 is dispensable for primitive macrophage development but becomes rate-limiting as reliance on HSC self-renewal and differentiation increases and becomes absolute following the onset of definitive haemopoiesis. The differential timing between neutrophil and macrophage depletion suggests Zbtb11 has an essential role in establishing a full complement of neutrophils, whereas with macrophage and other lineages, the later depletion is reflective of a broad failure of myelopoiesis. The positioning of ZBTB11 downstream of the master myeloid regulators PU.1, C/EBPα and GFI1 in the haemopoietic transcriptional hierarchy suggests ZBTB11 may potentially act at the level of progenitors to direct proliferation, differentiation and/or survival towards amplifying neutrophil number. This is supported by the data showing that Zbtb11 dysfunction results not only in a quantitative defect in neutrophils but also a qualitative defect manifest in the high proportion of immature neutrophil lineage cells in mne.We have identified a role for the previously enigmatic Zbtb11 protein in myeloid development. The Zbtb11 requirement for granulopoiesis is already apparent during primitive haemopoiesis reflected by the paucity of neutrophils in ZBTB11 within the haemopoietic transcription factor hierarchy, its precise mechanism of action remains elusive. Pathway analysis generated from RNAseq data from mne versus WT neutrophils reveals central roles for Zbtb11 in RNA processing, DNA replication and repair as well as cell death and survival, and we have presented functional data validating roles in both DNA replication and cell death, where Zbtb11 deficiency results in markedly increased apoptosis and virtually absent DNA synthesis. Studies describing a role for TP53 in haematopoiesis, specifically the granulocytopenia accompanying overexpression of Trp53 in the Mdm2 knockout mousetp53 in mne was in response to cell stress or whether it was due to derepression in the absence of fully functional Zbtb11. The biochemical evidence shows that not only does ZBTB11 repress TP53 but that it requires Cys116 for this function, suggesting that the high levels of Tp53 in mne may at least in part be due to derepression of tp53 by mutant Zbtb11. Van Nostrand et al.mne, and may partially explain the pleiotropism that results from Zbtb11 dysfunction and high levels of Tp53 during development in mne.Although we have clearly positioned tp53 overexpression in mne by a widely used and extensively characterized translation-blocking morpholino partially rescued neutrophil number, pointing to a novel Pu.1–Zbtb11–Tp53 pathway for regulation of neutrophil development. This observation was in contrast to the tp53 mutant data, which demonstrated that in the context of a tp53M214K/M214K allele encoding transcriptionally dead Tp53 protein, neutrophil number was not normalized in mne. Discrepancies between morpholino knockdown and stable genetic mutant phenotypes have been highlighted recentlymne to a subtle level that allows for amelioration of the neutrophil deficit, while genetic inactivation of tp53 removes the normal level of control achieved by low levels of Tp53 and presents a different regulatory landscape that prevents rescue of the neutrophil deficiency. It is also possible that compensatory mechanisms by TP53 family members p63 and p73 may come into play in the tp53 mutant3536383940mne phenotype be attributable solely to stress response overexpression of Tp53? Neither the genetic inactivation nor morpholino knockdown data can rescue mne gross morphological defects, which strongly supports the notion that the phenotype observed in mne is not due solely to activation of Tp53 stress response pathways and that Zbtb11 can exert its biological effects through a Tp53-independent mechanism. With regard to CNS cell death, the morpholino and genetic data are corroborative, demonstrating that CNS cell death is dependent on both an intact mne locus and functional tp53. This is consistent with an additional role for the ZBTB11–TP53 pathway outside of haemopoietic development.Knockdown of zbtb11 during zebrafish myeloid development was cell-autonomous by transient overexpression approaches. However, we were not able to confirm reliable, reproducible expression of Zbtb11–GFP targeted to myeloid cells from transient, mosaic expression, even using a Gal4/UAS approach in an attempt to amplify the signal. Future endeavours to address this issue experimentally will require stable transgenic approaches optimized for Zbtb11 reporter expression in zebrafish, or approaches in other animal models.We sought to investigate whether the requirement for 3–7H(X23–32)CX2C CX2C) zinc finger in the N-terminal domain of Zbtb11 that underpins its function as a transcriptional repressor provides the first functional data for the HHCC domain in a human protein. The evolutionary conservation of the HHCC motif across >22 vertebrate species of Zbtb11 further highlights its importance for function. The identification of a cellular function for the HHCC domain in Zbtb11 supports the previously untested hypothesis2+ ion identified in HIV-1 integrase and the functional consequences for enzyme activity, suggest a complex novel regulatory role mediated through this domain. This complexity is reflected in the Zbtb11 in vivo bioassay data, where the absence of the HHCC domain allows Zbtb11 to rescue the mne phenotype but mutation of any of the four metal ion-coordinating amino acids does not. The notion that steric hindrance by an incorrectly folded N terminus is potentially more detrimental than its complete absence has previously been documented47in vivo bioassay or a single previous in vitro studyThe non-catalytic HHCC domain that resides in the N-terminal domain of retrovirus and related retrotransposon integrases, such as HIV-1 (human immunodeficiency virus 1), is crucial for determining the conformation and therefore activity of the integrase and infectivity of the virusX2C ref. . StudiesZbtb11 is a previously under-studied protein to which we now ascribe a biological function squarely positioning it within the haemopoietic transcription factor hierarchy as a regulator of basal neutrophil development and emergency granulopoiesis. In addition, we have identified a genetic and biochemical pathway connecting ZBTB11 and TP53 that now merits consideration in all tissues in which both Tp53 and Zbtb11 are expressed. Lastly, we have identified a novel integrase-like HHCC domain in Zbtb11. To our knowledge, we have provided the first cellular function for this domain in a human protein, specifically the transcriptional repressor activity of Zbtb11, with potential functional implications for regulatory domains of other human proteins. Together, these studies provide a basis for understanding how Zbtb11 dysregulation may contribute to disease pathogenesis and opens a new window on virally derived cellular domain function.Marsanne (mnegl11) is a novel mutant isolated from an ethylnitrosourea (ENU) mutagenesis screenmpx:EGFP)i114 (ref. lyz:dsRed)nz50Tg (ref. gata1a:dsRed)sd2Tg (ref. ubi:secAnnexinV-mVenus)mq8Tg gl26 (ref. UAS:nfsb-mCherry)c264 . Compound and mutant lines were generated by intercrossing. Fish were housed in the Ludwig Institute for Cancer Research Aquarium and ARMI FishCore, and mice were housed in the WEHI mouse facility using standard husbandry practices. Experiments were performed according to protocols approved by the Animal Ethics Committees of the Ludwig Institute for Cancer Research, The Walter and Eliza Hall Institute of Medical Research and Monash University.Strains: St Kilda Wild Type , WIK, AB, AB*  and Tübingen . 114 ref. , Tg using exon 2 primers  transgenic animals. Erythrocytes were sorted first on the basis of gata1-dsRed fluorescence and remaining populations were sorted on the basis of their physical characteristics (forward and side scatter). Triplicate sample replicates obtained from single complementary DNAs were subjected to quantitative PCR using primers in gata1 and spi1, which demonstrated low/absent expression of gata1 in the myeloid population and low/absent levels of spi1 in the erythroid population (Haemopoietic populations were obtained from adult zebrafish whole kidney marrow from Tg(pulation .mne cDNA was used for PCR amplification of zebrafish zbtb11 cDNA. The PCR product was cloned into pBluescript II SK+ (Stratagene) then directionally cloned into pCS2+ ClaI/XhoI sites to generate mne-zbtb11-pCS2+. Mutagenesis was used to generate WT-zbtb11-pCS2+ from the mnezbtb11 clone. Human ZBTB11 was amplified from K562 cells and mouse Zbtb11 was amplified from mouse thymocyte cDNA (a kind gift from Dr Matthew McCormack). Human (2.9 kb) and zebrafish (2.3 kb) Zbtb11 promoters were cloned from K562 and zebrafish genomic DNA, using In-Fusion (Clontech) into pGL3-luc (Promega). All constructs were verified by nucleotide sequencing.Primers for cloning are shown in tp53 MO or control MO  and sequence verified (for primers see in vivo bioassay embryos from a mne+/− incross were injected with WT (rescue control) or test Zbtb11 mRNA and seeded at ∼40 embryos/dish. To determine rescue, gross morphological phenotype was scored at 48 h.p.f. for all embryos and the percentage of mutant versus WT embryos was determined for each Zbtb11 construct and compared against the non-injected controls (Mendelian ratio of ∼25% mutants).For the zbtb11-specific oligonucleotide containing a T7 polymerase recognition sequence at the 5′ end was annealed to the constant oligonucleotide (encoding the reverse complement of the tracrRNA tail) via an overlapping homologous regionmpx:EGFP) or Tg(lyz:dsRed) embryos. Injected embryos were screened genotypically by sequencing and phenotypically for the mne phenotype. F0 embryos were raised and out-crossed onto mne to determine founders containing non-complementing CRISPR alleles. Indel mutations were characterized in the F1 generation.Oligonucleotides for guide RNA (sgRNA) synthesis were designed using CHOP–CHOP (sequence in marsanne embryos using Trizol/chloroform extraction and isopropanol precipitation and treated with RNase-free DNase (Ambion). Any remaining phenol was removed using an RNeasy micro kit (Qiagen). Samples were provided to the Ramaciotti Centre for Genomics  for QC and processing. Input was 100 ng of total RNA, samples were processed using the Affymetrix WT Plus kit with no amplification and hybridization was to Affymetrix Zebrafish Gene Array 1.0 ST (Affymetrix). Data were analysed using Bioconductor (Bioconductor—Open Source Software for Bioinformatics ( http://www.bioconductor.org) Copyright 2017) and R version 3.2.5 ) packages. Expression values for all genes were calculated using the robust multi-array average methodmarsanne. For the identification of genes with differential expression between groups, fold-change cutoff (≥2.0) and P value cutoff (≤0.05) were used for differential expression.Total RNA was isolated from three biologically independent pools of WT and O-dianisidine was performed for 15 min at RT in 0.6 mg ml−1O-dianisidine, 0.01 M sodium acetate (pH 4.5), 0.65% H2O2 and 40% vol/vol ethanol. Embryos were imaged on an Olympus MVX10 microscope and processed in FijiWISH was performed using standard techniques2O) and incubated in acetone at −20 °C for 7 min, then permeabilized in 1% DMSO/1% Triton-X100/PBS for 1 h at RT. EdU incorporation was detected by incubation in PBS/0.3% Triton-X100 containing 0.2 mM Alexafluor-555 (Life Technologies), 0.1 M L-ascorbic acid, 100 mM Tris pH 8.5, and 2 mM CuSO4 for 2 h at RT and washed (5 × 5 min PBST) prior to imaging. Embryos were imaged on an Olympus MVX10 fluorescence microscope and the number of EdU positive cells in caudal haemopoietic tissue counted in Fiji using the Find Maxima algorithm.Embryos were phenotyped at 48 h.p.f. and scored prior to labelling. EdU incorporation was achieved by soaking embryos in 0.2 mM EdU , 10% DMSO in E3 medium on ice for 60 min. Following washing (3 × 5 min in E3) and fixation in 4% PFA/PBS for 90 min, embryos were again washed  marneffei at 48 h.p.f. Inducible macrophage ablation using Tg embryos generated by inter-crossing was achieved with 10 mM metronidazole (Sigma M3761) treatment beginning at 36 h.p.f. and continuing for 11 h. At 48 h.p.f., embryos were placed in fresh E3 medium and imaged (t=0 h post treatment (h.p.t.)) to enumerate macrophages and neutrophils, then incubated a further 53 h.p.f. (t=53 h.p.t.) and again imaged. Leukocyte numbers in the caudal haemopoietic tissue region (posterior to end of yolk extension) were quantified either by manual counting or by leukocyte unitsStimulation of granulopoiesis was with microinjection of 0.1 and 0.05 ng csf3a mRNA or intravascular injection of freeze-killed 7 cells grown to log phase and cross-linked with 1% formaldehyde for 10 min at RT then quenched in 0.125 M glycine. Sonication of the crude nuclear fraction was conducted for 10 rounds of 30 s on/30 s off using Bioruptor (Diagenode) to achieve chromatin fragmentation of ∼550 bp. A polyclonal anti-ZBTB11 antibody validated for immunoprecipitation  and Protein A Dynabeads were used for chromatin immunoprecipitation. This antibody was also validated in-house by immunoprecipitation of overexpressed human ZBTB11–GFP fusion proteins followed by detection with anti-GFP antibody (Roche) on immunoblot. Following reverse cross-linking overnight at 65 °C, DNA was isolated and a PCR mix containing SYBR green (Roche) was prepared according to the manufacturer's specifications using primer sets tiled across the TP53 promoter and negative control primer sets (TP53 locus. Antibody was prepared by immunization of rats with mouse ZBTB11 KLH-conjugated SSEESYRAILRYLTNERC peptide. ELISA positive supernatants were tested by immunoblot for reactivity with mouse ZBTB11 and cross-reactivity with human or zebrafish Zbtb11 (negative for both). The selected clone (IC2) was validated by immunoprecipitation of overexpressed mouse ZBTB11–GFP fusion protein , blocked for 2 h at RT in Odyssey blocking buffer (LI-COR) prior to incubation with ab84058 (abcam) at 1:500 dilution overnight at 4 °C. Signal was detected by anti-rabbit IRDye-680LT secondary antibody (LI-COR) on an Odyssey infrared detection system (LI-COR). See mne, and neutrophils FACS sorted into RNALater (Qiagen) on the basis of bright mpx:EGFP or lyz:dsRed fluorescence . To confirm correct gating, sample FACS-sorted populations were analysed under fluorescence microscopy and found to contain only fluorescent neutrophils. RNA was extracted from sorted cells using RNeasy Micro (Qiagen) and provided to the MHTP Medical Genomics facility  for QC, library preparation and sequencing. Details in brief: unstranded barcoded libraries were prepared using total RNA and Nugen Ovation RNA-Seq system V2 for amplification and cDNA generation, followed by Ovation Ultralow System V2 for library preparation. Hundred base pair paired-end sequencing was performed on Illumina HiSeq2 generating ∼20 M reads per sample. Data were QC'd using FastQC, ends trimmed using Trimgalore and sequence aligned to the zebrafish genome (GRCz10) using STAR. Counts were derived using HTseq-count and differentially expressed genes determined using limma+voom with a FDR=0.01 and logFC cutoff of 2.0 using Degust v0.21 .At 48 h.p.f., single-cell suspensions were prepared from either WT embryos or from phenotype-sorted +CD11b+ granulocytes from the bone marrow of C57BL/6 mice. ChIP samples were prepared according to the standard Millipore/Upstate protocol and using the polyclonal anti-Pu.1 IgG (T-21 X: sc-352 X) from Santa Cruz. Libraries were prepared and sequenced using the Illumina TruSeq workflow. Reads were aligned to the mm10 build of the Mus musculus genome using Subread alignerFACS was used to isolate Ly6GZbtb11 amino acids 1–200 were used in a blastP homology search against the Protein Data Bank (PDB) resulting in 5/14 hits with Cys at C116 position which aligned with Foamy virus intasome protein. Three structures  complexed with manganese were used as a template for homology modelling of Zbtb11 amino acids 77–123. Alignment was performed with Clustal version 2.0.9 ref.  and mode+) and activity normalized against vector alone. They were as follows: zebrafish: gata1 and scl  were co-transfected using Fugene 6 (Roche) into 293T HEK cells, maintaining the total amount of DNA constant. Transcription factors were all subcloned into the same vector . F-tests were used to determine variance, which was always similar for parametric tests presented. Where variance was significantly different between groups, non-parametric tests were used. Unless otherwise stated, data are from ≥3 experiments: (1) for luciferase assays: mean ±s.e.m.; two-way ANOVA; Dunnett's multiple comparisons to determine significance of transcription factor versus vector alone and Tukey's multiple comparisons to determine significance between doses of a given transcription factor; (2) for in vivo leukocyte studies: mean ±s.e.m., two-tailed t-test; (3) rescue experiments: χ2 analysis. NS, P>0.05; *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.Group sizes were planned to be >10 embryos/genotype/experiment. In practice, the number of embryos was often much greater and limited by the maximum practical number of randomly selected embryos that could be analysed. If group sizes were smaller, it was due to limited embryo availability or loss during the experiment, and these were assumed to be randomly distributed across groups unless otherwise stated. Where appropriate to the hypothesis being tested, embryos were assigned as mutant or wild-type, either by phenotype and/or The microarray data sets generated during the current study are available in the Gene Expression Omnibus repository, accession number GSE94532. The authors declare that all remaining data supporting the findings of this study are available within the article and its How to cite this article: Keightley, M.-C. et al. The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger. Nat. Commun.8, 14911 doi: 10.1038/ncomms14911 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables.


We currently have the knowledge and experience to prevent much of human papillomavirus (HPV)-related disease burden globally. In many countries where prophylactic HPV vaccination programs have been adopted as highly effective public health programs with good vaccine coverage, we are already seeing, in real-world settings, reduction of vaccine-related HPV-type infections, genital warts and cervical pre-cancers with potential reductions in vulvar, vaginal and anal pre-cancers. Moreover, we are seeing a change in cervical screening paradigms, as HPV-based screening programs now have strong evidence to support their use as more sensitive ways to detect underlying cervical abnormalities, as compared with conventional cervical cytology. This article describes the impact of prophylactic vaccination on these outcomes and in settings where these vaccines have been implemented in national immunisation programs. Given the successes seen to date and the availability of essential tools, there has been a global push to ensure that every woman has access to effective cervical screening and every girl has the opportunity for primary prevention through vaccination. A gender-neutral approach by offering vaccination to young boys has also been adopted by some countries and is worthy of consideration given that HPV-related cancers also affect males. Furthermore, vaccination of young boys has the advantage of reducing the risk of HPV transmission to sexual partners, lowering the infectious pool of HPV in the general population and ultimately HPV-related diseases for both genders. Therefore, it is appropriate that all countries consider and promote national guidelines and programs to prevent HPV-related diseases. Although most HPV infections are transient, persistent infections are a prerequisite for pre-cancerous lesions and ultimately cancer.5. It is highly likely that latent infection 6 or subclinical infections (or both) exist and this is particularly relevant when an individual is immunocompromised since it increases the risk of developing symptomatic disease. Oncogenic HPVs were shown by Harald zur Hausen to be the causative agent of cervical cancer in the early 1980s by virtue of molecular epidemiology; for this work, zur Hausen shared the Nobel Prize in Physiology or Medicine in 20087. Of the many genotypes specifically infecting the anogenital area, HPV16 and HPV18 are the commonest high-risk or oncogenic genotypes in cervical cancer and are responsible for approximately 50% of high-grade cervical dysplasias and 70% of cases of cervical cancer, the fourth most common cancer in females globally9. Oncogenic HPVs cause almost 100% of cervical cancers, 90% of anal, 70% of vaginal, 40% of vulvar, 50% of penile and 13% to 72% of oropharyngeal cancers, and HPV16 predominates in all of these non-cervical HPV-related cancers. HPV6 and HPV11, which are classified as low-risk genotypes, cause 90% of genital warts as well as the rare but debilitating recurrent respiratory papillomatosis (RRP)11.Human papillomavirus (HPV) is the commonest sexually transmitted infection, and the resultant diseases have significant morbidity and mortality15. Currently available vaccines now include bivalent (2vHPV and targets HPV 16/18)13, quadrivalent (4vHPV and targets HPV 16/18/6/11)15 and nonavalent  vaccines16. Whilst many countries have licensed these vaccines, worldwide introduction into national immunisation programs differs by country, and some vaccines are available only on a user-pays basis and therefore entail an out-of-pocket expense. This has contributed to the resultant generally poor and inequitable uptake. For the greatest impact of these vaccines, the aim is to vaccinate adolescent females prior to sexual debut and to gain high coverage of the target population9. Some countries have implemented catch-up programs for older females or routine vaccination of adolescent males (or both) to increase overall population coverage and enhance herd protection17.Given the heavy disease burden of cervical cancer, prophylactic HPV vaccines were developed to target the commonest high- and low-risk HPV genotypes. Two such vaccines were first licensed for clinical use in 2006 following phase 3 clinical trials, which showed efficacy, safety and immunogenicity against vaccine-related HPV types18. The true benefit of vaccination can be seen only from real-life impact and effectiveness once vaccination has been included in public health programs. To appreciate this requires good surveillance and observation of changes in genoprevalence within vaccine-eligible age cohorts as well as of disease outcomes. Some of the challenges here have included the fact that HPV cannot be cultured by traditional means, so DNA typing in the general community before and after vaccination is required to determine molecular genoprevalence. Although a number of assays to determine the presence of HPV are currently available19, their appropriateness is based on whether they are meant to be used for clinical needs  or for pure epidemiological purposes  21. Furthermore, as HPV infection is not notifiable, nor are many of the associated clinical manifestations in most countries, individual surveillance systems have been required to measure the impact and effectiveness of the vaccines. In addition, as the times from infection to the various disease manifestations differ and may be weeks to months  to years (pre-cancers) or decades (cancer), the impact of vaccination on each disease varies. Consequently, in the 10 years since the introduction of both 4vHPV and 2vHPV vaccines, measures of vaccine impact have largely been determined by individual observational studies in countries with higher vaccine coverage, although a number of large, countrywide surveillance activities are now occurring. Thus, a direct comparison between the multiple studies published to date is complicated as the population studied, the duration of study and the endpoints observed or measured (or both) differ. Monitoring impact is difficult, particularly as some endpoints such as pre-cancerous lesions require a robust pre-cancer screening program to be in place or are not notifiable or have a prolonged interval between infection and development of diseases, particularly for cancers which usually occur after decades of persistent infection with a high-risk HPV type.Clinical trials indicated efficacy of the vaccines against the HPV types included in the respective vaccines as well as some modest cross-protection against some non-vaccine but phylogenetically related strains 22. It is largely observational and determines the effect of vaccination observed in populations after a program commences and compares outcomes in those vaccinated with those unvaccinated.In this article, we define vaccine impact as the effect of public health programs of HPV vaccines at a population level and the measurement thereof of reduction in disease burden (before and after initiation of a vaccination program) and largely focus on this. In contrast, vaccine effectiveness is examined at an individual levelVaccine impact will be determined by the type of vaccine program (an ongoing routine vaccination program to a specific age group versus inclusion of a catch-up program which may be limited in time and/or target a narrow or wide number of age cohorts), vaccine coverage, which population is targeted , the duration of vaccine protection, the duration of follow-up after vaccination, and type of surveillance, if any, of various outcome measures.23. In 2013, the Australian government extended the program to include 12- to 13-year-old males, including a 2-year catch-up vaccination program for those ages 14 to 15 years25. Overall, the program has been well received and high vaccine coverage rates have been achieved; the greatest rates have been from those within the school-based cohorts: over 70% have received all three doses for young girls and just under 70% have received all three doses for boys26. Consequently, Australian researchers were among the first to report reductions in the prevalence of vaccine-type HPV infections, by 86% in 18- to 24-year-olds who had received three vaccine doses and 76% for those who had received one or two doses28. In general, Tabriziet al. found that the greatest decline in vaccine-type HPV prevalence corresponded to the age group which had the highest vaccine coverage, even in the unvaccinated but eligible-age population, highly suggestive of herd protection28. Furthermore, Australia is already seeing herd protection in young males as a result of the female program29.Australia was one of the first countries to implement a fully government-funded, population-based HPV vaccination program. It commenced in 2007 as an ongoing school-based program with a three-dose course of the 4vHPV vaccine, targeting females in the first year of high school at age 12 to 13 years, with a catch-up for those ages 12 to 26 years until December 200930. In contrast, in the USA, coverage of the 4vHPV vaccine has been much lower, at 40% for females and 22% for males; yet despite this, national impact studies from the USA 4 years after the introduction of HPV vaccines noted a 56% decrease in HPV vaccine types 32. Although the early vaccination initiation uptake rate was low (at 17%) for females 19 to 26 years old in 2009, by 2012 the uptake in the USA had doubled to 34%, resulting in a further 45% decrease in the prevalence of vaccine-type HPV33, or an overall 64% decrease in vaccine-type HPV prevalence after 6 years32. A less marked, but still significant, reduction of 34% prevalence over the same time frame was noted in the 20- to 24-year-old group32. In sexually active US women ages 14 to 24 in the post-vaccination era, those who had received at least one HPV vaccine dose had a 2.1% reduction of vaccine-type HPV prevalence compared with 16.9% in the same age group of unvaccinated women32.Other countries that have achieved high coverage with 4vHPV or 2vHPV vaccines also report rapid and large declines in vaccine-related HPV infections. For example, in Scotland, which also has a school-based program, which targets 12- to 13-year-old girls as an ongoing program and had a limited 3-year catch-up from 2008 to 2011 for 13- to 17-year-olds, coverage rates for the 2vHPV vaccine have been even greater, at up to 90%, and reported declines in vaccine-related infections were from 29.8% to 13.6%34. Australia was the first to report a reduction in genital warts, and the reduction was larger and faster than had been expected by researchers36, and the 4vHPV vaccine provided up to 92% reduction in HPV-associated genital warts37. Interestingly, observations from early use of 2vHPV vaccines noted an unexpected decrease in genital warts. It is speculated that this response is due to vaccination resulting in a cell-mediated immune response which confers a moderate amount of protection against some low-risk HPV types39.Genital warts are a common condition, which affects up to 10% of the female population under 45 years old and which usually develops 2 to 3 months after infection with low-risk HPV genotypes, primarily HPV 6 and 1143. A Belgian study noted a decrease of 8.1% in genital warts in the general population, and the highest decreases were in 16- to 22-year-old males and females between the pre- and post-vaccine era44. Australia, with its high vaccine coverage rates, reported the greatest worldwide decline, of up to 92% in the under-21 age group37. Several studies have highlighted that the greatest efficacy of vaccination against genital warts was when the first dose of vaccine was given at a younger age44 with less of an effect at an older age40.Since the commencement of 4vHPV vaccination, there has been a substantial reduction in reports of genital warts in countries with 4vHPV vaccination programs 35–37,45. Again, this was first reported in an ecological study in Victoria, Australia46 and was made possible because Australia had a HPV vaccine register, comprehensive cervical cytology reporting as well as the ability (following legislation) for the registers to be linked. Australia’s national HPV vaccination programs have led to a 34% decline in low-grade and a 47% decline in high-grade cervical intraepithelial neoplasia (CIN) and adenocarcinomain situ, and the largest reductions were in the vaccinated younger age group47. Women under the age of 20 had a prevalence decline from 10.9/1000 screened women to 5.0/1000 over a period of 10 years, and prevalence in 20- to 24-year-olds decreased from 21.5/1000 screened to 13.5/1000 in a similar time period. In the over-30-year-old age group, the prevalence of high-grade CIN has continued to slowly rise48. Similar declines have been observed in Scotland and Denmark52 mark52 . There a54, although the peak has shifted to the late 20s to early 30s in some countries55. One would expect to see reductions within the next few years in the under-30-year-olds, particularly in those countries with high coverage of catch-up programs extending to 26 years of age, such as Australia and Denmark.At present, it is too early to show clinical disease or population-based disease reduction for cervical cancer, as this is a disease peaking in the mid-30s and mid-40s in Australia56. With the greater coverage provided by the nonavalent vaccine, it is proposed that with high coverage, 90% to 93% of all cervical cancers would be prevented58.Based on the PATRICIA trial  of the 2vHPV vaccine, it has been estimated that with 50% vaccine coverage, there could be a worldwide reduction of cervical cancer incidence of 246,086 cases annually but that with 90% coverage, there could be up to 442,955 cases averted59. Thus, more time and large population-based effectiveness studies with various cancers as endpoints will be required before impact can be determined. There are early data sets currently reviewing the implication of vaccine-type HPV reduction and the effect on persistent anal infections and intraepithelial neoplasia60.The incidence of non-cervical HPV-related cancers is generally low and peaks at a later age compared with cervical cancerVaccine uptake and coverage. Although the effect of population-wide vaccination has been observed for 10 years, the reported findings may not accurately reflect future impact and this is for several reasons. Some countries have been slow to adopt HPV vaccination or vaccine coverage has been low (or both), even in high-income countries. Vaccine uptake rates in 13- to 17-year-olds in the USA in 2013 were 57% (one dose) and 38% (three doses)32. In Australia, between 2007 and 2015, vaccine uptake rates were 85.6% (one dose) and 77.4% (three doses) for females turning 15 years of age61. Successful strategies to increase coverage have included publically supported health promotion and missed-doses catch-up vaccine campaigns62, client reminders, recall programs, provider assessment and feedback interventions. Low- and middle-income countries often have a high HPV disease burden: however, only 15% of these countries have adopted a vaccination program18. Focusing on these countries and giving reassurance regarding vaccine safety will substantially impact HPV vaccination rates and outcomes63.Safety. Concern regarding vaccine safety is an issue that often arises. The US Centers for Disease Control and Prevention (CDC), through the Vaccine Adverse Event Reporting System, classified only 7% of adverse events as serious from over 90 million doses of HPV vaccines. The CDC found no causal association between HPV vaccines and ovarian failure, Guillain-Barré syndrome or postural orthostatic tachycardia syndrome. Overall, HPV vaccines have a good safety profile, but ongoing monitoring will be required as 9vHPV vaccines have been introduced67. In Japan, poor or inappropriate information (or both), including reports on complex regional pain syndrome, has resulted in the government withdrawing support for HPV vaccination programs70. Despite comprehensive independent evaluations of safety72, including endorsement of safety by bodies such as the World Health Organization (WHO)73, the International Papillomavirus Society66 and the CDC74, the Japanese government has continued to refuse to re-implement population-wide vaccination69.Ideal age for vaccination. In many respects, if the HPV vaccines could be administered in childhood, they could use infrastructure already in place for the Expanded Program on Immunization vaccines, as in general these see high uptake even in those countries with low resources. However, the immunity developed by HPV vaccination needs to be robust for several decades to ensure that infection does not occur when young people become sexually active and therefore to prevent diseases in the longer term. Although some studies have shown detectable neutralising antibodies 10 years after vaccination, there are not yet any data about antibody persistence beyond that22. It is currently believed that the antibody responses made by the vaccines are strong and long-lasting and that only small titres are in fact required for protection75.Over the last 10 years, there have been changes in sexual behaviour, such as lowered median age of sexual debut, increased exposure from the increase in the number of sexual partners, and choice of orientation of sexual partner; however, these trends vary from country to country. The ability of vaccine programs in different countries to be flexible will allow these changes to be accounted for in the future. As shown inDosing. New information regarding number of doses of vaccine necessary for long-lasting immunity is becoming available. The current WHO recommendation is that with respect to HPV-vaccine-related-type antibody responses, two doses of HPV vaccine for females 9 to 14 years old is non-inferior to three doses in adult women as long as the two doses are at least 6 months apart73. This decision was based largely on neutralising antibody outcome and more recent findings of reduced HPV-vaccine-type-related disease outcomes, but again good surveillance systems should be able to monitor impact on disease endpoints with longer time intervals from the point of vaccination. Furthermore, this may help to address concerns regarding reduced efficacy76 to ensure that a third dose could be administered if surveillance shows breakthrough disease. For females older than 15 years, those who are immunocompromised or HIV-positive, a three-dose regimen is still recommended77.Gender-neutral approaches. Although females have been the primary target of HPV vaccine, extending the program towards males may have some benefit. A 2011 Australian study predicted that, based on the female-only vaccination program, there could be up to a 68% decrease in male HPV infections by 2050 and a 14% decrease in head, neck and anogenital cancers. The effect on oropharyngeal cancers is unclear78. This prediction is based on heterosexual relationships offering herd protection. A high-risk HPV group, men who have sex with men, are less likely to benefit from a female-only vaccination program79. A gender-neutral approach may not only reduce HPV-related diseases in males but also reduce the infection and transmission to females, ultimately reducing the pool of infectious virus in a community. Male vaccination would confer an even greater benefit in settings where female vaccination rates are low. Hence, vaccination of males has recently been incorporated into several national programs. Measuring vaccine effectiveness in the context of gender-neutral programs will bring further challenges24.Multivalent human papillomavirus vaccines. Some countries have implemented or have plans to commence the use of 9vHPV vaccines to offer protection against the additional five next most common oncogenic HPV types after HPV 16/18 in an effort to further improve health outcomes. This move is supported by studies which indicate potential cost savings and health benefits that could result from universal nonavalent vaccination programs81.84. Moreover, in trials in younger women, it was shown that vaccines were not efficacious when a woman was HPV-positive for a particular type85, although it was likely for women who were HPV-negative but antibody-positive from natural infection that they would gain efficacy against later disease87. The importance of a coordinated screening and vaccination program cannot be overlooked and this has prompted the HPV-FASTER concept. This protocol intends to offer women up to the age of 45 years HPV vaccination and incorporates HPV testing after the age of 30 for screening, with ongoing follow-up and early management of high-risk HPV-positive women. Further studies will be required to investigate the acceptability and feasibility of such an approach, most appropriate cut-off age of offering vaccination, ideal screening commencement, cessation and interval times to refine the protocol88.Consideration for vaccinating women who are already sexually active but may not have been infected with all HPV types covered in the vaccines could well reduce the burden of disease more rapidly. This concept is based on data from phase 3 trials indicating that 2vHPV and 4vHPV vaccines are efficacious, immunogenic and safe in women ages 26 to 45 years old with a 90% vaccine efficacy in protection against cervical pre-cancer in HPV-DNA-negative women (regardless of serostatus) and 50% vaccine efficacy in women who had previously been exposed to HPVSurveillance. A necessary condition for accurately monitoring the impact and effectiveness of vaccine programs is the existence of diligent and ongoing surveillance89. For HPV, this requires surveillance of HPV vaccination coverage and safety, HPV infection genoprevalence, cervical dysplasia incidence, cervical  cancer registries, as well as surveillance for non-cancer outcomes such as warts and RRPs. Ideally, this would include the ability for the various data sources to be linked. Encouragement in participation in screening programs is essential, particularly as new cervical screening programs that incorporate DNA testing as the primary screen usually have longer intervals between screens compared with those with cervical cytology as primary. Also, the vaccinated population may not recognise that, despite vaccination, there is a need for ongoing monitoring. With so many changes, education of the community, clinicians and women in particular is critical.Overall, the implementation of HPV vaccine public health programs has resulted in major decreases in vaccine-type HPV infection prevalence as well as associated disease incidence in countries that have introduced them. Administration of vaccines is ideally done prior to sexual debut for both males and females for the greatest impact. Herd and cross-protection contribute further to the impact of the vaccines. More time for observation is required to determine the effect on cancer rates, as carcinomas develop decades after infection acquisition. Although we have the tools to markedly impact HPV-related disease, we also face the challenge of ensuring that all young people, regardless of their geography, social status, or immunocompromised status, are eligible for and can access these vaccines. To observe the vaccine impact and effectiveness of national HPV vaccination programs, support is required from accurate vaccine uptake surveillance, preferably through vaccine registers in addition to genoprevalence and HPV-related disease endpoint surveillance, including cancer registries and pre-cancer screening programs.


This study evaluated the Xpert HPV Assay in women attending screening in general practice by comparing Xpert with two established HPV tests, cytology and histology.A prospective study in women aged 20–60 years attending screening in Bristol, Edinburgh and London using residual Preservcyt cytology samples. Sample order was randomised between Roche cobas4800 and Cepheid Xpert assays with Qiagen hc2 third.N=63)), cobas was negative in two. Histology was available for 172 participants. 79 reported CIN2+, 47 CIN3+. All CIN3+ was positive on Xpert and hc2 and one case negative for cobas. One case of CIN2 was negative for all assays.3408 cases were included in the primary analysis. Positivity for Xpert was 19.6%, cobas 19.2% and hc2 19.9% with high concordance . Xpert, cobas and hc2 showed similar sensitivity  for CIN2+. All pairwise comparisons had high concordance (Kappa ≥0.78 with any abnormal cytology. Xpert and hc2 were positive for all cases of ≥moderate dyskaryosis (The performance of Xpert HPV Assay in a general screening population is comparable to established HPV tests. It offers simplicity of testing, flexibility with non-batching of individual samples and rapid turnaround time. •This is the first study to report on the performance of the Xpert HPV Assay in a screening population.•Xpert HPV Assay׳s performance in detecting HPV is comparable to two clinically validated HPV tests.•Xpert HPV Assay showed a relative sensitivity of 98.73% for CIN 2 or higher and 100% for CIN3 or higher. The external reference site gave blind testing and standardised conditions for the primary comparator – cobas 4800. The three clinical sites were Bristol, Edinburgh and London. Samples were originally collected from women attending for routine cervical screening as part of the UK NHSCSP in PreservCyt liquid cytology using a Cervex brush  or similar. A residual specimen was the remaining PreservCyt sample, kept at room temperature, after the completion of routine clinical testing . A satisfactory cytology result (with or without HPV triage) and sufficient remaining sample volume to carry out at least two HPV tests (minimum 8 ml PreservCyt) was required. Sample size calculations were done separately for women aged 20–29 years and aged 30–60. An HPV prevalence of 25% in the younger women and 8.7% in the older age group resulted in minimal sample sizes of 900 and 2600 respectively to be able to have 95% power to ensure that the lower 2-sided 95% CI for kappa would be above 0.75 when the true value was 0.82 for cobas and above 0.65 when the true value was 0.75 for hc2 for each age group. Assumed true kappa values were based on previous studies The samples were collected from women who had originally attended screening between July 2013 and January 2014. Populations were somewhat different as the screening algorithms were different in the 3 centres leading to different positivity rates. In order to obtain 900 women in the 20–29 age group, this was oversampled in Edinburgh, as screening begins at age 20 years in Scotland and 25 years in England. Also in Scotland women who had borderline or mildly dyskaryotic smears were followed by repeat short term cytology and not triaged with HPV to determine if they should be referred immediately, so that these women were also included in the sample from Edinburgh. However within each centre samples were collected at random from each age stratum.The primary comparison was between the Xpert HPV Assay and the Roche cobas 4800 HPV test  with a secondary comparison between Xpert HPV Assay and the hc2 test . Aliquot order was either by method 1, where odd numbered specimens had an aliquot for cobas detection taken first and Xpert second and vice versa for even numbers or method 2 where batches of 100 specimens were divided into 2 halves with the first 50 having an aliquot for cobas detection taken first and Xpert second and the next 50 having the Xpert first and cobas second. The aliquot for hc2 was always taken third. The original specimen in the ThinPrep pot was mixed either by inverting 8–10 times or briefly vortexing for 5–10 s prior to the removal of each aliquot. The cobas aliquot (volume 2 ml) was stored at room temperature (15–25 °C) and dispatched to the reference laboratory in Liverpool where testing was conducted in accordance with the manufacturer׳s instructions. The Xpert HPV Assay was conducted onsite in laboratories in Bristol, Edinburgh and London. The aliquot (volume 1.2 ml) was stored at room temperature (15–25 °C) prior to testing. Testing was carried out within six weeks of sample collection in accordance with manufacturer׳s instructions. The aliquot for hc2 testing  was stored at room temperature or refrigerated (2–30 °C) and tested onsite within six weeks, as per manufacturer׳s instructions.Detailed technical descriptions of the Xpert HPV Assay have been reported elsewhere The cut-off for positivity with the Xpert HPV Assay was<40Ct for HPV 16 and HPV 18/45, and <38 Ct for HPV 31/35/33/52/58, HPV 51/59, and HPV 39/68/56/66. Positivity for Cobas was <40Ct for HPV 31/33/35/39/45/51/52/56/58/59/66/68 and HPV16 and <40.5 Ct for HPV 18 and>1 RLU for hc2.Positivity rates and agreement of the Xpert Assay with the Cobas (primary comparator) and high-risk hc2 (secondary comparator) were evaluated. Referral to colposcopy was only based on the cytology result, with or without HPV triage according to local protocols. The triage tests used were Abbott RealTime in London and hc2 and Aptima in Bristol. Scotland, at the time of the study, started routine cervical screening at age 20 years whereas in England it was age 25 years. The Edinburgh centre therefore had a younger age distribution. Scotland is due to change to 25 years in 2016. Triage also differed between the two countries. In England, but not Scotland, borderline squamous or mildly dyskaryotic cytology is followed by HPV triage testing. Those women testing negative are followed up with repeat cytology whereas those testing positive are referred for colposcopic examination, where histopathology samples may be taken for further assessment. In Scotland there is currently no HPV triage and women are referred for colposcopy only after more than one consecutive low grade cytology result. Women with higher grade cytology results were referred for colposcopic assessment in all centres Ethics approval was received from National Research Ethics Service Committee East Midlands – Leicester (reference: 13/EM/0244) on 10th June 2013. Individual informed consent was not required as the study was non-invasive and the samples would otherwise have been discarded. In addition the residual samples were pseudo-anonymised and only identifiable to the research team by subject number. Cytology data and histopathology data were linked to the HPV result by the centre and then all data were pseudo-anonymised before release to the statistical centre and were only identifiable by subject number.3A total of 3529 samples were collected. Of these 15 women were found to be ineligible and another 106 samples did not produce a valid result for both Xpert and cobas leaving 3408 women in the primary analyses  with allConcordant and discordant outcomes between Xpert and the other two tests are shown in HPV 16 was reported separately for the Xpert and cobas assays. For Xpert positivity rates for HPV16 were 5% overall and 14% for women aged 21–29 and 2% for ages 30–60. For cobas the corresponding rates were the same 5%, 14% and 2%. 4This is the first study to assess the performance of the Xpert HPV Assay in a screening population. The data suggest that the performance of Xpert is highly comparable to two clinically validated tests (cobas 4800 and hc2). Xpert HPV Assay offers more extensive partial typing than cobas which reports HPV 16 and HPV 18 and a consensus positivity for 12 other high risk HPV types. Hc2 does not offer individual typing but a consensus result for 13 high risk types. The Xpert HPV Assay also has the advantage that results are available within a short timeframe (the assay run time is under 1 h) with the flexibility to run a single or multiple cartridges. The system is very accessible operationally, requiring simple vortexing of the ThinPrep pot and pipetting into the single cell cartridge which is then slotted into the GeneXpert machine. This flexibility with non-batching and rapid turnaround time of individual samples could prove particularly useful in laboratory and clinical settings where workload is variable.The sensitivity and specificity results in this study were similar across centres, despite their different disease rates. However these are relative measures since colposcopy referral was based only on routine procedures and so some disease in cytology negative women may have been missed.There were differences between centres in the proportion of abnormal histology and cytology and HPV prevalence. There are a number of possible explanations for this. Women under 25 years were targeted in Edinburgh whereas there were very few in London and Bristol where screening commences at 25 years, although invitations can be sent six months earlier. Subsequently there were differences between centres in the age distribution. In addition there were different referral policies between England and Scotland. For example Bristol has incorporated HPV triage as part of screening for substantially longer than London, and this was not used at all in Edinburgh at the time of this study so that those from Edinburgh with up to two low risk cytology results were kept within the ‘screening’ population. The higher HPV and disease rate in Edinburgh had the advantage or providing more CIN2+ cases overall which increased the accuracy of the sensitivity estimates. However, no differences between the performance of the three HPV tests was seen across the different centres. Overall the HPV prevalence was only slightly higher here than that previously recorded in a similar English population as part of the ARTISTIC Study (19.9% versus 16.1% using hc2), but ARTISTIC used a 2RLU cut-off for hc2. Cytology results were also virtually identical with 10.9% low grade and 1.9% high grade abnormalities for both studies In conclusion, this study shows that the Xpert HPV Assay is a useful additional to the HPV testing market and compares well in terms of performance with other more established HPV testing systems. A study in which the HPV result is used for clinical management is needed to demonstrate the added sensitivity of this test in cytology negative women and directly compare its performance to cytology.


Previous studies were conflicting regarding the associations between HPV vaccination, cervical cancer risk perceptions, high-risk sexual behaviors and STIs. This study compared the HPV-vaccinated and non-vaccinated young women in Uganda regarding cervical cancer risk perceptions, high-risk sexual behaviors, syphilis and HIV infections 5 years after vaccine implementation.This was a population-based comparative cross-sectional survey conducted in Uganda. The 438 participants were sexually active young women aged 15–24 years and mean age was 18.6 (SD 1.4). The majority (53.0%) were HPV-vaccinated in 2008 without assessment of sexual activity prior to HPV vaccination. Upon verbal assessment of sexual activity at the time of follow-up, data were collected using a questionnaire and laboratory testing of blood samples for syphilis and HIV infections.There were no significant differences between the HPV-vaccinated and non-vaccinated groups regarding the prevalence of high-risk sexual behaviors, syphilis and HIV infections. Cervical cancer risk perceptions and age at sexual debut were nonetheless significantly lower among the vaccinated group compared to their non-vaccinated counterparts. However, HPV vaccination was not significantly associated to cervical cancer risk perceptions and early age at sexual debut in multivariate logistic regression analysis.We found no associations between HPV vaccination, cervical cancer risk perceptions, high-risk sexual behaviors, syphilis and HIV infections among young women in Uganda 5 years after vaccine implementation. Young girls in the study population were found to be sexually active at a young age, affirming the importance of targeting girls of younger age for HPV vaccination.The online version of this article (doi:10.1186/s12905-017-0394-y) contains supplementary material, which is available to authorized users. Cervical cancer (CC) is the third most common cancer among women globally in terms of 5-year prevalence  with an estimated 527,624 new cases and 265,672 deaths in 2012 [An infection with sexually transmittable Human papillomavirus (HPV) is a necessary but not a sufficient cause of cervical cancer . There aImportant to note is that HPV vaccine alone would not completely prevent cervical cancer because the vaccine contains only 2 of the 14 HR-HPV types associated to CC. Secondly, it would be impossible to attain 100% vaccination coverage particularly in developing countries with weak health systems. Thirdly, potential epidemiological changes may occur as a result of HPV vaccination. The changes are most likely to arise because there are other HR-HPV types that are not covered by the current vaccines in developing countries  and so it is possible that epidemiological changes indeed may occur by transitioning to disease caused by HR-HPV types not covered by HPV vaccination . Such discussions of the limitations of the currently available HPV vaccines are frequent in the literature –7. The aThe status of high-risk sexual behaviors, syphilis and HIV infections in Uganda at the time of the study shows a decreasing trend in the prevalence of the above determinants among the 15–24 year-old young women from the pre-HPV vaccine introduction period to post-HPV vaccine period. For example, the percentage of the 15–24 year-old young women who initiated sexual intercourse before the age of 15 decreased from 14.4% in 2005 to 10.9% in 2011 [Regarding the association between HPV vaccination and high risk sexual behaviors, previous studies were conflicting in their findings. Specifically, there were previous studies including in US and Canada that indicated no association between HPV vaccination, risk perceptions and high-risk sexual behaviors among young women –18. For In view of the aforementioned conflicting previous studies on the association between HPV vaccination, CC risk perceptions, high-risk sexual behaviors and STIs, there is need for further research on the topic, more so from developing countries in Africa which carries the heaviest burden of CC. In this study, we compared the HPV-vaccinated young women and their non-vaccinated counterparts in Uganda regarding CC risk perceptions, high-risk sexual behaviors and STIs 5 years after HPV vaccine implementation.This was a population-based comparative cross sectional study. It was conducted in Ibanda district Uganda as part of a larger HPV16/18 (Cervarix®) vaccine follow up study. Ibanda district was the district in Uganda where the first pilot bivalent HPV 16/18 vaccination project which targeted young women in primary school class 5 (P5) was implemented in 2008.The study participants were HPV-vaccinated and non-vaccinated 15–24 year-old young women who were enrolled in secondary schools in Ibanda district. Young women were included in the study if they were sexually active and by 2008 were either in primary school class five (P5) in Ibanda district and were fully vaccinated with 3 doses of the bivalent HPV16/18 vaccine (vaccinated group) or were in primary school class six (P6) or higher in Ibanda district but were not vaccinated with bivalent HPV 16/18 vaccine (non-vaccinated group). Partially vaccinated young women were excluded from the study because they were expected to be very few as the 2008 HPV vaccination coverage in the area was above 95% . The 95%The high HPV vaccination coverage (95%) among the 2008 cohort of girls also made it impractical to establish the required sample size for the non-vaccinated control arm of the study from the same 2008 cohort of girls. Therefore, two different cohorts of girls (i.e. 2006 and 2007 cohorts of the participating schools) were used for the non-vaccinated control group. This however posed the problem of differential age distribution between HPV vaccinated and non-vaccinated control groups.The sample size was determined by power analysis calculation . Any STIWe performed multi-stage sampling procedure to select the study participants. We first developed a total list of senior secondary schools in Ibanda district where the 2008 cohort of HPV vaccinated young women and their non-vaccinated counterparts were expected to be studying at the time of the study. The total list comprised of 32 secondary schools. Being a school semester period, all the schools in the district were visited one after another by the research team. And young women found in senior secondary school classes three to six (i.e. S3-S6) were approached class by class, informed about the study, consented and screened for eligibility. Demographic data were obtained from all the young women approached. Appointment dates to visit Ruhoko health centre IV, a designated health facility within the district for data/sample collection were agreed with the selected young women and their teachers. On the appointment day, vehicles were sent to the schools to facilitate transportation of the young women to and from the health facility in company of their teachers. Figure Data/sample collection took place in 2014. At the health facility, data and samples were collected from consecutive young women reaching the facility until the required sample size was reached. The study procedures performed at the health facility included completing the CC questionnaire and collection of blood samples for HIV and syphilis testing.The standard questionnaire was used to collect data about the CC risk perceptions and sexual behaviors  and level of condom use . The sexual behavior section of the questionnaire was interviewer-administered to ensure proper phrasing or probing for correct answers and was coupled with health history taking, physical examination and prescription of treatment for those found with health problems.Both the risk perception and sexual behavior sections of the survey instrument were created by the Study Investigators specifically for the study. Face validity of the instrument was achieved by sending the questionnaire to a sample of 5 experts on HPV vaccination and all of them responded back with the judgement that the scale appears to be a good measure of CC risk perceptions. Armed with the criteria  . The samParts of the same venous blood samples drawn for the HIV tests were used for the Syphilis tests. Serum extracted from the venous blood from the laboratory at the study site were kept and transported in liquid nitrogen from the field site to Immunology laboratory at Makerere University College of Health Sciences where they were immediately transferred to minus 80C freezers until testing. The liquid nitrogen tanks ensured cold chain for samples during transportation from fieldwork site to the laboratory. Syphilis tests were performed using a commercially available kit, Human Treponema Pallidum Hemagglutination Assay (TPHA) liquid GmBH, Wiesbaden, German.). The TPHA test is a qualitative microhemagglutination test for the presence of IgG and IgM antibodies to Treponema pallidum, the causative agent for syphilis, in human serum and EDTA plasma. The tests were performed according to the manufacturer’s protocol. Women with positive TPHA test results were referred to clinician for treatment. All the Syphilis tests were performed by yet another independent laboratory technician who wasn’t part of the blood draw team from the Immunology laboratory at Makerere University College of Health Sciences Kampala Uganda.2) or Fisher’s exact tests for categorical variables which also generated p values, odd ratio (OR) and 95% confidence interval. Continuous variables were compared using independent sample t-test which also provided p-values. Additionally, using early age at sexual debut (<16 years vs 16+ years) as the categorical outcome variable, logistic multivariate regression was performed to further examine the association between HPV vaccination and early age at sexual debut after controlling for all other covariates . These covariate included in the logistic multivariate regression were the variables that were significantly associated to early age at sexual debut from the bivariate analysis and were theoretically relevant. Although Uganda national age at sexual debut among females was 15 years [Data were entered into Statistical Package for Social Sciences (SPSS) version 22.0 for analysis. The HPV-vaccinated and non-vaccinated groups were compared for the demographic data, CC risk perceptions, sexual behaviors and STI prevalence using Pearson’s’ chi-square . All the participants were female students attending secondary school in Ibanda district, the study area in rural southwestern Uganda. All the participants were non-smokers, with neither history of pregnancy, nor child birth, nor abortion nor hormonal contraceptive use. As shown in participants flow schema 1, non-eligibility rate was 63% and the major reason for non-eligibility was sexual inactivity.As shown in schema 1, of the 492 eligible young women whose demographic data were obtained for the study, 54 declined responding to the CC questionnaire. Of the 438 who initiated responding to the CC questionnaire, 38 didn’t turned up to the health facility for blood draw. Of the 400 who turned up for blood draw, 2 declined blood draw and finally of the 388 blood samples obtained for HIV tests, 1 was invalid for HIV test. The demographic characteristics of the 54 young women who declined the CC questionnaire plus the 38 young women who didn’t show up for blood draw and the 2 young women who declined blood draw were not significantly different from the rest of the study participants who completed the study (statistics not presented here).Table As shown in Table In multivariate analysis for the association between low cervical cancer risk perception and set predictors  as shown in Table In bivariate analysis Table , there wAs shown in Table In multivariate logistic regression analysis level shown in Table In bivariate analysis Table , there wThe HPV-vaccinated young women of our sample were significantly younger in age and were mostly from the lower senior secondary educational level (S1-S4) compared to their non-vaccinated counterparts. This was attributable to the participants sampling strategy used in the study which targeted the 2008 cohort of HPV-vaccinated young women who were expected to be younger and in the lower educational level compared to their non-vaccinated counterparts recruited mostly from the higher senior secondary class five to six (S5-S6).The ethnic tribal differences between the HPV-vaccinated and non-vaccinated groups observed in the sample for this study could also be attributed to the fact that the 2008 pilot HPV vaccine project in Uganda was implemented in Ibanda district (the study area), a district predominantly occupied by people of Ankole ethnic tribe and hence the HPV-vaccinated young women were more likely to belong to the Ankole ethnic tribe. Notably, the selection of Ibanda district and hence high participation of young women of Ankole ethnic tribe in HPV vaccine rollout program and the study was neither based on HPV and cervical cancer disease burden differences nor cultural differences between ethnic tribes with regard to sexual activity that may impact these findings.Interestingly, the CC risk perceptions demonstrated by the HPV-vaccinated young women in this study were significantly lower than that for the non-vaccinated control group although it wasn’t significantly associated to high-risk sexual behaviors. This finding was consistent with previous studies from Colombia and US that reported an inverse association between HPV vaccination and perceived risk of CC , 23 in bWith exception of age at sexual debut  starts sexual activity at an earlier age than young women of older generation (non-vaccinated group), emphasizing the importance of initiating HPV vaccination of young women at a younger age than 15 years before they start sexual activity.The HPV-vaccinated young women in our sample didn’t differ significantly from their non-vaccinated counterparts regarding the history of STD syndrome and the prevalence of both syphilis and HIV infections. These findings from our study were consistent with previous studies from the US and Sweden that both reported no significant differences between HPV-vaccinated and their non-vaccinated counterparts regarding STI prevalence rates , 14. SinThe strengths of our study include being one of the first HPV vaccine follow up studies conducted from developing country in Africa with high burden of HPV-related cancers to empirically examine the association between HPV vaccination, CC risk perceptions and high-risk sexual behaviors using a population-based comparative cross sectional survey design. We assessed for sexual behaviors both subjectively using a questionnaire and objectively by performing laboratory tests for syphilis and HIV infections which were the respectable biomarkers of high-risk sexual behaviors used in previous related studies among young women , 14. AndA weakness of our study was that some of the data were self-reported and hence subject to reporting bias. We minimized on reporting bias by using a standard questionnaire and ensuring that the sexual behavior section of the questionnaire was interviewer-administered which provided opportunities for clarification on questions and probing. Another potential weakness was the fact that we studied a largely rural in-school young women population shortly after their sexual debut and hence our results may not be widely applicable to a more diverse population.Further research on this topic in developing country particularly Africa should be a long term HPV vaccine follow up of the young women for cervical cancer secondary prevention outcomes such as attitudes and uptake of cervical cancer screening.We found that CC risk perceptions and age at sexual debut were both significantly lower among the HPV-vaccinated young women compared to their non-vaccinated counterparts in Uganda 5 years after vaccine implementation. However, HPV vaccination was neither associated to lower cervical cancer risk perceptions nor early age at sexual debut in multivariate analysis. Other high-risk sexual behaviors were rampant among both groups although the differences were not statistically significant. Our findings don’t support the notion that HPV vaccination is associated with high-risk sexual behaviors. Importantly, we found that young women in study area in rural Uganda were sexually active at a younger age and are engaged in high-risk sexual behaviors, affirming the importance of targeting young women of younger age for HPV vaccination.


Balaenoptera musculus intermedia to environmental variability is poorly understood. This study uses acoustic data collected from 586 sonobuoys deployed in the austral summers of 1997 through 2009, south of 38°S, coupled with visual observations of blue whales during the IWC SOWER line-transect surveys. The characteristic Z-call and D-call of Antarctic blue whales were detected using an automated detection template and visual verification method. Using a random forest model, we showed the environmental preferences pattern, spatial occurrence and acoustic behaviour of Antarctic blue whales. Distance to the southern boundary of the Antarctic Circumpolar Current (SBACC), latitude and distance from the nearest Antarctic shores were the main geographic predictors of blue whale call occurrence. Satellite-derived sea surface height, sea surface temperature, and productivity (chlorophyll-a) were the most important environmental predictors of blue whale call occurrence. Call rates of D-calls were strongly predicted by the location of the SBACC, latitude and visually detected number of whales in an area while call rates of Z-call were predicted by the SBACC, latitude and longitude. Satellite-derived sea surface height, wind stress, wind direction, water depth, sea surface temperatures, chlorophyll-a and wind speed were important environmental predictors of blue whale call rates in the Southern Ocean. Blue whale call occurrence and call rates varied significantly in response to inter-annual and long term variability of those environmental predictors. Our results identify the response of Antarctic blue whales to inter-annual variability in environmental conditions and highlighted potential suitable habitats for this population. Such emerging knowledge about the acoustic behaviour, environmental and habitat preferences of Antarctic blue whales is important in improving the management and conservation of this highly depleted species.Harvested to perilously low numbers by commercial whaling during the past century, the large scale response of Antarctic blue whales  Balaenoptera musculus intermedia were widely distributed in the Southern Ocean prior to commercial whaling )Here we present the results of analyses on the call type distribution  and acoustic behaviour  of Antarctic blue whales as a function of satellite-derived environmental parameters using the circumpolar passive acoustic measurements during the Southern Ocean Whale and Ecosystem Research (SOWER) program of the International Whaling Commission (IWC), hereafter IWC SOWER. The IWC SOWER program was a long series (1978–2009) of line-transect survey cruises directed primarily at obtaining abundance estimates for Antarctic minke whales  and for We analysed passive acoustic data collected using both sonobuoys and towed hydrophones during the IWC SOWER cruises conducted south of 55°S to the ice edge from December to February of 1996/97 to 2008/09. Some acoustic observations extended as far north as 38°S during transits to and from the Antarctic . AcoustiThe template detector method operates on an acoustic time series of spectrograms by constructing a correlation kernel for the vocalization . Calls wWe tested six different thresholds from 20% to 70% by increments of 10% to determine optimal thresholds for our analyses . The 20%Automated detection of animal vocalizations is effective at accelerating acoustic data analyses and producing more robust results than human visual sound detections ,34. The B. m. musculus calls, and recommended a new detection template for each year. More detailed estimates of the changes of start and end frequency of Antarctic blue whale calls over time from this SOWER acoustic dataset and the most recently recorded blue whale calls both in the Southern Ocean and low latitudes will inform on the long term behavioural changes in blue whale vocalizations and whether such templates for each year should be used to reduce false detections.Širović  observedin situ monitoring, the whale numbers and number of groups seen were assumed to be zero unless a matching time and position with a whale was found in the visual sighting forms.Acoustic stations  were differentiated into two types: type 1 stations were sonobuoy deployments in association with a visual sighting of blue whales (107 stations); type 2 stations were deployments without blue whales visually detected (377 stations). Estimates of numbers of individual blue whales and of blue whale groups observed in type 1 acoustic stations were sourced from the acoustic data forms completed during the IWC SOWER cruises as provided by the IWC Secretariat and from the line transect visual survey sighting forms, if whales were seen during the normal line transect visual surveys. The date, time of the day, and station number of each acoustic recording were used to link blue whale sightings to the acoustic results. In instances where blue whale calls were detected from the analyses but not heard during the The number of calls recorded from each sonobuoy and/or hydrophone deployment were allocated to each station as documented in the IWC SOWER cruise reports and acoustic forms. The recording duration of each deployment was determined during acoustic data analyses, and the station duration was calculated as the sum of durations of recordings from that station. Call rates of both the Z- and D-calls were calculated for each station as the total number of calls divided by the station duration to give calls per hour at each station. Call rates were not determined as calls per hour per individual, however, but as call rates per group(s) per hour due to the difficulty in determining the precise number of vocally active animals within any groups. Results of call rates were thereafter assigned into day  and night (18:00 to 06:00) depending on their recording time before fitting to the models, although such day/night differentiation reflected the survey mode of the vessel rather than Antarctic summer light regimes as visual monitoring only took place from 06:00–18:00 each day.www.menugget.blogspot.com was used to convert the binned GlobColour chl-a data to a grid format.Satellite chlorophyll-a (chl-a) measurements were obtained from the globally blended and binned high resolution level-3, case 1 water GlobColour project  that merin situ ship and buoy observations. The OI analysis is a daily average SST that is bias adjusted using a spatially smoothed 7-day in situ SST average and is therefore tuned to about 0.3 meter [Satellite-derived sea surface temperatures (SST) were obtained from the Group for High Resolution Sea Surface Temperature (GHRSST) global Level 4 sea surface temperature analysis produced daily on a 0.25° grid spatial resolution at the NOAA National Climatic Data Center . GHRSST .3 meter . The Ope.3 meter  and xtra.3 meter  packagesSea surface height (SSH) daily measurements were obtained from the Archiving, Validation and Interpretation of Satellite Oceanographic (AVISO) data program using the erdTAssh1day function in R. AVISO SSH product combines data from up to six multiple satellites, including Jason-1, TOPEX/Poseidon, the European Remote Sensing (ERS) Satellites 1 and 2 and their successor, the Environmental Satellite (ENVISAT), and the Geodetic Satellite (GEOSAT) . The monftp://eclipse.ncdc.noaa.gov/pub/seawinds/SI/ and processed in R using custom built functions. These wind speeds and directions are blended from multiple satellites , the Tropical Rainfall Measuring Mission (TRMM), Microwave Imager (TMI), the Quick Scatterometer (QuikSCAT) and the Advanced Microwave Scanning Radiometer-EOS (AMSR-E)) observations, on a global 0.25 degree spatial grid and a 6-hourly temporal resolution (see [-2. Distance in kilometres (km) to the nearest Antarctic coastline was calculated using the custom developed function defined as the shortest distance to the Antarctic coastline for each of the acoustic stations.Monthly blended vector sea surface wind speed and direction (at 10 m above sea level) and sea surface wind stresses were downloaded from ion (see ). The seftp://sidads.colorado.edu/DATASETS/NOAA/G02135/shapefiles/. Sea ice index version 2 uses V1.1 of the sea ice concentrations from Nimbus-7 SMMR and DMSP SSM/I-SSMIS Passive Microwave Data  as the input data source for the final portion of the Sea Ice Index record. The data are a level-3 gridded product, mapped to a polar stereographic grid at a spatial resolution of 25 km. Data were processed using the rgdal mapping package [Monthly sea ice extents were downloaded from the G02135 dataset  at the N package  in R. Dihttp://gcmd.nasa.gov/records/AADC_southern_ocean_fronts.html. The distance (km) of acoustic stations to the closest southern boundary of ACC calculated using a custom design R function.We used positions of the southern boundary of ACC  defined Before modelling the data, we determined the effects of multi-collinearity between predictor variables using the generalised variance inflation factors  implemeRF models are known to provide higher performance and have a number of advantages over standard regression methods like generalized additive models –55. RF uRF model was used to determine which environmental parameters influenced the acoustic occurrence  and acoustic behaviour  of the Antarctic blue whales during the Southern Ocean summer. The chl-a data were log-transformed before model fitting due to the skewness of their distribution. Pearson’s correlation coefficients (r) were calculated to measure the linear correlation (dependence) between the call rate of each call type.The relative importance of predictor variables in the model was assessed by computing the influence of each of the variables on the prediction error of the model. The relative importance of each of the variables in the model can be computed by permuting the Out Of the Bag (OOB)/test data sample or by determining the decrease in node impurity, as measured by the mean sum of squares, resulting from splitting of the variable of interest and averaging over all trees. For each tree the prediction error is computed on the OOB data and the permuted data are calculated and averaged across all trees and normalized by the standard deviation of the difference. This normalized index is calculated for each of the variables and used as index of relative importance. RF model was fitted to determine the importance of the following predictors on blue whale occurrence: longitude, latitude, log-transformed chl-a, SST, SSH, water depth, distance from the shoreline, distance from the nearest SBACC, wind stress, wind speed, wind direction, station duration, day/night, and month. RF model for call rates determined the importance of the following predictors: longitude, latitude, log-transformed chl-a, SST, SSH, water depth, distance from the shoreline, distance from the nearest SBACC, wind stress, wind speed, wind direction, station type, whale numbers, day/night, month and whale groups.The RF optimal parameter configurations for both call rate models were: the number of growing trees ntrees = 500 for Z-calls and 3,000 for D-calls; the splitting minimum size of terminal nodes of trees nodesize = 2 for Z-calls and 1 for D-calls; and the number of call rates randomly selected at tree node mtry = 3 for Z-calls and 2 for D-calls. The RF optimal parameter configurations for blue whale occurrence modelling: ntree = 500, nodesize = 1, and mtry = 6.Since the detection ranges of sonobuoys were not estimated here, we ran the RF model with and without sighting data using the above optimal parameter configurations to evaluate effects of detection ranges on blue whale call rates. The model produced relatively similar sets of outputs in both cases. The modelling was performed with the R statistical software package using libraries randomForest  and rangWe used the 5-fold cross-validation (5-foldCV) and the Leave Group Out Cross Validation , to quantify the predictive accuracy of our classification model type between the predicted values and observed values of blue whale occurrence . The areUsing 70% of the data for training and the remaining for validation, the performance of RF for call rate modelling was assessed. Root mean square prediction error (RMSPE) was used to measure the difference between values predicted by the model and observed values. In addition, Spearman's rank correlation coefficient (rho) was applied as a qualitative measure of the performance of the model. A low RMSPE value indicates a better model whereas the opposite is true for rho.The AUC values indicated better performance for RF than GBM . The cor-3  station had a higher effect on call rates than type 2  station Figs  and 11, lacialis . The reclacialis . AlthougEuphausia superba [In this study, Antarctic blue whales were commonly found in waters with SSTs between -1.4°C and 3.5°C, which suggests that this species has a broad temperature tolerance in the Southern Ocean. This could also explain the latitudinal dependence of blue whales, as the colder waters are generally found further south near the ice edge. D-call rates increased when SSTs were below 0.3°C illustrating a preference for temperatures associated with high zooplankton productivity of the ACC thermal fronts. Inter-annual variability in SST and mesoscale ocean circulation affect the inherent Antarctic ice cover that is critical to the distribution of blue whale prey species  superba ,18. SST The area between the ice edge and 800 km from the nearest Antarctic shore was determined by the model to be important for call occurrence and call rates of Antarctic blue whales in summer. This indicates that not all blue whales transit to the ice edge to feed, but that some animals remain well north of the ice edge. The SBACC is associated with high primary production, krill and whales, suggesting that it provides predictably productive foraging for many species; it is of critical importance to the function of the Southern Ocean ecosystem ,64. HoweWater depths between 380 and 5,900 m at the ice edge and open sea, respectively, were of importance for both the occurrence and acoustic behaviour of whales reflecting a wide bathymetric preference by the species. It is important to note that the location of the ice edge in summer generally approximates the continental slope front  and is normally a region of high chl-a  and prey-2 range observed by Nowlin and Klinck [-2 between 20°W and 110°E. Wind direction controls the supply of nutrient carrying sediments to the continental margin, thus is an important sign of ocean productivity which is closely coupled to climate variability [SSH values of less than -1 m were important to blue whale occurrence and call rates, indicating that blue whales occurred closer to the Antarctic Peninsula as SSH decreased closer to the Antarctic Peninsula . Specifid Klinck  and Thomd Klinck  for the d Klinck  observediability . The occ-1 were experienced across the survey regions, and these wind speeds were typical for the Southern Ocean [-1  and rn Ocean , and mayrn Ocean . Our RF [-1 Figs .-1) are needed to further predict the temporal and spatial effects of wind-induced noise on the acoustic behaviour of blue whales . High wind speeds are also known to introduce air bubbles in the upper water column that absorb and refract sound [Deployments of acoustic instruments in poor weather conditions , which could indicate that Antarctic blue whales may be acoustically more active when not feeding.Available evidence demonstrate that krill are most abundant in areas with the highest-productivity within their habitats (e.g. ), yet siBalaenoptera musculus population is considered highly vulnerable to climate change [The results of the RF model provide evidence that blue whale occurrence and acoustic behaviour are sensitive to annual variabilities of major environmental parameters such as chl-a, wind speed, wind direction and stress, SSH and SST. Consequently, blue whales might be vulnerable to either gradual long-term changes or abrupt and persistent short-term changes or variability (i.e. climate change) of those key environmental factors. This might hamper the recovery of this species and lead to habitat loss and local distributional shifts ,79 as cle change ; the vulThe RF modelling enabled the explicit interpretation of the complex relationships between blue whales and their environment from our long term acoustic data set. RF performed well for call rates and occurrence modelling, it holds perhaps the greatest promise for acoustic behaviour and occurrence modelling because of its wide versatility that allows it to assume simpler, faster and more interpretable forms with incorporable automatic predictor selection.Antarctic blue whales showed both latitudinal and longitudinal preferences in the Southern Ocean. Passive acoustic techniques provided useful information on blue whale occurrence and behaviour, however, more direct and continuous acoustic recording of blue whales are needed. Whales preferred relatively colder waters closer to the ice edge with potentially high krill abundances and occurred closer to the ice edge. The year 2007/08 was an environmental anomaly and blue whale call rates responded to the change, suggesting that future changes in environmental conditions have the potential to affect blue whale behaviour and occurrence. The link between environmental conditions and blue whale occurrence and behaviour revealed important biological information essential for improving the management and conservation of this depleted whale species. This study shows the potential influence of long-term systematic environmental change on Antarctic blue whales.