TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory dilated cardiomyopathy (DCM). TTN haploinsufficiency caused by TTNtv is suggested as the disease mechanism. However, it is unclear whether TTN insufficiency causes DCM. Moreover, it is unknown whether modulation of downstream pathways serves as a therapeutic strategy for DCM caused by TTN insufficiency. Here, we show that reduction of cardiac Ttn expression by adeno-associated virus mediated shRNA (Ttn shRNA) generated DCM in mouse, demonstrating impaired cardiac performance, enlarged left ventricle (LV) and reduced LV wall thickness. A screen of 10 dysregulated and selected genes identified that Yin Yang 1 (Yy1) significantly suppressed DCM caused by Ttn shRNA. Gene profiling by RNAseq showed Yy1 modulated cell growth related genes. Ttn insufficiency activated cardiomyocyte cell cycle reentry by upregulating of Ccnd1 and Ccnd2. Cardiomyocytes activated by Ttn insufficiency did not advance to S phase by EdU incorporation assay. Yy1 promoted cardiomyocyte cell cycle by further enhancing Ccnd1 and Ccnd2 and increasing DNA replication without undergoing cell division. Importantly, upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically promoting cardiac cell cycle.Truncating variants in  There are currently no approved therapeutic products indicated for DCM treatment. Typical treatments are those indicated for broader cardiovascular disease. As the disease progresses, patients have limited treatment options, such as surgical or other invasive interventions and heart transplant2. DCM will result in heart failure with reduced ejection fraction (EF), usually without prior ischemic. The walls of the left ventricle are thin and over-expanded, leading to improper contraction and insufficient blood being pumped by the dilated heart. DCM results from a variety of external factors, such as viral infection, alcohol abuse, exposure to cardiotoxic medications and pregnancy, as well as from genetic variants in a number of causal genes including TTN, LMNA, ACTC1, MHY7 and PLN9. Titin (coded by TTN) plays an important role in the contraction and relaxation of cardiac muscles by connecting Z-disc to the M-line in the sarcomere. TTN truncating variants (TTNtv) contribute up to 15% ambulatory DCM and 25% end-stage or familial DCM9. In DCM, TTNtv are significantly enriched most in A band as well as other regions including I-band, Z-disc or M-line with variable position-related odds ratios10. TTN haploinsufficiency caused by TTNtv is emerging as the potential disease mechanism. Rat models with TTNtv in Z-disc and A band did not result in change in titin protein levels and obvious cardiac performance under normal physiological conditions. It was not known whether Ttn insufficiency causes DCM in mouse.Dilated cardiomyopathy (DCM) occurs as many as 1 in 250 people11. Cell cycle reactivation is observed in a limited cardiomyocytes under physiological conditions13. As an emerging strategy for cardiac therapeutic regeneration, we and others showed that enhancing this process by removing cell cycle brakes or augmenting accelerators is beneficial for heart recovery from heart failure models caused by ischemia or pressure overload18. We previously observed that cardiomyocytes undergo an extra round of cell cycle in Mybpc3 deficient mice, suggesting cell cycle reactivation could compensate sarcomere insufficiency19. It was not known whether Ttn insufficiency could induce cardiac cell cycle reactivation. Moreover, it was unknown whether enhancing this process could be a therapeutic strategy for DCM caused by Ttn insufficiency. Here, we address these major gaps and identify therapeutic candidates that are vital for advancing potential hits into a therapeutic approach for DCM.Mammalian cardiomyocytes exit their cell cycle shortly after birth, preventing heart repair through cardiac regenerationTtn shRNA sequences targeting Ttn gene expression, we designed 4 unique shRNA constructs, designated Ttn shRNA-1, -2, -3 or -4  into 293 T human embryonic kidney cells. Two shRNA constructs, Ttn shRNA-1 and -2, significantly reduced EGFP signal  into the thoracic cavity of 1.5 week old male neonates, as described21. Three weeks after viral transduction, all mice injected with medium or high dose of Ttn shRNA-1or -2 viruses  died due to severe heart failure. Mice treated with low dose  were evaluated by echocardiography. Ttn shRNA-1 or -2 transduced mice reached the endpoint of showing impaired cardiac performance, enlarged left ventricle (LV) and reduced LV wall thickness  was significantly reduced compared to that of mice transduced with control shRNA   was significantly reduced compared to control . Histological analysis did not reveal myocardial disarray in both Ttn shRNA-transduced mice. Moreover, cardiac fibrosis, assessed by image analysis of Masson’s trichrome (MT)-stained heart specimens and commonly observed in other DCM models including PLN (R9C) and Lmna−/−, was not significantly induced in Ttn shRNA transduced mice  relative to control shRNA-transduced mice   for subsequent assessment. Cardiomyocytes with anti-Troponin I staining was EGFP positive after AAV-EGFP transduction, indicating the virus was distributed to most cardiomyocytes   Fig. 6,19,22. RNA Fig. . Ttn shRTtn shRNA, we profiled control (n = 3) and Ttn shRNA (n = 3) treated hearts (dose = 0.2E + 13 vg/kg) by RNAseq, and the significantly changed were analyzed by Gene Ontology  in human dermal fibroblasts , cardiac performance was assessed by echocardiography. When treated with most selected candidates, Ttn shRNA transduced mice developed a similar or even more severe DCM compared to those treated with control cDNA , indicating that re-introducing these pathway-associated candidates could not suppress Ttn shRNA-induced DCM  significantly improved cardiac performance of Ttn transduced mice compared to those treated with EGFP control .We further assessed the suppressive effect of Yy1 on DCM induced by Ttn shRNA for three additional timepoints  was significantly suppressed compared to that treated by EGFP control . Remarkably, Yy1 treated mice continued to maintain significantly rescue of the cardiac chamber dilation compared to Ttn shRNA transduced mice treated by EGFP control. Cardiac function (FS) of Ttn shRNA mice treated with Yy1 was significantly improved compared to Ttn shRNA mice treated by EGFP control, suggesting that Yy1 suppresses DCM induced by Ttn shRNA. At dose of 0.2E + 13 vg/kg, candidate Yy1 was induced ~ 4.5 fold by qPCR analysis. The expression of heart failure markers including Nppa, Nppb and Myh7 in mice treated with Yy1 was significantly reduced compared to that in DCM mice treated with EGFP control , Ttn shRNA + EGFP  and Ttn shRNA + Yy1 animals  four weeks after virus transduction by RNAseq. To uncover signaling pathways regulated by Yy1, we found 134 genes significantly changed in Yy1 treated group compared to Ttn shRNA group . Gene ontology (GO) analysis identified top significantly enriched gene sets in category III including “positive regulation of growth” and “small molecule metabolic process”  and Ankyrin repeat domain-containing protein 23 (Ankrd23) by Ttn shRNA was suppressed by Yy1  in Ttn shRNA group compared to control shRNA group (n = 10). Ccnd2, another cyclin family member, was significantly induced by ~1.71 fold  in Ttn shRNA group compared to control shRNA group (n = 10). Yy1 significantly enhanced additional Ccnd1 and Ccnd2 expression by ~ 1.33 fold  and ~1.38 fold  respectively compared to Ttn shRNA group (n = 12)  for cardiomyocytes (CM). Ccnd1 protein was predominantly detected in the nuclei of non-cardiomyocytes (non-CM)  compared to control group  , suggesting that Ttn shRNA activates CM cell cycle reentry. In addition, Ccnd1 + CM was further increased by ~3.76 fold  in Yy1 treated group compared to Ttn shRNA group. Ccnd2 protein was predominantly expressed and elevated in the nuclei of CM   compared to Ttn shRNA group. We also observed a significant increase of Ccnd2 + non-CM in Ttn shRNA group  compared to controls , and Yy1 further significantly increased Ccnd2 + non-CM  compared to Ttn shRNA group. We quantified protein levels of Ccnd1 and Ccnd2 in heart tissues by western blot analysis  and ~ 4.14 fold  increase of Ccnd1 and Ccnd2 proteins compare to control shRNA (n = 4). In comparison to Ttn shRNA group (n = 4), additional ~ 1.98 fold  and ~ 1.53 fold  of Ccnd1 and Ccnd2 was increased in Yy1 group  and ~ 1.93 fold  in CM derived from Ttn shRNA group compared to control shRNA group , suggesting CM activated cell cycle reentry by upregulating Ccnd1 and Ccnd2 expression in response to Ttn insufficiency  compared to Ttn shRNA group (n = 3). Yy1 did not further increase Ccnd2 expression, suggesting Ccnd2 is not transcriptionally regulated by Yy1 in CM. To assess whether activated cardiac cell cycle advanced to G1/S phase, we assayed DNA synthesis by EdU incorporation. Most EdU was detected in non-CM because they do not exit from cell cycle , suggesting CM cell cycle activated by endogenously upregulated genes including Ccnd1 and Ccnd2 does not advance to S phase  increase of EdU + CM was detected compared to Ttn shRNA group, indicating Yy1 promoted CM cell cycle from G1 to S phase. EdU incorporation also was increased by ~ 6.41 fold in non-CM of Yy1 group. To assess whether cardiac cell cycle further progressed to mitotic phase, we stained heart sections with M phase marker, phosphorylated histone (pH3)  compared to control shRNA group . Although Yy1 group showed an increased EdU incorporation in non-CM, upregulation of Yy1 did not further increase pH3 + signals  in non-CM of Yy1 group compared to Ttn shRNA group, suggesting upregulation of Yy1 does not increase non-CM mitosis. We did not observed pH3 + CM in the hearts of all three groups, indicating activated CM reaching to M phase are limited  Fig. . To loca 5) Fig. . Importa07) Fig. . Yy1 sigsis Fig. . Upon Ttoup Fig. . We furtncy Fig. . Yy1 sigase Fig. . Upon Yyted Fig. .Figure 4Ccnd1 and Ccnd2 was significantly enhanced by Yy1 in CM of mouse hearts. To assess whether enhancing of Cyclin D suppresses DCM caused by Ttn insufficiency, we co-introduced Ccnd1 or Ccnd2 (0.2E + 13 vg/kg) with Ttn shRNA (0.2E + 13 vg/kg). Cardiac performance was assessed by echocardiography. Cardiac function (FS) of mice treated with Ccnd1 or Ccnd2 was significantly improved compared to Ttn shRNA mice treated by EGFP. Ccnd1 or Ccnd2 overexpression significantly suppressed DCM induced by Ttn shRNA, suggesting cyclin D serves as a crucial downstream of Yy1 to enhance cardiac performance  and heart failure38.Ttn shRNA, indicating cardiomyocytes response to Ttn insufficiency by activating cell cycle reentry signals. We did not observed this reaction in another DCM model caused by Lmna insufficiency. Activated cardiomyocyte cell cycle induced by Ttn insufficiency does not advance to S phase. We found that upregulation of Yy1 promoted cardiac cell cycle reentry by further enhancing Ccnd1 and Ccnd2. Yy1 promoted cardiomyocyte cell cycle to S phase by a significant increase of EdU incorporation. However, we did not detect mitotic phase marker pH3 in cardiomyocytes, suggesting activated cardiomyocytes undergoing mitotic phase are limited. Importantly, Upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Taken together, Yy1 promotes cardiac cell cycle to facilitate to suppress Ttn shRNA-induced DCM.We detected over 35 fold increase of Ccnd1 and Ccnd2 in cardiomyocyte nuclei upon Mybpc3, another causal gene for sarcomeric cardiomyopathy, induces an extra round of cardiomyocyte cell cycle during neonatal stage19. It is now of great interest to know whether this reactivation of cardiac cell cycle is a common mechanism for sarcomere deficiency and enhancing of this process by Yy1 or other regulators is able to suppress cardiovascular disease related to sarcomere. Reactivation of cardiomyocyte cell cycle does not always lead to cell number increase. Cytokinesis is still rarely detected in adult hearts upon regenerative enhancement. Yy1 might promote hypertrophic growth coupled with cell cycle reentry as Ccnd2 is a mediator for Myc overexpression or exercise-induced hypertrophic growth in cardiomyocytes39. Recently, Yy1 is suggested to serve as a structural regulator between enhancer and promoter interactions and facilitates gene expression40. Apart from modulating cell growth, upregulation of Yy1 might indeed reinstate or promote enhancer - promoter interaction to restore gene regulatory network dysregulated by Ttn insufficiency.Our recent study showed insufficiency of Atp2a2, Bcl2, Sod2 and Sirt341. Most of them failed to protect DCM induced by Ttn shRNA, suggesting root causes and disease mechanisms should be taken into account for DCM prevention and treatment strategies. It is of great interest to know whether Yy1 is able to suppress DCM caused by other genes including LMNA, MYH7 and PLN. To translate our research, one concern is whether upregulation of Yy1 could cause any cardiac defects. Previous study showed overexpression of Yy1 induced a relative marginal hypertrophy cardiomyopathy only in male mice. Here, we specified the Yy1 expression by cardiac specific promoter, virus dose and postnatal transduction. In contrast to a previous study, no detectable impairment of cardiac structure and performance was observed after ~ 5 months of virus transduction37. Taken together, our findings provide a strong supporting evidence for translational research.Our selected candidates included many therapeutic targets for heart failure including All mice were maintained and studied using protocols approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore. Animal work was undertaken in accordance with Singapore National Advisory Committee for Laboratory Animal Research guidelines. Relevant national and institutional guidelines and regulations must be consulted before commencement of any animal work. All studies were conducted in male C57BL/6JINV (Jax) mice. For virus injection, 50 µl viruses were injected into thoracic cavity of 10 days old pups via insulin syringe, avoiding the heart and lungs. For EdU injection, EdU  was dissolved in saline and 5 mg/kg EdU was delivered to mice by intraperitoneal injection for two weeks after AAV transduction. For heart harvesting, mouse was anesthetized by 2% isoflurane and the heart was exposed by opening chest. After that, 15% KCl was injected into inferior vena cava to achieve asystole at diastole, then the heart was rapidly isolated and flushed with D-PBS through LV to wash out blood. Half of the apex was isolated and immersed in RNALater  at room temperature for RNA extraction, while the other half was snap frozen in liquid nitrogen for protein extraction. The rest part of heart was fixed in 4% paraformaldehyde for 24 hours and subsequently embedded by paraffin.Cardiac dimension and function of mice three and/or four weeks after virus transduction were achieved by echocardiography . All mice were shaved to expose chest area one day before experiment. During echo, 1.5% isoflurane with oxygen were applied to each mouse, and cine of 300 frames of both B mode and M mode  were recorded when heart rate was around 450–500 bpm. Measurements were processed by Vevo®LAB . LV tracings were averaged from at least 3 consecutive heart beats of M-mode. LVDD (LV diastolic dimensions), LVWT , EF (ejection fraction) and FS  were obtained from short axis images.HEK293T cells were cultured at 37 °C with 5% CO2 and maintained in DMEM (Hyclone) supplemented with 10% FBS, 1 mM sodium pyruvate and 10 µg/ml gentamicin. Transfection of shRNA constructs and other plasmids was performed using PEI  according to manufacturer’s instructions.et al. In brief, the heart was perfused with warm EDTA, perfusion buffer and collagenase buffer through LV when aorta was clamped, then heart tissue became softened and swollen. After that, heart tissue was teared into pieces and dissociated by pipetting. Cell suspension was then passed through 100 µm strainer and processed to three rounds of gravity settling. Cardiomyocytes, in the pellet, were harvested for RNA extraction.We adapted protocol from Ackers-Johnson et al. In brief, rAAVs were produced on HEK293T cells by transient triple plasmid transfection, including rAAV viral vector with gene to be delivered, helper plasmids pAdΔF6 and plasmid pAAV2/9 (Penn Vector Core). Three days after transfection, viruses were collected from cell pellets and were purified by Optiprep density gradient medium . After concentrating by centrifugal filter , viruses were aliquoted and stored at −80 °C. For virus titration, forward primer: gataaaagcagtctgggctttcaca and reverse primer: gagcccatataagcccaagctattg were designed to target at rAAV genome-containing particle, cTnT promotor region, and to determinate the physical titers by qPCR.Protocol was adapted from Grieger TM qRT-PCR Titration Kit was applied for titration. As for virus infection, human primary fibroblasts (ATCC PCS-201-010) were subjected to two rounds of overnight transduction at 50% confluency.Lentiviral plasmids teto-HAND2, FUW-TetO-GATA4, teto-MEF2C, teto-TBX5, teto-MYOCD and teto-YY1were obtained from Addgene or by cloning. Lentiviruses were produced on HEK293T cells. Two days after transfection, viruses were harvested from supernatant and concentrated by centrifugal filter . Lenti-XTtn shRNA candidates were designed to target 21 base-pair gene-specific regions (Invitrogen). The sequences of mouse Ttn shRNAs are as follows: shRNA-1: AGTACTTTCAGCTTAATGGTG (Exon 262); shRNA-2: TAAAGAAGCCGATTTCTTGGT (Exon 275); shRNA-3: ATTACTGGCACACTCAGTTGT (Exon 275); shRNA-4: ATAAGTTGGAGACAAGGAGCG (Exon 307). The sequence against LacZ, AAATCGCTGATTTGTGTAGTC, was used as control shRNA. The miR-155 backbone based shRNA cassette was inserted at 3 prime of AAV-cTnT-EGFP vector.Ttn shRNA) compared to control group (Ctrl shRNA). For dissecting molecular mechanism of Yy1, 134 genes (P < 0.005 and FDR <0.2) were selected from Yy1 treated group (Ttn shRNA + Yy1) compared to Ttn shRNA group (Ttn shRNA + EGFP). Differentially expressed genes were uploaded to Morpheus for Hierarchical clustering and color-coded heat-map. Gene ontology (GO) of each category were analyzed by Gene Set Enrichment Analysis .Total RNA from left ventricular tissue of male mice (n = 3 per group) was achieved to establish RNAseq library. RNA samples were pre-treated with Truseq Stranded Total RNA Library Prep kit  to remove abundant cytoplasmic rRNA. The remaining intact RNA was fragmented using a chemical mix, followed by first- and second-strand cDNA synthesis using random hexamer primers. End-repaired fragments were ligated with a unique illumina adapter. All individually indexed samples were subsequently pooled together and multiplexed for sequencing. Libraries were sequenced using the Illumina Hiseq. 2000 sequencing system and paired-end 101 bp reads were generated for analysis. RNAseq data was deposited to NCBI. For selecting potential therapeutic genetic candidates, 3101 genes  were identified from DCM group  and qPCR was carried out by KAPA SYBR Fast qPCR Master Mix kit . All qPCR primers are listed as follows:Heart samples were fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin and sectioned at 5 µm intervals. Paraffin samples were further treated with xylene (to remove paraffin), re-hydrated, and permeabilized in 0.1%(v/v) Triton-X100 in PBS. Hematoxylin and eosin (HE) was applied to observe myocyte architecture and Masson trichrome (MT) to identify cardiac fibrosis. Fibrosis was quantified by Image J. The percentage of fibrosis was calculated as the blue-stained areas divided by total ventricular area. As for immunostaining, boiled citric acid was used for Ccnd1, Ccnd2, EdU and pH3. Primary antibodies: Ccnd1 , Ccnd2 , pH3  and cTnI . Other labeling dyes: DAPI for nucleus  and EdU Imaging kit . All the positive signals from three completed cross sections were counted for each heart sample, and data were normalized to total nucleus number.Frozen heart tissues were lysed in cold RIPA buffer with protease inhibitor  and were homogenized with prechilled TissueLyser  by adding metal beads. After centrifuge, supernatants were collected to detect proteins with small molecular weight like Yy1, Ccnd1 and Ccnd2. 20 µg of each sample was loaded into SDS-page gel and further transfer to nitrocellulose membrane  for blotting. Primary antibody including: Yy1 , Ccnd1 , Ccnd2 , and Lamin B1 . Secondary antibody including: Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody .et al.42. Briefly, pellets were resuspended in solubilization buffer  and samples were further incubated on ice for 10 mins followed by boiling (95 °C) for 3 mins. 35 µg protein from each sample was added with 6 µM of bromophenol blue  and then loaded onto agarose-strengthened SDS-PAGE gel . Protein bands were visualized by SimplyBlue SafeStain  and band intensity were quantified using ImageLab (Bio-Rad).As for pellet fraction, protocol was adapted from Neagoe Statistical analysis was achieved by Prism Graphpad 7.0. P value between two groups was performed by two-tailed, unpaired T-test with Weltch correction, and one-way ANOVA with Tukey’s multiple comparisons test for multiple groups. Quantitative data were shown as mean ± SD. ns, non-significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.SUPPLEMENTARY INFO


To characterize high-risk human papillomavirus (HPV) infections in a sample of women in two small English-speaking Caribbean countries: Saint Kitts and Nevis and Saint Vincent and the Grenadines.Sexually active women ≥ 30 years old attending primary care health facilities participated in the study. Each participant had a gynecological examination, and two cervical specimens were collected:  a specimHigh-risk HPV was detected in 102 of 404 (25.2%) in Saint Kitts and Nevis and in 109 of 368 (29.6%) in Saint Vincent and the Grenadines. High-risk HPV genotypes 52, 35, 51, 45, and 31 were the most common high-risk types in Saint Kitts and Nevis. In Saint Vincent and the Grenadines, the most common high-risk HPV genotypes were 45, 35, 31, 18, and 51. Current age was found to be significantly associated with high-risk HPV infection in both countries. In addition, in Saint Vincent and the Grenadines, high parity (> 3 pregnancies) and having had an abnormal Pap smear were found to be independent risk factors for high-risk HPV.These results contribute to the evidence on HPV prevalence for small island states of the Caribbean and support the accelerated introduction of the 9-valent HPV vaccine in the two countries and elsewhere in the English-speaking Caribbean. Use of the study’s results to guide the development of policy regarding implementation of HPV testing as the primary screening modality for older women is recommended. Human papilloma viruses (HPVs) are very common sexually transmitted infections. Infection with high-risk HPV genotypes has been recognized as a necessary factor for the development of cervical cancer as well as cancers of the vagina, anus, vulva, penis, and oropharynx HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 have been identified as cervical carcinogens  and Saint Vincent and the Grenadines (2011 estimated population of 109 903). In addition, the study sought to determine whether HPV infection is associated with sociodemographic, behavioral, and reproductive factors in the population.The study was conducted by the Pan American Health Organization (PAHO) in collaboration with CARPHA and the Ministries of Health of Saint Kitts and Nevis and Saint Vincent and the Grenadines. Ethical approval for the study was granted by the Ministries of Health in the respective countries and by the Pan American Health Organization/World Health Organization Ethical Review Committee, based on the assurance of confidentiality of information and the potential benefit to participants.Participants for this cross-sectional study were recruited from February to November 2014. All islands in each country and all primary health care clinics on each island were included, as well as five voluntarily participating privatesector health sites (four in Saint Kitts and Nevis and one in Saint Vincent and the Grenadines), one nongovernmental organization (NGO) (Saint Vincent Planned Parenthood Clinic), and two outreach sites at garment factories in Saint Kitts and Nevis.A convenience sample of women 30 years or older attending primary care clinics, selected gynecologists, and NGO clinics in each country were invited to participate in the study. Informed consent was required and signed by each participant. An identification number was assigned and utilized on all study documentation for each respective participant. Participants were informed that strict confidentiality would be maintained and that no identification information would be reported, but instead used only for follow-up care. All persons meeting the study criteria and giving informed consent were entered into the study until 500 women from each country were recruited. No financial compensation was provided to participants for taking part in the study.Standard operating procedures (SOPs), which included specimen collection, were developed for the study. Midwives and nursing staff were trained in interviewing techniques. The midwives were also trained in specimen collection using the SOPs.Questionnaire. Each participant was interviewed privately and confidentially by a trained health staff member , using a questionnaire. The questionnaire collected information on the demographic, behavioral, and reproductive characteristics of study participants as well as knowledge of and attitudes toward immunizations against vaccinepreventable diseases, HPV in particular.Sample collection. Each participant had a gynecological examination done and cervical specimens collected by the trained midwives at the study sites. Specimens were collected for a Papanicolaou (Pap) test and for HPV DNA testing using the HPV High Risk Screen Real-TM  All cervical smears taken for Pap testing were immediately sprayed with a fixative prior to labeling and being packaged for transport to the laboratory. The cervical cells for HPV testing were taken from the endocervix, using a Cytobrush, which was then placed in a specimen transport medium in a 2-ml vial.Both specimens remained at room temperature until the end of the collection day, when they were then transported on ice packs to the central laboratory in each respective country for storage at 2 to 8 °C. The specimens collected for HPV DNA testing were later batch-shipped at 2 to 8 °C from each respective study country via air courier service to the Caribbean Genetics (CARIGEN) laboratory at the Department of Basic Medical Sciences, University of the West Indies, Mona, Kingston, Jamaica, for processing.Sample processing. The Pap smear tests were processed at the University of the West Indies Pathology Laboratory, using the Bethesda cytology classification system. In order to ensure high-quality cytology results, every 10th slide was reread by another cytologist, and all positive smears were reviewed by the pathologist. At CARIGEN, the specimens that were collected for HPV DNA testing were stored at 2 to 8 oC once received, and they were extracted within three days, using an automated QIAxtractor (QIAGEN) DNA extraction instrument. The final extract was either tested immediately or stored at –20 °C prior to processing. For every set of 16 samples processed on the QIAxtractor, a phosphate-buffered saline (PBS) blank was used to serve as a “contamination control.”β-globin gene used as internal control to check for extracted DNA. Some samples with a high quantity of mucous or insufficient quantity of epithelial cells caused problems and were not processed properly on the extractor. This resulted in the internal control not being detected. Samples with a small quantity of cells resulted in a “low cell” reading. Both low-cells samples and those with high mucous content were reextracted using the DNA IQ Manual Extraction kit .These specimens were tested for HPV using the HPV High Risk Screen Real-TM (Sacace), an in vitro Real Time amplification test for quantitative detection of high-risk human papillomavirus  in urogenital brushes. The test uses primers directed against regions of HPV A9 Group ; HPV A7 Group ; and HPV A5-A6 Group ; and with DNA was amplified using the RotorGene/6000 (Corbett Research)/Rotor-Gene Q (QIAGEN) Real Time PCR cycler, according to the manufacturer’s instructions. After amplification, data from each run was exported to an Excel spreadsheet macro provided by the manufacturer that allowed for the calculation of HPV group and viral loads. Known standards and contamination controls were run with each amplification for viral load determination and PCR amplification efficiency.The HPV typing was reported as high-risk HPV-positive with reference to the grouping  included in the HPV High Risk Screen Real-TM (Sacace Biotechnologies) test, as HPV-negative, or as low cells. HPV typing was reported as high-risk HPVnegative if the test was negative for the aforementioned groupings. HPV typing was reported as low cells if there were insufficient epithelial cells.β-globin gene used as an internal control.With the HPV typing, the specimens that were identified as positive were typed for HPV using also the HPV High Risk Type Real-TM (Sacace). This is an in vitro Real Time amplification test for quantitative detection of high-risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 in urogenital brushes. Each vial contains primers directed against regions of each HPV type and a DNA was amplified using the Rotor-Gene/6000 (Corbett Research)/Rotor-Gene Q (QIAGEN) Real Time PCR cycler, according to manufacturer’s instructions. After amplification, data from each run was exported to an Excel spreadsheet macro provided by the manufacturer. This allowed for the identification of the individual HPV types. Known positives were run with each amplification to check for PCR amplification efficiency. A blank sample of nuclease-free water was also run with each amplification as a contamination control. The respective HPV type was reported for all the HPV types included in the HPV High Risk Type Real-TM (Sacace) test.The analysis was restricted to the participants whose cervical specimens were adequate for HPV DNA isolation, which was 404 in Saint Kitts and Nevis and 368 in Saint Vincent and the Grenadines. Data were analyzed using EPi Info version 7 software. Cross-tabulations were used to explore associations of the presence of high-risk HPV infection with the several demographic, behavioral, and reproductive factors and the Pap smear results. Risk ratios and chi-squared statistics were used to determine the strength of associations. Using abnormal Pap smears and the presence of high-risk HPV infection as dependent variables, multiple logistic regression analyses were then used to model the relationship of possible risk factors from among the demographic, behavioral, and reproductive variables with these outcome variables.The results presented are for the women who had cervical specimens that were satisfactory for testing for HPV DNA. The number of cervical specimens that were not satisfactory for testing (due to problems with storage and transportation) and that were thus excluded from processing and analysis was 96 of 500 (19.2%) in Saint Kitts and Nevis and 132 of 500 (26.4%) in Saint Vincent and the Grenadines.In Saint Kitts and Nevis, of the 404 women who were tested for high-risk HPV, 56.5% of them were between 30 and 44 years old and 43.5% were 45 or older. In Saint Vincent and the Grenadines, of the 368 women tested, 51.7% were between 30 and 44 years old and 48.3% were 45 or over. The median age of the women in Saint Kitts and Nevis was 42 years , while in Saint Vincent and the Grenadines, the median age was 44 years .There were significant differences in the demographic profile of the participants from the two countries, as shown in Table 1.In both of the countries in the study, a little over one-quarter of those who were tested were positive for high-risk HPV: 102 of 404 (25.2%) in Saint Kitts and Nevis and 109 of 368 (29.6%) in Saint Vincent and the Grenadines.There were 12 high-risk HPV genotypes detected in the women tested in the two countries: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 . Figure 1 also compares the prevalence of those genotypes in the two Caribbean countries with the levels for the entire world Overall, HPV high-risk genotypes 45, 35, 31, and 52 were the most prevalent types detected in the two Caribbean countries in the study. HPV 52 and 35 were the most prevalent high-risk types in Saint Kitts and Nevis. In Saint Vincent and the Grenadines, the most prevalent types were 45, 35, and 31. HPV 16 was detected in 10 of 102 (9.8%) of the high-risk infections in Saint Kitts and Nevis and in 9 of 109 (8.3%) of those in Saint Vincent and the Grenadines. HPV 18 was identified in 9 of 102 (8.8%) of the high-risk infections in Saint Kitts and Nevis and in 13 of 109 (11.9%) of those in Saint Vincent and the Grenadines.In Saint Kitts and Nevis, of the 102 women with high-risk HPV infections, 6 of them (5.9%) also had abnormal Pap smears. Of those 6, 4 of them were classified as atypical squamous cell of undetermined significance (ASCUS) and 2 were classified as low-grade squamous intraepithelial lesions (LSIL). Of those 6, 2 of them had high-risk HPV 16 infections, and none of them had an HPV 18 infection.In Saint Vincent and the Grenadines, of the 109 women with high-risk HPV infections, 14 of them (12.8%) also had abnormal Pap smears. Of the 14 abnormal smears, 3 of them were classified as ASCUS, 5 were LSIL, and 6 were high-grade squamous intraepithelial lesions (HSIL). Of the 6 HSILs, 2 were associated with high-risk HPV 31, and 1 was associated with HPV type 16. Also, 1 HSIL was associated with HPV 18 in a multiple infection with HPV types 45, 33, and 52. The remaining 2 of the 6 HSILs were associated with HPV 52 and HPV 58. Overall in Saint Vincent and the Grenadines, 6 of the 14 (42.8%) of those with abnormal Pap smears were infected with high-risk HPV 16 or 18. Of those with abnormal smears, 4 of them (28.5%) were infected with HPV 16, and 2 of them (14.3%) with high-risk HPV 18.In the two countries, more than a fifth of the women with high-risk HPV were infected with multiple types of high-risk HPV. This was true in Saint Kitts and Nevis for 28 of 102 women (27.4%) and in Saint Vincent and the Grenadines for 23 of 109 women (21.1%). Just over a third of the total number of multiple infections in each country included high-risk HPV 16 or 18: in Saint Kitts and Nevis, 11 of the 28 (39.3%), and, in Saint Vincent and the Grenadines, 9 of 23 (39.1%). Multiple high-risk types were identified in 2 of 6 of those with abnormal smears (1 ASCUS and 1 LSIL) in Saint Kitts and Nevis and 6 of 14 (42.8%) of those with abnormal smears  in Saint Vincent and the Grenadines.P < 0.01). In Saint Vincent and the Grenadines, in addition to current age, number of sexual partners in the lifetime (≥ 3 partners), number of pregnancies (> 3 pregnancies), and having had an abnormal Pap smear were also significantly associated with high-risk HPV status (P < 0.05). In Saint Kitts and Nevis, participants with abnormal Pap smears also had a high prevalence of high-risk HPV. However, no significant association could be ascertained due to the small numbers (Table 2).In Saint Kitts and Nevis, only current age was found to be very significantly associated with high-risk HPV infection  of 2.38 and of 1.85, respectively). In Saint Vincent and the Grenadines, parity > 3 pregnancies and abnormal Pap smear result were also identified as independent significant risk factors, with such women more than twice as likely to have a high-risk HPV infection . The number of sexual partners, though associated, was not identified as an independent risk factor for high-risk HPV infection (Table 3).The study used a convenience sample, and the recruitment of study participants was done at sites associated with the provision of health care. As a consequence, women who do not readily access health care may have been excluded. This may have introduced bias into the study, so the results are not generalizable to the general population of women 30 years and older or those with other healthseeking behaviors in the two countries.The results reported are only for the women who had cervical specimens that were satisfactory for testing for HPV DNA. A large percentage had cervical specimens that were unsatisfactory for testing: 96 of 500 (19.2%) of study participants in Saint Kitts and Nevis, and 132 of 500 (26.4%) in Saint Vincent and the Grenadines. These specimens could not be processed and were therefore excluded from the analysis. As a consequence, there is a possibility that important information may have been lost by not being able to process the unsatisfactory cervical specimens. Issues such as the need for air courier shipment service from each respective study country to Jamaica and inadequate cold storage of cervical specimens from both countries may have impacted the quality of the specimens and resulted in loss of valuable information.The study sites used were in both the public and private health sector. However, the number of participants recruited from the latter were small and therefore did not yield enough data to detect differences in demographic, behavioral, and reproductive factors or HPV prevalence between the participants seen in public and private health care settings.To our knowledge, this is the first report on HPV prevalence and HPV genotypes done in any of the islands of the Organization of Eastern Caribbean States (OECS). The prevalence of high-risk HPV infections in Saint Kitts and Nevis (25.2%) and in Saint Vincent and the Grenadines 29.6%) was similar to the prevalence of high-risk HPV infections (20.3%) found in a study done in Tobago by Ragin et al. .6% was sThe distribution of the most prevalent (> 10%) high-risk HPV types showed similarities in Saint Kitts and Nevis and Saint Vincent and the Grenadines. HPV 52, 35, 51, 45, and 31 were the most prevalent in Saint Kitts and Nevis, whileHPV 45, 35, 31, 18, and 51 were the most common in Saint Vincent and the Grenadines. Table 4 compares the high-risk genotypes identified in this study with those identified from research in other Caribbean countries. HPV 45 was the most prevalent high-risk HPV type found in the Tobago study  infected with high-risk HPV had normal cytology. This has also been shown in other HPV studies done in the Caribbean , 6, 11.The contribution of HPV genotypes 16, 18, 31, 33, 35, 45, 51, 52, 56, 58, and 59 to cervical disease is well established  of multiple HPV infections (low risk and high risk) was identified in the Jamaica study In this study, younger age (30–44 years) was identified as a risk factor associated with high-risk HPV in both countries. Other studies, done in Belize and Trinidad, have identified the highest prevalence of HPV infections in the younger age groups , 9, withThis is the only known study of HPV prevalence and associated risk factors done in the countries of the Organization of Eastern Caribbean States. These results contribute to the evidence on HPV prevalence for small island states and other nations of the English-speaking Caribbean.This research supports the accelerated introduction of HPV vaccines in the immunization schedule of the two study countries, since most of the circulating HPV genotypes are in the 9-valent HPV vaccine. The two countries could use the school health system to implement this vaccination program. In addition, continued research to develop a HPV vaccine for protection against HPV 35, the second most prevalent high-risk HPV genotype in both countries, is strongly recommended.The study’s results can also be used to guide decision-making regarding the introduction of the 9-valent HPV vaccine and the use of HPV testing as the primary screening modality for older women in other English-speaking Caribbean countries. It should be possible to conduct the processing and testing of HPV specimens in-country so as to reduce possible negative outcomes relating to the quality of the specimens.The authors wish to thank Oswaldo Benitez, who assisted with the development of the proposal; Wendel Guthrie (Jamaica) and Derrek Jeffers (Saint Kitts and Nevis) for providing clinical updates for the nurses in primary health care and NGO facilities that collected cervical specimens for the HPV study; and the private health facilities and nongovernmental organizations that collaborated and participated in the study. For the processing of the cervical specimens, we recognize the Caribbean Genetics (CARIGEN) laboratory, Department of Basic Medical Sciences, University of the West Indies, Mona, Kingston, Jamaica, as well as the Pathology Department at that same university. Special thanks also go to the Ministries of Health of Saint Kitts and Nevis and Saint Vincent and the Grenadines and their respective steering committees for collaboration and execution of the study through nurses and doctors collecting the data and cervical specimens. In Saint Kitts and Nevis, the steering committee included: Patrick Martin, Judy Nesbett, Eulynis Brown, Jenevie Daniels, Rhonda Lowry-Robinson, John Essein, Candace Gumbs, Clester Roberts, Derrek Jeffers, and Patrice Lawrence-Williams. In Saint Vincent and the Grenadines, the steering committee included: Simone Keizer-Beach, Sherian Slater, Camille Nichols, Ferosa Roache, Arlitha Scott, Ronald Child, Claudette Williams, and Anneke Wilson.The study was supported by a grant from the Albert B. Sabin Vaccine Institute . This organization was not involved in the study development or its execution.RPSP/ PAJPH or PAHO.Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the


Introduction: This study assesses the sociodemographic facilitators and barriers to human papillomavirus (HPV) vaccination for diverse teens in a region with low HPV vaccination rates.Materials and Methods: In this community-based participatory research study, we surveyed adult family members of teens aged 11–17 years from African American, African refugee, American Indian/Alaskan Native, Hispanic/Latino, and Native Hawaiian/Pacific Islander community groups in Salt Lake City, Utah. Bivariate analyses assessed associations between sociodemographic characteristics and, respectively, HPV vaccine receipt and intentions for vaccination. Barriers to vaccination were also investigated.Results: Only 20% of participants had vaccinated at least one of their children with at least one dose of the HPV vaccine. HPV vaccination was significantly related to caregiver age (p=0.035), race/ethnicity (p=0.001), educational attainment (p=0.006), annual household income (p=0.0454), years in the United States (p=0.023), and caregiver parent birthplace (p=0.008). Among caregivers with unvaccinated children, intention to vaccinate was significantly related to race/ethnicity , caregiver parent birthplace (p=0.023 for sons), health insurance coverage , and type of health insurance coverage (p=0.008 for sons). The most frequently cited barriers to HPV vaccination were lack of knowledge about the HPV vaccine, costs, side effects, and child not being sexually active.Conclusions: Our results show substantially lower HPV vaccine coverage than both national and state rates, signaling the urgent need for multipronged HPV vaccination interventions within these communities; strategies are discussed. Therefore, in this study, we worked with five Utah community groups representing African American, African refugee, AI/AN, Hispanic/Latino, and NH/PI populations to probe the facilitators and barriers to HPV vaccination.To better serve these communities and achieve national HPV vaccination goals,This article is part of a larger research project in which adult family members of teens participated in focus groups and surveys about their HPV vaccine-related beliefs and behaviors. This article is focused on the survey results about HPV vaccine receipt and intentions for vaccination. We also explore reported barriers to vaccination.The larger research project was conducted in collaboration with Community Faces of Utah (CFU), a partnership between five racial/ethnic community organizations , the Utah Department of Health, and the Collaboration and Engagement Team of the Center for Clinical and Translational Science at the University of Utah. The development, implementation, analysis, and dissemination phases of this study were guided by a community-based participatory research approach; therefore, the CFU community leaders actively participated in developing data collection instruments, recruiting participants, facilitating data collection, interpreting findings, and presenting conclusions at conferences. The study was conducted between May 2014 and February 2015. All research was reviewed and approved by the University of Utah Institutional Review Board.17Participants were adult (aged 18 years or older) parents, legal guardians, or caregivers who were vaccination decision-makers for teens aged 11–17 years. CFU community leaders conducted convenience sampling through their organizational networks to recruit participants in two phases. In the first phase (May 2014–October 2014), participants were recruited to participate in a focus group and complete a survey; they were offered a $25 gift card incentive. In the second phase (October 2014–February 2015), participants were recruited to complete the (same) survey only; they were offered a $15 gift card incentive. This article presents results from the survey data only; other articles present the results from the focus groups.The 21-item survey was developed based on an internal literature review and the input of CFU community leaders. This survey assessed sociodemographic characteristics, acculturation indicators (such as birthplace and English language use), participants' awareness and knowledge of the HPV vaccine, and adolescents' receipt or intended receipt of the HPV vaccine. For receipt and intended receipt of HPV vaccination, participants were asked about adolescent sons and daughters separately, however, the question was not specific to individual daughters and sons if participants had more than one daughter or son. An English version of the survey was used by the African American, AI/AN, and NH/PI community organizations. A Spanish version of the survey was used by the Hispanic/Latino community organization. As the majority of the African refugees did not read in their native language, an English version of the survey was read aloud to participants by community translators and completed using a color-coded schematic. With the exception of the African refugee group, all surveys were self-administered with community translators available to answer questions.In this article, the primary outcomes of interest are receipt of at least one dose of the HPV vaccine and intentions for HPV vaccination. Additional outcomes of interest are reported barriers to HPV vaccination. These barriers were assessed separately for daughters and sons as well. The sociodemographic correlates assessed  were caregiver age, gender, race/ethnicity, marital status, educational attainment, annual household income, occupation, birthplace, caregiver parent birthplace, English usage, health insurance coverage, type of health insurance coverage, and whether adolescents have a primary care provider.p-values of <0.05 were considered significant. All statistical analyses were performed using R-2.15.1 .Frequency counts and percentages of selected characteristics are reported for the sample . For allThere were 228 participants; 93 were recruited during the first phase of data collection (completing a focus group and a survey), and 135 returned surveys during the second phase of data collection.n=148, 64.91%), female , married or living as married , and born out of the United States . Free text responses of country of origin for those who provided responses showed that n=74 (32.74%) reported being born in the United States, n=65 (28.76%) reported being born in Latin American countries, n=45 (19.91%) being born in Pacific Islands, n=39 (17.26%) from African countries, and n=3 (1.33%) from Australia . Most participants' parents were also born out of the United States . Despite the large proportion of participants who were immigrants, there was a fairly even distribution across English use . Most participants had health insurance  and had a primary care provider for their children . Among participants who reported having health insurance, the majority had private insurance .Demographic characteristics of participants are presented in p=0.0354), race/ethnicity (p=0.0003), educational attainment (p=0.0055), annual household income (p=0.0454), years in the United States (p=0.0229), and caregiver parent birthplace (p=0.0084) were all significantly associated with HPV vaccination.About 20% of participants had self-reported at least one of their children receiving a minimum of one dose of the HPV vaccine . In bivaN=144, p=0.0480) and health insurance coverage (p=0.0281) were significantly associated with intention to vaccinate daughters. In bivariate analyses of participants with unvaccinated sons (p=0.0025), caregiver parent birthplace (p=0.0232), health insurance coverage (p=0.0465), and type of health insurance coverage (p=0.0079) were significantly associated with intention to vaccinate sons.In bivariate analyses of participants with unvaccinated daughters N=144, , race/etted sons , race/etN=144), reasons for not vaccinating were assessed , costs , and side effects . The top 4 reasons given for not vaccinating eligible sons were as follows: not knowing about the HPV vaccine , son not being sexually active , costs , and side effects . Notably, very few caregivers reported concerns that the HPV vaccine would promote sexual activity , including national estimates associated with specific racial/ethnic groups .18 in the region and nationally, and suggests that interventions should target older parents (older than 35 years) in diverse communities, perhaps through platforms more frequently used by this demographic .19We found that caregiver age was significantly related to HPV vaccination, with younger participants composing a higher percentage of those who vaccinated their children than those who did not. This finding supports the previous literature that has consistently shown younger parent age (younger than 35 years) to be associated with children's HPV vaccine receipt20 and suggest that in addition to tailoring interventions to each racial/ethnic group, it will be necessary to give particular attention to immigrants, regardless of how long they have lived in the United States.Our results also showed that race/ethnicity, number of years in the United States, and caregiver parent birthplace were significantly related to HPV vaccination. The finding of differences in HPV vaccination by racial/ethnic group illustrates that there is no one-size-fits-all solution for these populations; rather, each minority group will require a unique set of tailored intervention strategies. While a larger percentage of participants who vaccinated their children had U.S.-native parents, among immigrant participants, a larger percentage of those who lived in the United States for fewer than 20 years had vaccinated their children compared with those who lived in the United States for more than 20 years. These findings highlight the “immigrant paradox,”15 and imply that HPV vaccination interventions should devote focus to these at-risk families.Finally, our findings included significant negative associations between HPV vaccination and both educational attainment and annual household income. We found that a larger percentage of participants who did not vaccinate their children had lower educational attainment and lower annual household income. These findings support previous literature documenting the same associations across many populations,21We found that among parents with unvaccinated children, race/ethnicity and caregiver parent birthplace were significantly related to intentions to vaccinate. We found that a greater proportion of Hispanic/Latino caregivers were willing to vaccinate their unvaccinated sons and daughters with the HPV vaccine compared with the other racial/ethnic groups. Comparatively, willingness to vaccinate sons and daughters was lower in the NH/PI community. A recent study comparing Hispanic/Latino and Asian and Pacific Islander parents found that while the awareness of the HPV vaccine increased most among Hispanic/Latino mothers between 2007 and 2011, Asian or Pacific Islander mothers continued to have lower awareness.11 and signal the need for interventions to be tailored to each racial/ethnic community group and to be sensitive to immigrants and first-generation Americans.In addition, a greater proportion of caregivers' whose parents were born outside of the United States indicated greater willingness to vaccinate their unvaccinated sons in the next 12 months. However, willingness to vaccinate was not significant for daughters. As with HPV vaccination, these findings support the previous literature documenting racial/ethnic and immigrant/native differences23 In addition, we found that having private insurance was associated with willingness to vaccinate sons but not daughters. Although the mechanisms underlying these findings are not fully understood, campaigns to raise awareness of low-cost or free HPV vaccination through local healthcare providers  may payoff.We also found that having health insurance coverage was significantly related to intentions to vaccinate sons and daughters with the HPV vaccine, which is supported by other studies as well.21 Racial and ethnic minorities are at greater risk for lower awareness about the HPV vaccine, suggesting the need for educational interventions among diverse communities.24 These efforts could take the form of educational media campaigns26 or collaborations with clinicians.27 The next most frequently reported barrier to vaccinating both sons and daughters was the cost of the vaccine, despite the availability of low-cost or free HPV vaccinations through the Vaccines for Children program. This finding suggests that caregivers may not be receiving the necessary information about these programs, so efforts at the community level, such as patient navigation programs or health fairs, may help.29The most frequently reported reason for not vaccinating male and female children was lack of knowledge about the HPV vaccine, which indicates the continued need for raising awareness of the existence of the vaccine, which is supported by other research.21Another top barrier common to both sons and daughters was the concern about side effects. Unlike the barriers of not knowing about the HPV vaccine and of perceived costs of the HPV vaccine, these barriers suggest that parents may be aware of the existence and availability of the HPV vaccine, but are misinformed about its safety and recommendation guidelines. Therefore, providers and community organizations should focus education efforts on the safety profile of the HPV vaccine and emphasize the benefits of early vaccination. Finally, a top barrier more common to caregivers of unvaccinated sons, which was the second-most frequently reported barrier for this group, was the perception that sons were not yet sexually active. This finding suggests that diverse caregivers of sons may have greater informational needs about the HPV vaccine recommendations, which has been described as a risk factor for lower awareness of the HPV vaccine in other studies.30 Moreover, we did not distinguish between HPV vaccine initiation (at least one dose) and completion . Another limitation of the study is the use of purposive sampling. Participants selected for the study may have been more active in their racial/ethnic community groups than other members of the populations. In addition, analyses were limited to a bivariate approach, so results do not control for confounding variables and do not assess interaction effects . Finally, our findings may not be generalizable to other geographic areas with different racial/ethnic history.This study is limited by the use of self-reported HPV vaccination; it is possible that actual HPV vaccination rates for these populations are higher or lower than those we report; a study by Dorell et al. on parental recall of vaccination status for children in the National Immunization Survey Teen dataset found that underreporting HPV vaccination status (16.6%) was more common than overreporting (7.8%) when comparing parental recall versus medical record data.To the best of our knowledge, this is the first study to assess the sociodemographic correlates of HPV vaccination of minority children in Utah, representing African American, African refugee, AI/AN, Hispanic/Latino, and NH/PI communities. Our findings support the need for community approaches to HPV education among diverse caregivers in Utah, particularly the need to better inform communities about the availability of low-cost or free HPV vaccines and about the benefits of the HPV vaccine, and to clarify misunderstandings about the vaccine. Our results also indicate target audiences—groups that are at particular risk for not vaccinating their adolescents—of older, immigrant, and socioeconomically disadvantaged caregivers. Furthermore, we recommend further research to develop targeted interventions for the NH/PI communities and diverse caregivers of vaccine-eligible sons.Finally, we recognize that the most vulnerable community of this sample, the African refugee community, may be affected by specific factors influencing HPV vaccination that are not directly assessed within the scope of this study; therefore, we recommend that future research relevant to this population be conducted.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.


Background and objectives: The aim of this study is to propose a methodology that combines non-invasive functional modalities electroencephalography (EEG) and single photon emission computed tomography (SPECT) to estimate the location of the epileptogenic zone (EZ) for the presurgical evaluation of patients with drug-resistant non-lesional epilepsy. Materials and Methods: This methodology consists of: (i) Estimation of ictal EEG source imaging (ESI); (ii) application of the subtraction of ictal and interictal SPECT co-registered with MRI (SISCOM) methodology; and (iii) estimation of ESI but using the output of the SISCOM as a priori information for the estimation of the sources. The methodology was implemented in a case series as an example of the application of this novel approach for the presurgical evaluation. A gold standard and a coincidence analysis based on measures of sensitivity and specificity were used as a preliminary assessment of the proposed methodology to localize EZ. Results: In patients with good postoperative evolution, the estimated EZ presented a spatial coincidence with the resection site represented by high values of sensitivity and specificity. For the patient with poor postoperative evolution, the methodology showed a partial incoherence between the estimated EZ and the resection site. In cases of multifocal epilepsy, the method proposed spatially extensive epileptogenic zones. Conclusions: The results of the case series provide preliminary evidence of the methodology’s potential to epileptogenic zone localization in non-lesion drug-resistant epilepsy. The novelty of the article consists in estimating the sources of ictal EEG using SISCOM result as a prior for the inverse solution. Future studies are necessary in order to validate the described methodology. The results constitute a starting point for further studies in order to support the clinical reliability of the proposed methodology and advocate for their implementation in the presurgical evaluation of patients with intractable non-lesional epilepsy. About 20%–30% of people with epilepsy have resistance to treatment with antiepileptic drugs . The surThe brain perfusion ictal and interictal single-photon emission computed tomography (SPECT) together with the subtraction of ictal and interictal SPECT co-registered with MRI (SISCOM) are valuable noninvasive tools in the presurgical evaluation of patients with medically intractable epilepsies. These images offer a high criterion of veracity in ictal onset detection represented by an increase in cerebral blood perfusion . PreviouThe objective of this article is to propose a methodology that combines non-invasive functional modalities EEG and SPECT in identifying and localizing the epileptogenic zone for the presurgical evaluation of patients with drug-resistant non-lesional epilepsy. This article presents a case series as an example of the application of this novel approach. A gold-standard is established as an initial assessment of methodology’s potential to localize EZ based on Engel scale and spatial coincidence of estimated EZ with the resection site. No previous studies were found using SISCOM output as a spatial restriction for the estimation of EEG sources.This article illustrates the methodology follow up for the presurgical evaluation in the first Cuban comprehensive surgery program carried out at the International Center for Neurological Restoration in Havana, Cuba .The proposed methodology is applied, retrospectively, to five patients  diagnosePostsurgical outcome one year after surgery was evaluated according to the modified Engel classification, Engel I outcome patients (favorable outcome) versus Engel II–IV outcome patients (unfavorable outcome). Additionally, each patient who underwent surgery had an MRI one year after surgery.Patients were included in this study if they (1) had undergone both ictal and interictal SPECT imaging before surgery, (2) had a volumetric preoperative MRI, and (3) had a volumetric postsurgical MRI 1 year after surgery.All the procedures followed the rules of the Declaration of Helsinki of 1975 for human research, and the study was approved by the scientific and ethics committee from the International Center for Neurological Restoration (CIREN37/2012).A brain perfusion SPECT was performed for all subjects, with the same gamma camera, and under the same conditions. Each patient underwent two studies  of brain perfusion SPECT using 99mTc-ethylene-cysteine dimer (ECD). In both studies during the administration of the radiopharmaceutical, the patient remained monitored by EEG. For inter-ictal SPECT, the dose is administered with the patient at rest and with a seizure-free period of more than 24 h. For ictal SPECT, the radiopharmaceutical was injected when the onset of a crisis with EEG is recognized. SPECT image acquisition was performed using a double-headed gamma camera  equipped with a fan-beam collimator. One hundred and twenty-eight projections in a circular orbit of 360° per subject were collected in matrices of 128 × 128 pixels and with an energy window of 20% centered on the photopeak of 99 mTc. Tomographic 3D reconstruction was performed using a DROSEM algorithm. The SISCOM methodology was carried out, according to O’Brien et al. . Ictal a(1)source space definition in order to model the link between the EEG measurements and cortical activity. Because the generators of the signals are believed to be mainly cortical, the source space is given- by the triangularized inner cortical surface (gray matter/white matter interface), segmented from individual patient’s T1-weighted 3D MRI;(2)the spatial co-registration of the measurement electrodes to the using the Montreal Neurological Institute  MNI152 template using translation and rotation;(3)forward modeling of the cortical currents: To model the relationship between the source space and the EEG measurements at the sensor level. The source space, the 3D electrode locations, and the individually defined boundaries are then combined to characterize the electric field propagation with a three-compartment boundary element method (BEM) ; and fin(4)inversion of the model for source estimation using the Multiple Sparse Priors (MSP) algorithm .The cortical generators of EEG measurements can be estimated by solving an inverse imaging problem where the unknown sources are distributed on an individual’s cortex. The methodology followed for the estimation of the inverse solution of ictal EEG using EEGLAB MATLAB toolbox consists of:(1)source space definition in order to model the link between the EEG measurements and cortical activity. Because the generators of the signals are believed to be mainly cortical, the source space is given- by the triangularized inner cortical surface (gray matter/white matter interface), segmented from individual patient’s T1-weighted 3D MRI;(2)the spatial co-registration of the measurement electrodes to the using the Montreal Neurological Institute  MNI152 template using translation and rotation;(3)forward modeling of the cortical currents: To model the relationship between the source space and the EEG measurements at the sensor level. The source space, the 3D electrode locations, and the individually defined boundaries are then combined to characterize the electric field propagation with a three-compartment boundary element method (BEM) ;(4)Functional area definition using SPECT: To localize the functional areas using SISCOM methodology that will be used to constrain the inverse procedure;(5)SISCOM-informed inverse modeling of the cortical currents: To use the ictal hyperperfusion area derived from SISCOM to constrain the estimation of the cortical activity generating the EEG measurements using MSP algorithm.Currently, the available priors in ESI gain from MRI, functional MRI and PET ,13. In tFor the present case series, a coincidence analysis was performed as an initial evaluation of methodology. The three EZ proposed by the methodology: SISCOM, ictal ESI and ictal ESI using SISCOM as a prior, were co-registered with the post-surgery anatomical image T1. Also, the results were co-registered with AAL atlas  to identA low sensitivity would indicate many false negatives. On the other hand, the specificity reflects the methodology’s capacity to not reporting false positives.Five patients were evaluated with this methodology, and three patients were subjected to surgical treatment. Two patients (patients 1 and 2) had a favorable outcome (Engel I). In these two cases, the EZ estimated by the proposed methodology shows spatial coincidence with the resection site represented by high values of sensitivity, as well as specificity . For botPatient 3 underwent surgery for the resection of the right frontal lobe. Unfortunately, the patient had an unfavorable outcome (Engel III), which indicates recurrence seizures. Two regions were identified with SISCOM methodology  in red, Patients 4 and 5 were diagnosed with extra-temporal epilepsy and, as the rest of patients, did not present appreciable lesions in the structural MRI. Both cases were diagnosed with multifocal epilepsy, and they were not subjected to surgical intervention. The estimation of the sources from ictal ESI revealed extensive cortical zones that includes bilateral temporal and frontal lobes for both cases  in blue.In the present work, we propose a methodology that combines different functional neuroimaging techniques to identify epileptogenic zone (EZ) in patients with drug-resistant non-lesional epilepsy. Single-photon emission computed tomography (SPECT), subtraction of ictal and interictal SPECT co-registered with MRI (SISCOM), and ictal electroencephalography (EEG) source imaging (ESI) are complementary diagnostic methods in the localization of seizure zone when MRI is negative. The novelty of the article consists in estimating the sources of ictal EEG using SISCOM result as a prior for the inverse solution. There are no research records that use SPECT-SISCOM as a prior for ictal ESI in the consulted literature. A case series is presented to implement the described methodology. A gold-standard of value I on the Engel scale and spatial coincidence of estimated EZ with the resection site was established for postsurgical evaluation of patients. The spatial coincidence analysis allows a preliminary quantitative evaluation of the methodology based on sensitivity and specificity measurements.Five patients were evaluated with this methodology, and three patients were subjected to surgical treatment. Two patients had favorable outcome (Engel I). In these two cases (patients 1 and 2) the methodology proposed EZ that coincide spatially with the resection site represented by high values of sensitivity, as well as specificity . The lowThe third patient underwent surgery did not have a favorable outcome with continued seizures ensue (Engel III). The methodology reported EZ that were not included in the resection site for this patient, instead were localized posterior it. For this particular case, low values of both sensitivity and specificity were obtained.The remaining two patients did not undergo surgery because they were diagnosed with multifocal epilepsy after the presurgical evaluation. The implementation of our methodology for these two patients proposed diverse and dispersed EZ that cover both hemispheres. This result demonstrates that the methodology can be used not only in the cases of focal non-lesional epilepsy for determination of EZ, but also in multifocal cases.The proposed methodology estimates EZ by ictal ESI, SISCOM and ictal ESI using SISCOM as prior. For the present case series, which was an initial test of the methodology, shows that the highest values of the sensitivity of localizing EZ were obtained for ictal ESI using SISCOM as a prior. Due to the spatial location of the neuronal sources of the scalp recorded EEG activity cannot be conclusively determined (inverse problem has no unique solution) by introducing reasonable priors from other modalities, inverse solution methods reveal the most probable sources and communication structures. Further studies are necessary in order to conclude that combining EEG and SPECT allows less false negatives, which would contribute to a better surgical outcome. Otherwise, SISCOM has the highest specificity providing reliability by false positives. The objective of this work is not to evaluate the veracity of the individual techniques used (ESI or SISCOM) to identify EZ in drug-resistant epilepsy, which has been the goal of some publications ,16,17,18The major limitation of this study is the small sample size. Therefore, we anticipate that future studies with increased sample size will be able to validate the proposed methodology. Nonetheless, the main value of this study is to outline a novel noninvasive approach combining MRI, SPECT and EEG for the location of the epileptogenic zone in pharmacoresistant non-lesional epilepsy.From the above discussion, it can be concluded that the proposed methodology may contribute to epileptogenic zone localization in non-lesion drug-resistant epilepsy. The case series, although limited by small sample size, suggests that combining ictal ESI and SISCOM has higher predictive value than individual techniques for good outcomes. Before implementing SISCOM methodology or ictal ESI methods, care must be taken their advantages and disadvantages. However, the fusion of the different methodologies is helpful and give better results than implement only individual techniques.The main aim of this paper is to a new methodology based on the integration of non-invasive functional neuroimages to an accurate estimation of the source generating epileptiform discharges for the presurgical evaluation of patients with drug-resistant focal epilepsy. The methods have been oriented to the identification of the epileptogenic zone by the processing of ictal EEG and ictal and inter-ictal SPECT. The methodology proposed offers relevant information about EZ in both temporal and extra-temporal, focal and multifocal epilepsy. The major limitation of this study is the small sample size. The case series results provide future study directions in order to validate the proposed methodology and advocate for their implementation in the presurgical evaluation of patients with intractable non-lesional epilepsy. The fusion of different non-invasive functional techniques on the magnetic resonance imaging of the patient provides valuable information in identifying and localizing the epileptogenic zone to be considered as a surgical resection site. Localizing information of one modality may enhance detection of subtle abnormality in another modality.


Twenty-five percent of all globaldeaths as a result of cervical cancer occur in India.1 Although cervical cancer rates in India are decreasing, there ishuge geographic variability, and the absolute number of patients is still expected toincrease over the next decade as a result of population growth.2Moreover, the decreasing incidence rate seems to be plateauing and is unlikely todecline further unless specific interventions are put in place.3With the highest burden of cervical cancer mortality in theworld, India faces enormous loss of life, productivity, and suffering from this disease.The age-standardized incidence ratio for cervical cancer in India is 22 per 100,000women per year, which is higher than the incidence in South East Asia and in the world .4 Recently, ascreening program has been recommended by the government that involves screening womenevery 5 years using visual inspection by acetic acid as the primary mode ofscreening.5 With properimplementation, this screening program would begin to address the prevention of cervicalcancer. However, it is imperative that along with this secondary prevention program,there is a strong focus on primary prevention.Although widespread and organized screening programs can facilitate earlier detection andmanagement of precancers and cancers, thereby leading to decreased mortality, suchscreening programs are logistically highly complex, resource intensive, and at a nascentstage in India. Less than 5% of the eligible women in India have ever beenscreened.3 Cost-effectivenessstudies on HPV vaccination have shown that spending on HPV vaccinations is more costeffective than treating cervical cancer.6Globally, it is now widely accepted that vaccination against high-risk strains of thehuman papillomavirus (HPV) is a safe and effective means of primary prevention ofcervical cancer. More than 80 countries have introduced HPV vaccination in theirnational immunization programs, of which 33 are low- and middle-income countries(LMICs).8A vaccine delivery and demonstration project led by an international nonprofitorganization, PATH, was started in 2009 in Andhra Pradesh and Gujarat but was suspendedin 2010 as a result of public concern, allegedly arising from the deaths of seven girlswho received HPV vaccine.10 Subsequent investigations concludedthat these deaths were not linked to vaccination but occurred as a result of variedunrelated causes such as snake bite, epilepsy, malaria, and suicide. Unfortunately, theproject was never restarted but, collaterally, led to the development of more stringentregulatory, ethical, quality control, and monitoring standards of clinical trials in thecountry, as well as serendipitously provided proof for the efficacy of a two-doseregimen in the 9- to 13-year age group, which was later also approved by theWHO.11Although HPV vaccines have been licensed for use in India by the Drug Controller Generalof India since 2008, until recently, these vaccines have not been included in theroutine government immunization program. The issue of HPV vaccine introduction intogovernment programs has been the subject of debate among academic, medical, publichealth, and advocacy groups; nongovernmental organizations; and parliament. Scientificpublications have argued for and against vaccine introduction in India.The experience of developed as well as developing countries around the worldin the past 10 years and WHO recommendations are unequivocally in favor ofintroducing HPV vaccination in the government’s immunizationprogram.Girls age 9 to 13 years, translating to girls in grade 6, would form thetarget group for protection. Both government and private schools should becovered to ensure herd immunity. Two doses of the vaccine should be given ata gap of 6 to 12 months.Both the bivalent and quadrivalent vaccines are equally effective in theIndian context, and price could be used as the selection criterion.Vaccination should be accompanied by cervical cancer screening programs formothers accompanying the female child.Parents should be informed and their consent obtained with no element ofcompulsion in case of any unwilling family.ICMR will assist in collecting evidence on HPV vaccine and developing apolicy brief.ICMR’s role in introduction of the program in states will be tomonitor and document feasibility of program implementation.The past few months have seen the beginning of the turning of the tide. The IndianCouncil of Medical Research (ICMR) organized multiple expert group meetings with variousstakeholders to review the state of knowledge and current global recommendationsregarding the HPV vaccine, as well as to prepare interested state governments forinitiating this activity. The expert group endorsed the following recommendations forthe introduction of HPV vaccine in the programmatic settings in India.12 This program invitedgirls age 11 to 13 years to get vaccinated at the Delhi State Cancer Institutes (Eastand West). A total of 1,200 doses have been administered as of March 2017 in a hospitalsetting along with mothers being offered the Papanicolaou smear and mammographyservices. No serious adverse events have been reported.Delhi was the first state in India to initiate a public HPV vaccination program forschool children, on the occasion of National Cancer Awareness Day .The Delhi government would like to expand the program to vaccinate all class 6 studentsthrough the school health program, which will include girls from both private and publicschools numbering approximately 250,000 per annum.13 A total of 261schools in Bathinda and 187 schools in Mansa were involved in the program. In total,5,851 girls were vaccinated at Bathinda and 4,002 at Mansa, constituting 97.5% and 98.5%coverage, respectively. Although 28 minor adverse events were reported, consisting offainting (n = 16), dizziness (n = 5), vomiting (n = 5), and headache (n= 2), these were managed locally with complete recovery, without any need forreferral to a higher center. In the second phase, plans are afoot to include five moredistricts, which have the next highest incidence rates of the disease, thereby coveringall districts that have a reported incidence of > 10 per 100,000 women. The programwill be gradually scaled up to include all girls in class 6 in both government andprivate schools across the state. The program is adopting both a facility-based andschool-based approach to vaccination in the second phase.On November 23, 2016, the government of Punjab also initiated HPV vaccination in acampaign in the Bathinda  and Mansa  districts. In phase 1, nearly 10,000 girls studying in class 6 of governmentschools were covered.14These initial programs mark the first steps toward elimination of cervical cancer burdenin India over the next decades. Along with vaccination, awareness regarding otherprimary preventive measures, such as tobacco cessation and safe sexual practices, shouldaccompany cervical cancer control.16 The key barriers evidenced by this research were sociocultural,health systems, and financial barriers. However, progress has been made throughfinancing mechanisms such as the Global Alliance for Vaccines and Immunization and otherorganizations that aim to improve access of eligible LMICs to vaccines throughnegotiating lower vaccine prices and cofunding until countries can afford thevaccines.17 The GlobalAlliance for Vaccines and Immunization is a global health partnership of public- andprivate-sector organizations that provides a unique opportunity for a wide range ofpartners to build consensus around policies, strategies, and priorities and to recommendresponsibility of implementation to the partner with the most experience and insight inthe area. These funding opportunities have opened the window for eligible countries toimplement national programs for countries that already have experience with deliveringthe vaccine.Past studies have reported the challenges and barriers in LMICs, and lessons have beenlearned from the implementation of HPV vaccine rollout in many LMICs.Given that these are the first large-scale HPV vaccination introduction programs inIndia, experiences gained at both the programmatic and the community level will be keyto scaling up the program within the states and in other parts of the country. Althoughthese primary prevention efforts are expected to have an enormous impact on the expectedburden of cervical cancer in regions where they are implemented, the comprehensivecontrol of cervical cancer will still require addressing all aspects of prevention andcare, including increasing awareness, screening and early detection of cervical lesions,appropriate treatment, and palliative care when necessary.


The purpose of the meeting was to provide a platform for students working with functional magnetic resonance imaging (fMRI) to present their research to an international audience of peers. This year’s meeting featured special lectures from Dr. Leslie Ungerleider  and Dr. Daniel Weinberger (“Genetic Variation and fMRI Response”).The fourth fMRI Experience meeting was held at the Bethesda, Maryland campus of the National Institutes of Health on May 13


To develop a new 18 high-risk human papillomavirus (HR HPV) detection and genotyping assay, which is important to evaluate the risk degree of HR HPV for causing cancers.All 18 HR HPV and β-globin relative DNA fragments were synthesized and cloned to a plasmid pUC57 to obtain their recombinant plasmids. Based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, each of the 18 HR HPV genotypes were investigated using their constructed recombinant plasmids. The new 18 HR HPV genotyping assay was tested using 356 clinical specimens and the results were compared to ones detected by the Roche Cobas 4800 HPV assay (Cobas). The discrepant results between two assays were resolved by sequencing and genotyping methods.3 to 102 copies/reaction for the all 18 HR HPV. This new developed HR HPV genotyping test was used to detect the clinical specimens. When the results on clinical samples detected by the new MALDI-TOF MS HPV test were compared with ones detected by the Roche Cobas 4800 HPV assay in terms of 14 HR HPV, the concordance was 80.1% . The discrepant results were resolved by sequencing and genotyping and suggests that the developed HR HPV assay is more sensitive and specific.The new 18 HR HPV MALDI-TOF MS genotyping assay was developed using HPV recombination plasmids. The sensitivity was 10The new developed 18 HR HPV detection method based on MALDI-TOF MS platform is a high-throughput assay for the all 18 HR HPV genotypes and a powerful complement to current detection methods.The online version of this article (10.1186/s12885-019-6036-z) contains supplementary material, which is available to authorized users. Cervical cancer is the second most common malignant cancer in women worldwide. Persistent infection with high-risk types of human papillomavirus (HR HPV) has been established as a necessary, but insufficient, factor in the development of high-grade cervical dysplasia and cervical carcinoma . HPV DNAThe strong causal relationship between HR HPV and cervical cancer revealed that screening for HR HPV types is necessary for the prevention and control of cancers . The HybMultiplex polymerase chain reaction (PCR) together with the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform  is a novel method for type specific detection of HR oncogenic HPV types. This assay contains a three-step process consisting of multiplex PCR, primer extension with a single nucleotide, and MALDI-TOF mass separation of extended products on a matrix-loaded silicon chip array. Based on the MALDI-TOF MS platform, a genotyping method of the 14 HR HPV types aforementioned in the Roche Cobas 4800 HPV assay kit was developed . A 15 HRThe aim of this study was to develop a detection method for genotyping of the 18 HR HPV types, namely all currently known HR and probable HR HPV types  based on the MALDI-TOF platform. The relative DNA fragment of each 18 HR HPV was firstly cloned into a plasmid pUC57 to obtain their recombinant plasmids, which was used as standard type templates, and a high-throughput HR HPV genotyping method was investigated. The effectiveness of this method was compared with a commercial kit, the Roche Cobas 4800 HPV assay, which detects 14 HR HPV types in a total of 356 cervical clinical specimens. Discrepant results between them were analyzed by sequencing and genotyping.https://www.ncbi.nlm.nih.gov/)  agar plates containing 100 μg/ml of ampicillin [DNA L1 regions of all 18 HR HPV mentioned above and human β-globin DNA used as an internal control were searched out from the NCBI website  and 1 μl of recombinant HPV plasmid DNA (1 ng/μl) using as templates in a 384 well plate format . Water was included as a negative control in every test. The consensus primer pairs GP5+/GP6+ [http://frodo.wi.mit.edu) [http://www.oligoevaluator.com/OligoCalcServlet) was used to detect secondary structure and primer dimer itself; (3) the MassARRAY Assay Designer software 4.0  was used to check whether the formation of primer dimer among different extension primers or not in the same well. Sequences and molecular weights of extended primers were listed in Additional file The assay was designed for simultaneous detection and genotyping of 18 HR HPV types in two separated wells, one of which contained 9 HPV-plex and β-globin primer pairs. The multiplex PCR was performed in a total of 5 μl reaction volume with 2.5 mM MgClP5+/GP6+  with somP5+/GP6+ . The extmit.edu)  was applA total of 356 clinical samples used for this study were from women visiting the gynecology outpatient clinics of Jiading District Maternal and Children Health Hospital of Shanghai, China. The median age of the patients was 39.8 years (range from 22 to 68 years old). According to the protocols of practice, the cervicovaginal cells at the transformation zone of the uterine cervix were collected by a gynecologist or a trained gynecologist assistant with a standard cytobrush (with spatula), and suspended in PreservCyt® .The collected samples in PreservCyt medium for routine liquid-based cytology (LBC) were processed in a room of the laboratory physically separated from where the PCR amplification was performed. Briefly, 2 ml of cellular liquid in PreservCyt medium was removed into an Eppendorf tube for sedimentation. Then the gravity-sedimentary cellular material was lysed in a 400 μl of digestion solution containing 5ul of proteinase at 65 °C for 30 min, followed by treatment in 150 μl NaCl solution. The DNA in the lysed supernatant was precipitated by water-free alcohol, and the pellets were dissolved in TE buffer. The clinical DNA specimens were detected and genotyped using the developed 18 HR HPV MALDI-TOF MS assay. To avoid cross-contamination, the laboratory spaces were separated into three parts: the rooms for pre-PCR processes , for PCR processes (PCR reaction system preparation and reactions), and for post-PCR processes (PCR product extensions and conducts of mass spectrometry), respectively.The Roche Cobas 4800 HPV test was carried out according to the manufacturer’s protocol for detection of 14 HR HPV genotypes including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68  [For the samples with discrepant results between two HPV detection tests, their HPV genotypes were identified using PCR and sequencing. Nested PCR method was performed by using the general primer pair MY09/11 for primary PCR and the GP6+/MY11 or GP6+/GP5+ primer pairs for nested PCR . The visp < 0.05 was considered significant. Discordant samples were analyzed in sequencing and genotyping using the pearson χ2-test.Concordance was calculated assuming a standard 2 × 2 contingency table . The degTo develop the 18 HR HPV genotyping assay, each of their relative HPV DNA was synthesized and cloned into a plasmid pUC57 to obtain their recombination plasmids. The obtained 18 HR HPV DNA recombinant plasmids were sequenced to confirm the existence of each of HPV DNA fragments. Next, multiplex PCR reactions were carried out using their HPV recombinant plasmids as templates based on the MALDI-TOF MS platform. Each type-specific assay was optimized and then all the 18 HR HPV primers were combined into a two-well reaction. Each of the 9-plex HPV including HPV16, 31, 35, 45, 52, 56, 59, 68 and 82 was identified in one well  and slightly less than the positive rate of 48.0% which was produced by MALDI-TOF MS assay. In terms of 14 HR types, which could be detected by the Roche Cobas 4800 HPV assay, the concordance between them was 80.1%   of them were identified as HPV16 by sequencing and genotyping, the remaining 6 samples were identified as 2 HR HPV including 52 and 82, and 5 LR HPV including HPV6, 11, 54 and 81, and 2 negative Table . The typPersistent infection of HR HPV can lead to high-grade pre-cancer and cervical cancer . It is tUsing recombination plasmids containing HPV DNA, one group developed 14 HR HPV genotyping method including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 based on the MALDI-TOF mass spectrometry platform . In anotThe concordance was substantial agreement between the new developed MALDI-TOF MS 18 HR HPV genotyping method and the Roche Cobas 4800 HPV assay for clinical samples HR HPV detection in terms of 14 HR HPV types detected by the later. The Roche Cobas 4800 HPV test detects only HPV16, HPV18 and 12 other HR HPV as a pooled result, which does not identify individual HPV genotype. The new developed MALDI-TOF MS HPV assay can identify each of 18 HR HPV individually, meaning that it has more range of HR HPV detection spectrum than the Roche Cobas 4800 assay. Moreover, the sensitivity in HPV detection by MALDI-TOF MS was superior to that of real-time fluorescence PCR-based assays including the Roche Cobas 4800 assay in the present study and the previous report .Sequencing has the ability to detect all known HPV types. Discrepant results between the new MALDI-TOF MS and the Roche Cobas 4800 HPV assays in detecting clinical samples were analyzed by sequencing and genotyping. In the MALDI-TOF MS−/Cobas+ samples, LR HPV including HPV 6, 11, 40, 42–44, 71, 72, and 81 were found, while in the MALDI-TOF MS+/Cobas- samples HPV 6, 11, 54 and 81 were seen by sequencing and genotyping. Much more LR HPV genotypes were found in Cobas-positive samples compared with MALDI-TOF MS-positive ones. The consequence of LR HPV genotype cross-reactivity in the Roche Cobas 4800 HPV test caused an increased number of false-positive results, leading to over-treatment for women who possessed only LR HPV genotype. This suggested that the new developed MALDI-TOF MS HPV test demonstrated much less cross-reactivity than the Roche Cobas 4800 test. Taken together, this MALDI-TOF MS HR HPV assay is an evolving tool with exciting potential to study the epidemiology and clinical management of HPV-associated diseases . The croHPV genotype distribution has been shown to vary by race and geographic region , 24. A mThis study demonstrated that a two-well assay was developed to detect and type all 18 HR HPV based on the MALDI-TOF MS platform. The new developed method was able to provide more exact individual HPV type information compared to the Roche Cobas 4800 HPV assay. Other advantages of the MALDI-TOF MS method were the high-throughput detection of clinical samples in 384-well plate format and readily amenable to automation, with more sensitivity and specificity.Additional file 1:Table S1. The sequences of the 18 HR HPV L1 regions. (XLSX 20 kb)Additional file 2:Table S2. The sequences of the 18 HR HPV extended primers. (DOCX 36 kb)