Young adult survivors of childhood acute lymphoblastic leukemia (ALL) treated with protocols including cranial radiotherapy demonstrate a persistent weight gain and reduced final height. Published reports on the effects on growth of different oncologic therapies are conflicting and difficult to interpret because they combined children treated with both cranial irradiation and multi-agent chemotherapy. Our study investigated the effect of chemotherapy alone on body mass index (BMI) and on growth at the achievement of final height in a homogeneous cohort of Italian childhood ALL survivors.We retrospectively studied 162 Caucasian patients treated on the Italian Association of Pediatric Hematology and Oncology protocols without radiotherapy between 1989 and 2000 at five Italian centers with 107 inclusions . Height- and BMI-standard deviation score (SDS) were collected at diagnosis of ALL, at the end of treatment and at the achievement of final height. Changes in height SDS and BMI SDS with time were analyzed using dependent sample Student's t-test.A significant reduction of height-SDS was documented during treatment in both genders. This reduction of height-SDS was not followed by an appropriate catch-up growth, despite the achievement of a mean final height within the normal range. At diagnosis females showed a lower mean BMI-SDS than males. During treatment, in the whole population, BMI-SDS increased significantly. After it, while males lost BMI-SDS, females showed its persistent increase.Survivors of childhood ALL generally seemed to achieve a normal final height with a BMI within the normal range. These parameters appeared to be only minimally affected by chemotherapy. Nevertheless, height catch-up growth was not completed after chemotherapy in both genders and all patients experienced an increase of BMI-SDS during chemotherapy that only females seemed to conserve until the achievement of final height. Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and represents 80% of all leukemia cases. Recent Nowadays, ALL survivors form the largest group of long-term survivors from childhood cancer, even if they are a heterogeneous group considering therapeutic protocols. In last decades, in most of them, cranial radiotherapy has been replaced by intrathecal chemotherapy, as standard central nervous system prophylaxis and treatment, and a reduced percentage of children have received cranial radiation with a cTo evaluate the impact on growth, we performed a retrospective multicenter study in a large pediatric ALL population treated without radiotherapy, followed until the achievement of final height.This study was a longitudinal, retrospective and multicenter study: we reviewed the clinical notes of patients treated for ALL at five Italian pediatric oncologic centers  on Italian Association of Pediatric Hematology and Oncology (AIEOP) protocols, without radiotherapy, from 1989 to 2000, and followed until the achievement of adult height. Inclusion criteria comprised the successful completion of the treatment (standard risk protocols) and a continued first remission. Exclusion criteria included: relapse, exposure to radiotherapy and/or bone marrow transplant, the presence of other diseases potentially influencing growth  and of chronic treatment with any other medication apart from the AIEOP protocols which might affect growth  and lack of complete auxological data. Details concerning ALL treatment protocols were published elsewhere and summProvincial Ethical Committee approved the protocol study (practice 185/11).Age, height and weight at diagnosis of ALL (sT), at the end of treatment (EoT), and at final height (FH) were extracted from the record of each eligible patient. Uniformly, in every center, height was measured to the nearest 0.1-cm with a wall-mounted stadiometer ; body weight was measured to the nearest 0.1-kg and body mass index (BMI) was obtained from the weight in kg/height in meters squared and expressed as standard deviation-score (SD-S) with respect to chronological age. The auxological instruments were routinely checked and calibrated. Height- and BMI- SDS were calculated for each value using age- and sex- specific World Health Organization (WHO) growth chart 2007. ParentaFor each participant, demographic and therapeutic information was obtained, including gender, ethnicity, diagnostic white cell count (WBC) at sT and treatment modalities.All results, apart from ages expressed by median, were reported as the mean ± SD. Parametric statistical analysis  was performed using dependent sample Student’s t-test to detect mean changes in height SDS and BMI SDS from diagnosis of ALL to final height.Data were also analyzed according to gender and to groups of age (younger or older than four years at diagnosis). Independent t-test was performed to detect differences between groups. Gender, WBC, age at diagnosis, height at diagnosis and BMI at diagnosis, genetic target height were analyzed in a univariate regression model with final height SDS as dependent variable through Pearson’s correlation. Potential predictors of final height SDS and BMI SDS at FH were analyzed by multivariate regression models.A P value below 0.05 was considered statistically significant.Of 162 Caucasian patients treated on AIEOP ALL87, 88, 91, 95 and 2000 protocols without radiotherapy from 1989 to 2000, 107 met inclusion criteria. Causes of exclusion enclosed: relapse , exposure to radiotherapy (6.7%) and lack of data (19.7%). None was excluded because of the presence of other diseases or of other chronic treatment potentially influencing growth. Only auxological parameters at sT were available in the 32 non-participants patients excluded because of lacking data. No statistical differences were detected between included and excluded patients among available variables .Table At every step-time, both males and females had a mean height- and BMI-SDS within the normal range Figures  and2. AIn the whole population, a significant reduction of height-SDS was documented during treatment (from sT to EoT) in both genders Table . This revs TH SDS – FH SDS: -0.59 vs. -0.16; -0.70 vs. -0.35; -0.44 vs. -0.09 in the whole population, males and females; respectively). Only females achieved a FH SDS significantly reduced than data at sT . Other 60 presented at sT a median age of 7.79 years (range 4.15 – 13.73 years). There was no significant difference between groups in sex distribution. During chemotherapy, a significant reduction of height-SDS was registered in both groups and it still persisted at FH . Height SDS together with BMI SDS at sT was also identified as independent predictive factors for final BMI SDS .According to our knowledge, this is the first study examining long-term growth outcomes in a wide cohort of un-irradiated Italian survivors from childhood ALL.Growth impairment has already been reported as a frequent complication of the treatment of ALL. PatientOur data demonstrate that chemotherapy minimally affected final height, confirming some short-term previous published findings, 25, 27.In irradiated population, girls typically experienced greater growth impairment than boys, 28. AlsHow chemotherapy can affect growth is still unclear, even because hypothalamic-pituitary cells together with liver and chondrocyte do not have a high multiplicative capacity and, therefore, they have not usually been considered vulnerable to the action of chemotherapeutic agents. A reduction of growth hormone (GH) secretion induced by chemotherapy could be supposed. Rose and colleagues documented a hypothalamic-pituitary abnormality after chemotherapy in 83% of 18 hematological malignancy survivors (17 ALL) who were referred to their Clinic because of slow growth. GH defiObesity is probably the most worrisome and common late effect in survivors of childhood ALL. RazzoukExposure to chemotherapeutic medications seemed to differ between genders: females seemed more sensitive to effects of chemotherapy and they might be more at risk to present greater and more persistent metabolic alterations than males. Up to now, none of therapeutic choices and/or doses could explain this sexual dimorphism. Others factors should be considered. Female survivors are already known to be less active and to have a more unbalanced caloric intake than males. NeverthIn our cohort, age at diagnosis did not correlate with final BMI SDS and it was not identified as its independent predictive factors. Nevertheless, examining population according to age-subgroups (younger and older than 4 years at sT), only younger children presented a persistent increase of BMI SDS after the end of chemotherapy. This might support that chemotherapy acts similarly to radiotherapy in altering body composition later-in-life via blunting hypothalamic leptin sensitivity and via modification of metabolism.In conclusion, our study demonstrates that chemotherapy alone, as in AIEOP protocols, minimizes the loss in FH SDS together with the increase in BMI SDS in adult survivors of childhood. These results could be positively influenced by the fact that, in all the included Italian centers, the management of oncologic patients involves pediatric endocrinologists from the early stages of treatment, allowing early evaluation of pathological conditions and, consequently, early interventions. Nevertheless, both males and females experienced a loss in height-SDS during chemotherapy that was not followed by an appropriate catch-up growth, and an increase of BMI-SDS. Physicians have to keep in mind that cancer survivors, because of treatment , are prone to develop cardiovascular disease. Obesity can increase the risk for morbidity and mortality due to diabetes, coronary artery disease, atherosclerosis and other diseases related to overweight in adulthood, especia


Using resin cast etching, osteocyte canaliculi were observed directly approaching the laser-modified implant surface. Transmission electron microscopy showed canaliculi in close proximity to the laser-modified surface, in addition to a highly ordered arrangement of collagen fibrils aligned parallel to the implant surface contour. It is concluded that the physico-chemical surface properties of laser-modified surfaces  promote bone bonding which may be of benefit in situations where large demands are imposed on biomechanically stable interfaces, such as in early loading and in compromised conditions.Osseointegrated implants inserted in the temporal bone are a vital component of bone-anchored hearing systems (BAHS). Despite low implant failure levels, early loading protocols and simplified procedures necessitate the application of implants which promote bone formation, bone bonding and biomechanical stability. Here, screw-shaped, commercially pure titanium implants were selectively laser ablated within the thread valley using an Nd:YAG laser to produce a microtopography with a superimposed nanotexture and a thickened surface oxide layer. State-of-the-art machined implants served as controls. After eight weeks’ implantation in rabbit tibiae, resonance frequency analysis (RFA) values increased from insertion to retrieval for both implant types, while removal torque (RTQ) measurements showed 153% higher biomechanical anchorage of the laser-modified implants. Comparably high bone area (BA) and bone-implant contact (BIC) were recorded for both implant types but with distinctly different failure patterns following biomechanical testing. Fracture lines appeared within the bone ~30–50 μm from the laser-modified surface, while separation occurred at the bone-implant interface for the machined surface. Strong correlations were found between RTQ and BIC and between RFA at retrieval and BA. In the endosteal threads, where all the bone had formed  For more than 30 years, osseointegrated implants inserted in the temporal bone have served as an important component of bone-anchored hearing systems (BAHS) . Low agein vitro [in vivo [Implant surface modification is a main strategy that can optimise the surface properties of an implant and promote its integration in the recipient bone. The physical (e.g. topography) and chemical (e.g. oxide thickness) properties of an implant surface play a critical role in modulating the tissue response . The addin vitro . Moreove[in vivo .The hierarchical structuring of titanium surfaces can be achieved by site-specific laser ablation  and the In the clinical situation, an objective measurement of implant-bone stability is required. The stiffness of the bone-implant unit can be assessed non-invasively by resonance frequency analysis (RFA), whereby oscillations are induced by a piezoelectric element and the corresponding resonance frequency is recorded. RFA values are mainly influenced by the firmness of the fixation, healing time, extent of osseointegration, stiffness of the surrounding bone and implant geometry . ClinicaIt has previously been demonstrated that the rabbit tibia  or femur (mainly trabecular bone) serve as suitable experimental animal models corresponding to the clinical insertion sites in the temporal bone  and the maxilla (mainly trabecular bone). After six to eight weeks in this model, it is possible to determine differences in bone-implant contact (BIC) , bone arScrew-shaped implants  machined from commercially pure titanium . For half the implants, selective laser ablation was employed to produce site-specific surface modification confined to the thread valley, reaching approximately 30% of the thread height on each flank, leaving the majority of the implant as machined. The laser ablation process was performed using a Q-switched Nd:YAG laser 120W  with 1064 nm wavelength and 125 μm spot size on rotating implants (15 RPM) in ambient air. One millimetre at each end of the implants was left untreated. The remaining half of the implants were left as machined. The implants were cleaned in Extran MA01®  prior to sterile packaging and subsequent autoclaving.Surface chemical analysis was performed using Auger electron spectroscopy (AES) (PHI 700 Scanning Auger Nanoprobe) on two implants of each type, where four locations were analysed on each implant. The oxide thickness was determined by AES depth profiling using two implants of each type, where three areas with dimensions of 10 μm x 10 μm were analysed on each implant. The surface morphology was evaluated by scanning electron microscopy (SEM) using a Leo Ultra 55 FEG SEM  in the secondary electron mode operated at 5 kV accelerating voltage, using a regular secondary electron detector at low resolution and an in-lens detector at high resolution, at ×50–200,000 magnifications. The surface topography was analysed by white light 3D interference microscopy using a Wyko NT1100 optical profiler  using a ×20 magnification lens in the vertical scanning interferometry mode with a vertical resolution of 3 nm. Two implants of each type were used and four locations were analysed on each implant. The collected data were processed (Weeco Vision 32 v3.43) and the roughness data parameters were calculated .The experiment  was approved by the local Animal Ethics Committee at the University of Gothenburg, Gothenburg, Sweden (Dnr 291–2012). Ten adult female New Zealand white rabbits  received one implant of either type in each proximal tibial metaphysis for a healing period of eight weeks. The animals were kept on a 12-hour day/night cycle with ad libitum access to food and water. Prior to the surgery, the animals were anaesthetised with a 1 mL intramuscular injection of Hypnorm  and a 1 mL intraperitoneal injection of Stesolid . Additional Hypnorm  was given at 20–30 minute intervals to maintain anaesthesia. Post-operatively, 0.4 mL (0.3 mg/mL) of Temgesic  was administered subcutaneously. The animals received two additional 0.4 mL doses of Temgesic a day, for three days.TM 200 drill unit . The implant stability quotient (ISQ) was measured prior to attaching the cover screw by means of resonance frequency analysis (RFA) with the Osstell Mentor system . Two measurements were made at 90° to each other and their mean values were considered for statistical analyses. Eight weeks post-implantation, the ISQ was measured again. To minimise measurement errors, each rabbit had its individual ISQ probe  for measurements made at implantation and retrieval.The surgical procedure was performed in a sterile environment. The surgical site was shaved and cleaned using chlorhexidine digluconate . The limbs were covered with sterile drapes, leaving only the surgical area exposed. The bone surface was exposed by incision and blunt dissection of the underlying tissue, including the periosteum. The drill holes were prepared by a stepwise enlargement, starting with a 2 mm diameter round burr, followed by a 2 mm twist drill, 3 mm pilot drill and a 3 mm twist drill under copious irrigation with saline. The final preparation was made with a screw tap. The implants were installed at slow speed (15 rpm) with an OsseoSetThe animals were euthanised by an intravenous overdose of sodium pentobarbital (60 mg/mL). A removal torque (RTQ) evaluation was made using a torque-testing machine connected to the implants via a custom fabricated adapter. The system was aligned and calibrated. The RTQ value was monitored in real time while rotating the implants at a constant angular speed of 0.2°/s.en bloc and processed by fixation in 4% paraformaldehyde, stepwise dehydration in a graded ethanol series, followed by embedding in plastic resin . The embedded blocks were bisected. One half-block of each specimen was used to prepare a 50 μm thick central ground section  [The implants and surrounding bone were retrieved Germany)  stained de-novo-formed bone was being analysed.The resin embedded bone-implant blocks were wet polished with 400–4000 grit SiC grinding paper. The samples were air-dried overnight prior to low-vacuum backscattered electron scanning electron microscopy (BSE-SEM) imaging in a Quanta 200 environmental SEM  operated at 20 kV and 0.5 Torr water vapour pressure. Five test and control pairs  were used. For each sample, the first thread located below the level of the original cortical bone  was imaged in order to ensure that only imagej.nih.gov/ij).The mineralised bone area (B.Ar) in the first thread located below the level of the original cortical bone was quantified on BSE-SEM images recorded at ×300 magnification. All unmineralised areas in each recorded image were excluded by segmentation using ImageJ (imagej.nih.gov/ij) and the osteocytes were quantified using the Cell Counter plug-in [The osteocyte density, i.e. the average number of osteocytes per mineralised surface (N.Ot/B.Ar), was quantified on BSE-SEM images recorded at ×300 magnification. All unmineralised areas in each recorded image were excluded by segmentation using ImageJ  detector connected to a Dilor XY 800 spectrometer , equipped with a 676 nm Ar/Kr laser operated at ~100 mW, with a 50x objective, 600 groove/mm grating, 300 mm focal length and pinhole size of 100 μm. Five test and control pairs , the same as for prior BSE-SEM, were used. For each sample, three spectra each were recorded in areas of mineralised bone, approximately 50–100 μm from the implant surface at the inner 1/3 , the outer 2/3 (thread flank) and immediately outside the thread. For each sample, the first thread located below the level of the original cortical bone  was imaged in order to ensure that only -1 and background fluorescence subtraction was performed by fitting a fourth-order polynomial function using the Background Correction program [1 PO43- peaks in all spectra corresponded to ~959 cm-1. The baseline-corrected spectra were then normalised using Plot (http://plot.micw.eu/) to show equal intensities of the ν1 PO43- band in all spectra. Curve fitting, using mixed Gaussian and Lorentzian functions, and the quantification of integral areas were performed using MagicPlot (www.magicplot.com).At each position, five acquisitions were made with an integration time of 20 s each. Raw spectra were truncated between 300–1800 cm1 PO43- peak [2 PO43-/Amide III) [1 CO32-/ν1 PO43- [1 CO32-/ν2 PO43- [1 PO43- (∼930–980 cm-1), ν2 PO43- (∼410–460 cm-1), Amide III (∼1215–1300 cm-1) and ν1 CO32- (∼1050–1100 cm-1) [The investigated Raman metrics included mineral crystallinity, taken as the reciprocal of the full-width at half-maximum (1/FWHM) of the ν43- peak , the apaide III) , 33, andν1 PO43- ; ν1 CO32ν2 PO43- . The int00 cm-1) .3PO4 and 5% NaOCl, rinsed and allowed to air-dry overnight. The samples were Au sputter-coated (~10 nm) for high-vacuum secondary electron SEM imaging in an Ultra 55 FEG SEM  operated at 5 kV accelerating voltage, 5 mm working distance and 30 μm aperture size.Resin embedded bone-implant blocks were prepared for the direct visualisation of osteocytes adjacent to the implant surface using a resin cast etching procedure , 38. TheOne selected block from the laser-modified group was sputter coated with Au/Pd and transferred to a dual-beam instrument, Versa 3D FIB-SEM , for the preparation of an electron transparent lamella for transmission electron microscopy (TEM) , 40. TheTransmission electron microscopy was performed in an FEI Titan 80–300  equipped with a Probe Cs corrector and operated at 300 kV. Bright-field TEM and high-angle annular dark field-scanning TEM (HAADF-STEM) imaging was performed to study the ultrastructure of the bone-implant interface. Further, site-specific, energy-dispersive X-ray spectroscopy (EDX) was performed across the interface zone using a nanoprobe in STEM mode.The Mann-Whitney U test was used for the surface characterisation data . The non-parametric Wilcoxon’s signed rank test for pair-wise analysis was used for the biomechanical tests (RFA and RTQ), histomorphometry (BIC and BA) and Raman spectroscopy. Pearson correlation analysis was used to detect possible positive or negative correlations between the different test parameters of the various analyses performed.n = 20). First, the Pearson correlation analysis was performed between parameters anticipated to measure implant stability: RTQ and RFA at retrieval (rRFA) and the parameters that describe the degree of bone-implant contact: BIC , as well as the amount of bone filling in the implant thread: BA . The type of inserted implant, whether machined or laser-modified, and the RFA at insertion (iRFA) were included. Next, parameters that demonstrated significant correlations according to the Pearson analysis were entered in a multiple, stepwise, linear regression analysis. In the multiple regression model, RTQ and rRFA were regarded as dependent variables, whereas the significantly correlated inner BIC, total BIC, inner BA or total BA were used as independent variables that may or may not predict the dependent variables. Whenever the implant type and/or the iRFA revealed a significant correlation, in the Pearson model, they were then also controlled for as confounding factors in the linear regression model.Pearson correlation and linear regression models were used to determine the relationship between different parameters of osseointegration. Analyses were performed on merged groups, machined and laser-modified implants  and Auger electron spectroscopy (AES) demonstrated a predominance of Ti, O and C on both implant surfaces, with only minor contamination by Ca and S. AES depth profiling confirmed a difference in surface oxide thickness, 53 nm for the laser-modified surface and 13.8 nm for the machined surface .The machined surface was relatively smooth, with a repetitive pattern of machining ridges . A distiiRFA) and retrieval (rRFA)  revealed significantly increased values over time between insertion (l (rRFA) . The ISQAs demonstrated , the lasHistological evaluation of the implants  showed that they were vertically and unicortically positioned in the tibia . FurtherFor both implant types, mature bone occupied the cortical and endosteal threads, consisting of densely packed osteons, which were made up of central blood vessels surrounded by concentric bone lamellae and mature osteocytes with a typical oval shape . Areas ode novo and not as an extension from the endosteum. Moreover, new bone was formed directly on the laser-modified surface   and bonerRFA showed a significant positive correlation with the total BA, total BIC and iRFA  contrast than the original cortical bone. The endosteal threads containing only de-novo-formed bone showed as much as 80 ± 3% and 82 ± 4% mineralised bone area for the machined and laser-modified surfaces, respectively. Furthermore, the osteocyte densities within these threads were also comparable, 1019 ± 118 mm-2 and 1005 ± 126 mm-2 for the machined and laser-modified surfaces, respectively.The newly formed bone within the implant threads was highly mineralised , but it 1 PO43-), the apatite-to-collagen (ν2 PO43-/Amide III), and the carbonate-to-phosphate  ratios were comparable for the two implant types.At each analysed location, i.e. the valley, flank and outside  Figs, thFollowing resin cast etching, the vast osteocyte lacuno-canalicular network (Ot.LCN) could be visualised . Osteocy2 layer , the extracellular matrix composition (using Raman spectroscopy) and the structure at the implant surface (using SEM and TEM), as well as the stiffness of the bone-implant unit (by RFA).RTQ measurements revealed a completely different load-deformation curve for laser-modified implants compared with the machined implants. The attainment of higher applied torque at break-point and the abrupt loss of strength indicate a fracture-type failure pattern. A structural correlate to this failure pattern was suggested from histological and BSE-SEM observations, showing the appearance of fracture lines in the bone, approximately 30–50 μm from the laser-modified surface. Taken together, these findings indicate that the interfacial adhesion between the implant surface and bone was stronger than the bone itself. In agreement with previous observations , 20, thede novo, comprised remodelled, osteonal, lamellar bone. These endosteal threads contained a high proportion of well-mineralised tissue, irrespective of the type of implant. Further, the molecular structure of bone, evaluated by Raman spectroscopy, had a similar appearance for both implant surfaces. The degree of tissue maturation was comparable for the machined and the laser-modified implant surfaces, with respect to mineral crystallinity, the apatite-to-collagen ratio and the carbonate-to-phosphate ratio. Collectively, the quality of the bone formed within the threads, as related to the composition and structural organisation of the tissue, was similar for the two types of implant and most probably did not constitute a determinant of the observed difference in RTQ. It must be underlined that it was not possible to perform a thorough characterisation of the interfacial tissue directly adjacent to the implant surface (i.e. the first 5–10 μm) due to the prior biomechanical testing and, for this reason, local variations in extracellular matrix composition as a function of implant surface properties, as reported in other studies [An alternative explanation of the marked difference in biomechanical anchorage could be a difference in the quality of bone formed at the implant surfaces. Histology and BSE-SEM imaging showed that the newly formed bone, particularly within the endosteal threads (of both implant types) and therefore formed  studies , 37, couA structural cue for the strong biomechanical anchorage of the laser-modified implants was instead provided by high-resolution analysis (using TEM and HAADF-STEM imaging modes) of the implant-bone interface. The bone-implant interface showed well-mineralised collagen fibrils aligned parallel to the implant surface, closely following the micro-scale surface contour. This ultrastructural observation corroborates previous observations in humans , 42 and For both implant types, the ultrastructural findings indicate that the tissue side of the bone-implant interface is in fact mineralised bone. Furthermore, the close adaptation of the well-organised, mineralised bone to the laser-modified surface suggests direct bone bonding to the implant surface and provides a possible explanation for the increase in RTQ and its associated load-deformation pattern. This functionally graded interface could therefore be regarded as the next level of osseointegration, rather than a passive, mechanically interlocked cement-line matrix. It was not, however, possible directly to compare the organisation of the bone-implant interface with the machined surface. It is well documented that sample processing procedures, e.g. fixation, dehydration and resin embedding, induce tissue distortion . At the Since the present study aimed to elucidate the biomechanical and morphological correlates of osseointegration, i.e. the relationships between commonly used techniques to evaluate osseointegration, no emphasis was placed on the cellular and molecular mechanisms and this could be regarded as a limitation of the work. In order to acquire a further understanding of the underpinning mechanisms, additional tools such as quantitative polymerase chain reaction (qPCR), immunohistochemistry, protein analysis and so on need to be applied. Moreover, studies of this kind will enable an understanding of the temporal course of the cellular response to machined and laser-modified surfaces.Although not proven, one possible explanation of the increase in RTQ and the ultrastructural bonding of laser-modified implants is the increased thickness of the surface oxide layer. The oxide film is critical for implantable biomaterial applications of titanium, as it limits the release of ionic or molecular Ti species from the surface, thereby protecting the biological environment from the extremely reactive Ti metal . While tEarly studies of the role of surface oxide indicated that implant surface modification with respect to increased oxide thickness results in a high degree of bone contact . MoreoveThis work demonstrates the osseointegration of commercially pure titanium implants with site-specific laser ablation within the deepest part of the implant threads to achieve improved biomechanical anchorage. Laser ablation in the inner 1/3 of the implant thread enabled the site-specific restructuring of the implant surface, inducing the development of a distinct microtopography comprising 1–10 μm diameter globules of resolidified metal, which predictably enlarge the surface area several times over. Superimposed on this microtopography is a relatively thick (compared with machined implants) nanostructured titanium dioxide layer, which further enhances the osteoconductive/bioactive potential of the laser-modified implant surface. The macro-shape of the screw, a distinct microtopography and the fine nanotexture give rise to a three-level surface topography, thereby matching the building blocks of bone in its hierarchical architecture.The clinical context in which the present observations will be implemented is the improvement of the bone fixation of bone-anchored hearing aids. Although implant loosening predominantly occurs during the first year , recent rRFA) correlated to implant type, RFA at insertion (iRFA), total BIC, inner BIC, total BA and inner BA. Interestingly, the RTQ showed a significant correlation with the implant type, which supports the comparative analysis showing a higher RTQ for the laser-modified surface vs. the machined surface. Moreover, an association was also revealed between the RTQ and the bone-implant contact . According to the linear regression model, when excluding the effect of surface modification (i.e. the implant type), the total BIC explained about 84% of the increase in RTQ. This means that, when evaluating osseointegration, irrespective of the type of implant surface modification, the degree of total bone-implant contact, as measured by histomorphometry, is a major predictor of RTQ and thereby of implant stability. Finally, when the effect of surface modification was not excluded, the linear regression model mainly showed that the inner BIC was a predictor of RTQ.Based on the parameters that are commonly used to support the functional and clinical assessment of implant stability (i.e. osseointegration), we hypothesised that the RTQ and the RFA at retrieval .Another important observation was that the In overall terms, the results suggest that the RTQ is a method that is able to discriminate between the degree of osseointegration as influenced by differences in implant surface properties. On the other hand, RFA at retrieval only reflects the amount of mineralised bone within the implant threads (BA) but not the actual adaptation of the bone to the implant surface contour (i.e. BIC).The present study shows that osseointegration was attained for two macroscopically similar yet differently structured titanium surfaces. Bone-implant contact, bone area in threads, extracellular matrix composition and osteocyte densities were similar for the machined and laser-modified implants. In contrast, laser-modified surfaces demonstrated a higher biomechanical torque, a distinctly different load-deformation pattern and a fractured interface in the surrounding bone rather that at the implant-tissue interface during torque. Taken together with the ultrastructural observation of a highly ordered arrangement of collagen fibrils aligned parallel to the surface, these findings suggest that the physico-chemical surface properties of laser-modified surfaces  promote bone bonding. Attaining ultrastructural and biomechanical effects of this kind is likely to be of benefit in situations where large demands are imposed on biomechanically stable interfaces, such as in early loading and compromised conditions. From a methodological perspective, the present results also suggest that removal torque analysis has greater predictability, when it comes to the degree of osseointegration and implant stability, than resonance frequency analysis.S1 Fig(PDF)Click here for additional data file.S2 FigOverview BSE-SEM images of the (a-e) machined and (f-j) laser-modified implants.(PDF)Click here for additional data file.S3 FigRaman spectra recorded at the valley regions of the (top) machined and (bottom) laser-modified implant threads.(PDF)Click here for additional data file.S4 FigRaman spectra recorded at the flank regions of the (top) machined and (bottom) laser-modified implant threads.(PDF)Click here for additional data file.S5 FigRaman spectra recorded outside the (top) machined and (bottom) laser-modified implant threads.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


EGF, rs4444903), patatin-like phospholipase domain-containing protein 3  genes, and near the interleukin-28B  gene are linked to treatment response, fibrosis, and hepatocellular carcinoma (HCC) in chronic hepatitis C. Whether these SNPs independently or in combination predict clinical deterioration in hepatitis C virus (HCV)-related cirrhosis is unknown. We genotyped SNPs in EGF, PNPLA3, and IL28B from liver tissue from 169 patients with biopsy-proven HCV cirrhosis. We estimated risk of clinical deterioration, defined as development of ascites, encephalopathy, variceal hemorrhage, HCC, or liver-related death using Cox proportional hazards modeling. During a median follow-up of 6.6 years, 66 of 169 patients experienced clinical deterioration. EGF non-AA, PNPLA3 non-CC, and IL28B non-CC genotypes were each associated with increased risk of clinical deterioration in age, sex, and race-adjusted analysis. Only EGF non-AA genotype was independently associated with increased risk of clinical deterioration  after additionally adjusting for bilirubin, albumin, and platelets. Compared to subjects who had 0–1 unfavorable genotypes, the HR for clinical deterioration was 1.79 (95%CI 0.96–3.35) for 2 unfavorable genotypes and 4.03 (95%CI 2.13–7.62) for unfavorable genotypes for all three loci (Ptrend<0.0001). In conclusion, among HCV cirrhotics, EGF non-AA genotype is independently associated with increased risk for clinical deterioration. Specific PNPLA3 and IL28B genotypes also appear to be associated with clinical deterioration. These SNPs have potential to identify patients with HCV-related cirrhosis who require more intensive monitoring for decompensation or future therapies preventing disease progression.Single nucleotide polymorphisms (SNPs) in the epidermal growth factor ( Chronic hepatitis C (CHC) is the most common cause of liver-related death and liver transplantation in the United States IL28B) locus. The CC IL28B genotype is associated with spontaneous clearance of HCV and predicts interferon and ribavirin treatment response IL28B genotype and disease course are conflicting EGF) gene polymorphism rs4444903 has been associated with EGF levels PNPLA3) SNP rs738409 has mainly been studied in nonalcoholic fatty liver disease (NAFLD) Attempts have been made to develop risk scores to predict the risk of disease progression for individual patients. Such scores have incorporated both clinical variables and genetic data IL28B, EGF, and PNPLA3 genotypes on the natural history of HCV-related cirrhosis or examined these SNPs in the same population. We therefore sought to evaluate the association between these SNPs and clinical deterioration in a cohort of patients with HCV-related cirrhosis.These data suggest that these SNPs may be useful for the prediction of the natural history of CHC. However, no study has evaluated the influence of EGF genotype and HCC The patient cohort was identified from pathology reports at Massachusetts General Hospital. A natural language search for biopsies performed between 1990 and 2007 was previously performed for the keywords: HCV, HBV, NAFLD, NASH, and hepatitis. Pathology reports were reviewed to identify patients whose biopsies were consistent with HCV-related cirrhosis. Inclusion criteria were age ≥18 years at time of biopsy, positive HCV antibody or HCV RNA, and presence of cirrhosis (Ishak stage 5 or 6/6 or Metavir stage 4/4). Exclusion criteria included co-infection with human immunodeficiency virus or hepatitis B virus (HBV), liver transplantation, ascites, variceal hemorrhage, HE, or HCC prior to or within one month of the biopsy, and lack of follow-up data following the biopsy. The electronic medical records of patients identified by the pathology database search as having biopsies consistent with HCV-related cirrhosis were manually reviewed by two independent reviewers. The keyword search of the pathology database yielded 370 patients whose reports were consistent with a diagnosis of HCV-related cirrhosis. After review of the medical record, 220 patients were eligible. Formalin-fixed, paraffin-embedded (FFPE) blocks were not available for 38 patients, and genotyping could not be performed on 13 patients. Thus, the final cohort included 169 patients . Nine paThe follow-up period for each patient was defined as the date of the index biopsy until the occurrence of the first episode of clinical deterioration, death, loss to follow-up, or December 31, 2012, whichever came first. Clinical deterioration was defined as the development of ascites, HE, variceal hemorrhage, HCC, or liver-related death. Outcomes were identified by manual chart review performed by two independent reviewers. The primary outcome was the time to the first episode of clinical deterioration after the index biopsy.We collected the following baseline variables from the electronic medical record: age, gender, race, medical history, body mass index, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, albumin, platelet count, creatinine, international normalized ratio IL28B rs12979860, EGF rs4444903, and PNPLA3 rs738409  according to the manufacturer's instructions. Genotypes were assigned using Sequence Detection System (SDS 2.4) software with manual review by two independent investigators, blinded to subject phenotype.DNA was extracted from FFPE liver tissue (Five 10 µm sections from each patient) using the QiaAMP FFPE Tissue Kit . Genotyping was performed on 5 ng of DNA using the 7900HT Fast Real-Time PCR System with commercial TaqMan SNP Genotyping Assays for We calculated the person-years of follow-up for each individual from the date of the biopsy to the development of the first episode of clinical deterioration, date of death, or end of follow-up, whichever came first. Kaplan-Meier method was used to analyze the time to clinical deterioration. The log rank test was used for comparison between genotypes. The crude and adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for the effect of the SNPs on the rate of clinical deterioration were estimated with Cox proportional hazards regression models. The proportional hazard assumption was checked via the statistical significance of the interaction between log (follow-up time) and genotype. For our primary analysis, we examined each SNP in an age, sex, and race-adjusted model. In a secondary analysis, we also examined the association of each SNP in a multivariable model additionally adjusting for established predictors of clinical deterioration including baseline albumin, platelets, and total bilirubin IL28B genotype and SVR. Third, we performed an analysis excluding the one subject who had an outcome within 6 months of the index biopsy. Finally, we performed an analysis excluding subjects who were Child-Pugh Class B or missing a Child-Pugh score because of missing laboratory data. A 2-tailed P-value <0.05 was considered statistically significant. SAS  version 9.3 was used for statistical analyses.We performed several sensitivity analyses. First, we performed an analysis excluding HCC and HCC-related death as a first outcome. For this analysis, we calculated the person-years of follow-up for each individual from the date of biopsy to the development of the first episode of ascites, variceal hemorrhage, HE, date of death, or end of follow-up, whichever came first. Second, we performed an analysis incorporating sustained virologic response (SVR) at any point prior to the censor date into the model given the known association between EGF, IL28B, and PNPLA3 genotypes are shown in Among the 169 patients with biopsy-proven HCV-related cirrhosis, the baseline demographic and clinical data according to EGF non-AA genotype was associated with an age, sex, and race-adjusted HR of 3.20  for clinical deterioration. Compared with CC genotype, IL28B non-CC genotype was associated with an age, sex, and race-adjusted HR of 1.78  for clinical deterioration. Compared with CC genotype, PNPLA3 non-CC genotype was associated with an age, sex, race-adjusted HR of 1.79  for clinical deterioration.To evaluate the association of genotype independent of other clinical factors, we used multivariable models adjusted for age, sex, and race ; Fig. 2.EGF non-AA genotype remained an independent predictor of increased risk of clinical deterioration, multivariable-adjusted HR = 2.87   and PNPLA3 genotype  and clinical deterioration.In multivariable models with age, race, sex and established predictors of disease progression including albumin, platelets, and total bilirubin, = 0.008) . In contEGF non-AA, IL28B non-CC, and PNPLA3 non-CC, HR 4.03; 95%CI 2.13–7.62) and 2 unfavorable genotypes  were associated with a significantly increased risk of clinical deterioration compared to the presence of one or zero unfavorable genotypes (Plineartrend<0.0001)  .EGF non-AA genotype remained a significant predictor of increased risk of clinical deterioration . The association between PNPLA3 genotype and clinical deterioration also persisted . However, the association between IL28B genotype and clinical deterioration was no longer significant . We also conducted analyses restricted to those with Child-Pugh Class A cirrhosis, and found that EGF non-AA genotype was associated with the risk of clinical deterioration in Child-Pugh Class A subjects . The significant association between PNPLA3 genotype and risk of clinical deterioration also persisted . The association between IL28B genotype and risk of clinical deterioration did not remain statistically significant .When we excluded a single subject who developed an outcome within 6 months of the index biopsy, the HRs for the association of the SNPs with risk of clinical deterioration were not materially altered. We performed a sensitivity analysis limiting the outcome to the time to development of ascites, variceal hemorrhage, or HE. EGF non-AA genotype remained a significant predictor of increased risk of clinical deterioration . There were no longer significant associations between IL28B genotype  and PNPLA3 genotype  and clinical deterioration. The addition of SVR to the age, sex, and race-adjusted model with all 3 loci did not materially alter the results.One hundred twenty-six patients (75%) received some course of antiviral therapy with either interferon alfa alone or interferon alfa and ribavirin following the index biopsy. Among these, forty patients (32%) achieved a SVR to antiviral therapy. Among these forty patients, only 3 patients developed clinical deterioration. One patient had an episode of variceal hemorrhage and two patients developed HCC. When we incorporated achievement of SVR into the age, sex, and race-adjusted model, EGF, IL28B, and PNPLA3 genotypes and the risk of clinical deterioration in patients with HCV-related cirrhosis. The risk associated with EGF genotype persisted, even after adjustment for known clinical predictors of progression, including albumin, bilirubin, and platelets. These results suggest that genetic variation at the EGF locus is independently associated with clinical deterioration in patients with CHC and provides prognostic information beyond known clinical predictors. In contrast, genetic variations at IL28B and PNPLA3 loci were associated with prognosis in age, sex, and race-adjusted models, but the association was no longer statistically significant after adjustment for albumin, bilirubin, and platelets. This may reflect either a weaker association of these polymorphisms with clinical deterioration compared with the EGF polymorphism or the possibility that the effect of the IL28B and PNPLA3 polymorphisms on clinical deterioration may be mediated by these clinical factors. Thus, controlling for these variables may mask a true biological relation. Our combined analysis shows that the risk of clinical deterioration significantly increases with the presence of each unfavorable genotype and suggests that all three SNPs in concert could be useful in predicting disease progression.We found significant associations between EGF locus and clinical outcome in liver disease. We previously found that the EGF AG and GG genotypes are associated with a two to four-fold increased risk for HCC EGF locus independently or in concert with two additional SNPs in not only the development of HCC but also the progression of HCV-related cirrhosis. Consistent with these results is a cross-sectional study showing that the G allele of EGF rs4444903 is associated with higher degrees of liver fibrosis in younger subjects with CHC Our findings are consistent with prior data regarding the EGF rs4444903. We have previously reported increased stability of EGF 61*G allele transcripts compared to EGF 61*A allele transcripts in human hepatoma cell lines and primary human hepatocytes as well as increased levels of serum and liver EGF in subjects with cirrhosis who have the EGF GG versus AA genotype Our results are consistent with the functional nature of IL28B non-CC genotype in age, sex, and race-adjusted analysis is supported by evidence that carriage of the T allele is associated with fibrosis severity, cirrhosis and HCC in CHC IL28B genotype and fibrosis progression. The difference in our findings may be related to the fact that their cohort consisted of patients who were prior non-responders to interferon and ribavirin and had advanced fibrosis or cirrhosis. Of note, the association between IL28B genotype and clinical deterioration was not significant in our analysis when restricted to Child Pugh Class A subjects or after excluding HCC from the definition of the primary outcome. This may reflect limited power in this smaller cohort and potentially a more limited role for the IL28B genotype in hepatic decompensation compared with development of HCC. The association between IL28B genotype and clinical deterioration was also no longer significant after adjustment for SVR. This likely reflects the fact that the impact of IL28B on clinical deterioration is mediated through response to therapy or potentially limited power to detect the association in the larger model.Our finding of an association between increased risk of clinical deterioration and the PNPLA3 non-CC genotype and risk of clinical deterioration in age, sex, and race-adjusted analysis is supported by a study showing an association between the PNPLA3 C>G polymorphism and fibrosis progression in CHC Our results showing an association between PNPLA3 and IL28B polymorphisms and clinical deterioration in analyses adjusted for established laboratory predictors of outcome. Second, we did not have information on disease duration prior to the biopsy. However, no patients had evidence of decompensated liver disease prior to the biopsy and our results were consistent even among those with Child-Pugh Class A disease. Third, because the index biopsies were performed between 1990 and 2007, we did not have reliable information quantifying the amount of steatosis on each biopsy, and thus could not incorporate this into our multivariable model. While steatosis has been shown to predict fibrosis progression prior to the development of cirrhosis, it has not been shown to predict decompensation once cirrhosis has occurred. Fourth, our population was predominantly Caucasian males, limiting the external generalizability of our findings. However, recent meta-analyses revealed that although allele frequencies of the EGF polymorphism vary according to race, the association between EGF genotype and HCC risk appears independent of race EGF G allele and cirrhosis in 62 subjects with chronic HBV infection Our study has several limitations. First, we excluded sixteen subjects from the multivariable model adjusting for known predictors of disease progression due to missing laboratory variables. This may have limited our power to detect the association between the EGF and possibly IL28B and PNPLA3 genotyping in identifying persons with CHC at high risk for disease progression. In light of our group's finding that pharmacological inhibition of EGFR with erlotinib prevented progression of cirrhosis and regressed fibrosis in animal models of progressive cirrhosis EGF gene with EGF levels, our data support the potential for EGF genotyping to identify patients who may be candidates for strategies to modulate EGF.In conclusion, our findings support a role for


Salmonella Typhi and DENV/Mycobacterium leprae co-infection were identified. Entomological surveys implicated water storage containers and discarded tires as the most important development sites for Aedes aegypti and Ae. albopictus, respectively. Although this is the first documented dengue outbreak in RMI, the age groups of cases and high prevalence of secondary infection demonstrate prior DENV circulation. Dengue surveillance should continue to be strengthened in RMI and throughout the Pacific to identify and rapidly respond to future outbreaks.Dengue is a potentially fatal acute febrile illness caused by four mosquito-transmitted dengue viruses (DENV-1–4). Although dengue outbreaks regularly occur in many regions of the Pacific, little is known about dengue in the Republic of the Marshall Islands (RMI). To better understand dengue in RMI, we investigated an explosive outbreak that began in October 2011. Suspected cases were reported to the Ministry of Health, serum specimens were tested with a dengue rapid diagnostic test (RDT), and confirmatory testing was performed using RT-PCR and IgM ELISA. Laboratory-positive cases were defined by detection of DENV nonstructural protein 1 by RDT, DENV nucleic acid by RT-PCR, or anti-DENV IgM antibody by RDT or ELISA. Secondary infection was defined by detection of anti-DENV IgG antibody by ELISA in a laboratory-positive acute specimen. During the four months of the outbreak, 1,603 suspected dengue cases (3% of the RMI population) were reported. Of 867 (54%) laboratory-positive cases, 209 (24%) had dengue with warning signs, six (0.7%) had severe dengue, and none died. Dengue incidence was highest in residents of Majuro and individuals aged 10–29 years, and ∼95% of dengue cases were experiencing secondary infection. Only DENV-4 was detected by RT-PCR, which phylogenetic analysis demonstrated was most closely related to a virus previously identified in Southeast Asia. Cases of vertical DENV transmission, and DENV/ Aedes species mosquitoes, can result in dengue, an acute febrile illness characterized by headache, body pain, retro-orbital pain, rash and leukopenia Dengue is the most common mosquito-borne viral illness in the world, and is endemic throughout the tropics and subtropics where ∼96 million cases occurred in 2010 Recent dengue outbreaks have been reported in the Pacific islands, including Fiji Ae. aegypti and Ae. albopictus were detected in RMI during mosquito surveys . Although dengue activity was not above baseline in the Western Pacific Region of the World Health Organization (WHO) in 2011, country-specific rates were highest in RMI Dengue was apparently first detected in the Republic of the Marshall Islands (RMI) during an outbreak in 1989 in which DENV-1 was isolated from cases on Majuro, Kwajalein and Ebon atolls . In 1990 and 2004, DENV-2 and -1, respectively, were detected in serum specimens collected from RMI residents reported to CDC as having dengue-like illness . In 2010, To enable early detection of dengue and other outbreak-prone diseases, in 2009 a surveillance system was initiated in RMI that included implementation of dengue rapid diagnostic tests (RDTs) RMI is composed of 29 atolls and five islands with a total land mass of 70 square miles (sq mi) spread across ∼750,000 sq mi of ocean . The 201Surveillance data were collected during the outbreak, summarized weekly, and reported to WHO. After the outbreak had ended, a retrospective analysis of surveillance data was performed to: 1) describe the epidemiology of the 2011–2012 outbreak, including disease severity; 2) estimate the proportion of secondary DENV infections; 3) describe the molecular epidemiology of the DENV(s) responsible for the outbreak; and 4) identify the water containers producing vector mosquitoes.Suspected cases identified at Majuro and Ebeye Hospitals were reported directly to MOH via the Dengue Surveillance Form DSF; , which wSerum specimens were collected from all suspected dengue cases upon presentation to Majuro and Ebeye Hospitals and tested by RDT . Convalescent specimens were collected upon discharge or during follow-up evaluation. For suspected dengue cases that presented to other health facilities in RMI, whole blood was collected at presentation and tested by RDT at the health facility. A convenience sample of both RDT-positive and-negative serum specimens were sent to CDC for confirmatory testing by multiplex, DENV-type-specific, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) A suspected dengue case was an individual for whom a health care provider suspected dengue as the cause of illness and reported the case to MOH. A laboratory-positive case was a suspected dengue case for which: 1) DENV nucleic acid was detected by RT-PCR; 2) DENV non-structural protein 1 (NS1) was detected by RDT; or 3) anti-DENV IgM antibody was detected by RDT or ELISA. A laboratory-negative case was a suspected dengue case that had no laboratory diagnostic evidence of DENV infection. Dengue, dengue with warning signs, and severe dengue were defined according to 2009 WHO guidelines 450≥0.15 A representative sample of all laboratory-positive cases was selected to estimate the rates of primary and secondary infection. Cases were stratified by age group with optimal allocation for comparison between age groups. Sample size was calculated to estimate the proportion of secondary infections by age group based on data from the 2010 dengue epidemic in Puerto Rico Serum specimens in which DENV nucleic acid was detected and with CT values <25 were inoculated into cultures of C6/36 cells, in which growth of DENV was detected by RT-PCR and indirect immunofluorescence Relative abundance and species composition of juvenile mosquito populations in aquatic container habitats were assessed in 155 households on Majuro atoll during 3–8 November, 2011. Sampling focused on four communities  where the incidence of suspected dengue was high during 10 October–5 November 2011. All outdoor container habitats at selected households were inspected for the presence of standing water, juvenile mosquitoes, and if >20 pupae were present in the container. Mosquito specimens were retained from a subset of samples from each container habitat class and preserved in ethanol (larvae) or reared to eclosion (pupae) for identification to species.All surveillance data were entered into a Microsoft Access  database at MOH. Rates of suspected and laboratory-positive dengue cases were calculated using population denominators from the 2011 RMI Census This investigation underwent institutional review at CDC and was determined to be public health practice and not research; as such, Institutional Review Board approval was not required. All specimens tested in RMI were not anonymized to facilitate reporting of diagnostic test results to clinicians. All specimens sent to CDC were anonymized prior to shipment.The first identified laboratory-positive case had onset of illness on October 14, 2011 and resided in Majuro atoll . The numth–75th quartile: 1–3) post-onset of illness. Paired acute and convalescent specimens were available for 59 (3.7%) cases.Serum specimens were collected upon presentation from all patients with dengue-like illness. Specimens were collected a median of two days , 657 (41%) were laboratory-negative, 79 (4.9%) were not tested, and 867  were laboratory-positive. Of 1,379 specimens tested with the RDT in RMI, 626 (45%) were positive: 536 (86%) by detection of NS1, 73 (12%) by detection of anti-DENV IgM antibody, and 17 (2.7%) by detection of both NS1 and anti-DENV IgM antibody. From a convenience sample of 845 RDT-positive and -negative specimens sent to CDC and tested by RT-PCR, DENV-4 was detected in 482 (57%) and was the only DENV detected during the outbreak. Of the 363 specimens that tested negative by RT-PCR and were further tested by IgM ELISA at CDC, anti-DENV IgM antibody was detected in 45 (12%).Laboratory-positive cases were identified from seven of the 25 inhabited atolls and islands that comprise RMI . Among tMost (72%) laboratory-positive cases were aged 10–29 years . The higFrom a representative sample of 194 laboratory-positive acute specimens, 184 (95%) had evidence of secondary infection . Rate ofFollowing virus isolation and E gene sequencing, Bayesian phylogenetic analysis demonstrated that the virus circulating in RMI is distinct from other DENV-4 previously identified in the Pacific islands, and instead belongs to clade II of the Indonesian genotype that is geographically associated with Southeast Asia . The mosLaboratory-positive cases were not significantly associated with sex, age, or ethnicity . AlthougFour infants aged <1 year tested positive with the RDT: three by detection of NS1, and one by detection of anti-DENV IgM antibody; all were hospitalized. One infant was a neonate male born to a febrile mother in whom NS1 was detected on the day of parturition. On day seven of life, the infant developed severe dengue with cyanosis and severe bleeding requiring blood transfusion. Both the baby and mother recovered and were ultimately discharged. As 111 pregnant women gave birth during the outbreak, the rate of documented vertical DENV transmission resulting in dengue during the outbreak was 0.9% (1/111). Four additional pregnant, laboratory-positive females were identified during the outbreak; all recovered without complication. No reported fetal losses or perinatal deaths were associated with maternal DENV infection during the outbreak.Salmonella typhi was identified in a blood culture the following day. He was discharged home in stable condition after seven days of hospitalization.An 11-year-old boy from Utrik atoll had onset of fever in early November and subsequently had streaks of blood in his stool. After being transported to Majuro Hospital, he was given intravenous ampicillin, cephazoline, and metronidazole for a presumptive diagnosis of typhoid fever; however, his fever persisted. White blood cell count, platelet count, hemoglobin, and hematocrit were normal. NS1 was detected on day 8 of illness, and An 18-year-old male in his third month of treatment for leprosy was admitted with a two-day history of fever and malaise. On examination he was febrile with patchy nodular skin lesions on the trunk and extremities. NS1 and DENV-4 were detected in a serum specimen. His hospital course was unremarkable and he continued treatment for leprosy, including a decreased dose of prednisone. He was discharged on day six of hospitalization.Aedes spp. mosquitoes (data not shown). Large water storage receptacles were the most abundant water-filled container (235 per 100 households inspected), and accounted for 32% of mosquito-positive containers and 50% of containers with >20 pupae. Discarded tires, domestic utensils  and small refuse items  collectively represented 44% of mosquito-positive containers and 29% of containers with >20 pupae.Mosquito larvae or pupae were detected at 87% of inspected homes  and in 64% of containers with standing water outside homes , with an estimated density of 413 mosquito-positive containers per 100 households  . Indoor Aedes spp. juveniles collected from 109 containers with larvae or pupae were identified to species .A total of 738  species . SpecimeThe epidemiologic characteristics of this outbreak were captured by improving public and clinical awareness of dengue, enhancing surveillance to enable rapid case reporting, and use of a RDT followed by confirmatory testing to define dengue cases. Pertinent findings include 3% of all RMI residents being reported as a suspected dengue case, laboratory confirmation of dengue in 3% of Majuro residents, hospitalization of 13% of dengue cases, and identification of water storage containers as prominent sources of vector mosquitoes. The prevalence of secondary infection in all age groups demonstrates that DENVs regularly circulate in RMI.The rate of suspected dengue cases observed during this outbreak (3%) was similar to that recently reported from FSM and the Solomon Islands Several studies have demonstrated that primary infection with DENV-4 infrequently causes clinically apparent illness Interestingly, the DENV-4 that caused the RMI outbreak was distinct from other DENV-4 circulating previously in the Pacific, but was nearly identical to viruses isolated from dengue patients in 2012 from FSM and another with travel history to RMI. Moreover, the genetic relatedness of the DENV-4 from RMI to DENV-4 from Southeast Asia suggests that human travel from Southeast Asia resulted in the RMI outbreak, which subsequently spread to FSM. A similar chain of events enabled spread of DENV-4 throughout Pacific islands in 2009 Ae. aegypti was likely the primary mosquito vector responsible for DENV transmission during the RMI outbreak, Ae. albopictus is also a competent vector and may have contributed to DENV transmission, as was recently suggested to be the case in FSM Ae. albopictus has since been collected from multiple locations within RMI . Our results confirm that this species is now well established on Majuro atoll, representing the vector species most frequently recovered from domestic container habitats other than large water storage and refuse items. Ae. marshallensis may also have contributed to DENV transmission, although the vector competence of this species is unclear Although Ae. aegypti suggests they were the most important source of vector mosquitoes. Since only 5% of households in RMI have access to piped water, 79% of households store rainwater and the remainder rely on well or bottled water Ae. albopictus, was similar to observations from a recent dengue outbreak in FSM The high abundance of water storage containers infested with This investigation was subject to several limitations. Because confirmatory diagnostic testing was not performed for nearly half of suspected cases that were tested only by RDT, which may yield false-negative test results This outbreak demonstrated the acute burden that a dengue outbreak can have in a Pacific island population. To reduce the likelihood of future outbreaks, evidence-based behavioral and interventional approaches to reduce mosquito vector populations should be developed and routinely implemented. Dengue surveillance should continue to be strengthened in RMI and other Pacific island countries to rapidly detect and respond to outbreaks. In anticipation of a dengue vaccine, the age group of individuals affected by dengue in the Pacific should be elucidated to better inform vaccine campaigns.Figure S1Map depicting the geographic location of the Republic of the Marshall Islands. Source: http://www.travelpod.com/bin/graphics/maps/country/large/mh-map.gif. Published under the Creative Commons-BY license.(TIF)Click here for additional data file.Figure S2Dengue Surveillance Forms used to capture demographic, clinical and laboratory data for suspected dengue cases reported to the Ministry of Health during a dengue outbreak in the Republic of the Marshall Islands, October 2011–February 2012. A: Surveillance form used to capture data at presentation. B: Surveillance form used to capture data at discharge or during follow-up evaluation.(TIF)Click here for additional data file.


The use of multi-parametric (MP) MRI to diagnose prostate cancer has been the subject of intense research, with many studies showing positive results. The purpose of our study is to better understand the accessibility, role, and perceived accuracy of MP-MRI in practice by surveying practicing urologists. Surveys were sent to 7,400 practicing American Urological Association member physicians with a current email address. The survey asked demographic information and addressed access, accuracy, cost, and role of prostate MRI in clinical practice. Our survey elicited 276 responses. Respondents felt that limited access and prohibitive cost of MP-MRI limits its use, 72% and 59% respectively. Academic urologists ordered more MP-MRI studies per year than those in private practice . Urologists who performed more than 30 prostatectomies a year were more likely to feel that an MP-MRI would change their surgical approach . Only 25% of respondents agreed or strongly agreed that MP-MRI should be used in active surveillance. For patients with negative biopsies and elevated PSA, 39% reported MP-MRI to be very useful. Our study found that MP-MRI use is most prominent among practitioners who are oncology fellowship-trained, practice at academic centers, and perform more than 30 prostatectomies per year. Limited access and prohibitive cost of MP-MRI may limit its utility in practice. Additionally, study participants perceive a lack of accuracy of MP-MRI, which is contrary to the recent literature. Prostate cancer is the second leading cause of cancer-related death in men, claiming almost 30.000 lives in 2013  members. All survey participants were practicing urologic physicians in the United States had current email addresses as of September 01, 2013. An initial email with a brief explanation of the study and an invitation to complete the survey was sent on September 12, 2013, and a reminder email for those who had yet to complete the survey was sent on September 25, 2013. Respondents were not required to answer all questions for submission of their survey. Each email was embedded with a personalized link to ensure that each respondent could only submit a single survey, and all responses were confidential.Each survey was de-identified and responses were collected using our institutional RedCap electronic data-capturing tool . Survey Categorical responses to survey questions were assessed using Chi-square test of independence analysis and Fisher’s exact test. Continuous variables were compared with the Student’s t-test, and statistical analyses were two-sided using a significance of 0.05.Our survey elicited 276 (3.8%) responses from practicing AUA member physicians. Unfortunately, not all participants answered every survey question. The demographics that were obtained from the survey question can be found in When respondents were stratified by their type of practice, there was a statistically significant increase in the reported number of MP-MRIs ordered by physicians who practice in an academic setting compared those who do not . Eighty-nine percent of respondents endorsed having local access (less than 1hr) to facilities with MP-MRI capabilities (245/276). However, when all respondents were asked if they felt that access to qualified imaging centers and radiologists limited their use of MP-MRI, 72% of respondents agreed or strongly agreed (192/268). When asked if the cost of MP-MRI was prohibitive for its use, 59% of all respondents agreed or strongly agreed (156/263).Overall, 34% of respondents reported using MP-MRI targeted biopsies, either ultrasound fusion or cognitive techniques (91/270). When these responses were stratified by fellowship training, we found that respondents with oncology fellowship training performed significantly more MP-MRI targeted biopsies compared to those who did not . In our survey, 38% of all respondents found MP-MRI to be helpful in a patient with an elevated PSA or abnormal prostate exam prior to biopsy (102/270). When these responses where stratified by fellowship training, we found 48.2% of respondents with oncology training found MP-MRI somewhat or very helpful in this situation compared to 33.5% of respondents without oncology training. This difference was found to be trending towards significance with a p value of 0.057.In patients with a negative biopsy and an elevated PSA/abnormal prostate exam, 39% of all respondents reported MP-MRI to be very helpful (88/225). Fourteen percent of respondents reported MP-MRI to be very useful when utilized prior to definitive treatment with either prostatectomy or radiation (32/225). In patients with intermediate or high risk prostate cancer who appear to be candidates for a nerve sparing prostatectomy, 26% of respondents report that getting an MP-MRI will sometimes or often change their surgical approach to a non-nerve sparing prostatectomy (66/253). When stratified based on average number of prostatectomies performed per year (<30 versus >30); we found that surgeons who perform more than 30 prostatectomies per year are more likely to change their surgical approach based on MP-MRI than those who perform less than 30/year .Regarding the use of MP-MRI in active surveillance, 25% of all respondents agreed that it should be used in all patients (69/276). When asked if MP-MRI should be used to evaluate patients for active surveillance, 30.7% of respondents who were 10 years or less out of residency reported MP-MRI to be very helpful compared to 24.8% of respondents 11 years or more out of residency (30/98 versus 45/183).When asked approximately what proportion of patients with positive MP-MRI findings who subsequently undergo MP-MRI targeted prostate biopsies are found to have biopsies positive for prostate cancer, 28% reported positive biopsies “often” or “very often” (65/233), 33% reported positive biopsies “sometimes” (77/233), and 39% reported positive biopsies “rarely” or “never” (91/233). Similarly, when asked about how closely respondents find MP-MRI results to correlate with final pathology after prostatectomy, 11% reported strong correlation (26/233), 62% reported moderate correlation (145/233), 20% reported weak correlation (46/233), and 7% reported no correlation (16/233).The use of multi-parametric (MP-MRI) to diagnose prostate cancer has been the subject of intense research, with many studies showing positive results. To our knowledge, no other studies have attempted to better understand the accessibility, role, and perceived accuracy of MP-MRI in practice by surveying practicing urologists. We found practicing in an academic center to be strongly associated with increased use of MP-MRI (p=0.0001), which is likely due to a combination of increased access, familiarly with recent literature, and interdisciplinary efforts within academic institutions.There is very little within the literature evaluating practitioner access to MP-MRI, particularly outside academic centers. While 89% of our respondents reported to be within one hour of a facility with MP-MRI capabilities, 72% still felt that their use of MP-MRI was limited by lack of access. While the exact reasons for this discrepancy could not be gleaned from the survey data, causes may relate to perceived lack of quality in the MP-MRI provided, difficulty with patient scheduling, or lack of strong multi-disciplinary relationship with radiologists.Another possible reason that MP-MRI has been slow to gain wide spread popularity is that many (59% within our study) feel that the cost of these studies is prohibitive, and that issues surrounding insurance reimbursement negate the perceived value of the study. As the presence of MP-MRI becomes stronger in national and international management guidelines, insurance approval and payment for these studies may become more streamlined -9. LikewThe use of MP-MRI in the management of prostate cancer has gained a lot of attention in recent literature; however, the appropriate use in clinical practice has not yet been established. Despite the growing body of evidence supporting the use of MP-MRI guided biopsies in lieu of saturation biopsies, many of our respondents did not echo this sediment. We found that only 42% of respondents felt that the current literature provided evidence for some role for MP-MRI in localized prostate cancer patients, while 31% disagreed and 27% could not decide. These results further prove the level of controversy surrounding MP-MRI.MP-MRI can be used to provide guidance for tissue sampling either directly, via a cognitive approach, or through fusion software. MP-MRI guided biopsies have been shown to upgrade Gleason grade and detect otherwise undiagnosed anterior gland tumors in a significant number of patients -13. DespIn intermediate or high-risk prostate cancer patients who appear to be candidates for a nerve sparing prostatectomy, MP-MRI can be helpful for surgical planning -15. Of oIt has also been proposed to incorporate MP-MRI into active surveillance protocols -17. WhilWhile several studies have shown the high accuracy of MP-MRI, respondents seemed to largely express skepticism , 22-23. There are several limitations to our study. The response rate of only 3.8% is clearly less than ideal, and it would have been preferable if all respondents had answered every survey question. Never the less, with 276 respondents exhibiting a wide range of demographics, we feel that the responses provide an adequate sample size. The survey was designed to provide data that could be easily analyzed, yet multiple choice answers carry the risk of being leading. It might be anticipated that the survey would have promoted a bias toward MP-MRI, yet the results indicate otherwise. Further directions for research include a survey administered to a large number of practitioners across different vendor platforms and in various institutional settings. Also, it would be helpful to obtain insight into why practitioners chose their various answers, would could be accomplished with a more in-depth amended to include the option for free response.Prostate MRI has been shown within the literature to be a highly useful diagnostic tool. However, our study indicates a relatively low level of support for the use of MP-MRI in clinical practice. This indicates the need for improved education, access, and standardization of treatment recommendations to address the challenges of implementing this new technology into practice. Further research is needed to confirm the favorable results of MP-MRI across different vendor platforms, in various institutional settings, and using available radiologic-pathologic correlation.


Loss to follow-up (LTFU) is a cause of potential bias in clinical studies. Differing LTFU between study groups may affect internal validity and generalizability of the results. Understanding reasons for LTFU could help improve follow-up in clinical studies and thereby contribute to goals for prevention, treatment, or research being achieved. We explored factors associated with LTFU of mother-child pairs after inclusion in the ANRS 12140-Pediacam study.From November 2007 to October 2010, 4104 infants including 2053 born to HIV-infected mothers and 2051 born to HIV-uninfected mothers matched individually on gender and study site were enrolled during the first week of life in three referral hospitals in Cameroon and scheduled for visits at 6, 10 and 14 weeks of age. Visits were designated 1, 2 and 3, in chronological order, irrespective of the child’s age at the time of the visit. Mother-child pairs were considered lost to follow-up if they never returned for a clinical visit within the first six months after inclusion. Uni- and multivariable logistic regression were adjusted on matching variables to identify factors associated with LTFU according to maternal HIV status.LTFU among HIV-unexposed infants was four times higher than among HIV-exposed infants . Emergency caesarean section (adjusted Odds Ratio (aOR) = 2.46 95% Confidence Interval (CI) [1.47-4.13]), young maternal age , and absence of antiretroviral treatment for prophylaxis  were independently associated with LTFU among HIV-exposed infants. Factors associated with LTFU among HIV-unexposed infants included young maternal age , low maternal education level  and housewife/unemployed mothers .Failure to return for at least one scheduled clinical visit is a problem especially among HIV-unexposed infants included in studies involving HIV-exposed infants. Factors associated with this type of LTFU included maternal characteristics, socio-economic status, quality of antenatal care and obstetrical context of delivery. Enhanced counselling in antenatal and intrapartum services is required for mothers at high risk of failure to return for follow-up visits.The online version of this article (doi:10.1186/s12889-015-1555-2) contains supplementary material, which is available to authorized users. HIV/AIDS remains a disease of public health importance and mother-to-child transmission (MTCT) is one of the major problems . Recent Poor socioeconomic conditions have been identified as the main reason for LTFU in such studies and PMTCT programs. They include poverty, lack of paternal support, poor mobility, long distances between residence and care structures, and the cost of transport . TherefoData used in this analysis were obtained from the ANRS-Pediacam cohort based in three referral hospitals in Cameroon , Essos Hospital Center in Yaoundé (EHC) and the Laquintinie Hospital in Douala (LH)) and coordinated by the Centre Pasteur of Cameroon. The ANRS 12140-Pediacam study was designed to assess the feasibility of early diagnosis of HIV and early antiretroviral multitherapy in HIV-infected infants, and to evaluate the humoral response of these children to vaccines of the Expanded Program on Immunization (EPI).The ANRS 12140-Pediacam is an ongoing prospective cohort study involving two consecutive phases. The first phase of the study included all infants born live to HIV-infected mothers with documented serostatus (group 1) identified before the 8th day of life from November 2007 to October 2010 and an equivalent number of infants born to HIV-uninfected mothers matched individually on gender and recruitment site. All newborns were expected to attend a clinical visit, according to the Cameroon National EPI calendar, at ages 6, 10 and 14 weeks for routine vaccination. Infant follow-up in the first phase was scheduled to coincide with this EPI timetable to minimise the number of visits to the hospital required. However, visits (follow-up visits) are designated hereafter as the first, second and third, independent of the age of the child. Samples for HIV virological testing were collected from HIV-exposed infants at the first follow-up visit (scheduled for 6 weeks), as previously described ,14. HIV Incentives, including free medical support for consultation, biological analysis, additional vaccines and reimbursement of transport costs, were provided to parents/caregivers by the project during follow-up visits.All HIV-infected children and control groups of HIV-uninfected infants followed since birth, born to either HIV-infected or non-infected mothers were eligible for the second phase of the Pediacam project planned from 14 weeks to 5 years. This phase is not described here as it is not relevant to this study.Socioeconomic and demographic characteristics of the families and obstetrical characteristics were collected at enrolment by questionnaire-based interview with mothers and examination of their hospital booklets. Questionnaires were also used subsequently to collect data on infant vital status, pathologies, vaccinations and HIV testing process as appropriate. Reminder phone calls were made to families who did not return for a follow-up visit within one week of the planned date.The ANRS 12140- Pediacam study has received ethical approval in Cameroon from the National Ethic Committee and in France from the Biomedical Research Committee of the Pasteur Institute of Paris. An administrative authorization was also delivered by the Cameroonian Ministry of Public Health. Written informed consent was obtained from parents or guardians prior to inclusion of infants into the research project.th week of life were eligible for this analysis.All infants born live to HIV-infected mothers and infants born to HIV-uninfected mothers matched on gender and recruitment site, enrolled from November 2007 to October 2010 into the first phase of the ANRS-Pediacam study planned from the first to 14The main outcome was loss to follow-up (LTFU) or “failed to return for at least one scheduled clinical visit” defined as mother-child pairs who never returned for a clinical visit during the first 6 months of age after inclusion in the ANRS-Pediacam study. Those who attended clinical visits, even if only once, were not considered to be lost to follow-up. The threshold of six months was chosen because several studies indicate that knowing the HIV status of the child before age 6 months favours early initiation of HAART ,16. RetuThe covariables considered included variables pertaining to infant’s characteristics at birth , maternal characteristics and socio-economic status , quality of antenatal care and obstetrical context of delivery  and paternal characteristics .Maternal and infant characteristics are described using frequencies for categorical variables, medians and interquartile ranges for continuous variables. Their relation to LTFU was evaluated in each group defined by maternal HIV serostatus because of the differences observed between the two groups. For multiple births, only the first infant was included in the analysis. To examine covariables associated with LTFU, logistic regression models were adjusted on site and gender (matching variables) as appropriate for the matched study design . The iniIn total, 4104 mother-child pairs were enrolled in the first phase of the Pediacam study between November 2007 and December 2010, including 2051 HIV-exposed infants and 2053 HIV-unexposed infants. Among the 4104 mother-infant pairs initially enrolled, 68 were excluded because data for mother and infant characteristics at enrolment were not available. Further, 123 multiple gestation births were excluded to avoid duplication of maternal characteristics. Our analyses were thus conducted on the remaining 3913 mother-infant pairs (1964 HIV-exposed and 1949 HIV-unexposed). About 85.4% (3342/3913) of all the infants enrolled were born in the three maternities participating in the study, 13.9% (n = 542) in other maternities in the same cities and 0.7% (n = 29) at home or on their way to a healthcare facility. The two groups of infants differed significantly at enrolment . For HIV-infected mothers, univariable analysis indicated that the following factors were associated with LTFU: prematurity, low birth weight, infant hospitalization at birth, young maternal age <25 years), few antenatal visits (fewer than 4), delivery by emergency caesarean section, late HIV diagnosis (during pregnancy or at delivery), absence of PMTCT prophylaxis, and TCD4 lymphocyte cell count never determined , few antenatal visits (fewer than 4), low maternal education level, economically disadvantaged family, absence of running water at home, absence of a functional fridge at home, both lower maternal and paternal education level, and absence of both maternal and paternal socio-professional activity were associated in univariable analysis with LTFU  than HIV-uninfected mother-infant pairs (36.7%). Few relevant studies that have addressed the problem of LTFU included a control group. In a nested case control study including 594 HIV-uninfected and 456 HIV-infected mothers in Zimbabwe , dropout.2-24.8, et al. [The factors associated with LTFU differed according to maternal HIV status. HIV-uninfected mothers aged ≤ 35 years (<25 years and 25–35 years) more likely than those >35 years old to be LTFU. Similarly, Kaplan et al.  reportedet al. . In Afriet al. . Lower met al. . In Indiet al. . The diret al. reported an association in Malawi between LTFU and teaching/student occupations: infants born to HIV-infected mothers who were teachers or students were less likely to attend follow-up visits. This might have been due to concerns about stigmatization from being identified as HIV positive. Our analysis indicated that HIV-unexposed infants born to employed mothers were more likely to be LTFU possibly because they do not have enough time off from work to attend visits. In US, Krishnan et al. reported that activities such as work or school prevent participants from adhering to follow-up [Ioannidis ollow-up .et al. [LTFU of HIV-infected mother-infant pairs was also significantly and independently associated with the absence of ART prophylaxis. This observation is consistent with findings in Uganda presented by Ahoua et al. ; they reWe found an association between LTFU and emergency caesarean section, and this contrasts with a study in Malawi where this factor was not reported to be associated with LTFU . HoweverA study in Malawi identified low birth weight as a significant factor influencing return to follow-up visits . This waIn this study, 67 HIV-infected mother-infant pairs who were LTFU were traced: 26 of the infants were deceased Figure .Previous studies identified poor socio-economic conditions as a reason for LTFU. For example, HIV-infected women in India staying in families with higher economic status were found to be less likely than women living at a lower economic status to be LTFU both before and after delivery . A studyThe factors associated with LTFU in the HIV-infected mothers group pertained to antenatal care and the obstetrical context of delivery; those associated with LTFU in the HIV-uninfected mothers group were largely relevant to socioeconomic status. For both groups of maternal HIV status, young maternal age was associated with LTFU.One of the strengths of this study is its multicentre design including three referral hospitals with different working practices and recruitments, reflecting the diversity of practical management of PMTCT programmes. Moreover, these centres are located in two large cities of Cameroon with heterogeneous populations, albeit mostly living in urban areas. In contrast, recruitment of HIV-infected and HIV-uninfected women in the same health centres may have affected the acceptability to, and follow-up of, HIV-uninfected women, ad this is a limitation of the study. In addition, the low rate of LTFU among HIV-infected mother-child pairs, relative to other sub-Saharan Africa studies, could have reduced the power of the study to identify factors associated with LTFU.Factors associated with LTFU  of mother-child pairs differ according to maternal HIV serostatus. For HIV-infected mother-child pairs, these factors pertain to the quality of antenatal care and the obstetrical context of delivery whereas for HIV-uninfected mother-child pairs, they are related to maternal characteristics and socio-economic status. Ensuring the quality of antenatal and intrapartum services could help in improving follow-up. Provision of training for clinicians in antenatal and intrapartum services may help them identify mothers at high risk of missing follow-up visits. Identification of such cases would allow targeted counselling and education about the importance of follow-up.