Vertebral body replacement is well-established to stabilize vertebral injuries due to trauma or cancer. Spinal implants are mainly manufactured by metallic alloys; which leads to artifacts in radiological diagnostics; as well as in radiotherapy. The purpose of this study was to evaluate the biomechanical data of a novel carbon fiber reinforced polyetheretherketone (CF/PEEK) vertebral body replacement (VBR). Six thoracolumbar specimens were tested in a six degrees of freedom spine tester. In all tested specimens CF/PEEK pedicle screws were used. Two different rods (CF/PEEK versus titanium) with/without cross connectors and two different VBRs (CF/PEEK prototype versus titanium) were tested. In lateral bending and flexion/extension; range of motion (ROM) was significantly reduced in all instrumented states. In axial rotation; the CF/PEEK combination (rods and VBR) resulted in the highest ROM; whereas titanium rods with titanium VBR resulted in the lowest ROM. Two cross connectors reduced ROM in axial rotation for all instrumentations independently of VBR or rod material. All instrumented states in all planes of motion showed a significantly reduced ROM. No significant differences were detected between the VBR materials in all planes of motion. Less rigid CF/PEEK rods in combination with the CF/PEEK VBR without cross connectors showed the smallest reduction in ROM. Independently of VBR and rod material; two cross connectors significantly reduced ROM in axial rotation. Compared to titanium rods; the use of CF/PEEK rods results in higher ROM. The stiffness of rod material has more influence on the ROM than the stiffness of VBR material. Vertebral body replacement (VBR) combined with dorsal instrumentation is the preferred treatment option to achieve decompression and restore stability of the spinal column either in patients with a traumatic fracture (>AO Spine A3) ,3,4,5,6 Various biomechanical ,6,11,12 n = 6) fresh frozen human thoracolumbar spine units (Th11–L3) were tested with a mean age at death of 59.2 years (ranging from 56 to 65 years). The bodies were donated by people who had given their written informed consent prior to death to use their bodies for scientific and educational purposes. For standardization and homogeneous study conditions, all tested vertebral bodies were analyzed via pre-interventional CT scan . Trabecular bone mineral density (BMD) was measured between 66.8 mg/ccm and 100.4 mg/ccm . Spinal specimens with deformities, previous spinal surgery, structural disorders or post traumatic disorders were excluded. The vacuum sealed, frozen specimens (minus 20 °C) were thawed overnight at 6 °C before all soft tissues were removed, preserving supporting spinal ligaments and joint capsules. Six  were inserted in all specimens at the Th11/12 level, as well as at the L2/3 level for all investigated cases  and a 6.0 mm standard titanium rod system . Titanium cross connectors  completed the setup.For posterior instrumentation, pedicle entry points were identified according to the anatomical landmarks and controlled using bi-planar fluoroscopy. Nonmetallic, radiolucent 6.5 × 45 mm CF/PEEK pedicle screws  and the caudal vertebra (L3) were embedded in polymethylmethacrylate  with sufficient clearance for pedicle instrumentation and with the midline of all five vertebrae aligned horizontally. Flanges in the upper and lower PMMA blocks provided a rigid fixation to the spine simulator. To measure intersegmental motion of the treated (Th12–L2) and adjacent segments (Th11/12 and L2/3), an ultrasound-based 3D motion analysis system  was fixed to the ventral side of the vertebrae . AccordiAt first, the intact specimens (Th11-L3) were loaded with pure moments of 7.5 Nm to record a baseline. Afterwards a corpectomy of the target vertebra L1 with removal of the cranial and caudal discs was conducted with standard surgical tools, according to clinical routine. For the reconstruction of the anterior spinal column, either an expandable, nonmetallic radiolucent CF/PEEK VBR   or an exAll surgical procedures were performed by the first author (experienced senior spine surgeon). Two plane  native radiographs were taken to control and document correct positioning of pedicle screws and VBRs a,b. FinaFor each specimen, the following states were tested with alternatives for the VBR (CF/PEEK or titanium), the posterior rod material (CF/PEEK or titanium), and the application of cross connectors.(1)native):Flexibility test, Native (Corpectomy and instrumentation with VBR and pedicle fixation with CF/PEEK rod.(2)VBR instrumented with CF/PEEK rod (CF_CF):Flexibility test—CF/PEEK Change of posterior rod fixation to titanium rod.(3)VBR Instrumented with titanium rod, (CF_Ti):Flexibility test—CF/PEEK Change of VBR to titanium.(4)VBR instrumented with titanium rod (Ti_Ti):Flexibility test—titanium Addition of two cross connectors to the posterior rods dissecting the ligamentum supraspinale/interspinale.(5)VBR instrumented with titanium rod and 2 cross connectors (Ti_Ti_cc):Flexibility test—titanium Change to CF/PEEK VBR.(6)CF/PEEK VBR instrumented with titanium rod and 2 cross connectors (CF_Ti_cc):Flexibility test—Change rods to CF/PEEK.(7)CF/PEEK VBR Instrumented with CF/PEEK rod and 2 cross connectors (CF_CF_cc).Flexibility test—p-values were calculated with adjustment for multiple corrections.Statistical analysis of the ROM was performed using the SPSS software . Data and results of the ROM were evaluated for the three motion directions, normalized and compared to the motion of the native segment. A general linear model (GLM) with repeated measures was used for statistical comparison. Results of the ROM are displayed in absolute metrics  and as bp < 0.05) reduced ROM compared to the native state. All instrumented states in all planes of motion showed a significantly (p = 1.0) in ROM were measured for the different VBR material (CF/PEEK versus titanium), posterior rod material (CF/PEEK versus titanium) or additional cross connectors. In lateral bending, the mean ROM in the native state was 11.2° (SD 3.26). The various instrumentations reduced ROM to 0.24°–0.30° (SD 0.18–0.21). No significant differences  in ROM were measured for the different VBR material (CF/PEEK versus titanium), posterior rod material (CF/PEEK versus titanium), or additional cross connectors.In flexion/extension, the mean ROM in the native state was 10.3° (SD 2.4). Varying instrumentations reduced ROM to 0.29°–0.35° (SD 0.12–0.31). No significant differences  the ROM in axial rotation. Posterior rod material (CF/PEEK or titanium) had greater effects to the ROM than VBR material (CF/PEEK or titanium) in axial rotation.In axial rotation the mean ROM in native state was 4.83° (SD 0.95). The various instrumentations reduced ROM to 1.84°–3.95° (SD 0.33–0.9). Less rigid CF/PEEK rods combined with CF/PEEK VBR without the use of cross connectors showed the smallest ROM reduction to 3.95° (SD 0.9) in axial rotation. Titanium rods with titanium VBR and the use of two cross connectors reduced ROM to 1.84° (SD 0.33). Independent of type of VBR or rod material (CF/PEEK or titanium), two additional cross connectors reduced significantly, (p < 0.05) reduced ROM compared to native state. Equally to the index level, all instrumented states showed in all planes of motion a significantly (p > 0.78) in ROM were measured for the different VBR materials (CF/PEEK versus titanium), posterior rod materials (CF/PEEK versus titanium) or additional cross connectors. Similar to the index segment, mean ROM of the L2/3 segment in lateral bending was slightly higher (0.2°) with the use of two cross connectors.In lateral bending the mean ROM in native state was 7.9° (SD 2.64). The various instrumentations reduced ROM to 1.38°–1.76° (SD 0.66–1.02). No significant differences . The various instrumentations reduced ROM to 1.13°–1.50° (SD 0.58–0.95). No significant differences in ROM were measured for the different VBR material (CF/PEEK versus titanium), posterior rod material (CF/PEEK versus titanium) or additional cross connectors. Similar to lateral bending mean ROM was slightly (0.2°) higher with the use of two cross connectors.p < 0.05). Posterior rod material (CF/PEEK or titanium) had greater effects to the ROM than VBR material (CF/PEEK or titanium) in axial rotation.In axial rotation the mean ROM in native state was 3.88° (SD 2.33). The various instrumentations reduced ROM to 1.27°–1.96° (SD 0.48–0.85). Less rigid CF/PEEK rods combined with CF/PEEK VBR without the use of cross connectors again showed smallest ROM reduction to 1.96° (SD 0.85) in axial rotation. Titanium rods with titanium VBR and the use of two cross connectors reduced ROM to 1.27° (SD 0.48). Independently to VBR or rod material (CF/PEEK or titanium) two cross connectors significantly reduced ROM in axial rotation (p < 0.05) reduced ROM compared to native state. Equally to the index and caudal adjacent level, all instrumented states showed, in all planes of motion, a significantly . The various instrumentations reduced ROM to 2.61°–2.96° (SD 1.41–1.63). No significant differences in ROM were measured for the different VBR material (CF/PEEK versus titanium), posterior rod material (CF/PEEK versus titanium) or additional cross connectors. Similar to the index and caudal adjacent level mean ROM of Th11/12 segment was slightly (0.3°) higher with the use of two cross connectors in lateral bending.In flexion/extension the mean ROM in native state was 6.59° (SD 2.80). The various instrumentations reduced ROM to 1.87°–2.28° (SD 1.34–1.90). No significant differences in ROM were measured for the different VBR material (CF/PEEK versus titanium), posterior rod material (CF/PEEK versus titanium) or additional cross connectors. Similar to lateral bending the mean ROM was slightly (0.3°) higher with the use of two cross connectors in lateral bending.p < 0.05) ROM in axial rotation slightly. Posterior rod material (CF/PEEK or titanium) had greater effects on the ROM than VBR material (CF/PEEK or titanium) in axial rotation.In axial rotation the mean ROM in native state was 4.27° (SD 1.93). The various instrumentations reduced ROM to 2.70°–3.25° (SD 1.27–1.47). Less rigid CF/PEEK rods combined with CF/PEEK VBR without the use of cross connectors again showed smallest ROM reduction to 3.25° (SD 1.45) in axial rotation. Titanium rods with titanium VBR and the use of two cross connectors reduced ROM to 2.70° (SD 1.42). Independently to VBR or rod material (CF/PEEK or titanium) two cross connectors reduced . For this purpose a human cadaveric corpectomy defect model was tested in a spine tester with three-dimensional motion measurement of each segment. The implants presented in this study were designed to reconstruct spinal stability after corpectomy in traumatic or malignant vertebral fractures. Reconstruction was varied by anterior VBR material (CF/PEEK versus titanium) and dorsal instrumentation’s material (CF/PEEK versus titanium) with or without additional cross connectors. Our results indicate the less rigid CF/PEEK rods combined with CF/PEEK VBR without the use of cross connectors reduces the ROM in axial rotation by only 18%  compared to an intact specimen . In all other modes  the CF/PEEK rods combined with CF/PEEK VBR provided comparable reduction in ROM when compared to titanium rods in combination with a titanium VBR.p < 0.05) the ROM in axial rotation. Posterior rod material (CF/PEEK or titanium) had greater effects on the ROM than VBR material (CF/PEEK or titanium) in axial rotation (p < 0.05) reduced mean ROM by 97% and 96% compared to the intact specimen, respectively. There were no significant differences between different VBR and posterior rod system (CF/PEEK versus titanium).Titanium rods in combination with a titanium VBR and the use of two cross connectors demonstrated significantly more stiffness in axial rotation with a decrease of ROM of 62% . Independently to VBR or rod material (CF/PEEK or titanium) two cross connectors significantly reduced  reduced ROM in all planes of motion. No significant differences were detected between the VBR materials (CF/PEEK versus titanium) in all planes of motion. Less rigid CF/PEEK rods in combination with the CF/PEEK VBR without cross connectors showed the smallest reduction in ROM. Independently of VBR and rod material (CF/PEEK versus titanium), two cross connectors significantly reduced ROM in axial rotation and are therefore highly recommended. Compared to titanium rods, the use of CF/PEEK rods results in higher ROM. The stiffness of rod material has more influence on the ROM than the stiffness of VBR material.Compared to the native state, all instrumented states showed a significantly (


In each subject, ten LVETs were obtained from ten heartbeats selected properly from one-minute recording. The differences of the measured LVETs between the PCG and one of the ICG, PPGneck and PPGfinger were −68.2 ± 148.6 ms, 4.8 ± 86.5 ms and −7.0 ± 107.5 ms, respectively. As compared with the PCG, both the ICG and PPGfinger underestimated but the PPGneck overestimated the LVETs. Furthermore, the measured LVET by the PPGneck was the closest to that by the PCG. Therefore, the PPGneck may be employed to improve the LVET measurement in applying the ICG for continuous monitoring of SV in clinical settings.Cardiac stroke volume (SV) is an essential hemodynamic indicator that can be used to assess whether the pump function of the heart is normal. Non-invasive SV measurement is currently performed using the impedance cardiography (ICG). In this technology, left ventricular ejection time (LVET) is an important parameter which can be determined from the ICG signals. However, the ICG signals are inherently susceptible to artificial noise interference, which leads to an inaccurate LVET measurement and then yields an error in the calculation of SV. Therefore, the goal of the study was to measure LVETs using both the transmission and reflection photoplethysmography (PPG), and to assess whether the measured LVET was more accurate by the PPG signal than the ICG signal. The LVET measured by the phonocardiography (PCG) was used as the standard for comparing with those by the ICG and PPG. The study recruited ten subjects whose LVETs were simultaneously measured by the ICG using four electrodes, the reflection PPG using neck sensors (PPG Cardiogenic shock refers to a group of syndromes in which heart function is extremely decreased, resulting in a significant reduction in cardiac output and causing severe acute peripheral circulatory failure. Consequently, cardiogenic shock is the ultimate manifestation of heart pump failure. When the heart pump is insufficient, it cannot maintain its minimum cardiac output, leading to a drop in blood pressure. Seriously insufficient blood supply to vital organs and tissues can lead to systemic circulatory dysfunction. Thus, a series of pathological and physiological processes characterized by ischemia, hypoxia, metabolic disorders, and vital organ damages will emerge as a result ,2. In trKubicek et al. have proposed a non-invasive measurement technique to measure the cardiac hemodynamic parameters using the impedance cardiography (ICG) method . High-frLeft ventricular ejection time (LVET) is an important parameter in the SV determination with the ICG technology. As we known, detection of the time difference between the first and the second heart sound in the heart sound signal is a standard method for the LVET measurement. In the LVET measurement with the ICG technique, the ICG waveform is first differentiated and the time difference is determined between the first zero crossing point and the lowest point in the differential ICG signal. However, both low signal-to-noise ratio and body motion-induced interference noise frequently make the ICG signal instable, which in turn causes a decrease in the accuracy and preciseness of the LVET measurement. Further, this leads to a big error in the assessed SV. In recent years, arterial blood pressure waves have been derived through the PPG method. The PPG method is mainly divided into two types: one is transmission and the other is reflection . The traThe purpose of this study was to evaluate whether the measured LVET at different locations using the PPG technique was more reliable than that by the ICG technique, and to see whether the transmission PPG or the reflection PPG could produce more accurate LVETs as compared with the phonocardiography (PCG). In the study, ten healthy adult males were included to simultaneously perform four kinds of LVET measurement. The results showed that the LVET estimated by using the PPG technique was more accurate and stable than the ICG technique. LVETs measured from the arteries close to the heart by the transmission PPG were more stable than those measured from the peripheral arteries by the reflection PPG.neck (reflection PPG sensor placed on the neck), PPGfinger (transmission PPG sensor placed on the finger), and PCG signals. The PPGneck sensor was embedded in a digital chip  and it communicated with a microcontroller unit (MCU)  by means of the I2C interface. The analog to digital conversion (ADC) for the other signals was processed by the MCU. The resolution of ADC was 12 bits, and the sampling frequency was 500 Hz. After the signal conversion was completed, the MCU transmitted these signals to a computer for display and storage through a USB port.The hardware circuit diagram of this study for physiological signal acquisition is shown in neck sensor is placed on the left side of the neck, the PPGfinger sensor is clamped to the finger of subject’s left hand, and the PCG sensor is placed over the chest and near the heart.The electrodes of the measurement system and the placement of the PPG sensors are shown in brho is the blood impedance that is assumed to be a constant value of 150 ohm-cm, L is the distance between the B and C electrodes, Z0 is the impedance of thoracic cavity, and dZ/dt(max) is the maximum change of the ICG impedance signal. According to Equation (1), the SV has an absolute linear relationship with the LVET. Z/dt of ICG signal. The B and X points in the heart sound signals represent the opening and closure of the aortic valve, respectively. The time difference between these two points (B and X) is defined as the LVET. In the differential ICG signal, dZ/dt, the LVET is defined the time duration between the first zero crossing point and the minimum point.From the ICG signal, cardiac SV can be calculated by the following equation proposed by Kubicek :(1)SV=rhIn the PCG signals, the B and X points are the time points of the maximum peak of the first and second heart sound, respectively. The LVET is determined as the time duration between the B and X points. Therefore, the LVET is initiated by the opening of the aortic valve, and is terminated by the closing of the aortic valve. During the LVET, the blood is actually pumped from the left ventricle to the aorta. Thus, in the blood pressure waveform, the point B represents the time for the first zero crossing point of its differential waveform and the point X the time for the first maximum valley of its differential waveform. Moreover, the point X should occur just before the dicrotic notch, and the location of the dicrotic notch may be used to estimate the point X. According to the ICG theory, the B and X points in the differential ICG waveform are used to determine the LVET, as show in The experiment in this study was divided into three steps, including the collection of experimental data, data processing and analysis, and statistical analysis. This study recruited ten healthy male subjects with no cardiac diseases or injured limbs. Their age was between 22 and 27 years (23.5 ± 1.7 years), weight was between 49 and 96 Kg (66.0 ± 13.1 Kg), and height was between 163 and 185 cm (172.6 ± 5.8 cm). This experiment was approved by the Research Ethics Committee of China Medical University & Hospital (No. CMUH107-REC3-061), Taichung, Taiwan.finger, and PPGneck signals were measured at the same time. After the signals were filtered by the 20 Hz third-order Butterworth low-pass filter and the 0.2 Hz first-order Butterworth high-pass filter, the best 10 heartbeat signals for each subject were properly selected by visual inspection. The 10 LVETs corresponding to the 10 heartbeats in the PCG signal were uncovered and used as the standard values. Furthermore, LVETs corresponding to the 10 heartbeats in the ICG and PPG signals were detected, respectively. Finally, the means and the standard deviations of the difference between the PCG-measured LVET and the other techniques-measured LVET  were used to investigate their accuracy and stability.The measurement duration for each subject was one minute. During the measurement, ECG, PCG, ICG, PPGneck (short dash line), PPGfinger (dash-dot-dot line), and their differential signals, ICG (long dash line), PPGfinger (dash-dot line), and PPGneck (medium dash line). It can be seen that both PPG signals are more stable than the ICG signal, and there is a significant delay of about 78 ms between the PPGfinger signal and the PPGneck signal.Typical physiological signals measured from one subject in this study are shown in finger, and PPGneck signals. r2, between the standard LVET measured by the PCG signal and the three LVETs measured by the ICG, PPGneck, and PPGfinger signals for the ten subjects. The correlation coefficients were 0.178 ± 0.162, 0.270 ± 0.199 and 0.129 ± 0.149 for the ICG, PPGneck, and PPGfinger signals, respectively. The PPGneck technique had a highest correlation coefficient, 0.680, for subject 5. The ICG technique had a worst correlation coefficient, 2.31 × 10−6, for subject 4. neck and PPGfinger) and the LVETs measured by the PCG technique are −68.2 ± 148.6 ms, 4.8 ± 86.5ms and −7.0 ± 107.5 ms. In brief, both the ICG and PPGfinger underestimated but the PPGneck overestimated the LVETs as compared with the PCG. Besides, it can be seen that the LVETs measured by the PPGneck were the closest to those measured by the PCG. In addition, the measured LVETs were significantly smaller with the ICG than the PCG (p < 0.01), but the measured LVETs with the PPGneck and PPGfinger did not significantly differ from those with the PCG . neck and PPGfinger. We found that the larger the LVET measured by the PCG, the more the underestimated amount of the LVET measured by the ICG and PPGfinger. The accuracy of LVET measurement is the lowest for the ICG technique, as shown in neck techniques are beyond the −1.96 SD, as shown in For each subject, 10 heartbeats were selected from the ICG, PCG, PPG® technique.The ICG technology was developed about half a century ago . But, thneck and PPGfinger, are found to be more stable than the ICG signal. As shown in It is usually difficult to obtain a stable ICG signal, since during the ICG measurement the tissue impedance is vulnerable to a variety of body motions, such as the body shaking, breathing and other human disturbances . Moreoveneck signal in individual subject are properly selected to estimate the LVET. Then, the same ten-beat segments from the ICG and PPGfinger signals are used to determine the LVET. A best correlation coefficient (0.68) exists between the LVETs measured by the PPGneck and PCG techniques in Subject 5. On the contrary, the correlation coefficient of the LVET measured by the ICG and PCG technique is only 0.153 in that subject. neck and PPGfinger for Subject 5, in which there is an obvious dicrotic notch point on individual heartbeat in the PPGneck signal. But, the dicrotic notch point in the ICG signal only shows up on some heartbeats. neck, and PPGfinger for Subject 4, in which the dicrotic notch point does not appear in the ICG signal. As a result, the correlation coefficient of his LVET measured by the ICG and PCG techniques is pretty low (only 2.31 × 10−6). Although the PPGfinger signal usually shows the clear dicrotic notch points, these dicrotic notch waves possess serious distortion, and their happening time can be easily affected by the presence of hydrostatic pressure in large arteries or vena cava, and the vascular characteristics.In our experiments, we find that the PPG signal appears more stable than the ICG signal. Therefore, ten-beat segments from the PPGneck, with a relatively large difference of −7.0 ± 107.5 ms by using the PCG and the PPfinger in the study. Also, with the ICG, the measured LVET is found to be far from that with the PCG, with a big difference of −68.2 ± 148.6 ms.The second issue is about why the measured LVETs with the reflection PPG sensor have more accurate than those with the transmission PPG sensor, as compared with the standard (that is the LVETs determined by the PCG). This is because the transmission PPG sensor is put on the fingers during the measurement, and the blood vessels in the fingers are remote to the heart and belong to the peripheral circulation. As blood is pumped from the heart, it will be delivered to the periphery after several times of bifurcations and conversions. The peripheral arterial blood pressure waves are really affected by the characteristics of the arterial blood vessels along the delivering path. Thus, alteration and transformation in the peripheral pressure waveform finally results in inaccurate LVET assessment. In the reflection PPG, when the sensor are positioned at some place close to the aorta , the measured PPG signal seems less affected by the characteristics of the conduit arteries from the heart to the neck. Nevertheless, the present result shows that the measured LVETs with the reflection PPG are more accurate than those with the transmission PPG. Furthermore, a relatively small difference of 4.8 ± 86.5 ms exists between the LVETs measured by using the PCG and the PPGLVET measurement with the PCG signalshas been considered as the standard method . The senAccording to the LVET measurement with the arterial pressure signal, the time of the arterial pressure beginning to rise is the start time for the LVET, whereas the time of the arterial pressure commencing to fast decline is the end time for the LVET.In previous studies, most investigators have applied this criterion in the ICG signal to detect the LVET ,5,12. Thneck signal is used to determine the LVET. Through the experiments, compared with the LVET by the standard method, the LVET measured by the PPGneck signal is indeed more accurate than that by the ICG. Secondly, the closer the artery detected by the PPG sensor is to the heart, the more accurate the measured LVET will be. Although the LVETs measured by PPGneck signals only has a moderate correlation with those by the PCG signals, and still has a large variability for the correspondent difference with the PCG signals, they appear more accurate and higher correlation than those measured by the ICG signals. In the future, according to the results of this study, the reflection PPG sensors may be utilized in the ICG instrument to enhance the stability and accuracy of the LVET measurement in clinical settings.This paper proposes two concepts to improve the accuracy in calculating cardiac SV with the ICG technique, in which PPG


Influenza incidence and seasonality, along with virus survival and transmission, appear to depend at least partly on humidity, and recent studies have suggested that absolute humidity (AH) is more important than relative humidity (RH) in modulating observed patterns. In this perspective article, we re-evaluate studies of influenza virus survival in aerosols, transmission in animal models and influenza incidence to show that the combination of temperature and RH is equally valid as AH as a predictor. Collinearity must be considered, as higher levels of AH are only possible at higher temperatures, where it is well established that virus decay is more rapid. In studies of incidence that employ meteorological data, outdoor AH may be serving as a proxy for indoor RH in temperate regions during the wintertime heating season. Finally, we present a mechanistic explanation based on droplet evaporation and its impact on droplet physics and chemistry for why RH is more likely than AH to modulate virus survival and transmission. The amount of water vapour in air can also be expressed as a partial pressure in units such as atmospheres or millibars. A related concept is specific humidity, which is the mass of water vapour per mass of air, such that absolute and specific humidity are related by the density of air.There has been a surge of interest in understanding transmission and seasonality of influenza and other infectious diseases. Humidity, among other factors, appears to play an important role in influenza transmission, and a confluence of recent studies has suggested that absolute humidity (AH) rather than relative humidity (RH) modulates influenza virus survival, transmission and seasonality –4. While−3, while at 0°C and 100% RH, the corresponding AH is 4.8 g m−3. On the other hand, an AH of 4 g m−3 corresponds to an RH of 83% at 0°C and 17% at 25°C. Greater detail on different measures of atmospheric moisture and their relationship to human health can be found in Davis et al. ) [et al.  present Furthermore, we can calculate the deposition efficiency in different regions of the respiratory system to contrast where the droplets might deposit if inhaled . Table 2−1, or 40 times the initial concentration of 9 mg ml−1. Thus, we expect it to crystallize at an ambient RH of approximately 90% (concentration factor of 65) and lower, and indeed, we have observed crystallization of salt in model respiratory fluid droplets exposed to varying RH [Besides its effects on physics of a droplet, evaporation also affects its chemistry, which could affect the stability of a virus contained within the droplet. The mass of solutes in a respiratory fluid droplet containing one virus is expected to be at least five orders of magnitude larger than the mass of the virus , so thesrying RH ,56. Underying RH . In studrying RH –81. Whilrying RH  or pH-serying RH , but mor4.Nature in 1960, Hemmes et al. [The debate over the importance of AH versus RH for influenza virus survival and transmission dates to the mid-twentieth century. Writing in s et al.  claimed,s et al. . RH can The story is not yet complete, but we know that RH determines the extent of evaporation of an airborne droplet and thus the resulting chemical microenvironment to which the virus is exposed. If virus survival in the environment is a dominant factor in influenza transmission, then RH is expected to influence incidence and seasonality, too.


In response to infectious disease, hosts typically mount both constitutive and induced defences. Constitutive defence prevents infection in the first place, while induced defence typically shortens the infectious period. The two routes to defence, therefore, have very different implications not only to individuals but also to the epidemiology of the disease. Moreover, the costs of constitutive defences are likely to be paid even in the absence of disease, while induced defences are likely to incur the most substantial costs when they are used in response to infection. We examine theoretically the evolutionary implications of these fundamental differences. A key result is that high virulence in the parasite typically selects for higher induced defences even if they result in immunopathology leading to very high disease mortality. Disease impacts on fecundity are critical to the relative investment in constitutive and induced defence with important differences found when parasites castrate their hosts. The trade-off between constitutive and induced defence has been cited as a cause of the diversity in defence, but we show that the trade-off alone is unlikely to lead to diversity. Our models provide a framework to examine relative investment in different defence components both experimentally and in the field. This The distinction between constitutive and induced defence is important to the individual because if infection is prevented through constitutive defence, there is no damage caused by the parasite directly and little risk of damage due to the use of the immune system (immunopathology). However, it is important to understand that the distinction also has fundamentally different population-level epidemiological impacts: constitutive defence prevents onward infection, while induced defence will only shorten the infectious period. As a consequence, in order to understand the optimal use of constitutive against induced defence, we need to take into account epidemiological impacts and population dynamics as well as individual impacts of the relative investment in the two forms of defence. We know that organisms have both constitutive  and induced (activated at infection) defence mechanisms to infectious disease, but we lack a clear understanding of what determines the relative investment and diversity of these two arms of the immune system.The key to this understanding is not only the effectiveness and costs of the immune component for the individual but also the population-level impact on the epidemiology of the disease such as the prevalence or the force of infection . ClearlyAnother important distinction between constitutive and induced defence is the way in which costs to defence are likely to be manifested. Without costs whether they are associated with either the development and maintenance or the use of the defence mechanism, we would clearly expect maximal defence in all individuals. There is considerable empirical evidence that higher resistance to disease is associated with fitness costs in terms of life-history characters such as development time or competitive ability –20. ThesHere, we develop theory that makes general predictions on how both host and parasite characteristics have an impact on the relative investment in constitutive against induced defence in the context of both the impact on the individual and the population-level feedback on the epidemiology of the disease. There has been theory on the evolution of inducible defences to predators and herbivores –32 and n2.constitutive defence as reduced susceptibility to infection (avoidance). Both susceptible and infected hosts are assumed to pay the cost of the constitutive defence, which occurs through a reduced reproductive rate. We define induced defence as an increased ability to clear disease (an increased recovery rate). Only infected hosts pay the cost of an induced defence, which is an increase in mortality while infected (immunopathology). These assumptions are deliberately simplistic, because there may be costs in the absence of disease to induce defences, and the costs of use of the immune system may be manifested in reduced fitness once recovered [β = f(b) and that induced costs only occur on action but on fecundity γ = f(f), and thereby allow both costs to act either on mortality or fecundity while maintaining the distinction between when costs act. This would allow us to theoretically examine the impact of this additional difference between the way we have modelled constitutive and induced defence, that the costs in one acts on fecundity, while the other acts on mortality. However, there is no evidence of trade-offs with constitutive resistance and natural mortality, and action costs of immunity on fecundity while infected have not been measured. Sublethal costs on fecundity for recovered individuals may be immunopathological and further theoretical work will examine the impact of these different costs, in the context of coevolutionary models.We make the simplifying assumption that constitutive defence mechanisms are costly in terms of trade-offs with other life-history parameters—evolutionary costs—while costs of defence post infection are manifested in the costs due to the use of the immune system. In our models, we define ecovered . Howevera, which is reduced due to crowding by a factor qN (where N = S + I). Infected hosts potentially suffer a reduced birth rate by sterilizing factor f  and all hosts die at rate b. Transmission is a density-dependent mass-action process, with a constant part (kβ) and a part limited due to constitutive defence, c. Infected hosts suffer increased mortality, also made up of a constant part (kα) and an immunopathological part, α. Finally, infected hosts can recover at rate γ.To allow us to explore the epidemiological feedbacks to the evolution of the different defence mechanisms, we base our analysis on a classic compartmental epidemiological model:a is a function of c) and that induced defence incurs immunopathological costs (such that α is a function of γ). By considering the stability of the resident–mutant equilibrium, the fitnesses of each mutant type (either with a mutation in the level of constitutive or induced defence) can then be given byγ is a function of c), both with and without further life-history costs .We conduct our analysis within the evolutionary invasion framework of adaptive dynamics ,35. As sContinuously Stable Strategies (CSS), an attracting and uninvadable endpoint of evolution. Theory has shown that, generally, such points exist, provided that evolving increased defence incurs accelerating costs [Our focus is on how investment in constitutive and induced defence varies across ecological and epidemiological gradients. From an adaptive dynamics perspective we are, therefore, interested in ng costs ,37. We, repeller . For all3.(a)(i)a). A key implication of this is, given the immunopathological costs of induced defence, the result of higher investment in induced defence is that individuals have a very high chance of death due to the combined effects of high virulence from the pathogen and strong immunopathological costs. Naturally virulent diseases are, therefore, doubly problematic from an individual host's point of view because they not only kill you faster but may select for stronger self-harm. Overall, the ratio of investment in constitutive and induced defences remains fairly constant when virulence increases . There are relatively minor impacts of the parasite transmission rate and minor impacts on fecundity investment in constitutive and induced defence (results not shown).In response to parasites that cause higher virulence  there is selection for investment in both higher constitutive and induced defences a. A key (ii)a) and a lower susceptibility to crowding (figure 2b). Lower susceptibility to crowding means denser populations characteristic of pest species and our models show that the ratio of investment in the two arms remains fairly constant as overall investment falls (figure 2b). With lifespan, although investment in both arms falls in longer-lived organisms , there is a faster fall in constitutive defence such that short-lived organisms invest relatively more in constitutive rather than induced defences (figure 2a). For these parameters the overall investment in immunity falls as lifespan increases. This somewhat counter-intuitive result is parameter-dependent, but has been found in similar models that assume only evolutionary costs to constitutive defence [When we look at characteristics of the host , we see figure 2 defence . Longer  defence . With ou(b)There can be a fundamental distinction between parasites that castrate the host and those that allow reproduction in terms of the ecological feedbacks that they create and, therefore, how they impact disease resistance ,41. As a(i)a). As such, high virulence again leads to relatively higher investment in induced defence despite its immunopathological costs, leading to more lethal disease interactions due to parasites with high virulence selecting for defence responses that lead to higher mortality. With transmission rate, generally induced defences are rather insensitive to transmission rates. By contrast, constitutive defences peak in investment at relatively low-to-intermediate transmission rates (b) due to low initial risk of infection when transmission is very low and the high costs of defence increasingly not been worth paying when transmission becomes higher. These effects are generally rather minor.In castrators, virulence has little impact on investment in constitutive defence, but higher virulence selects for higher induced defences a. As sucon rates b due to (ii)a). We would, therefore, predict that longer-lived organisms invest relatively more in constitutive rather than induced defences that are costly to use when faced with castrators. Hosts with relatively low self-limitation, characteristic of pest species, will invest relatively more in constitutive and less in induced defences when faced with castrators, while more self-limited species are predicted to invest in induced rather than constitutive defences (b). More generally, the prediction is that hosts with high population densities will invest in constitutive defences, while hosts at low densities will rely more on induced defences. Higher densities selecting for constitutive defence , while constitutive defence investment increases a. We woudefences b. More gaterial, ) is consaterial, , but onl(c)c) and constitutive (γ) defence, with no further life-history costs to either defence mechanism, we find that c) and constitutive (γ) defence in our model . As such, this trade-off alone is unlikely to generate and maintain diversity in defence traits apart from in the case of coexistence of genotypes with equal fitness.So far, we have assumed that constitutive and induced defences are evolutionarily independent. However, there is increasing evidence that across a range of systems there is a trade-off between constitutive and induced defences. This trade-off has been suggested to be an explanation for the diversity in host defence both within and between species. Trade-offs are a compelling process by which coexistence can occur because different genotypes or species can use different combinations of constitutive and induced defence in this case to achieve the same outcome. However, this argument often relies on an assumption of equalizing trade-offs such that all the different genotypes have exactly the same fitness. Robust coexistence, however, requires the ability to invade each other from rare and can be examined by the potential for evolutionary branching in evolutionary ecological models ,44. The per se , there is the potential for evolutionary branching leading to protected polymorphisms  as opposed to induced (activated and costly only on infection) immunity is determined by host and disease characteristics. Fundamental to our approach is that we consider the selection that is due to not only the direct effects on the individual but also the impact of the different defences on the epidemiology of the disease. Moreover, we assume that the two defence mechanisms have different types of costs: constitutive defences are always present and, therefore, costly in the absence of disease while, in contrast, costs occur only when induced defences are used in response to infection. It is important to note that there is a fundamental emergent second cost to induced defence that is the damage to infection before the immune response occurs. Both the nature of the disease and the characteristics of the host determine the optimal investment. A key result is that in response to a highly virulent parasite, induced defences are favoured even though that results in immunopathology leading to extremely high infected mortality. There is a clear distinction between parasites that castrate their hosts and those that don't, and we show that the trade-off between constitutive and induced defence is unlikely on its own to generate and maintain diversity.We show an increased investment in induced defences in response to more virulent  disease. In the case of castrators, the relative investment in induced rather than constitutive defence also increases in response to more virulent parasites. This is an important result because it follows from this that because induced defences are assumed to be costly through immunopathology, virulent diseases become doubly harmful, both through the damage they cause directly and the immunopathology of the immune system that they select for. Critically, this suggests that the observed pathology of highly virulent disease may be a consequence, in part, of the way in which they favour investment in induced defence. Intuitively, we may think that in response to a highly lethal disease there would be selection against the use of an immune defence that causes immunopathology, but the opposite is true due to epidemiological feedbacks that alter prevalence and, therefore, the risk of infection.The characteristics of the host also determine the investment in constitutive and induced defences. In castrators, longer-lived hosts are selected to invest relatively more into constitutive rather than induced defences than short-lived hosts. For longer-lived organisms, there is more chance of being challenged during a longer life and, therefore, more reason to invest in a constitutive defence. However, against non-castrators, the pattern reverses, with constitutive defences relatively higher in shorter-lived hosts. In general, there is a clear distinction in the qualitative outcomes between castrators and more classical diseases that only increase the death rate of their hosts that results from our explicit consideration of the ecological feedbacks to selection on defence. In particular, the relationship between virulence and prevalence of the disease in the population is different in castrators ,44,45. FWe show that there is overall less investment in defence in hosts with high carrying capacities. Moreover, particularly in response to castrators, we show that relatively fast, high carrying capacity pest species will invest more in constitutive than induced defences. This relationship has been demonstrated experimentally in a bacterial phage laboratory system that possesses an induced defence based on CRISPR . At highper se may allow a form of neutral diversity with exactly equal fitness for a range of species or genotypes along the trade-off, but this form of equalizing trade-off is less likely to lead to persistence of diversity in the face of stochasticity and genetic drift [A key hypothesis to explain the immense variability both within and among species in defence strategies is that there is a trade-off between constitutive and induced defence . Althougic drift . AnotherThroughout, we have made the assumption that induced defences leading to recovery are only costly while infected. Such costs have been shown directly particularly in insect experiments  and are Existing theoretical work on inducible defences in nature has tended to focus on predator–prey ,51 or plWe have made a series of predictions of the impact of host and disease characteristics on relative investment in constitutive and induced defence. Epidemiological feedbacks on the evolutionary outcome are critical and this work emphasizes that a full understanding cannot be gained without considering the population as well as the individual impacts of different disease threats and host responses. The fundamental difference in the nature of the costs is also critical to the outcome and it is important to emphasize that the emergent costs of induced defence is critical to both the individual and these population-level impacts. Our predictions need experimental testing and as more comparative data become available, we will better understand their relevance in nature. We have not included uncertainty and variability in the environment or the infectious disease threat, but still show that there are key differences in the selection pressures for constitutive and induced defences in nature.


We estimated the economic impact of concurrent measles and rubella outbreaks in Romania during 2011–2012. We collected costs from surveys of 428 case-patients and caretakers, government records, and health staff interviews. We then estimated financial and opportunity costs. During the study period, 12,427 measles cases and 24,627 rubella cases were recorded; 27 infants had congenital rubella syndrome (CRS). The cost of the outbreaks was US $9.9 million. Cost per case was US $439 for measles, US $132 for rubella, and US $44,051 for CRS. Up to 36% of households needed to borrow money to pay for illness treatment. Approximately 17% of patients continued to work while ill to pay their treatment expenses. Our key study findings were that households incurred a high economic burden compared with their incomes, the health sector bore most costs, and CRS costs were substantial and relevant to include in rubella outbreak cost studies. Outbreaks of measles and rubella constitute serious public health events and entail a vigorous response from public health agencies. A typical response could include a range of complex activities, including isolation of case-patients, case–contact tracing, assessment of disease or vaccination history of each case-patient, identification of potentially susceptible persons, and, if required, vaccination or quarantine   and opportunity costs  by using the societal costing perspective. The health sector included government entities from primary to tertiary level  involved in providing treatment to measles or rubella case-patients and in designing and implementing the public health response to the outbreaks.To collect household costs, we conducted a survey among a purposely selected sample of households that had recent cases  drawn from the national measles and rubella surveillance database  Figure. Reimbursement data were obtained from the National Health Insurance House , which covered all registered government healthcare providers in Romania . There wTo assess national outbreak response costs, personnel involved in outbreak response activities were interviewed on the basis of referral from the National Institute for Public Health, Romanian Ministry of Health, which was charged with coordinating the national outbreak response. These open-ended interviews were conducted by members of the research team in Romanian at the offices of respondents and focused on collecting information about actual expenses incurred in measles and rubella outbreak response activities, including costs of enhanced surveillance, outbreak investigation, and outbreak response vaccination and vaccine stockpiling .We requested reimbursement data for health services received by the 27 identified congenital rubella syndrome (CRS) cases from birth up to the date of data collection (at which point the surviving 15 CRS case-patients were 3–4 years old) from the National Health Insurance House; we obtained records for 18 (11 surviving and 7 who had died) of the 27 case-patients. The projection of their lifetime productivity losses used the same data sources and definitions as the main analysis of costs during the outbreak . These 2All data collection activities occurred from mid-January through the end of May 2014. A post-hoc analysis of the costs of CRS cases was conducted in March–May 2016.https://www.microsoft.com), and Stata version 13 . We calculated district-level weights on the basis of the number of measles and rubella cases as a proportion of the total population in each of the 42 districts of Romania. We then applied these weights to the household survey data, with the costs reported by a respondent multiplied by the weight of the district in which that respondent resided, to estimate the mean, median, and interquartile range of treatment expenditures per household for measles and rubella infections and the proportion of cases receiving treatment by admission status.We analyzed data by using Access and Excel . We then assumed that each of these employed case-patients and caregivers >18 years of age lost the average number of workdays missed on the basis of the household survey. We valued each day of work missed at the minimum wage of Romania.We calculated the indirect costs of measles or rubella infections by first calculating the number of measles and rubella case-patients and caregivers We used a human capital approach to estimate the lifetime productivity losses for CRS case-patients  into US dollars by using 2013 exchange rates (3.32 Lei/1 US dollar). Costs are presented in 2013 US dollars . Weighted median inpatient costs for measles were 2.2 times higher than for rubella, and outpatient costs for measles were 1.5 times higher than for rubella  and US $1.5 million for 24,627 rubella cases (US $61.74/case) , 5.Healthcare providers received direct reimbursement fees from health insurance. These totals were US $3,275,757 for services provided to treat measles and US $674,633 for services provided to treat rubella infections during the outbreak .National response activities included enhanced surveillance, laboratory diagnostic testing, and immunization of at-risk populations at a total cost of US $1,559,975, of which ≈60% were for activities related to diagnosis and containment of rubella . VirtualEstimated health service reimbursement costs for the 27 CRS cases up to May 2016 were US $130,143 . EstimatTotal societal costs of the measles and rubella outbreaks were US $9.9 million, of which US $5.5 million was for measles-related activities and US $4.4 million for rubella-related activities (the rubella-related activities included US $1.2 million in CRS-related costs) . These cThe large concurrent outbreaks of measles and rubella in Romania provided a major opportunity for an economic assessment of both diseases, as well as for CRS, in a middle-income country setting. Our key study findings were that households incurred a high economic burden of measles or rubella infection compared with income, the health sector bore most of the economic cost of the measles and rubella outbreaks, and CRS case costs were substantial and relevant to include in rubella outbreak cost studies. Our study contributes to the limited literature on measles and rubella outbreak costs in middle-income countries and to the evidence gap in this global research priority.The economic burden of the measles and rubella outbreaks on the outbreak-affected households was substantial. Almost every household incurred costs relating to measles or rubella treatment. The direct household cost per case of measles or rubella was high compared with the average income in Romania in 2012: 30% of monthly income and 3% of annual income for measles, 9% of monthly income and 1% of annual income for rubella. Previous studies indicated that, when illness-related economic costs are >10% of annual household income, it is considered catastrophic because it potentially forces households to cut consumption of necessities, such as food and water, and leads to increased debt or greater poverty , which might introduce selection bias; household costs might be underestimated because of the smaller proportion of case-patients <18 years of age in our sample compared with the outbreaks overall. We only recorded the provider reimbursement claims that were actually paid by the national insurance companies. However, anecdotal information suggested that claims were sometimes reimbursed at lower standard amounts based on limited availability of funds at the national level. National outbreak response costs were based on interviews with key informants from the agencies responsible for response implementation and are believed to be exhaustive of the costs incurred. However, other agencies might have also provided outbreak response and might not have been known to the national health authorities we used as key informants for the agencies involved in outbreak response.For the CRS cost analysis, we did not obtain information on 9 of the reported cases (response rate 67%), which might introduce selection bias in our results, although no systematic differences in clinical profiles between included and excluded cases were observed. Information on some types of future CRS care costs  were not available and are therefore not included in the analysis. The CRS productivity losses assume that all surviving case-patients are fully unable to work; this assumption might overestimate the indirect costs of CRS if some of these case-patients are ultimately able to work for income. Because of the nonrepresentative sampling techniques used and nonresponse, no uncertainty bounds were calculated for these cost estimates. Finally, the estimation of economic costs was not fully comprehensive. The opportunity costs of healthcare workers devoting their time to doing other public health activities was not estimated because of incomplete data.In conclusion, the costs of the 2011–2012 measles and rubella outbreaks in Romania were high, especially when compared with total average household incomes and national health expenditures per capita. In addition to the health consequences of these outbreaks, households faced major threats of financial insecurities during these outbreaks and the long-term economic impacts of productivity losses. Preventing outbreaks through routine vaccination also reduces the economic burden on the health sector. This study makes a major contribution to identifying the overall societal economic burden of measles and rubella outbreaks in Romania and improves our understanding of the magnitude of the costs of measles and rubella outbreaks in middle-income economies.Additional information on assessment of economic burden of concurrent measles and rubella outbreaks, Romania, 2011–2012.


Physical inactivity (PIA) is an important risk factor for many chronic conditions and therefore might increase healthcare utilization and costs. This study aimed to analyze the association of PIA using device assessed and self-reported physical activity (PA) data with direct healthcare costs.n = 2279). Self-reported PA was assessed with two questions regarding sports related PA in summer and winter and categorized into “high activity”, “moderate activity”, “low activity” and “no activity”. In a subsample (n = 477), PA was assessed with accelerometers and participants were categorized into activity quartiles  according to their mean minutes per day spent in light intensity, or in moderate-vigorous PA (MVPA). Self-reported healthcare utilization was used to estimate direct healthcare costs. We regressed direct healthcare costs on PA using a two-part gamma regression, adjusted for age, sex and socio-demographic variables. Additional models, including and excluding potential additional confounders and effect mediators were used to check the robustness of the results.Cross-sectional data was retrieved from the population based KORA FF4 study  that was conducted in southern Germany from 2013 to 2014  contains supplementary material, which is available to authorized users. Physical inactivity (PIA) is prevalent in 31% of the world’s population, and has been identified as the fourth leading risk factor for global mortality causing 6% of deaths worldwide  in reference to the subjects with high activity in sports . In Model B, the difference between individuals with high and no PA was smaller and also insignificant. In the analysis with Model C, where subjects with stroke, MI, diabetes, cancer, and asthma were excluded, inactive participants had the lowest overall costs, but this difference was also insignificant .When adjusted for age, ageTable 2, sex, education and equivalence income (Model A) the excess costs of participants with very low MVPA overall direct healthcare costs were €986  in reference to participants with very high MVPA . The results were similar when adding wear time (Model E) or light intensity PA (Model F) as covariates.In this domain, results showed consistently that participants with very low MVPA had higher direct healthcare costs. When adjusted for age, ageThe association with light intensity PA was qualitatively similar to the association with MVPA, with people with very low light intensity PA having the highest direct healthcare costs. However, this association was only significant for Model C, where we excluded participants with chronic conditions .Concerning steps per day , inactive participants had higher direct healthcare costs as compared to active subjects (Model A). However, this difference was not statistically significant.The direct healthcare costs of those participants, who achieved the WHO recommendation , i.e. thThis study aimed to analyze the association between device assessed and self-reported PA and direct healthcare costs in a population-based sample. Device assessed but not self-reported PA was associated with lower direct healthcare costs. The size of the effect was dependent on the type of model used to calculate the costs attributable to PIA.It is difficult to compare the findings of this study with other studies assessing PIA and direct healthcare costs because of the numerous different methods that have been used to calculate direct healthcare costs and to assess or estimate PIA , 11, 28.Model D. In Model D the observed difference in direct healthcare costs was not statistically significant but still present.Another important issue of interest we tried to address was the various sources of confounding in the relationship between PIA and direct healthcare costs. Our assumption is that inactive subjects cause more direct healthcare costs than active participants, but we cannot exclude the fact that there might be participants who are inactive due to a sickness or disability (exposure) that influence their PA (outcome). One way to address this issue is to exclude participants who report having problems with walking or being unable to do PA . TherefoAnother way to address the confounding problem would be to use longitudinal panel data. By having information about the timely order of PA and direct healthcare costs it would be easier to disentangle the direction of the association and to control for confounding factors. Ideally, for this one would combine representative population-based data from various follow up examinations with health insurance claims data that provide granular information on the trend of direct healthcare costs over time.This paper has several limitations. First, the present results, including subjects of KORA FF4 with a response of 68% limiting the general external validity. Second, our results are based on cross-sectional data, which does not give information about long-term PIA behavior or change of PIA during the time that plays an important role in relation to direct healthcare costs.In addition, this study did not include indirect costs, which covers loss of productivity from premature death or work absenteeism. The authors are also aware that a constant utilization of health services was assumed in the present study when reported utilizations were extrapolated to a one-year period, which tends to underestimate the actual utilization over one year .A major strength of this study is, that it provides insight into the relationship between self-reported and device assessed PIA and direct healthcare costs and that it is based on a relatively large population-based sample. Another strength of this study is the estimation of various alternative models to assess the robustness of the results with regard to confounding factors.The results indicate that device assessed but not self-reported PIA is associated with higher direct healthcare costs. Larger studies with device assessed PA and longitudinal design are needed to better quantify the impact of PIA on direct healthcare costs.Additional file 1:Matrix-transformation for self-reported sports physical activity. The matrix shows how the participants’ responses regarding their physical activity during summer and winter (1–4) were combined and categorized (I-IV). (DOCX 14 kb)Additional file 2:Cost calculation for physician visits. The table shows the German unit costs according to Bock et al. .


The management of retained products of conception (RPOC) may be medical or surgical. Surgical options include blind curettage, ultrasound guided curettage, or curettage under direct vision via hysteroscopy. The definitive management of patients presenting with retained products of conception will depend on several factors: severity of bleeding, presence of hemodynamic instability or infection, and patient preference. Optimal management of retained products of conception should result in complete evacuation of the uterine cavity while minimizing endometrial trauma. This is of utmost importance in patients with reproductive desires. We report patients with RPOC managed via hysteroscopic removal using the Bigatti Morcellator. Both patients had complete evacuation of the visualized RPOC. The purpose of this paper is to present this approach as an effective management option particularly in patients with a history of subfertility and failed blind curettage. Retained products of conception (RPOC) refer to the presence of placental and/or fetal tissue remaining in the uterus after a gestational event . Most coMore recently, hysteroscopic removal has been proposed in the literature as being a superior option to blind curettage –7. In thWe present two (2) patients successfully managed at the Centre for Assisted Reproduction (CARE) Unit with RPOC using the Bigatti hysteroscopic tissue removal system after previous surgical treatment with blind curettage. Benefits of this approach included a short set up and procedure time and no intraoperative or postoperative complications.0+1 underwent a successful ovulation induction with an intrauterine conception but subsequently suffered a missed miscarriage. She initially underwent a suction curettage for the miscarriage but re-presented 3 months later with abnormal uterine bleeding: prolonged menstrual bleed and intermenstrual bleed. Ultrasound findings were suggestive of retained products on conception. She was offered and consented to hysteroscopic removal  to minimize the risk of repeat retention. Intraoperatively, a 1.5 cm area of retained products of conception was seen close to the right ostium. Complete product removal was achieved during a 7-minute procedure with minimal blood loss ((1) A 30 y.o. Pood loss .(2) A 38 y.o. nulliparous female after in vitro fertilization and embryo transfer: The patient had a successful implantation but was subsequently diagnosed with a missed miscarriage. She had a spontaneous expulsion of products of conception and was scheduled for a repeat frozen embryo transfer. However, during ultrasound, she was noted to have retained products of conception and was offered hysteroscopic removal of the same. She had preoperative cervical ripening with misoprostol 400 mg per vaginum followed by hysteroscopic morcellation under general anaesthesia. Intraoperatively, a 1 cm area of product of conception was visualized at the posterior wall of the uterine cavity which was otherwise normal. The procedure was uncomplicated and lasted 6 minutes.The optimal management of RPOC necessitates complete evacuation of the retained products. This is important as failure to do so is associated with significant short and long term complications. Short term complications include incomplete evacuation and need for repeat procedure, infection, sepsis, haemorrhage, and uterine and cervical trauma , 9. LongSince its introduction in 1997, the expertise and use of hysteroscopy in the management of endometrial pathology have significantly increased. Pathology may be removed using a resectoscope loop, morcellation, or a hysteroscopic grasper , 13. WheAdvantages of blind resection in the management of RPOC do exist. There is a shorter learning curve and preprocedure setup compared to operative hysteroscopy. It may be associated with lower costs in the short term as hysteroscopy requires the purchase of an additional resectoscope for each patient. Also, of significance is the avoidance of potential fluid overload associated with operative hysteroscopy.In their meta-analysis of 392 patients with endometrial pathology, Li et al. concluded that hysteroscopic morcellation was associated with a higher operative success (odds ratio 4.5) rate and a shorter operative time among patients with endometrial lesions compared to resectoscopy . With sp0 angled telescope with an integrated sheath and working channel into which a rigid shaver is inserted. The diameter of its outer sheath is 8 mm (24F) and an inner tube connected to a handpiece oscillates within the inner piece [The Intrauterine Bigatti Shaver (IBS) was recently introduced as an option for hysteroscopic morcellation. It consists of a 6er piece . Descripis 8 mm 2F and an er piece , 23, 24.


Bagaza virus (BAGV) has not been reported in birds in South Africa since 1978. We used phylogenetic analysis and electron microscopy to identify BAGV as the likely etiology in neurologic disease and death in Himalayan monal pheasants in Pretoria, South Africa. Our results suggest circulation of BAGV in South Africa. Flaviviridae consists of 53 virus species, including arboviruses of medical and veterinary relevance, such as West Nile virus and Bagaza virus (BAGV). BAGV was isolated in 1966 from Culex mosquitoes in the Bagaza district of Central African Republic  and 1 tragopan pheasant  suddenly died on a property northeast of Pretoria, Gauteng Province, South Africa. In June 2017, this property had another 4 monal pheasants that displayed signs of lethargy and ataxia and died within a day. Around the same time, a residence in the northern suburbs of Pretoria had 5 monal and 2 tragopan pheasants that exhibited neurologic signs and died. That residence had another incidence in 2018 when a monal pheasant exhibited neurologic disease. Also in 2018, a monal pheasant was found dead in North West Province, South Africa. The cause of these deaths was unknown. All the birds were adults that were locally bred from parents imported from Belgium >2 years before.In April 2016, two Himalayan monal pheasants (https://www.qiagen.com) according to manufacturer’s instructions under Biosafety Level 3 conditions. We used nested real-time reverse transcription Pan-Flavi assay targeting the nonstructural coding gene 5 (NS5) (https://www.thermofisher.com) and the MAMD , University of Pretoria , for virus isolation and to the Centre for Viral Zoonoses (CVZ), University of Pretoria, for zoonotic arbovirus investigations. At the CVZ, we extracted RNA from the brain tissues by using the RNeasy Mini Kit  and performed multiple sequence alignments using the online version of MAFFT  with default parameters. We used MEGA 6.06 (https://www.megasoftware.net) to view, edit, and truncate the datasets. We downloaded reference sequences for the flavivirus genus from GenBank  with an effective sample size value >200. We used TreeAnnotator version 1.8 (http://beast.community) to generate the MCC tree and discarded 15% as burn-in. We displayed bootstrap and posterior probabilities on RAxML topology.We assembled and edited sequence data by using CLC Main WorkBench  containing 2% fetal bovine serum and 0.1 mg/mL gentamycin. We harvested cells and supernatant when 80% of the cell monolayer showed cytopathic effect and sent these to the Electron Microscopy Unit of The University of Pretoria and to the CVZ for molecular identification.We performed virus isolation on all PCR-positive samples. We inoculated brain tissue supernatant onto a confluent monolayer of baby hamster kidney fibroblast cells (BHK-21 line) in 25 cmhttps://www.inqababiotec.co.za) performed Sanger sequencing; we confirmed all positive samples as BAGV using phylogenetic analysis of the flavivirus genus NS5 PCR regions at CVZ. Phylogenetic analyses were based on partial nucleotide sequences of NS5 from genomic positions 9091–9280 (166 nt) and 9030–10109  and were used to compare the identified strains with other flaviviruses. Analyses confirmed the molecular results from all 8 Himalayan monals as BAGV in the Ntaya virus group with a bootstrap value of 92, sister to BAGV strains from Spain   Himalayan monal pheasants but not in the 3 tragopan pheasants tested. Neurologic signs were reported before death in 7/8  positive birds, but 1/8  was found dead and its clinical signs are unknown. Inqaba Biotec   Figure. alue 68) .Flaviviridae particles   Table frarticles . We obseCx. quinquefasciatus mosquito (GenBank accession no. LC318701.1; We detected BAGV in the offspring of monal pheasants imported from Belgium to South Africa. We sequenced BAGV strains and found they monophyletically clustered with strains from Spain rather than strains from West Africa. However, nucleotide similarities in the large gene segment were highest when compared with a strain from Zambia that was isolated from a We used virus isolation and electron microscopy results to confirm the etiology of the agent as a flavivirus. The causative link between the clinical symptoms of the monal pheasants and evidence of BAGV infection should be regarded with caution because we did not exclude other possible infectious and noninfectious etiologies. However, detection of BAGV in the brain suggests crossing of the blood–brain barrier and exclusion of other flaviviruses, arboviruses, and orthobunyaviruses suggests BAGV as a probable cause. Future work will focus on next-generation sequencing to obtain full genomes because initial attempts were unsuccessful. More data are needed to determine the endemicity of BAGV and the reservoir host and vectors of BAGV in South Africa and to define the seroprevelance of these infections in birds and possibly in humans.Additional information on detection of Bagaza virus in Himalayan monal pheasants, South Africa, 2016–2017.


Current oncolytic virotherapy is primarily administered by intratumoral injection. However, systemic delivery is desirable for treating patients, particularly for those who have developed metastatic diseases. Several components are impeding the systemic delivery efficiency of oncolytic viruses. Chief among them is the rapid clearance of viral particles by the host’s mononuclear phagocyte system (MPS). We explored the possibility of genetically engrafting CD47, a “don’t eat me” signal molecule, to the membrane envelop of an oncolytic herpes simplex virus (HSV) to enable it to escape from the MPS for systemic delivery. Our results show that this modification indeed allows the virus to be more efficiently delivered to local tumors by the systemic route. Moreover, this modification also prolongs the virus persistence in local tumors after it arrives there. Consequently, systemic delivery of the modified virus produced a measurable antitumor effect against a murine tumor model that is otherwise resistant to the parental virus delivered by the same route. Our data thus suggest that engrafting enveloped oncolytic viruses such as those derived from HSV with CD47 molecule represents a conceivable strategy to enhance the efficiency of systemic delivery. All animal experimental procedures were approved by the University of Houston Animal Care and Use Committee. CT26 and 4T1 cells were cultured in DMEM in standard conditions until 70% confluence. Cells were then trypsinized, pelleted and resuspended in DMEM at a concentration of 2 × 106 pfu of either FusOn-CD47-Luc or FusOn-Luc when tumor at a size of 8 mm in diameter (n = 6). In vivo imaging was performed under an IVIS Spectrum Pre-clinical in vivo Imaging System  for the observation of luciferase activity. Briefly, mice were injected intraperitoneally with 150 mg/kg D-luciferin  and then anesthetized with 1–3% isoflurane. Bioluminescence images were taken 10 min after the luciferin injection. Images were analyzed using Living Image version 4.2 software (Perkin Elmer) and represented as total flux measurements in photons/second.For evaluating viral delivery and persistence at the tumor site, mice received intravenous or intratumoral injection of 2 × 10t-test. p-value < 0.05 was considered to be statistically significant.All quantitative data are reported as mean ± standard deviations (SD). Statistical analysis was made for multiple comparisons using analysis of variance and Student’s


In addition, a novel viral genome was detected in all four datasets, consisting of a single-stranded, positive-sense RNA molecule of 6033 nucleotides. This genome displays an organization similar to Tymoviridae family members in the Tymovirales order. Nonetheless, the new virus shows enough differences to be considered as a new species defining a new genus. Detection of this new agent in the original grapevines proved very erratic and was only consistent at the end of the growing season. This virus was never detected in the spring period, raising the possibility that it might not be a grapevine-infecting virus, but rather a virus infecting a grapevine-associated organism that may be transiently present on grapevine samples at some periods of the year. Indeed, the Tymoviridae family comprises isometric viruses infecting a wide range of hosts in different kingdoms . The present work highlights the fact that even though HTS technologies produce invaluable data for the description of the sanitary status of a plant, in-depth biological studies are necessary before assigning a new virus to a particular host in such metagenomic approaches.In the past decade, high-throughput sequencing (HTS) has had a major impact on virus diversity studies as well as on diagnosis, providing an unbiased and more comprehensive view of the virome of a wide range of organisms. Rather than the serological and molecular-based methods, with their more “reductionist” view focusing on one or a few known agents, HTS-based approaches are able to give a “holistic snapshot” of the complex phytobiome of a sample of interest. In grapevine for example, HTS is powerful enough to allow for the assembly of complete genomes of the various viral species or variants infecting a sample of known or novel virus species. In the present study, a total RNAseq-based approach was used to determine the full genome sequences of various grapevine fanleaf virus (GFLV) isolates and to analyze the eventual presence of other viral agents. From four RNAseq datasets, a few complete grapevine-infecting virus and viroid genomes were  Grapevine is one of the oldest domesticated crops and has been cultivated for more than seven millennia in a wide range of geographical areas  total RNA or total DNA, with or without specific enrichment steps . Many viruses infecting grapevine have been either confirmed or identified via HTS techniques, with at least five of which  are part of the Tymoviridae family. While Tymoviridae, similar to the Tymovirales order in general, is predominantly a plant-infecting virus family , (ii) a mono-partite, positive, single-stranded RNA genome (6.0–7.5 kb in length), (iii) a generally high cytosine content (32–50% range), and (iv) a genomic RNA with a 5′ cap and a 3′-end with either a tRNA-like structure (Tymoviruses) or a polyA stretch (Marafiviruses and Maculaviruses). Other than members of the genus Maculavirus and the unassigned mycovirus Fusarium graminearum mycotymovirus 1 (FgMTV1)  and the new genus tentatively named Gratylivirus (GRApevine TYmo-LIke).Here, using an RNAseq approach, our goal was to better characterize the sanitary status of Gewurztraminer scions grafted onto Kober 5BB rootstocks mono-infected with specific grapevine fanleaf virus (GFLV) isolates. Other than the near ubiquitous grapevine rupestris stem pitting-associated virus (GRSPaV) and viroids, we confirmed the presence of GFLV in the inoculated vines. Surprisingly, a thorough analysis of the RNASeq datasets revealed the presence of a new virus. Phylogenetic analyses showed this new virus to cluster within the core-collection maintained in an open-field by the Institut National de la Recherche Agronomique (INRA) in its Colmar research center   rootstocks using a heterologous grafting technique. Finally, certified virus-free Vitis vinifera cv. Gewurztraminer (clone 643 from INRA Colmar collection) were grafted onto healthy and GFLV-infected rootstock for further study  Table . Virus s.) Table . Viral iNicotiana benthamiana  in 5 ml of a modified Sorensen's phosphate buffer  without or supplemented with 2.5% nicotine. In each case, 20 plants of Total RNAs were extracted from 100 mg of leaf tissue, or 100 μl of mycelial homogenate, using the RNeasy Plant mini kit , as per manufacturer's recommendations. Post extraction, for cDNA library preparation and Illumina sequencing, purity criteria (A260/A230 and A260/A280 both >1.8) and quality levels (RIN>8) were assessed via Nanodrop™  and Bioanalyzer . The cDNA libraries were then prepared and processed at IGBMC “microarray and sequencing platform” facility . Paired-end 2 × 150 pb RNAseq was performed on a Hiseq 2500  following the manufacturer's instructions.For fungal DNA extraction, a CTAB-based extraction method was used . After the trimming and quality check procedure, only reads above 70 nucleotides (nts) were kept . Contigs were then tested against GenBank reference sequences using BlastN/BlastX .The sanitary status of the grapevine samples was assessed by mapping reads onto a curated collection of grapevine viruses reference . Correlation coefficients were determined using the statistical software package Statgraphics Centurion version 15.1.02 .Protein structure, modeling, and structural similarity match to Protein Data Bank (PDB) were performed using the I-TASSER suite  and GSP rev (5′-GATTACGCCAAGCTTCGCGGTACCAAACGTTCACGCTCACC) were used along with oligo UPM (Clontech Lab.) to amplify the genome 3′ and 5′ ends, respectively. Resulting PCR products were Sanger-sequenced, confirming already known sequences and allowing termini to be resolved.A 3′/5′ RACE kit  was used for the amplification and confirmation of the viral genome termini, as per manufacturer's recommendations. The virus-specific primers , GSP fwd , into successively pDONR/Zeo donor vector and pEAQ- HT-Dest1 binary plasmid  upstream and downstream of the coding region (with start and stop codon underlined): fwd 5′-For fungal identification, a barcoding analysis based on the internal transcribed sequences (ITS) which consisted in amplifying a genomic region with primers ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITF4 (TCCTCCGCTTATTGATATGC) followed by Sanger sequencing.RNASeq datasets obtained from a collection of Gewurztraminer grapevines singly infected by GFLV isolates were first analyzed by directly mapping total cleaned reads Table  onto a cde novo assembly was performed, allowing for contigs and scaffolds reconstruction. In this way, near-complete genomes for each of the viruses/viroids infecting all four samples tested, consisting in three new GFLV-RNA1 sequences, four GFLV-RNA2, one GFLV-RNA3, 11 GRSPaV as well as four HSVd, and three GYSVd1 viroid sequences were obtained , a peptidase (PRO), a helicase (Hel), and a polymerase (RdRp) with very strong e-values (1 e−8 or lower) but with only low amino acid sequence identity levels, close to 30%. The sequence was further manually extended by several rounds of read mapping, until no more reads mapped against it. This allowed to obtain a continuous contig of 6,030 nt (including a stretch of seven adenines ending the sequence at its 3′ end). To confirm terminal sequences, 3′- and 5′-RACE reactions were performed. The complete genome sequence of the new virus was thus established to be 6,033 nt (without the polyA tail), indicating that only nine bases at the 5′-end had been missing from the original completed assembly. For all tested samples, final read counts mapping onto this new complete virus genome are shown in Table To confirm this initial analysis, a ed Table . For eac953GYPGCGKT960) analogous to the Hel motif I [GxxGxGK(T/S)] of many viral NTP-binding proteins, well conserved within the Tymovirales order. The second ORF does not display any similarities with any GenBank sequences at the nucleic acid level nor at the amino acid level. Also no putative conserved domains could be detected in ORF2. More elements on this second ORF are detailed in the next section.Analyses of the new virus genome organization revealed two open reading frames (ORFs) Figure . The firTymovirales members  The genome organization and order of the conserved domains along the large ORF1 are most consistent with those of Tymoviridae members  being identified and represented. For example, the RdRp motifs are well conserved, especially the REP motif IV (1517ANDYTSFDQSQTGE1530) and REP-VI (1605VSGDD1609), with that of other members of the Tymoviridae. This was confirmed by looking at amino acid identity levels of all conserved domains, with the Tymoviridae percentages being generally higher than with any other Tymovirales  Tes Table . (iv) ThTymoviridae family are not observed in the new virus. For example, the “Tymobox” or “marafibox,” a very distinctive 16 nt region thought to control subgenomic RNA synthesis and present in most Tymoviridae , was not identified in the genome of the new virus. In addition, unlike for other Tymoviridae, for which an unusually high cytosine (C) content (32–50%) is observed, the new virus sequence exhibited a strong unbalanced content in C but in the opposite direction, with only 15.2 % .However, some typical features of the In silico modeling of this protein was performed via the I-TASSER suit website and models were then compared to the Protein Data Bank (PDB) library (data not shown). While partial sequence identity were fairly low (<18%), the best model hits corresponded to the capsid proteins of a few Tymoviruses    was cloned in a pEAQ-based expression vector in order to produce N. benthamiana or C. quinoa, as well as N. benthamiana B2, and NbDCLx, both accessions highly susceptible to virus and known to greatly promote virus multiplication) displayed any symptoms nor tested positive for the virus by RT-PCR.To better decipher the origin and the biological significance of the new virus, mechanical inoculation of crude leaf extracts were undertaken. None of the 80 plants tested , the virus was detected in two out of the tree tested samples . Conversely, a statistically valid and very robust positive correlation was observed between the new viral sequence and the presence of contigs identified as originating from fungi , strongly suggesting that this new virus could in fact be a novel mycovirus. Following this hypothesis, a total of 15 fungal species were isolated from grapevine leaves and berries at a time when the virus was detected   as well as two viroids (HSVd and GYSVd1)  Table . All 11 1) Table . Altoget1) Table . Surpris1) Table . No othede novo assembled for each grapevine sample and after comparison against the NCBI database using BlastN/BlastX, some contigs showed a distant relationship (average aa identity close to 30%) to several viruses belonging to the Tymoviridae family and the Marafivirus genus in particular. Further steps ultimately yielded a genome of 6,033 nt (excuding the polyA tail). From this sequence, two ORFs were predicted. Phylogenetic analyses of the replicase-based polypeptide encoded by the ORF1, placed this new virus within the Tymovirales order, however no unambiguous assignment to a particular family could be attained as the new virus REP clustered away from members of the five families currently defined in the Tymovirales. Although not biologically confirmed, ORF2 was computationally described as coding for the coat protein after modeling and comparison to the Protein Data Bank (PDB) library. The viral coat proteins to which distant homologies were identified in this way all have icoseadral particles, suggesting the new virus could also have paraspherical particles, similar to members of the Tymoviridae family  disqualifies it to be part of the Tymovirus genus  to tentatively name it ‘grapevine-associated tymo-like virus’ or GaTLV and (ii) to consider that it defines a new genus  that will remain in the order but unassigned to a particular family for the time being. More viruses closely related to GaTLV need to be described before a decision can be made to decide whether the genus Gratylivirus should be included in the Tymoviridae or be included in a novel family within the Tymovirales. The sequence of GaTLV was deposited to GenBank under accession number MH383239.Considering the originality of the features of this new agent, and the inability to unequivocally assign it at this time in any existing families in the Tymovirirales order are known to infect many different species covering different Kingdoms. They are mostly found in Plantae, but lately many have been described infecting Fungi as well as the class of Insecta in the Animalia Kingdom , this virus displayed a very high identity percentage (>99.6%) along the genome. While the sampling size might be too small to be certain, this lack of diversity could underscore either a slow-evolving virus, a virus infecting a new host which did not have the time to accumulate substantial divergence or a virus highly specialized to its host. While none of the aforementioned experiments were conclusive, directly identifying a host, only correlative results were accumulated, tentatively pinpointing GaTLV as a mycovirus belonging to the grapevine phytobiome. More experiments are needed in order to uncover the host of GatLV, such as graft experiments that, if negative, would additionally support the notion that GaTLV is not a grapevine-infecting virus or controlled fungicide treatment of grapevines that could lend support to the hypothesis that GaTLV may be a mycovirus.This work highlights the fact that even though HTS technologies produce an invaluable sum of information describing the sanitary status of a plant, a careful etiological and epidemiological study is necessary before assigning a new virus to a host. Nonetheless, in this work and as it is often the case following HTS analysis, even after a careful scientific investigation, it is still not possible to designate without any doubt the host of an infectious entity. Our study also confirm that grapevine phytobiome is probably richer than anticipated, with the use of HTS allowing for the detection of not only grapevine pathogens but also grapevine associated-ecosystem (Al Rwahnih et al., de novo assembled have been submitted to GenBank (Table All sequences nk Table . The rawJ-MH, TC, EV, and OL designed the experiments. J-MH, TC, SGa, MT, VK, GB, AA, SGi, GH, MB, and AM performed the experiments. J-MH, TC, EV, and OL wrote the paper. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.