We present a method and setup that provide complementary three-dimensional (3D) images of blood oxygenation (via quantitative photoacoustic imaging) and blood flow dynamics (via ultrasound Doppler). The proposed approach is label-free and exploits blood-induced fluctuations, and is implemented on a sparse array with only 256 elements, driven with a commercially available ultrasound electronics. We first implement 3D photoacoustic fluctuation imaging (PAFI) to image chicken embryo, and obtain full-visibility images of the vascular morphology. We obtain simultaneously 3D ultrasound power Doppler with a comparable image quality. We then introduce multispectral photoacoustic fluctuation imaging (MS-PAFI), and demonstrate that it can provide quantitative measurements of the absorbed optical energy density with full visibility and enhanced contrast, as compared to conventional delay-and-sum multispectral photoacoustic imaging. We finally showcase the synergy and complementarity between MS-PAFI, which provides 3D quantitative oxygenation (SO In this work, we focus on the synergistic combination of ultrasound Doppler and photoacoustic imaging for three-dimensional (3D) vascular imaging, without contrast agents. Ultrasound Doppler techniques have been used intensely in clinical practice for decades in various disciplines1, and are still undergoing major conceptual and technological advances for vascular imaging such as ultrafast Doppler2 and functional ultrasound imaging3. Ultrasound Doppler techniques indeed provide a broad range of information such as vasculature morphology, blood content, flow direction and quantification of blood flow speed and pulsatility. However, ultrasound cannot probe molecular information, such as the blood oxygenation, which can only be accessed through complementary techniques such as diffuse optical tomography (DOT)10, blood-oxygen-level-dependent (BOLD) magnetic resonance imaging (MRI)5 or photoacoustic imaging9. Photoacoustic/optoacoustic imaging is an emerging noninvasive imaging modality that provides optical absorption contrasts with the depth and resolution of ultrasound imaging8, at least within the light penetration depth. It is particularly suited for vascular imaging thanks to the strong optical absorption of hemoglobin11. In addition, photoacoustic spectroscopy (involving multi-wavelength acquisitions) has the ability to provide quantitative maps of chromophore concentrations and blood oxygenation13. Compared to other non invasive techniques that are sensitive to blood oxygenation such as DOT10 and BOLD-MRI5, the advantages of vascular photoacoustic imaging include its large depth-to-resolution ratio13 (on the order of 200) and its relatively low cost. Moreover, because ultrasound imaging and photoacoustic imaging both rely on detecting ultrasound signals, with similar acquisition hardware and similar image reconstruction principles, they have a huge potential to be combined synergistically to provide a comprehensive characterization of vascular networks, including vascular morphology, blood flow dynamics and blood oxygenation.Vascular imaging is an essential tool in medicine, and is provided by a number of imaging modalities, including state-of the art clinical techniques such as ultrasound imaging19, whereas state-of-the-art label-free Doppler techniques, such as implemented in our work, provide access to functional ultrasound imaging and have never been coupled to photoacoustic imaging. In addition, many of these investigations are restricted to 2D imaging18, while most structures including vascular networks are inherently three-dimensional. Three-dimensional vascular imaging in general has been the topic of intensive research for both ultrasound imaging and photoacoustic imaging25, in each specific community. Strategies for 3D imaging are commonly based on the assumption that reconstructing full-visibility images  involves full-view detection with a Nyquist-based spatial sampling, leading to approaches that require mechanical scanning and/or fully populated matrix arrays . Another issue arises for photoacoustic imaging, as the limited-view and the limited bandwidth situations lead to the well known visibility artefacts28. In the context of vascular imaging, such visibility artefacts consist of partially or totally invisible vessels. Model-based reconstructions29 can effectively restore a good image quality in the limited view/bandwidth case and when the number of elements remains limited . However, there are drawbacks in using such approaches: (1) to compensate for the limited-bandwidth of the probe, the signals are usually deconvolved by the impulse response of the probe which may increase the effect of low frequency noise. (2) Model-based reconstruction algorithms have very high computational costs. (3) Applying model-based approaches on experimental data often requires prior knowledge and subtle tuning of regularization parameters, which increases complexity and may limit its practical use. Overall, model-based reconstructions, especially in 3D, require very specific developments and expertise. (5) Finally, while these approaches can partially compensate for limited-bandwidth artefacts, they can not recover all the structures in the limited view scenario30.The combination of ultrasound and photoacoustic imaging has been the topic of earlier investigations, but with significant limitations as compared to the approach proposed here. Most of the investigations currently reported in the literature are restricted to conventional pulse-echo ultrasound imaging coupled with photoacoustic imaging30. However, ultrasound images were obtained by injecting microbubbles as ultrasound contrast agent, and the system remained limited in terms of aperture detection and limited view effects were observed in the mouse brain. Currently, none of the proposed system for 3D photoacoustic imaging can provide full-visibility images without mechanical scanning or contrast agents.To date, only one publication reported both 3D ultrasound and 3D photoacoustic imaging with the same system, based on a custom 512-element spherical array31 (PAFI), as a powerful method that provides full-visibility photoacoustic images of the blood vasculature, even under limited-view conditions. In particular, we obtained preliminary label-free full-visibility 3D photoacoustic images in a chicken embryo, at a single optical wavelength. However, single-wavelength PAFI provides purely morphological images of vascular networks, thus with little or no complementary information as compared to that provided by ultrasound Power Doppler (US-PwD). To fully exploit the potential of photoacoustic imaging, multispectral acquisition is required to access quantitative molecular information such as blood oxygenation12. Multispectral 3D photoacoustic imaging was demonstrated in early investigations34, but either required mechanical scanning or suffered from visibility artefacts when implemented with a fixed array30. In this work, we extend the PAFI approach by introducing multispectral photoacoustic fluctuation imaging (MS-PAFI), and show that it can provide 3D, full-visibility and quantitative maps of blood oxygenation  in an interleaved manner with For the results presented in this work, we used N repeated sequences of interleaved ultrasound and photoacoustic acquisitions, as represented schematically in Fig. mentclass2pt{minimboth 3D photoacoustic fluctuation images (PAFI) and 3D ultrasound Power Doppler images (US-PwD), in addition to conventional DAS photoacoustic images and conventional ultrafast ultrasound images. Both PAFI and US-PwD rely on selectively extracting the temporal fluctuation of a series of single-shot delay-and-sum (DAS) 3D images. From a full acquisition of N repeated sequences, M photoacoustic fluctuation images are obtained from the N DAS 3D photoacoustic images acquired for each wavelength, and one 3D Power Doppler image is obtained from up to 31 and US-PwD36 are strictly identical, and we will refer to fluctuation images for both modalities. Briefly, a singular-value-decomposition (SVD) filtering approach is used to selectively extract the fluctuations induced by flowing red blood cells from all other parasitic sources of fluctuations, following the approach proposed in earlier works36. For both PAFI and US-PwD, two parasitic sources of fluctuations are electronics noise and tissue motion. Additional sources of fluctuations in PAFI include the laser pulse energy fluctuations31, and the choice of the SVD-filtering parameters thus depends on each modality. All data-processing steps are detailed in the “We first illustrate the unique capability of the proposed setup to provide simultaneously 37. For each modality, the conventional DAS images, (a) and (d), computed from the average of N photoacoustic images and 31: structures oriented along the z-axis, emitting waves mostly outside the detection aperture are absent from the reconstructed images. The strong background is a direct consequence of the probe sparsity: while the non-periodic spatial distribution of the transducer elements prevents from grating lobes, the unfocused energy due to spatial sub-sampling remains present as a significant pseudo-random background35. As a major consequence, our proposed setup has a relatively low contrast to background ratio for conventional DAS imaging. However, when fluctuation imaging is considered , the visibility artefacts are eliminated and the parasitic background is significantly reduced: vessels oriented along the z-axis are clearly revealed in fluctuation images, whereas they are invisible in conventional DAS images. In addition, as expected from previous results39, the resolution of fluctuation images is increased as compared to conventional DAS images, with pair of close vessels clearly resolved only on fluctuations images. As a key consequence, fluctuation imaging enables 3D label-free imaging of blood vessels with a sparse matrix array and a commercially available 256-channel ultrasound electronics, which we demonstrate here simultaneously for PAFI and US-PwD.Figure 31 their volume fraction 40, and a binary function 31. Under the assumption that both the light fluence and all the physical parameters of blood are constant at the scale of the PSF, but may vary from one vessel to another, the theoretical expressions for the mean photoacoustic image  MS-PAFI provides images that are specific to blood, as the contributions from static chromophores are inherently filtered out by the approach, (3) PAFI is compliant with 3D imaging with a low number of transducers.By use of a sparse transducer with only 256 elements, connected to a commercially available 256-channel electronics, we have demonstrated both ultrasound and photoacoustic 3D imaging of the blood vasculature in chicken embryos, without contrast agents. We introduced multispectral photoacoustic fluctuation imaging (MS-PAFI), and demonstrated that it provides the same quantitative information as DAS photoacoustic imaging, with several major advantages: (1) MS-PAFI provides 45 with a similar transducer geometry , however ultrasound contrast agents were injected in the embryo to perform imaging, presumably because of sensitivity limitations. Ultrasound contrast agents were also used in the only prior publication reporting combined 3D ultrasound and 3D photoacoustic imaging30 but the authors chose the transcranial mouse brain model that probably led to a limited sensitivity and to the need for contrast agents. By implementing fluctuation imaging, for both photoacoustic and ultrasound data, we were able for the first time to provide simultaneously ultrasound Doppler and oxygenation 3D images of blood in vivo, without contrast agents.The very good sensitivity of our transducer, that allowed working with a small number of channels, comes from the relatively large size of the individual elements and their distribution over a spherical surface, at the cost of a limited field-of-view. A recent study implemented Power Doppler imaging in chicken embryos12. It was however out of the scope of this work to perform a full inverse problem that would include the reconstruction of The reconstruction of quantitative oxygenation value maps requires a quantitative measurement of 46, our values are in agreement with the low values reported for veins, but lower than the ones reported for arteries. We hypothesize that the chicken embryos were in a hypoxic state caused by the way the samples were imaged, with the chorioallantoic membrane (responsible for oxygen exchanges) immersed in water for acoustic coupling. Measuring regular arterial oxygenation values would require imaging through whole intact shells in air, which remains a challenge out of the scope of our work. Chicken embryos however provided a clear in vivo demonstration that even very slow capillary blood flows could yield quantitative measurements of the blood oxygenation, sensitive enough to discriminate small differences in oxygenation states, as illustrated by Fig. Although the in vitro validation involved SOa) is made user-independent and has as little influence as possible in the derived oxygenation values. Accordingly, we used an automated method  and the outer diameter D of the array are F, d and D) result from several compromises between resolution, field-of-view and detection sensitivity, for a given number (256) of elements. The corresponding field of view for both PA and US imaging is located at the center of curvature of the array, and overs approximately a 39.The ultrasound transducer is a 256-channel matrix array designed in our laboratory and fabricated by Imasonic . The surface of the transducer is a spherical shell with a 10-mm diameter hole on its main axis (z-axis). The individual elements are circular elements with diameter The transducer was connected to a commercially available 256-channel ultrasound electronics , capable of both emission and reception of 256 ultrasound signals in parallel, with repetition rates up to several kHz. For all experiments, the signals were digitized with a sample rate of 31.25 MHz. The laser source for the photoacoustic modality is an optical parametric oscillator (OPO) pumped by a diode-pumped Nd:YAG laser , with a wavelength tunable for each individual laser pulse (range 680–900 nm). The output beam was coupled to the probe through a custom fiber bundle , attached to the transducer through its central hole  were obtained from a local farm and placed in an incubator for 9 days. After making a small hole through the shell by use of a scalpel, 1.5 mL of egg white was removed with a syringe and the top part of the shell was cut with scissors. Warm PBS was added on the top of the sample to ensure acoustic contact with the transducer coupling cone without touching the membrane of the embryo. The sample was held on a hammock made with a plastic foil, attached to a bowl containing water kept at 36 50. Each of these M wavelengths, were acquired at 100 Hz (limited by the laser repetition rate) in an interleaved manner with ultrasound RF data. Ultrafast ultrasound sequences of The acquired radio-frequency (RF) data for a complete measurement corresponded to N repeated sequences of interleaved ultrasound and photoacoustic acquisitions, as represented schematically in Fig. mentclass2pt{minimhttps://www.mathworks.com/), which was also used to drive all the instruments.The various data processing steps leading to 3D ultrasound Doppler and photoacoustic fluctuation images from RF data are summarized in Fig. 51. No apodization was applied. One 3D ultrasound image M estimates of All acquired real-valued RF signals were transformed into complex-valued RF signals through a Hilbert transform, before further processing. For both modalities, standard delay-and-sum beamforming was implemented to reconstruct complex-valued 3D images from each elementary set of RF data. The delay-and-sum (DAS) algorithm used in this study consists in summing the complex-valued Hilbert-transformed signal along curves given by theoretical time-of-flights, using a phase rotation approximation at the probe central frequency for interpolation, such as performed by Bertolo and colleagues for instance36. For photoacoustic imaging, a single SVD filtering step was applied to the whole stack of a, after the singular vectors have been sorted by decreasing singular values. For both modalities, filtering out singular vectors with index smaller than the lower bound a turns out to remove dominant spatially coherent parasitic fluctuations (including tissue motion for ultrasound Doppler36 and laser pulse energy fluctuation for PAFI31). Because PAFI involves additional laser-induced fluctuations, as compared to ultrasound Doppler, a has to be chosen specifically for each modality. In ultrasound Doppler imaging, the choice of a is usually determined empirically based on a qualitative assessment of the image quality, which is the approach that we implemented to produce all the Doppler images shown in our work. For MS-PAFI, because we propose a novel method to produce quantitative SOa allows efficiently extracting the fluctuations of interest while producing user-independent SOa used for all the results presented in this work.Out of the maximum numbers of 3D images available from a complete measurement of N sequences, the numbers lues see . Table Son table  for eachEstimation of the fluctuation images were computed from the stacks of SVD-filtered images. The processing steps to obtain ultrasound Doppler images are standard ones, briefly summarized in the absolute estimation of the absorption coefficient is not possible as it would require the knowledge of 52, are provided in the By use of Eq. , the phofminsearch Matlab function. All the wavelength-dependent curves for the optical absorption coefficient shown in the manuscript correspond to The minimization was implemented by use of the m 7 Fig.  to 10  or 3D rendering produced with AMIRA 6.7 (https://www.thermofisher.com/fr/fr/home/electron-microscopy/products/software-em-3d-vis/amira-software.html). For non quantitative images such as in the Fig. Three-dimensional data were visualized either through maximal intensity projections (MIP) computed with Matlab 2020 (Supplementary Information.


COVID-19 infection is normally followed by several post-COVID effects. This study aimed to investigate to evaluate menstrual changes in females following COVID-19 infection, and to evaluate female perception about the effect of COVID-19 on their menstrual cycles.During this cross-sectional survey-based study, a convenience sample of 483 women from Jordan and from Iraq, who had infected with COVID-19 were invited to fill-out the study questionnaire.The study was conducted on the females, with a median age 31 years old. Results showed that 47.2% of them (n = 228) suffered from a change in the number of days between two consecutive periods, as well as from a change in the amount of blood loss. Also, more than 50% of them believed that COVID-19 infection may cause changes in the amount of blood loss during the cycle , and changes in the number of days between the two consecutive periods .Regression analysis showed that participants with higher educational level (bachelor or higher) , and those living in Iraq  believed that COVID-19 has lower tendency to cause menstrual changes. In addition, non-married females , and those who are current smokers  believed that COVID-19 has higher tendency to cause menstrual changes.his study revealed that COVID-19 infection could affect the menstrual cycle for the females. Further prospective studies should be done to confirm these findings and evaluate how long these menstrual irregularities lasted. In December 2019, particularly in Wuhan China, several patients suffered from pneumonia of unknown etiology but their clinical implications were similar to SARS outbreak which spread in 2003. This new virus was named the severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2), and currently the Coronavirus 19 (COVID-19). This disease has infected millions of victims worldwide [This infection can be asymptomatic or may range from mild to very severe symptoms, which might lead to death, especially for the elderly people as they are more susceptible to severe symptoms; while children tend to have mild symptoms –5. COVIDMenstrual cycle is a biologically fundamental cycle for women controlled tightly by endocrine, autocrine and paracrine factors, with high variability in length (26–35 days) and variations in hormones levels during the cycle . It is iRecently, researchers highlighted the relationship between COVID-19 infection and menstrual disturbances in females . We hypoth January-1st February 2022 in order to assess the menstrual changes in females following COVID-19 infection. During the study, a convenience sample of women were invited to participate on this study by sending the survey through social media . The inclusion criteria were adult females who had infected with COVID-19. The survey required five minutes to be filled, and the participation was voluntary. Participants were asked to provide their electronic consent before filling the survey. If they want to participate on the survey, they will choose agree option. On the other hand, if they did not want to participate, they will choose disagree option.This is a cross-sectional survey-based study; that was conducted between 17The sample size was calculated based on the number of subjects per predictor needed to conduct linear regression analysis as recommended by Tabachnick and Fidell’s (5–20 subjects per predictor) . Using 2An authenticated questionnaire that asses the effects of COVID-19 on the menstrual cycle were developed based on previous study . The facS1 Appendix) consists of four sections: the first section assesses the demographic information of participants. The second section determines the medical information of participants. The third part assesses the menstrual cycle changes which occurred after COVID-19 infection (6 questions). And the last domain contains females’ perception toward the impact of COVID-19 infection on the menstrual cycle changes (7 statements). Perception section was evaluated using the following Likert scale . For each patient, a perception score out of 5 was calculated for the seven perception statements. The survey was translated to Arabic and back translated to English, and it was distributed in the Arabic form, since Arabic is the mother tongue of the recruited women.The final questionnaire  approval at The Hashemite University . The World Medical Association Declaration of Helsinki guidance was followed in the study . ParticiThe final results were directly downloaded from the Google-Forms platform into the computer database as an Excel sheet. Then data file was converted and analyzed using IBM statistical package for social sciences (SPSS) . Descriptive analyses were presented as median ± interquartile range (IQR) for continuous variables, while frequency and proportions were used for categorical variables.Simple linear regression was carried out to initially screen the independent variables that affect participants’ perception about the effects of COVID-19 on the menstrual cycle. Variables that were found to have P-value< 0.25 using univariate linear regression analysis, were entered into multiple linear regression analysis. Variables were selected after checking their independence, where tolerance values > 0.1 and Variance Inflation Factor (VIF) values were < 5 were selected to indicate the absence of multicollinearity between the independent variables in regression analysis. In the multiple linear regression analysis, variables that were independently affecting participants’ perception about the effects of COVID-19 on the menstrual cycle were identified. A P-value of ≤0.05 was considered statistically significant.Table 1.During the study period, 483 female participants who were infected previously with COVID-19 were recruited, with a median age of 31 years (IQR = 10). The majority of the participants had a diploma or bachelor degree , and around half of them were married . Participants were recruited from both Jordan  and Iraq . Regarding the individual monthly income, around 54% of the females  reported to have a monthly income less than 500$. Finally, 55.1% of the females  reported to have a medical related degree. For more details about the socio-demographic of the study participants, refer to Table 2), around half of the females  have abnormal body weigh as following; 142 were overweight (29.4%), 93 were obese (19.3%), and one female was morbidly obese (0.2%). Moreover, around three-quarters of the females were non-smokers , and the majority were vaccinated against COVID-19 . Most of the participants  were infected before receiving the vaccine while the remaining 137 females (28.4%) were infected after receiving the vaccine. Moreover, around one-third of the participants  had received the seasonal influenza vaccine, and only 13.3% of them (n = 64) reported to have chronic diseases.Regarding participants’ medical information (Table 3), and results showed that 47.2% of them (n = 228) suffered from a change in the number of days between two consecutive periods whether an increase or decrease, as well as from a change in the amount of blood loss. In addition, 41.8% of the participants (n = 202) reported an increase or decrease in the length of menses. In addition, a lower percentage of females experienced a change in the pain which occurs just before or during menstruation , missed period or suffered from period cessation after the infection , and experienced bleeding between periods .The participated females were asked to describe the menstrual changes they experienced following their COVID-19 infection , and results showed that more than 50% of them believed that COVID-19 infection may cause changes in the amount of blood loss during the cycle , changes in the number of days between the two consecutive periods , and changes in the length of menses . Also, 47.8% of the females (n = 231) believed COVID-19 infection may cause changes in the pain which occurs just before or during menstruation. Moreover, lower percentages of females agreed/strongly agreed that COVID-19 infection may cause missing of some cycles , bleeding between periods , cessation of menses .Females were asked to express their perceptions towards the impact of COVID-19 infection on the menstrual change (Table 4). Results showed that participants with higher educational level (bachelor or higher) , and those living in Iraq  have lower perception that COVID-19 causes menstrual changes. In addition, non-married females , and those who are current smokers  have higher perception that COVID-19 causes menstrual changes.Lastly, the perception of the females towards the factors affecting the impact of COVID-19 infection on the menstrual change were investigated using univariate and multivariate linear regression analysis  control these disturbances, and at the same time they have an important role in defending against microbial infectious included COVID-19 ,17. VitaFurthermore, vitamin B6 plays a role on suppressing the severity of COVID-19 infection through activation of adaptive and innate immunity, reduces pro-inflammatory cytokines, enhance respiratory function, and prevent the hypercoagulability ,26. TherAlmost half of the females participated, experienced changes in the menstrual cycle after COVID-19 infection, either in the number of the days between the consecutive cycles or amount of blood or the length of the menses . NormallMore than half of the females believe that COVID-19 infection may cause changes in the amount of blood loss during the cycle, changes in the number of days between the two consecutive periods, and changes in the length of menses . Highly The study has few limitations. First, the questionnaire was distributed through social media platforms, so, people without access to these applications would not be able to access this questionnaire. Second, this is a cross-sectional in nature and susceptible to recall bias; thus, further prospective longitudinal studies should be done to confirm these findings and evaluate how long these menstrual irregularities lasted. Third, in this study the psychological status of females during the COVID-19 pandemic was not assessed. Finally, we recruited a convenience sample of participants via social media, which may have introduced selection bias, limiting the generalizability of results to the general population.This cross-sectional survey-based study revealed that COVID-19 infection could affect the menstrual cycle for the females within the age 21–42 years old. Further prospective studies should be done to confirm these findings and to evaluate how long these menstrual irregularities lasted.S1 Appendix(DOCX)Click here for additional data file.


The T and B cell repertoire make up the adaptive immune system and is mainly generated through somatic V(D)J gene recombination. Thus, the VJ gene usage may be a potential prognostic or predictive biomarker. However, analysis of the adaptive immune system is challenging due to the heterogeneity of the clonotypes that make up the repertoire. To address the heterogeneity of the T and B cell repertoire, we proposed a novel ensemble feature selection approach and customized statistical learning algorithm focusing on the VJ gene usage. We applied the proposed approach to T cell receptor sequences from recovered COVID-19 patients and healthy donors, as well as a group of lung cancer patients who received immunotherapy. Our approach identified distinct VJ genes used in the COVID-19 recovered patients comparing to the healthy donors and the VJ genes associated with the clinical response in the lung cancer patients. Simulation studies show that the ensemble feature selection approach outperformed other state-of-the-art feature selection methods based on both efficiency and accuracy. It consistently yielded higher stability and sensitivity with lower false discovery rates. When integrated with different classification methods, the ensemble feature selection approach had the best prediction accuracy. In conclusion, the proposed novel approach and the integration procedure is an effective feature selection technique to aid in correctly classifying different subtypes to better understand the signatures in the adaptive immune response associated with disease or the treatment in order to improve treatment strategies. The adaptive immune system is responsible for recognizing and eliminating antigens originating from infection and disease. It recognizes antigens via an immense array of antigen-binding antibodies  and T-cell receptors (TCRs), the immune repertoire. The interrogation of immune repertoires is highly relevant for understanding the adaptive immune response in autoimmunity, malignancy, and infection . AdaptivIdentification of prognostic and predictive features among high-throughput sequencing data is of high clinical relevance. Because individuals share almost no exact TCR/BCR nucleotide sequencing, TCR/BCR sequencing cannot be directly compared between different patient groups on the clonal level. However, TCR and BCR are the products of somatic V(D)J gene recombination, plus the addition/subtraction of nontemplated bases at recombination junctions. Thus, individuals share V(D)J genes, which allows for direct comparison of V and J gene usage across different patients. Therefore, it will enable researchers to directly obtain statistical inferences across subjects to provide insight into TCR/BCR repertoire with clinical characteristics and outcomes.Though preliminary analysis using Random Forest reveal has been used to identify differentially expressed VJ genes in distinct disease types such as melanoma and prostate cancer , it is ln = 19) and a cohort of age-matched healthy donor cohort (n = 39) that tested negative for COVID-19 antibodies. The clinical characteristics of the patients and sequencing information were shown in . The median number of unique clonotypes was 9,431 (ranging from 589 to 35065) for healthy donors. Recovered patients had a median read depth of 72,152 ranging from 21,683 to 290,424. There was a total of 708 unique VJ gene combinations across both cohorts. VJ gene usage was defined as the number of clonotypes that utilize a particular combination of V and J genes normalized by the number of unique clones. The TCR-seq data used includes a cohort of patients who recovered after COVID-19 with mild to moderate disease courses  patients receiving durvalumab enrolled in a Phase I trial  were included for analysis. The median number of unique clonotypes was 4,994 . In order to explore the treatment effect, here we considered using logshown in . Table 1We use a modified version of the simulation strategy as in . The binThe three base variables (n = 50 and 100). Under each scenario, the We consider several different simulation scenarios  includinFeature selection methods are often categorized into four classes: filters, wrappers, embedded, and hybrid methods. Filter methods evaluate and rank the importance of a single feature (univariate filter) or an entire subsect of features (multivariate filter) based only on their inherent characteristics, without incorporating any learning algorithm. Wrapper methods evaluate a specific subset of variables by training and testing a specific learning model   or SuppoEnsemble feature selection by combing the outputs from different feature selection methods can solve the problem mentioned above. Here, we propose a new ensemble feature selection procedure based on pseudo-variables, which has two major steps: 1) aggregating the feature selection results from multiple feature selectors  and 2) fAggregating the results from different feature selection approaches is a critical step in ensemble learning. The outputs of the different approaches can be various, either the subsets of selected features, the rankings of all features, or both. We consider the following general scheme to obtain the candidate feature set depending on the types of outputs . Supposep-dimensional vector and p features are quantitative variables, but the method can be applied for categorical variables or a mixed type. By using the correlation structure of the p variables, we can define blocks bth block includes p-dimensional vector bth block. Denote the complete parameter vector by Now we introduce a group lasso model  on the c methods  fit the And K times and report the feature sets selected more than a certain percentage threshold The selection of the tuning parameter clusions . The objclusions  to facilWe then feed the selected features (based on six existing methods and the novel ensemble feature selection approach) into eight different classifiers, including SVM with linear (SVM linear), polynomial (SVM poly) and radius kernels (SVM rad) , K-neareFor simulation studies, we assess and compare the performance of the different variable selection approaches by using the following measures: false discovery rate (FDR), sensitivity, stability, F-1 score , and emp = 1-FDR . For eac = 1-FDR . The ave = 1-FDR . The empplicates . The preplicates . The parIn the real data analysis, we use 5-fold cross-validation to evaluate the prediction accuracy and AUC . Featurehttps://www.r-project.org).All the analyses were performed by R , respectively. Because the lung cancer patients received durvalumab, we selected the VJ genes based on their usage changes from baseline to the post-treatment, which was defined as the ratio of VJ gene usages from post-treatment vs. the usages from baseline. We identified 9 genes: TRBV5-3/J1-1, TRBV1/J2-7, TRBV1/J1-5, TRBV20-1/J1-4, TRBV7-4/J2-3, TRBV11-1/J2-6, TRBV7-7/J2-2, TRBV1/J1-1, and TRBV5-7/J1-6 when long survivors compared to short survivors .In general, the ensemble feature selection approach consistently outperforms the other state-of-the-art feature selection methods in terms of both stability and accuracy. It possesses consistent higher stability and sensitivity but lower FDR independent of the sample size choices, the sparsity of the causal genes, and the prevalence of the outcomes . As expeOverall, the ensemble feature selection also improves the classification performance. The ensemble feature selection approach has the best prediction accuracy when integrating with LASSO, Random Forest, ridge, and SVM classification methods. While combing with KNN, the ensemble feature selection approach occasionally is not as good as information gain performs, but most of the time is worse. When working with xgb, the ensemble feature selection approach has competitive performance compared to LASSO . The ensWe formulated a novel ensemble feature selection approach with a customized statistical learning algorithm focused on VJ gene usage in repertoire-sequencing data. Using the proposed approach and algorithm, we identify the VJ genes with significantly different usage in COVID-19 recovered patients and healthy donors. Wang et al. analyzed the TCR repertoire in patients with COVID-19 using single-cell sequencing and found that the frequencies of TRAV4, TRAJ2-7, TRBV7-9, and TRBJ2-3 were significantly higher compared to healthy patients . We founIn the real data analysis, we found that none of the feature selection approaches (including the feature ensemble method) have substantial improvements in terms of classification performance comparing to the results without feature selection. This is not surprising, since feature selection is not guaranteed to improve the prediction accuracy. However, feature selection is able to reduce the dimensinsionality and complexity of the predictive models, which eventually leads to a faster model trainning time and convergency. Our ensemble method, though is not driven by the prediction accuracy as some other feature selectors  is still able to exhibit a competitive performance in spite of a small sample size and even with highly correlated features included.In addition, we carry out intensive simulation studies in different scenarios. We found that the ensemble feature selection approach surpasses the other commonly used feature selection methods based on efficiency and accuracy. When integrating with varying types of classification methods, in most cases, the ensemble feature selection approach has the best prediction performance. These results indicate that the ensemble feature selection approach not only identifies the most stable, highest sensitive features with low false discovery rates but also greatly improves the prediction performance. Sample size, sparsity of causal genes, and the prevalence of the outcomes influence the performance but are relatively small for the ensemble approach.In the similation studies shown above, the base learners used in the first phase were information gain, SVM-RFE, Vita and Boruta. We have conducted additional simulation studies, where the different base learners were used. The results shown that our proposed ensemble method is neither sensitive to the number nor to the choice of base learners . In addip case is always challenging due to the minimal information observed in the high dimensional space. However, our simulation shows that the performance of both feature selection and classification is still appealing.Though the repertoire-sequencing data was used to illustrate this approach, the proposed approach can be applied to any other feature selection work. In a real application, we could perform stratified feature selection within the strata defined by the important covariates or consider the covariates as additional features. Although the ensemble feature selection was currently applied to a binary outcome, it can also be extended to different types of outcomes  by changing the log-likelihood in the objective function in optimization. Not surprisingly, the proposed ensemble method, which aggregates the output from multiple feature selectors, takes longer than a single feature selection method. However, it has been demonstrated in the simulation studies that the feature selection performance can be significantly boosted. Moreover, we want to point out that the proposed method can be applied as an independent feature selection method by setting the rank matrix with all elements equal to a constant. In addition, by using parallel computing, the computational time can be dramatically decreased. Furthermore, learning in a small n large In conclusion, the proposed novel approach and integrated procedure can help us pursue an effective feature selection technique to aid in correctly prioritizing the important features and classifying different subtypes.


In this Commentary, the article by Rosenfeld et al. “Cross-Reactive Antibody Responses against Nonpoliovirus Enteroviruses” is put into context of the historic poliovirus epidemics and resultant vaccination success story as it compares to the current state of acute flaccid myelitis; the relationship to nonpoliovirus enteroviruses (EVs), in particular EV-D68 and EV-A71; and the potential for successful vaccination strategies. The discovery of cross-protective antibody neutralization among polio and nonpolio enteroviruses, specifically EV-D68, opens future questions about EV-D68 vaccination strategies, circulation patterns of nonpolio enteroviruses, and the interpretation of EV-D68 serostudies. Nearly 70 years ago, in February of 1954, a group of elementary school children were the first recipients of the Salk poliovirus vaccine down the street from the building where this article was written in Pittsburgh, Pennsylvania. The Salk inactivated poliovirus vaccine (IPV) contained all three types of poliovirus, types 1 to 3 (Acute flaccid myelitis (AFM) has recently emerged as a poliomyelitis-like syndrome temporally associated with the summer-autumn circulation of nonpoliovirus enteroviruses (NPEVs), specifically enterovirus D68 (EV-D68) and enterovirus A71 (EV-A71). Outbreaks of EV-D68 as well as AFM in the United States peaked in 2014, 2016, and 2018, affecting hundreds of patients. Ninety percent of AFM cases are in children, with a median age of onset of 5 years. Another AFM peak was expected in the year 2020; however, the circulation of SARS-CoV-2 as well as nonpharmaceutical interventions of masking and distancing changed the circulation patterns of multiple respiratory viruses, including Respiratory Syncytial Virus (RSV), influenza, and EV-D68 , 4. It iThe degree to which EV-D68 has circulated historically has been challenging to ascertain. It was discovered in 1962 as a cause of viral pneumonia. While EV-D68 was recognized as a reemergent pathogen in 2012 as a cause of severe respiratory disease, molecular diagnostic techniques were not widespread. Most commercially available PCR-based multiplex diagnostic assays for upper respiratory viruses combine and report rhinoviruses and EV from a single target, such that further deconvolution is required to define an EV-D68 infection based on available clinical data. This strategy is how outbreaks were discerned in 2014 and 2016. Since 2017, the CDC has included EV-D68 in its active, prospective surveillance program . In 2018mBio report by Rosenfeld et al. , genetically similar rhinovirus genotypes (>160), common circulation, and near-universal poliovirus vaccination, there is great potential for a robust and confounding cross-reactive antibody repertoire. This leads to reevaluation of conclusions drawn from previous serologic studies regarding NPEV circulation. The cross-reactivity between antibodies generated against poliovirus and EV-D68 neutralization is interesting and reminiscent of the recent discoveries of cross-reactive monoclonal antibodies that can neutralize multiple types of influenza , 10., geneticin vivo.In the United States, since the year 2000, children receive four IPV doses in the primary childhood series. The first three doses occur in the first year of life, followed by a booster dose between four to 6 years of age. Greater than 90% of children develop protective antibodies after two doses, with at least 99% immunity after three doses. Studies of polio vaccine-induced antibodies over time have demonstrated that most children continue to have protective levels prior to their 4 to 6 year old booster. If antibodies produced as a result of poliovirus vaccination alone protected against the development of AFM, we should not be seeing this as a disease of young children. Given that poliovirus vaccination had been widely adopted in the United States by the time EV-D68 was discovered, it is possible that the contemporary genotypes of EV-D68 have developed a mechanism of immune evasion against the most commonly generated polyclonal anti-polio antibodies. Alternatively, perhaps a high degree of cross-neutralization in cell culture is not replicated when a patient encounters a high viral load The knowledge gained by this study may inform us about how an individual’s accumulated antibody repertoire, even acquired after poliovirus vaccination, may help protect against the development of AFM when exposed to EVs. Serostudies of children have suggested the capacity to neutralize EV-D68 reaches a nadir at 1 year of age but climbs to over 90% of participants by the teen years . ExposurThis potentially cross-reactive or cross-protective antibody profile for EV may also help explain why EV-D68 seems to circulate in a biennial pattern. Circulation in 1 year could generate antibodies that provide mucosal surface protection for the following year, or it is possible that there is a partner NPEV or multiple NPEV that boost antibody response in the non-EV-D68 year and prevent circulation/infection in the off year. There is much that we do not understand about the ecology of circulating human respiratory viruses, again demonstrated by the altered circulation patterns of RSV, influenza, and others during the SARS-CoV-2 pandemic. How might the circulation of one NPEV affect the circulation of others? If this is the mechanism that prevented a large EV-D68 year in 2020 as well as a peak in AFM, then should we be prepared for a potentially even larger spike of EV-D68 and resultant AFM in the coming season due to more immunologically naive hosts?in vitro against all others. If we are to turn vaccination against NPEV and prevention of AFM into the next great vaccination success story, there is much work ahead to understand both the specific antibody responses required for protection and the immunologic interplay between poliovirus and NPEV.The study by Rosenfeld et al.  also ill


In addition we found that fewer yearly visits were performed in our Cardio-oncology unit in 2021 compared to 2019 (300 patients/year in 2019 vs. 144 patients/year in the COVID era). Conclusion: COVID-19 pandemic seems to influence the onset of CTRCD in cancer patients by indirectly reducing hospital access of cancer patients and cardiological checks. In addition our data reflect the impact of the COVID-19 pandemic in the late diagnosis of cancer, in the reduction of hospital admissions and regular medical checks, in the increase of comorbidities and cardiovascular complications.Aim: COVID-19 pandemic had a big impact on our life, it has revolutionized the practice of cardiology and the organization of hospital and outpatient activities. Thus the aim of our study was to assess the impact of the COVID-19 pandemic on the development of cancer therapy-related cardiac dysfunction (CTRCD). Methods and results: A single center retrospective study was carried out evaluating 96 cancer patients treated with anthracyclines and admitted to our Cardio-Oncology unit from June to August 2019 and 60 patients from June to August 2021. The incidence of CTRCD was assessed performing an echocardiogram at the time of the enrollment. We found a significantly higher incidence of CTRCD in the second period compared to first period (13% vs. 2%,  Anticancer drugs can cause several cardiovascular complications such as arrhythmias, arterial hypertension, pulmonary hypertension, myocardial ischemia, thrombovascular events, left ventricular dysfunction and heart failure ,2,3.2022 ESC Guidelines on cardio-oncology introduced the definition “cancer therapy-related cardiovascular toxicity” (CTR-CVT) to include all cardiovascular toxicity effects of anticancer-drugs. For cardiac injury, cardiomyopathy, and heart failure, the descriptive term cancer therapy-related cardiac dysfunction (CTRCD) is recommended .CTRCD is a common complication of anticancer drugs, especially during treatment with high doses of anthracyclines .Instead thrombovascular events are mainly caused by tyrosine kinase inhibitors, vascular endothelial growth factor inhibitors. Especially anti BCR-ABL can cause accelerated atherosclerosis, myocardial infarction, peripheral arterial disease .2020 ESMO consensus recommendations defined CTRCD as an absolute decrease in the left ventricular ejection fraction (LVEF) of >20 percentage points or an absolute decrease in the LVEF of 10 percentage points to a value of <50% or an absolute decrease in the LVEF to a value of <50% .Subclinical cardiac dysfunction was defined as an absolute decrease from baseline in the left ventricular global longitudinal strain (GLS) of 5% or a relative decrease from baseline in the GLS of 12% or troponins elevation from baseline .2022 ESC Guidelines on cardio-oncology defined aymptomatic CTRCD mild as a relative decline in GLS by >15% from baseline AND/OR new rise in cardiac biomarkers .Cardiac biomarkers (troponin and natriuretic peptides-NP) should be measured in patients at risk of CTRCD where available and results should be interpreted according to the patient clinical status, type of cancer treatment, and kidney function.In the past, Cardinale et al. showed that troponin was able to identify patients at risk of future cardiotoxicity, if treated with high dose of anthracyclines .Nevertheless there is a lot of evidence that cardiac biomarkers cannot predict cardiotoxicity of anticancer agents as well. The predictive value of cardiac biomarkers  still needs exploration.Over the years various strategies have been proposed in the Cardio-Oncology field in order to identify the early signs of cardiotoxicity during anticancer treatment ,10. For example, several studies showed the usefulness of GLS alone or combined with high- sensitivity troponin to predict future cardiotoxicity in women with breast cancer ,12.Today the role of the GLS is consolidated in the diagnosis of subclinical cardiac dysfunction during chemotherapy; other imaging modalities such as cardiac magnetic resonance ,14 and nRecently, the Cardio-Oncology Study Group of the Heart Failure Association (HFA) of the European Society of Cardiology (ESC) in collaboration with the International Cardio-Oncology Society (ICOS) proposed a baseline cardiovascular risk stratification proformas that can be used by oncology and haemato-oncology teams to stratify cancer patients for cardiovascular risk before initiation of potentially cardiotoxic cancer therapies .This risk stratification model is very important because it allows medical teams to devise the best surveillance protocol for patients on the basis of their baseline cardiovascular risk and chemotherapy used.The proposed surveillance protocols are based on the monitoring of LVEF, GLS and biomarkers. A more intensified surveillance protocol with closer medical checks is envisaged for high and very high risk patients .Unfortunately, the COVID-19 pandemic has impacted us, revolutionizing the practice of cardiology and the organization of hospital and outpatient activities.The risk of COVID-19 infection reduced patients access to hospitals, cardioncological and other medical checks and for this reason the use of telemedicine has grown in the field of cardiology and Cardio-Oncology. New surveillance protocols have been proposed for monitoring cardiotoxicity in cancer patients during the COVID-19 pandemic ,19. The monitoring of Cardiotoxicity was carried out through fewer hospital visits, fewer check-ups, and telemedicine: echocardiography was expected in high-risk patients and in patients with symptoms and signs of heart failure, and it was postponed in asymptomatic and low-risk patients during chemotherapy; also, a baseline cardiological evaluation was not performed in low-risk patients ,19. CalvThe impact of the pandemic on ischemic heart disease is already known. For example, the pandemic has led to increased incidence of mechanical complications resulting from late presentation of patients with ST segment elevation myocardial infarction . Little A single centre retrospective study was carried out evaluating patients admitted to Cardio-Oncology unit after the third wave of the COVID-19 pandemic (from June to August 2021) compared to patients admitted during a 2 year period prior to the COVID-19 pandemic (from June to August 2019). 300 patients/year in 2019 vs. 144 patients/year in the COVID era (2021) performed a cardiological visit in our Cardio-Oncology unit. Inclusion criteria were: patients with hematological (lymphoma) or solid tumor (breast cancer) referred to our Cardio-Oncology unit for baseline cardiological examination or follow-up; current or previous (<3 months) treatment with anthracyclines and/or anti HER2 ; minimum cumulative dose of ≥250 mg/mq of doxorubicin or epirubicin > 125 mg/mq. Exclusion criteria were: pre-existing heart failure with mildly reduced or reduced LVEF, previous myocardial infarction or other heart diseases causing a reduction in LVEF, severe valve diseases. From June to August 2019, in our Cardio-Oncology unit, 96 patients underwent cardiological clinical and instrumental assessment blood pressure measurement, an electrocardiogram with QTc interval measurement and a complete Color-Doppler echocardiogram with speckle tracking echocardiography (STE) and myocardial deformation indices measurement. Echocardiographic evaluation was carried out using a GE Vivid E95 ultrasound system prime echocardiography machine  and a 4Vc-D (1.4–5.2 MHz) linear transducer . An assessment of the cardiac chamber dimensions, an evaluation of the systolic and diastolic ventricular function was carried out according to the ASE/EACVI recommendations ,22. MyocDuring baseline cardiological visit and subsequent cardiological checks, the patient’s cardiovascular risk profile was optimized and treatment with angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) or beta blockers were started in patients with arterial hypertension, statin was started in patients with dyslipidemia; smoking cessation was recommended in patients who smoked. In addition, in this first period, patients performed baseline cardiological evaluations or cardiological check-ups after previous cardiological checks scheduled on the basis of a rigorous surveillance protocol that included not only cardiological visits with electrocardiogram but also echocardiography every 3 months in all patients and every 2 cycles in patients at high risk of cardiotoxicity.From June to August 2021, 60 patients were referred to our Cardio-Oncology ambulatory for baseline cardiological evaluation or follow-up visit. Unfortunately, due to the COVID-19 pandemic, many patients who came for a cardiological visit after starting chemotherapy had never performed a baseline cardiological evaluation in our ambulatory (they had performed only an electrocardiogram before starting chemotherapy or focus echocardiogram without GLS measurement); in addition many patients had not followed a standard surveillance protocol in the previous months of the pandemic.In this second period, Cardio-Oncology visits were performed by vaccinated physicians; both operators and patients wore FFP2 masks. Considering the risk of COVID-19 transmission, in this second period, we performed a focused echocardiogram assessing the main questions such as right and left ventricular function, the presence of pericardial effusion. We avoided using ECG leads in some patients to reduce the risk of COVID-19 transmission and we did not perform GLS analysis in all patients but only in 50% of patients, especially new patients performing baseline cardiological evaluation before starting chemotherapy.On the other hand, the other patients who had started chemotherapy during the pandemic did not have a baseline GLS to use as a comparison. In addtion protocols of chemotherapy were comparable between 2019 and 2021; cardiological examination was performed by the same group of specialist in our department; echocardiogram was performed with the same machine.t-test for independent samples. Qualitative variables were reported as a percentage. Chi-square test was used for the comparison of percentages. A p value < 0.05 was considered statistically significant. Qualitative variables were reported as a percentage. The analysis was performed using MedCalc® software .Quantitative variables were reported as mean and standard deviation; the differences between the analyzed groups were studied with the two-tailed Student A 156 patients were referred to our Cardio-Oncology unit during two different periods (before the COVID-19 pandemic and after the third wave of the COVID-19 pandemic) were enrolled. Of which, 96 patients  were enrolled during the first period and 60 patients  were enrolled during the second period. The characteristics of the study population are summarized in We analyzed cardiovascular risk factors of patients, previous chemotherapy or radiotherapy, type of tumor, median duration of chemotherapy, time of the cardiological visit at the time of the enrollment (baseline or follow-up).During the first period, 21% of the enrolled patients had performed previous chemotherapy and 21% previous radiotherapy. In the second period, 25% of patients had performed previous chemotherapy and 16% previous radiotherapy. In addition, hematological tumor was present in 40% of patients assessed before the pandemic and in 50% of patients assessed after the third wave pandemic. Solid tumor was present respectively in 60% and 50% of patients. At the time of the enrollment, 59% of patients were undergoing a follow-up visit after a standard and rigorous surveillance protocol during the first period; during the second period 50% of patients were undergoing a follow-up visit without a previous check-up or baseline cardiological evaluation including echocardiography. Other patients enrolled performed baseline cardiological evaluation before starting chemotherapy. The median duration of chemotherapy in patients assessed during follow-up check-ups was 12 ± 4 months in the first period and 12 ± 2 months in the second period. All patients were treated with antrhacyclines. In addition, in the second period, at least 50% of the patients had received a first dose of the SARS-CoV-2 vaccine without notable symptoms. 45% of the population had SARS-CoV-2 infection.p value < 0.0001). We did not find a significant difference regarding the incidence of cardiovascular risk factors, the type of tumor and the number of baseline visits between the two periods , we did not find significant differences regarding the general characteristics of the population. Only median age of the population was significantly higher in patients evaluated during COVID-19 pandemic compared to population evaluated before COVID-19 pandemic   .Patients that developed CTRCD before the COVID-19 pandemic were 2 patients (a 70 years old women and a 75 years old women) with metastatic breast cancer treated with previous radiotherapy (5 years earlier), subsequently high doses of anthracyclines, trastuzumab and taxol for another year.7 patients (4 patients with breast cancer and 3 patients with lymphoma) developed CTRCD earlier during anticancer treatment with anthracyclines after a median period of 1 year; these patients have not performed previous radiotherapy or chemotherapy; their median age was 65 ± 10 years old; 3 patients had arterial hypertension and 1 patient had diabetes as cardiovascular risk factors; only electrocardiogram and focus echocardiogram without GLS measurement were performed before starting chemotherapy in these patients.only 1 patient  with breast cancer and previous chemotherapy with high doses of anthracyclines, had late cardiotoxicity developing CTRCD 2 years after the end of the treatment.1 patient with lymphoma (a 72 year old man), who developed early CTRCD after anthracycline treatment was hospitalized for COVID-19 pneumonia during the second wave; 4 patients who developed CTRCD had asymptomatic COVID-19 infection and were isolated.Analyzing 8 patients that developed CTRCD after the third wave COVID-19 pandemic we observed that:Our data reflects the experience of a single Italian Cardio-Oncology centre showing the impact of the COVID-19 pandemic causing increased cardiotoxicity in cancer patients.A higher incidence of CTRCD was found in our Cardio-Oncology unit after the third wave of COVID-19 pandemic compared to the same period in 2019 before the COVID-19 pandemic. Patients analyzed before the COVID-19 pandemic had the same general characteristics of the patients analyzed after the third wave COVID-19 pandemic; the same type of tumor and the same chemotherapy treatment with antracyclines. They had no significant differences regarding cardiovascular risk factors or the duration of cancer treatment. Despite the absence of significant differences regarding factors affecting a greater risk of cardiotoxicity between the 2 populations, a higher incidence of CTRCD was found after the third wave COVID-19 pandemic, indirectly showing the effects of the pandemic. In fact, during the first period analyzed, two cases of late onset cardiotoxicity were found in patients with previous radiotherapy and high dose of anthracyclines and thus in patients at higher risk of cardiotoxicity. Before the COVID-19 pandemic, patients underwent standard and rigorous surveillance protocols, echocardiography was performed every 3 months during treatment with anthracyclines or every two cycles in patients at higher risk, or every year after the end of chemotherapy, in accordance with previous and current guidelines ,6,7. TheAfter the third wave COVID-19 pandemic, we found more cases of CTRCD .Surprising was the higher incidence of early CTRCD in 7 patients without previous chemotherapy and/or radiotherapy or previous cardiovascular diseases and therefore at lower risk of cardiotoxicity.Due to the pandemic and the risk of COVID-19 transmission these patients had not undergone a standard surveillance protocol; they had performed an electrocardiogram and focus echocardiogram without GLS measurement before starting chemotherapy and then at 1 year without performing frequent/regular cardiological checks due to the COVID-19 pandemic or careful optimization of cardiovascular risk factors.Thus, we assumed that the execution of less regular/frequent cardiological checks during chemotherapy, a focus echocardiogram during the pandemic without GLS measurement only in high-risk or symptomatic patients as well as the reduced access of patients to the hospital due to the risk of infection, the lack of a guided GLS approach contributed to the greater development of cardiotoxicity.Rules and guidelines changed between 2019 and 2022. On the basis of 2022 ESC guidelines on cardioncology, the patient surveillance protocol should be planned on the basis of the baseline cardiovascular toxicity risk; echocardiogram after anthracyclines is recommended at baseline and every 2 cycles in high and very high-risk patients; echocardiography should be considered after a cumulative dose of 250 mg/mq of doxorubicin or equivalent in moderate-risk patients; echocardiography may be considered in low-risk patients (IIb) after a cumulative dose of 250 mq/mq of doxorubicin or equivalent.In all adults receiving anthracycline chemotherapy, an echocardiogram is recommended before starting chemotherapy and within 12 months after completing treatment. In patients receiving neoadjuvant or adjuvant HER2-targeted therapies, echocardiography is recommended every 3 months and within 12 months after completing treatment. In low-risk HER2+ EBC patients who are asymptomatic and with a normal assessment after 3 months, reducing monitoring to every 4 months may be considered. In high- and very high-risk HER2+ EBC patients, more frequent echocardiography monitoring should be considered during treatment. Troponin and NT-pro BNP monitoring before every cycle during anthracycline chemotherapy and 3 and 12 months after therapy completion is recommended in high- and very high-risk patients. Cardiology referral is recommended in high-risk and very high-risk patients before anticancer therapy. In patients categorized at moderate CV toxicity risk, cardiology referral may be considered. It is recommended that patients categorized to be at low CV toxicity risk should proceed to anticancer therapy without delay. Discussion of the risk/benefit balance of cardiotoxic anticancer treatment in high- and very high-risk patients in a multidisciplinary approach prior to starting treatment is recommended [In our study, in 2019, all patients treated with anthracyclines received intensive follow-up every 3 months. In COVID-19 era follow-up was not performed regularly and cardioprotective measures were implemented to a lesser extent.In addition, the annual number of cardio-Oncology visits performed in 2021 was lower than in 2019. The number of patients that had received therapy in 2021 was half of the patients treated in 2019. These data are very important, they reflect the impact of the COVID-19 pandemic in the late diagnosis of cancer, in the reduction of hospital admissions and regular medical checks, in the increase of comorbidities and cardiovascular complications. Furthermore our study showed that population in 2021 was significantly older.It is known that elderly patients have a greater risk of developing cardiotoxicity especially patients >65 years. So certainly older age in association with less monitoring during the COVID-19 pandemic may have contributed to the increased risk of developing CTRCD.In fact before anthracyclines chemotherapy, age between 65 and 79 years old represents a risk factor “medium 2” and age ≥80 years represents a “high” risk factor using the HFA/ICOS risk stratification tool . ProbablIn addition, 5 patients that developed early CTRCD had COVID-19 infection (1 patient COVID-19 pneumonia and other patients asymptomatic infection). Thus, we hypothesized that COVID-19 infection could influence and favor the onset of early cardiotoxicity in patients treated with anthracyclines, either directly by inducing myocardial damage or indirectly by reducing regularly scheduled hospital check-ups of cancer patients. In fact, the COVID-19 infection contributed to the reduction in outpatient visits and hospital follow-ups delaying administration of therapies (both chemotherapy and cardioprotective drugs).In addition, it is known that the COVID-19 infection is often accompanied by myocardial damage, myocarditis, thrombosis and other cardiovascular complications .In fact, COVID-19 can cause myocardial injury and myocardial infarction with several mechanisms. For example, COVID-19 may lead to an increased propensity for plaque disruption and thrombus formation  leading Another potential mechanism leading to acute non-ischaemic myocardial injury is direct injury by COVID-19 through ACE2 receptors, which are present in the myocardium and are functional receptors for COVID-19. Patients with heart failure have a higher expression of ACE2, which may explain their increased risk for myocardial injury following COVID-19 .Therefore to reduce hospital admissions and infections, follow-up visits in cancer patients were drastically reduced during COVID-19 pandemic, also considering that the cardiologists were employed in the COVID wards. Thus only urgent cardiology visits in symptomatic cancer patients were performed during first wave COVID-19 pandemic. Probably the reduction of surveillance in the COVID era influenced the subsequent greater access of patients with cardiotoxicity to our Cardio-Oncology center after the third wave COVID-19. Our observational study want to underline the sharp impact of the COVID-19 pandemic in the hospital organization and in the health field. In recent years COVID-19 has turned life around the world upside down.The COVID-19 pandemic seems to influence and favor the onset of CTRCD in patients treated with anthracyclines; indirectly reducing access of cancer patients to the hospital and performing less regular/frequent check-ups and focus echocardiography without GLS measurement during chemotherapy, directly through myocardial damage induced by COVID-19. Our data are preliminary and needs to be confirmed by other studies.Certainly, our study wants to underline the importance of an adequate cardiological follow-up in cancer patients during and after chemotherapy in order to prevent cardiotoxicity, also during the COVID-19 pandemic.First, this study is monocentric, retrospective and the population is heterogeneous.Second, we did not perform blood chemistry tests or cardiac magnetic resonance to demonstrate a direct link between heart damage and COVID-19.Therefore, our conclusions are not supported by objective data but simply describe what we have observed in our cardio-Oncology center.Certainly, our study wants to underline the importance of adequate follow-up in cancer patients based on their risk of cardiotoxicity, showing the impact of COVID-19 pandemic. It is certainly recommended to resume regularly scheduled follow-ups in cancer patients as soon as possible, also during the COVID-19 pandemic, ensuring adequate protection from SARS-CoV-2 infection.It cannot be denied that COVID-19 has revolutionized clinical cardiology practice, but adequate monitoring in Cardio-Oncology is necessary to prevent cardiotoxicity and to start cardioprotective drugs.


Generalized anxiety disorder (GAD) is a chronic mental health condition that results in a significant individual and societal burden. While cognitive behavioral therapy (CBT) is well established as an efficacious treatment for GAD, many patients experience logistical barriers when accessing face-to-face CBT. Remotely delivered treatments remove many of these barriers. Despite emerging evidence demonstrating the efficacy of remotely delivered CBT for GAD, studies examining the efficacy of remote methods for GAD that are analogous to standard face-to-face treatment, in particular synchronous treatments such as CBT delivered via online videoconferencing (VCBT), are needed.The authors propose a two-group randomized controlled trial comparing the efficacy of VCBT for GAD against a waitlist control group. The recruitment target will be 78 adults with primary GAD of at least moderate severity. The manualized high-intensity VCBT intervention will be delivered weekly over a 10-week period. After treatment completion, waitlist participants will receive the same VCBT delivered in a brief format . Treatment for both groups will be delivered in real time via an online teleconferencing platform. Outcome measures will be administered at baseline, mid-treatment, post-treatment, and 3-month follow-up.This trial will report findings on the efficacy of a remote synchronous high-intensity VCBT intervention for GAD. The results have the potential to contribute towards advancing our knowledge on the evidence base for GAD, as well as increase the dissemination of VCBT for GAD.ACTRN12621000786897. Registered on 22 June 2021Australian New Zealand Clinical Trials Registry  Generalized anxiety disorder (GAD) is characterized by excessive and uncontrollable worry that is accompanied by a number of physical and/or cognitive symptoms . The disRemotely delivered treatments do not require the clinician and the client to be in the same location and these interventions can be provided in either a low-intensity or high-intensity fashion . Low-intOne promising remote high-intensity approach to treatment includes Internet videoconferencing-delivered CBT (VCBT). VCBT involves the therapist and client working together over video-link, maintaining the visibility of the therapist, and clients’ non-verbal behaviors. Recent research has demonstrated that high-intensity remote CBT results in equivalent outcomes compared to traditional face-to-face treatment across a number of common mental health disorders . While tThis study describes the procedures and methodology of a randomized controlled trial (RCT) investigating the efficacy and acceptability of VCBT for GAD. Based on the limited existing literature, it is hypothesized that high-intensity VCBT will (1) be acceptable to individuals with GAD; (2) result in significant reductions in symptoms, resulting in large within-group at pre-treatment and follow-up and large between-group effect sizes at 3-month follow-up; and (3) brief VCBT will result in outcomes similar to those seen in standard length face-to-face treatment. It is anticipated that the results of the proposed study will inform best-practice psychological treatment for GAD.Seventy-eight individuals will be recruited for this study. To be included in the trial, participants are required to 1) be an Australian resident, (2) be aged 18 or above, (3) meet criteria for GAD as the primary disorder, (4) experience symptoms of at least “moderate severity,” and (5) be on a stable dose of psychotropic medication. Participants will be excluded if they have symptoms that will put them at risk of harming themselves or others or will confound results of the treatment. Participants will also be excluded if they do not have regular access to the Internet and camera. A complete list of inclusion and exclusion criteria is outlined in Table  be an Aun = 39) or a waitlist control group (n = 39). Group 1 will receive immediate access to a manualized high-intensity VCBT intervention. Group 2 will receive treatment after group 1 completes treatment. Given this is the first controlled trial investigating VCBT for GAD, a waitlist control group was considered appropriate. The content and design of this RCT are in accordance with the guidelines for clinical trial protocols as specified by the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 statement [A CONSORT-R compliant 2-group RCT superiority trial will be used to investigate the research questions. Participants will be randomly assigned to an immediate treatment group n = 3 or a waiParticipants will be recruited via advertising on social media, posts on professional networking sites, and direct email/letter to community-based clinicians, general practitioners, and psychiatrists. Hardcopy flyers will be posted on community noticeboards. Interested participants will complete a two-stage screening process to assess eligibility criteria involving, firstly, an online screening process, followed by a telephone interview. Interested participants will initially be directed to an online screening questionnaire consisting of a participant information sheet and consent form, demographic questionnaire, and symptom screeners. Participants are informed in the participant information sheet and consent form that de-identified data may be used for ancillary studies. Participants who meet criteria based on the online screening questionnaire will then complete a diagnostic interview via telephone to confirm their diagnostic status and assess comorbid conditions. Telephone interviews will be audio-recorded to determine interrater reliability. Eligible participants will then be randomized to one of the two groups. Allocation sequence will be sequentially numbered based on completion date of screening interview and randomization will be conducted by the chief investigator (BW) using a random number generator.A 15-item standard demographic questionnaire will be used to collect self-reported information on age, location, gender, marital, employment and education status, medication use, and access to technology required for the study.Risky behaviors including deliberate self-harm and regular alcohol and/or illicit drug use will be assessed with the Risk Questionnaire, a 5-item questionnaire that has been used as a screening tool in other remote CBT treatment studies .The DIAMOND screener is a 30-item self-report questionnaire that indicates to the clinician which disorders from the DSM-5 require further investigation. Participants who endorse GAD items on the DIAMOND screener will progress onto the second screening stage.The DIAMOND is a structured clinical interview that systematically assesses the DSM-5 diagnostic criteria for anxiety disorders, mood disorders, obsessive-compulsive and related disorders, trauma- and stressor-related disorders, schizophrenia spectrum disorders, eating disorders, somatic symptom and related disorders, substance use disorders, and selected neurodevelopmental disorders. The DIAMOND demonstrates very good interrater reliability (kappa = .71) and test-retest validity (kappa = .68) for the GAD diagnosis .The C-SSRS is a standardized assessment of suicide risk and can be used to measure the severity of suicidal ideation and behaviors . The sca to 3 (nearly every day) and a total score is calculated by summing each of the seven items. The scale has demonstrated good psychometric properties in previous samples [The GAD-7 is a 7-item measure of symptoms of generalized anxiety disorder. Each of the seven items are rated on a 4-point scale from 0  samples , 24, 46.The GAD-D is a 10-item measure of generalized anxiety symptoms. Participants rate the frequency with which they have experienced GAD symptoms over the past month on a 5-point Likert scale ranging from 0 (never) to 4 , resulting in a total score ranging between 0 and 40. Previous studies have established acceptable psychometric properties .The PSWQ-3 is a 3-item, self-report questionnaire designed to assess the core features of worry in GAD . Participants rate items on a 5-point scale and responses are summed, with higher scores indicating greater worry. The PSWQ-3 has demonstrated good psychometric properties in previous samples .The OASIS is a 5-item transdiagnostic self-report measure of anxiety symptoms. The OASIS has been shown to have strong psychometric properties in previous studies , 39, andnot at all) to 3 (nearly every day) and symptoms are assessed over the previous 2 weeks. Scores are summed and total scores ≥ 10 are used to indicate clinically significant depressive symptoms [The PHQ-9 is a 9-item measure of depressive symptoms. Each item is assessed on a 4-point Likert scale from 0  to 5 (entirely characteristic of me). The IUS-12 has demonstrated robust psychometric properties in community  and treatment-seeking samples .α = .96) [The CBQ is a 17-item measure of core beliefs. It instructs participants to rate how much they believe each belief item  on a 6-point Likert scale from 1 (strongly disbelieve) to 6 (strongly believe). Higher scores indicate greater endorsement of negative core beliefs about the self. The CBQ-Trait version has demonstrated adequate validity and reliability and shown to have excellent internal consistency (Cronbach’s α = .96) .not at all) to 4 . The CPQ has been shown to have acceptable reliability and validity in both clinical and community samples [The CPQ is a widely used 12-item measure of perfectionism. Participants are asked to rate the degree to which each item describes them over the past month on a scale from 1  to 7 (severely ill) and improvement scores range from 1 (very much improved) to 7 (very much worse). The CGI has been shown to be a valid and reliable clinical outcome measure in previous studies , 56.The SDS is a commonly used 5-item measure that assesses how much psychiatric symptoms have interfered with work, social, and home life functioning. A cut score of 5 on any subscale has been used to identify individuals with clinically relevant symptoms in previous studies .α of 0.91 [The WAI-SR is a shortened version of the Working Alliance Inventory  under the supervision of an experienced clinical psychologist. Treating psychologists will be in their final year of a Master of Clinical Psychology degree at the University of Technology Sydney. All treating psychologists will be familiar with delivering manualized treatments and thoroughly trained in the administration of the treatment protocol by the project investigators. All sessions will be recorded and at least 10% of sessions will be randomly selected for treatment compliance and integrity checking. Treating clinicians will receive weekly supervision to review client progress and address clinical issues arising from sessions.In order to maintain confidentiality, all electronic data (including session recordings and other identifiable information) will be stored on a password-protected computer that is only accessible to members of the research team. All hardcopy data will be stored in a locked filing cabinet in the chief investigators locked office.t-tests with Bonferroni-corrected p-values (continuous measures) and chi-square tests . Treatment acceptability will be examined using descriptive statistics. The main analyses comparing the treatment group to the control group will be carried out using conservative intention-to-treat principles and using mixed-linear models with an unstructured covariance structure. Multiple imputation will be used to handle missing data. Effect sizes using Cohen’s d will be calculated for within-group and between-group differences, based on pooled standard deviations for both the entire sample using the estimated marginal means and completer sample . All analyses will be conducted based on the total score of the relevant outcome measure. The efficacy and acceptability of the brief treatment will be examined in the same manner described above. Comparisons between standard treatment and brief treatment will be analyzed using benchmarking analyses using the procedure outlined by Minami et al. [Group differences in demographic data and pre-treatment measures will be analyzed with independent samples i et al. . All anad = 0.80) differences in symptoms, which would be the minimum expected reduction in the RCT based on existing research [With alpha set at 0.05, power set at 0.80, and a sample size of 34 in each group, the study is powered to enable the detection of large effect size . The trial is registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12621000786897) and includes the full study protocol and participant information sheet and consent form. While not anticipated, any changes to the protocol will be updated through the ANZCTR registry.GAD is a chronic and impairing mental health condition . CBT is The protocol version number is v1.0 which was approved on 22 July 2021. Recruitment will commence in October 2021 and is expected to be completed by July 2023.