It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress human immunodeficiency virus (HIV) replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI).+ count, plasma viral load zenith, duration of virological suppression, NRTI backbone composition, low-level plasma HIV RNA detectability, and electronically measured adherence to ART.CA HIV unspliced RNA and total HIV DNA were quantified in two cohorts  of individuals treated with triple ART regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either an NNRTI or a PI. To compare CA HIV RNA and DNA levels between the regimens, we built multivariable models adjusting for age, gender, current and nadir CD4adj = 0.02 in both cohorts), with a similar but weaker association between the ART regimen and total HIV DNA (padj = 0.048 and padj = 0.10). No differences in CA HIV RNA or DNA levels were observed between individual NNRTIs or individual PIs, but CA HIV RNA was lower in individuals treated with either nevirapine or efavirenz, compared to PI-treated individuals.In both cohorts, levels of CA HIV RNA and DNA strongly correlated (rho = 0.70 and rho = 0.54) and both markers were lower in NNRTI-treated than in PI-treated individuals. In the multivariable analysis, CA RNA in both cohorts remained significantly reduced in NNRTI-treated individuals  and FP7 Health (305522). In individuals who are able to adhere to combination antiretroviral therapy (ART), therapy suppresses human immunodeficiency virus (HIV) replication, restores immune function, and prevents the development of acquired immunodeficiency syndrome (AIDS) . More thDespite the efficient suppression of HIV replication, ART is not curative and has to be sustained lifelong. Persistence of viral reservoirs forms the major obstacle to an HIV cure . Viral rIt is also a matter of debate whether different ART regimens are equally potent in suppressing residual HIV replication. All current antiretroviral drugs act by preventing the infection of new cells and are not expected to inhibit HIV RNA transcription or virus production in the long-lived reservoir cells that were infected prior to ART initiation, or in the progeny of such cells. Therefore, if regimens are equally potent in stopping the infection of new cells, one would not expect to detect a difference in residual viremia or CA RNA levels between ART regimens. Consequently, finding such a difference would suggest that some regimens are more potent in suppressing residual replication than others, arguing that virus suppression is less complete with at least some of the regimens. A number of studies reported lower levels of residual viremia in individuals treated with NNRTI-based, compared to PI-based, ART regimens . HoweverParticipants for the COmorBidity in Relation to AIDS (COBRA) cohort were recruited at two clinical sites in Amsterdam (The Netherlands) and London (UK) from ongoing prospective cohort studies on co-morbidity and aging in HIV, the AGEhIV Cohort Study in Amsterdam , and thehttps://www.isrctn.com (ISRCTN97730834). All participants provided written informed consent.Participants for the Adherence Improving Self-Management Strategy (AIMS) randomized trial were recruited at the HIV outpatient clinic of the Academic Medical Center (Amsterdam). Adherence to ART in this cohort was measured electronically using Medication Event Monitoring System (MEMS)-cap pill bottles , which record the moments of bottle opening. Adherence was defined as the percentage of doses taken within a specified time interval (11–13 hr for twice-daily and 22–26 hr for once-daily regimens) during the assessment period. For the present study, the adherence was assessed during 1-month periods that finished less than 20 days before or after the HIV sampling moments. The randomized trial, the results of which were reported previously , assesseHistorical plasma HIV RNA measurements, CD4+ T-cell counts, and treatment data were retrieved from the outpatient medical records. The duration of continuous virological suppression was calculated as the duration of the latest period with undetectable plasma HIV RNA prior to the measurement, allowing isolated ‘blips’ of 50–999 copies/ml. The duration of cumulative suppression was calculated by adding together all such periods of continuous suppression. The duration of the current regimen was calculated as the period during which the participant had been receiving combination ART that included their current NNRTI or PI drug and no other NNRTI or PI.Plasma HIV RNA was measured using commercial assays with detection limits of 40 or 50 copies/ml. For CA HIV RNA and total HIV DNA measurements, total nucleic acids were extracted from PBMC using the Boom isolation method . ExtractVariables were compared between NNRTI- and PI-based ART by using Mann-Whitney tests for continuous variables and Fisher’s exact tests or Chi-square tests for categorical variables. Strength of the associations between CA RNA or DNA and other variables was initially assessed by nonparametric Spearman or Mann-Whitney tests, as appropriate, and subsequently by fitting generalized linear models (GLMs) on rank-transformed dependent variables. Binary explanatory variables were included in the models if the representation of the least frequent category was >5%. Therefore, gender and plasma HIV RNA detectability were not included in the model in the COBRA cohort. Similarly, a threshold of 5% was used for inclusion of the NRTI backbone categories in the analysis, resulting in the inclusion of three most frequent categories for each cohort. The most frequent NRTI backbone category was used as a reference category. Explanatory variables that were associated with the dependent variables with a sufficient strength (p < 0.1) in univariable GLM analyses were included in multivariable models. Individual tests are described in the legends to figures and tables. Data were analyzed using Prism 8.3.0 (GraphPad Software) and IBM SPSS Statistics (version 25). All tests were two-sided. p-values <0.05 were considered statistically significant.We measured CA HIV unspliced RNA and total HIV DNA in PBMC samples from participants of the COBRA cohort . COBRA i10 copies/μg total RNA and 2.50 (1.84–2.77) log10 copies/106 PBMC, respectively. 10 copies/μg total RNA, p = 0.03; total DNA: 2.46 (1.78–2.64) vs 2.60 (1.93–2.90) log10 copies/106 PBMC, p = 0.07).The median CA HIV RNA and total HIV DNA levels in the COBRA cohort were 2.15 (1.58–2.52) logadj = 0.02 and padj = 0.0001, respectively; ART regimen: padj = 0.02 and padj = 0.048, respectively).To assess the association of CA HIV RNA and DNA with ART regimens, we built multivariable GLMs, adjusted for a number of demographic and clinical variables . Higher Having established an association between CA HIV RNA and DNA and the ART regimen in the COBRA cohort, we sought to validate these observations in another cohort. To this end, we used PBMC samples from participants of the AIMS randomized controlled trial that investigated the effects of a behavioral intervention to increase adherence to ART . Partici10 copies/μg total RNA and 2.41 (1.88–2.79) log10 copies/106 PBMC, respectively. 10 copies/μg total RNA, p = 0.007; total DNA: 2.36 (1.76–2.74) vs 2.57 (2.22–2.98) log10 copies/106 PBMC, p = 0.04).The median CA HIV RNA and total HIV DNA levels in the AIMS cohort were 1.71 (1.25–2.01) logadj = 0.04; ART regimen: padj = 0.02). The shorter duration of continuous suppression was also significantly associated with higher total HIV DNA (padj = 0.03), while the association of ART regimen with HIV DNA did not achieve statistical significance (padj = 0.10). We also built three alternative models, in which either duration of cumulative suppression or the duration of current regimen was included instead of the duration of continuous suppression, or the duration of continuous suppression was included together with the duration of current regimen. The adjusted associations of CA HIV RNA with the ART regimen remained significant in these alternative models  . In accoNext, we assessed the associations of individual drugs with CA HIV RNA and DNA levels . As the In this study, we demonstrated in two independent cohorts of individuals on suppressive ART that NNRTI-based triple ART regimens are associated with lower levels of CA HIV RNA compared with PI-based regimens. To the best of our knowledge, this is the largest study comparing CA HIV RNA levels between individuals on different ART regimens. Although several studies have compared residual viremia between ART regimens and most have found lower levels in NNRTI-treated than in PI-treated individuals , very feAmong all demographic and clinical variables, only duration of current regimen and duration of virological suppression were associated with the ART regimen, being longer in NNRTI- than in PI-treated individuals. This can be explained by the fact that while efavirenz and nevirapine, the drugs used by the absolute majority of our NNRTI-treated individuals, were approved for medical use in the late 1990s, modern PIs like atazanavir and darunavir, which were used by most of our PI-treated individuals, were only approved in the mid- or late 2000s. This means that most of the NNRTI-treated individuals in this study started ART, or switched to NNRTIs from the first-generation PIs, earlier than the PI-treated individuals. Because the HIV reservoir generally decays with time on ART , this asAnother variable that could have confounded the association of the ART regimen with the HIV reservoir measures such as CA HIV RNA is the adherence to ART. Earlier studies have reported lower adherence among individuals treated with older PI-based ART regimens . HoweverNotwithstanding the associations of CA RNA with other factors, in both cohorts, NNRTI-based ART was independently associated with lower CA RNA levels as compared with PI-based ART, as revealed by the multivariable analysis. This analysis revealed very similar effect sizes of the ART regimen on CA RNA in both cohorts, despite the fact that several factors, such as duration of virological suppression and the PI drugs, differed between the cohorts. On average, CA RNA levels were 1.75- to 2-fold lower in the NNRTI-treated participants. This confirms numerous reports that measured lower residual plasma viremia in NNRTI- compared to PI-treated individuals . In factInterestingly, some NNRTIs, such as rilpivirine, efavirenz, and etravirine, but not nevirapine, can promote selective apoptosis of infected cells by inducing HIV protease-mediated cytotoxicity . If thesReplenishment of the HIV reservoir by residual virus replication has been proposed as one of the mechanisms of HIV persistence . The asshttps://aidsinfo.nih.gov/guidelines/html/1/adult-and-adolescent-arv/11/what-to-start), because such individuals were rare (<5%) in the COBRA cohort and absent from the AIMS cohort. Studies in more recent cohorts are necessary to elucidate the differences in CA HIV markers between INSTI-based ART and other regimens. It must be noted that although NNRTI- and PI-based ART regimens are no longer recommended as the first-line therapy in all settings, millions of infected individuals are still treated with these regimens. Thus, our results are relevant for the clinical management of these individuals. Second, although our results provide strong evidence that individuals treated with PI-based ART undergo residual HIV replication, this evidence remains indirect as the direct demonstration of infection of new cells in an ART-treated individual is extremely difficult if not impossible. Therefore, although studies have used various approaches to prove or disprove the existence of residual replication on ART  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This study examines measures of the HIV reservoir in two cohorts treated with different ART regimens. The findings indicate that the HIV reservoir is lower in virally suppressed individuals on NNRTI-based than PI-based regimens. This was observed in both cohorts, further supporting this finding.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Julie M Overbaugh as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jos van der Meer as the Senior Editor.Thank you for submitting your article "Non-nucleoside reverse transcriptase inhibitor-based combination antiretroviral therapy is associated with lower cell-associated HIV RNA and DNA levels as compared with therapy based on protease inhibitors" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:This is a well-written study that will be of interest to many investigators working in the field of HIV persistence during ART. The strengths of the study include the analysis of samples from two relatively large cohorts of individuals (n = 100 and 124) and the use of multivariable models to adjust for numerous parameters. One weakness is the fact that the authors do not consider alternative models that may explain their results. This includes the interaction between viral reservoir and longer viral suppression, both of which were associated with NNRTI-based regimens. The data is important and shows that NNRTI usage is associated with lower levels of HIV persistence markers, but it does not provide a mechanistic explanation for that. Overall, this is a well-conducted and important study, with new findings that have potential clinical implications.1) A main concern is the interaction between duration of viral suppression and the reservoir given the reservoir decays with time. This could contribute to the results and appears to do so for the AIMs cohort; it feels like it is a bit of a chicken and egg issue with this which probably should be discussed more in terms of limitations. The authors do one analysis where they restrict the cases to those with more the 6 months of viral suppression to try to get at the impact of suppression. But I think this would be more telling if they did an analysis around intervals of suppression  as the analysis they did still could be driven by the long duration of some individuals and the associated decay due to that.2) At the end of the abstract and in a large part of the discussion, the authors propose that their results are explained by the fact that NNRTI have a greater ability to suppress HIV replication. There are alternative explanations: It may be that people on NNRTI clear their transcriptionally active reservoir continuously: The authors should discuss their findings in connection with the recent study by the group of Dr Shan . In this study, the authors demonstrated that the use of NNRTI may lead to the clearance of transcriptionally active HIV-infected cells through autophagy. Interestingly, the use of PI would block this phenomenon. Lang et al. found that both efavirenz and rilpivirine can lead to the death of transcriptionally active HIV-infected cells, whereas nevirapine has no effect (because it does not enhance dimerization of the gag-pol polyprotein intracellularly). Although this contrasts with the results of the present study, this should be extensively discussed. Also, although the number of participants receiving rilpivirine is probably small, they may be included Figure 3C.3) The authors should perform a more exhaustive review of the literature in the introduction by quoting the studies that have compared the effects of different ART regimens on HIV DNA and cell-associated HIV RNA levels. See for example Nicastri et al., Curr HIV Res 2008. Some of these studies are included in the discussion, but there are probably many more that should be included in the introduction (including comparisons with integrase inhibitors). I suggest to briefly review these studies after the sentence "However, to date, few studies have compared levels of CA HIV reservoir markers between different ART regimens".4) Table 1 shows that in the COBRA cohort, the participants on PI regimens have a shorter duration of continuous viral suppression compared to those taking NNRTI. This is likely an important bias here and although the adjusted analysis shows that the differences between the two groups persist after adjusting for this parameter, this should be discussed . Same remark for the AIMS cohort.5) In all correlations, it would be nice to represent the participants PI VS NNRTI using two different colors. This will help the reader determining whether these two groups from separate clusters in all these associations.6) The differences between Figure 3 A and 3B should be visible in the figure (add a title/label at the top of the graphs or change the Y axis label).7) Figure 3 C (and D) should be modified: The authors compared each NNRTI  but each PI to the other PIs only (no comparison with NNRTI). It would be much simpler to redraw this figure and represent all drugs individually (PIs and NNRTIs) and use multiple comparisons.8) Have the authors attempted to calculate the HIV RNA/HIV DNA ratio in both groups? This would be a better indicator of the viral transcriptional activity.9) The authors should discuss the need for a clinical trial in which participants would switch from PI to NNRTI to definitely confirm their findings in a longitudinal way.10) in one of their study cohorts, the authors do not observe an association between duration of ART and cell-associated HIV DNA/RNA levels; this could be due to clonal expansion of viral reservoir cells that amplify and expand viral reservoir size during long-term ART. The authors may wish to discuss this.11) the authors cite Li et al. (ref 60) to argue that NNRTI treatment is associated with delayed viral rebound after treatment interruption; however, this is likely unrelated to viral reservoir size, and rather caused by altered PK and the longer half-life of NNRTIS relative to PIs. Essential revisions:This is a well-written study that will be of interest to many investigators working in the field of HIV persistence during ART. The strengths of the study include the analysis of samples from two relatively large cohorts of individuals (n = 100 and 124) and the use of multivariable models to adjust for numerous parameters. One weakness is the fact that the authors do not consider alternative models that may explain their results. This includes the interaction between viral reservoir and longer viral suppression, both of which were associated with NNRTI-based regimens. The data is important and shows that NNRTI usage is associated with lower levels of HIV persistence markers, but it does not provide a mechanistic explanation for that. Overall, this is a well-conducted and important study, with new findings that have potential clinical implications.We thank the Editor and the Reviewers for their positive assessment of our study. We now included a discussion of possible alternative explanations of our results (see response to comment 2) and an additional figure showing that the differences in CA RNA and DNA between NNRTI- and PI-treated individuals are independent of the duration of suppression (see response to comments 1 and 4).1) A main concern is the interaction between duration of viral suppression and the reservoir given the reservoir decays with time. This could contribute to the results and appears to do so for the AIMs cohort; it feels like it is a bit of a chicken and egg issue with this which probably should be discussed more in terms of limitations. The authors do one analysis where they restrict the cases to those with more the 6 months of viral suppression to try to get at the impact of suppression. But I think this would be more telling if they did an analysis around intervals of suppression  as the analysis they did still could be driven by the long duration of some individuals and the associated decay due to that.We understand the concerns of the reviewer and feel several points need to be clarified. First, the duration of suppression could have potentially confounded the association of the ART regimen with CA RNA and DNA only in the AIMS cohort but not in the COBRA cohort, because only in the AIMS cohort the duration of suppression was associated with CA RNA and DNA. We extensively discuss this finding, as well as possible reasons behind it, in the Discussion. Second, to exclude this potential confounding, we performed multivariable analysis, adjusting for the duration of suppression and other variables, and found an independent association of the ART regimen with CA RNA and DNA in both cohorts. We even built three alternative models for the AIMS cohort, which did not change the results. Therefore, the duration of suppression does not confound the association of the ART regimen with CA RNA and DNA. To illustrate this further, we now performed the analysis suggested by the reviewer, assigning the participants into four groups according to the duration of suppression , and comparing CA RNA and DNA between NNRTI- and PI-treated individuals in every group separately. In every of the four groups, CA RNA levels were lower in NNRTI- then in PI-treated participants, with a similar but weaker effect observed for CA DNA, in a complete agreement to the results obtained in the total cohort. This is an additional demonstration that the duration of suppression is not a confounding factor in our analysis.2) At the end of the abstract and in a large part of the discussion, the authors propose that their results are explained by the fact that NNRTI have a greater ability to suppress HIV replication. There are alternative explanations: It may be that people on NNRTI clear their transcriptionally active reservoir continuously: The authors should discuss their findings in connection with the recent study by the group of Dr Shan . In this study, the authors demonstrated that the use of NNRTI may lead to the clearance of transcriptionally active HIV-infected cells through autophagy. Interestingly, the use of PI would block this phenomenon. Lang et al. found that both efavirenz and rilpivirine can lead to the death of transcriptionally active HIV-infected cells, whereas nevirapine has no effect (because it does not enhance dimerization of the gag-pol polyprotein intracellularly). Although this contrasts with the results of the present study, this should be extensively discussed. Also, although the number of participants receiving rilpivirine is probably small, they may be included Figure 3C.The reviewer is referring to the recent paper of Wang et al. . Their findings concerning this NNRTI action are not new, several groups studied this in the past . In essence, some NNRTIs, such as rilpivirine, efavirenz, and etravirine, but not nevirapine, can promote selective apoptosis of infected cells by inducing HIV protease-mediated cytotoxicity. If these NNRTIs are present in cells that are actively producing viral proteins, they may bind to the reverse transcriptase portion of a newly translated Gag-Pol polyprotein and promote its homodimerization, resulting in premature protease activation. This leads to a decrease in virus production and non-specific cleavage of multiple host proteins, including proteins that induce apoptosis/pyroptosis .However, we think this alternative explanation for the effects of NNRTIs on CA RNA and DNA that we see in our cohorts is very unlikely, for two reasons. First, for this mechanism to work, a cell has to express HIV Gag-Pol, and cells that express HIV proteins without ex vivo stimulation are exceedingly rare in ART-treated individuals. In fact, Pardons et al. (Plos Pathogens 2019) could not find any Gag-expressing cell in any of their patients unless they reversed latency ex vivo. Second, nevirapine, in comparison with other NNRTIs, has absolutely no such activity – and in our study, nevirapine was associated with the same levels of CA RNA and DNA as efavirenz . In fact, some studies showed even lower levels of residual plasma viremia in nevirapine-treated compared to efavirenz-treated patients (Haim-Boukobza AIDS 2011), which might be because nevirapine better penetrates some anatomical sanctuaries. Therefore, this mechanism cannot really explain our findings. We discuss this in the revised manuscript (pages 20-21). We did not include rilpivirine in Figure 3C because there were only 2 participants treated with this drug in our study, which is insufficient to draw any conclusions about the effects of this drug.3) The authors should perform a more exhaustive review of the literature in the introduction by quoting the studies that have compared the effects of different ART regimens on HIV DNA and cell-associated HIV RNA levels. See for example Nicastri et al., Curr HIV Res 2008. Some of these studies are included in the discussion, but there are probably many more that should be included in the introduction (including comparisons with integrase inhibitors). I suggest to briefly review these studies after the sentence "However, to date, few studies have compared levels of CA HIV reservoir markers between different ART regimens".We would like to draw the reviewer’s attention to our recent review published in Viruses , where we extensively review the available evidence on differences in HIV markers between infected individuals treated with different ART regimens. We performed a very rigorous literature search for this review (see Table 1 in the review). This review is cited in the present manuscript (ref. 28). The absolute majority of such studies measured only residual plasma viremia and only a very few studies measured cell-associated HIV markers: we discuss these studies in the Discussion  but we followed the suggestion of the reviewer and now refer to them in the Introduction as well (refs. 29-31).4) Table 1 shows that in the COBRA cohort, the participants on PI regimens have a shorter duration of continuous viral suppression compared to those taking NNRTI. This is likely an important bias here and although the adjusted analysis shows that the differences between the two groups persist after adjusting for this parameter, this should be discussed . Same remark for the AIMS cohort.Please see the response to the comment 1. Yes, in both cohorts the duration of suppression was lower in PI-treated than in NNRTI-treated participants, but in the COBRA cohort this effect could not even potentially have confounded the association of the ART regimen with CA RNA and DNA, because the duration of suppression was not associated with CA RNA and DNA in this cohort. Such potential confounding could have occurred in the AIMS cohort and we performed an extensive multivariable analysis to rule it out. Moreover, we now performed an additional analysis, in which participants were grouped according to the duration of suppression . Combined, our results show that CA RNA and DNA are associated with the ART regimen independently of the duration of suppression.5) In all correlations, it would be nice to represent the participants PI VS NNRTI using two different colors. This will help the reader determining whether these two groups from separate clusters in all these associations.We thank the reviewer for this suggestion, which we followed. Figure 1 now shows NNRTI- and PI-treated participants in different colors.6) The differences between Figure 3 A and 3B should be visible in the figure (add a title/label at the top of the graphs or change the Y axis label).Thank you, titles added on top of the graphs.7) Figure 3 C (and D) should be modified: The authors compared each NNRTI  but each PI to the other PIs only (no comparison with NNRTI). It would be much simpler to redraw this figure and represent all drugs individually (PIs and NNRTIs) and use multiple comparisons.Representing all drugs individually results in comparing too many groups with very unequal sizes (e.g. 68 participants were treated with efavirenz but only 12 with lopinavir), which considerably reduces the statistical power. In addition, we wanted to check whether both EFV and NVP are associated with lower CA RNA levels compared to PIs (and whether there is a difference between EFV and NVP), to rule out the alternative explanation for our results (see comment 2). Therefore, in figure 3C we compare EFV, NVP, and PIs, and in figure 3D we compare different PIs between each other. We think this comparison is more informative, and the results suggest that the effects of ART regimens on the CA RNA and DNA levels are ART class-specific and not drug-specific. However, we now also included the multiple comparisons suggested by the reviewer .8) Have the authors attempted to calculate the HIV RNA/HIV DNA ratio in both groups? This would be a better indicator of the viral transcriptional activity.We thank the reviewer for this suggestion and now included the comparison of CA RNA/DNA ratios between NNRTI- and PI-treated participants . Although there was a trend towards lower CA RNA/DNA ratios in NNRTI-treated participants, the difference was not significant (p=0.19), which is in line with the observations that both CA RNA and DNA levels were significantly lower in NNRTI-treated participants .9) The authors should discuss the need for a clinical trial in which participants would switch from PI to NNRTI to definitely confirm their findings in a longitudinal way.This is added to the Discussion (page 23).10) in one of their study cohorts, the authors do not observe an association between duration of ART and cell-associated HIV DNA/RNA levels; this could be due to clonal expansion of viral reservoir cells that amplify and expand viral reservoir size during long-term ART. The authors may wish to discuss this.This is a nice suggestion. We did discuss that the lack of association between duration of suppression and CA RNA and DNA in the COBRA cohort is probably due to the fact that after several years on ART, CA RNA and DNA reach a plateau and do not decline further. The reviewer provides a plausible explanation for this plateau effect. We agree and have included this in the Discussion (page 18).11) the authors cite Li et al. (ref 60) to argue that NNRTI treatment is associated with delayed viral rebound after treatment interruption; however, this is likely unrelated to viral reservoir size, and rather caused by altered PK and the longer half-life of NNRTIS relative to PIs.We agree that this could contribute to the results of Li et al. and included this in the Discussion (page 22).


We designed a prospective registry. Paroxysmal atrial fibrillation patients who underwent cryoballoon PVI were included. Capnography was performed. Occlusion was tested by injection. A comparison between ΔetCO2 and occlusion was performed. Eighteen subjects (138 injections) were included. A drop of >3.5 mmHg predicted occlusion of the PV . A ΔetCO2 of ≥3.5 mmHg during inflation of the cryoballoon in each PV directly correlates with PV balloon occlusion.The purpose of this study was to quantify the relationship between a drop in end-tidal carbon dioxide (etCO Despite good progress in its management, it remains one of the major causes of stroke, heart failure, sudden death, and dementia.1 This is most commonly achieved by radiofrequency current application in a point-by-point mode, leading to cellular necrosis by tissue heating. An alternative method is the use of cryogenic energy applied with a balloon in a single-shot mode, which leads to necrosis by freezing the tissue. Radiofrequency ablation for AF requires only limited use of fluoroscopy and no use of contrast media because catheter guidance is achieved with the use of an electroanatomic mapping system, but this approach demands extensive training.3 In contrast, cryoablation for AF requires more extensive fluoroscopic guidance and the use of iodinated contrast to position the balloon catheter, but it creates a circular lesion around the PV in a relatively simple manner.4PV isolation (PVI) is the primary target of ablation.4 demonstrated that the cryoballoon ablation technique was non-inferior to radiofrequency ablation with regard to the primary efficacy endpoint . There was no difference in the primary safety endpoint between the groups.Kuck et al.5 PV occlusion is verified in a standard method by injecting contrast through the cryoballoon when it is insufflated into the vein, followed by observing the retention of the contrast agent in the distal PV branches by fluoroscopy.6 Other methods for PV occlusion testing include PV pressure waveform monitoring7 and intracardiac echocardiography (ICE) examination under Doppler flow display.8Applying a cryoballoon for PVI requires fluoroscopic guidance to navigate the balloon catheter to the left atrium. When occlusive circumferential contact is established at the PV ostium, the cryoballoon leads to durable PVI.9 Second, during freezing, the time to acute intra-ablation PVI (time to isolation [TTI]) as recorded by an entrance and/or exit block test with a dedicated inner lumen circular diagnostic catheter is the most important parameter.9 However, not all PVs can be monitored for TTI because of the short (or missing) muscular sleeve extension into the PV.10 Finally, after delivering the freezing energy, the interval thaw time between the cryoballoon and the PV is another hallmark of an effective cryoablation procedure, with longer thaw durations favoring a more durable lesion as a consequence of proper balloon-to-tissue contact.11When examining the long-term efficacy of a cryoballoon procedure, there are 3 important acute balloon-related biophysical parameters to consider. First, it is critical to ensure complete PV occlusion between the balloon and the antral PV surface.Any technique able to predict the occlusion of the PV without the use of contrast media and fluoroscopy would be advantageous not only in terms of efficacy but also in terms of less radiation and contrast agent applied. If this can be achieved, the cryoballoon technique can be considered suitable for patients with renal failure. In addition, another measure of efficacy can potentially increase the reliability of the procedure.2) in patient-exhaled gases by side-stream gas sampling measured by infrared technology to detect CO2. It is part of standard monitoring during general anesthesia,12 and capnography is utilized to monitor systemic, pulmonary, and coronary blood flow.13 During capnography monitoring, cardiac output and end-tidal CO2 (etCO2) have a logarithmic relationship.14 In consequence, etCO2 is a sensitive indicator of pulmonary blood flow.15Capnography determines the concentration of carbon dioxide  value able to predict occlusion of the PV.The main objective of this study was to quantify the relationship between the drop in etCO2 were performed for all patients as part of the standardized monitoring protocol for patients undergoing a cardiovascular intervention who required general anesthesia in our institution. All patients gave informed consent before the cryoablation procedure, and the confidentiality of all patients was protected. According to the institutional protocol, risk-free studies do not require approval by an ethics committee.We designed a prospective single-center registry in which all patients who underwent PVI using cryoballoon ablation in a cardiovascular-dedicated center were potential candidates for inclusion. The information of 18 patients treated between October 2019 and March 2020 was recorded consecutively in a pre-determined database added to by the researchers during every procedure. Patients assessed in our outpatient clinic, during admission because of AF or other cardiovascular conditions, or patients referred from other institutions, were considered to be enrolled in an “all-comers” way if they met the inclusion criteria of an age of >18 years, a diagnosis of paroxysmal AF despite ≥1 anti-arrhythmic drug, and suitability for a cryoballoon ablation procedure. Patients were excluded in cases of permanent or persistent AF, a severely dilated atria, non-favorable PV anatomy , an intracavitary thrombus, mechanical valves, pregnancy, lack of patient information, or severe comorbidities. Capnography and etCO® cryo-catheter . Pre-procedural work-up included contrast chest computed tomography (CT) imaging to evaluate the PVs and left atrium anatomy. A transesophageal echocardiogram was obtained ≥24 hours before the procedure to rule out intracardiac thrombi.All cryoablation PVI procedures were performed using a second-generation 28-mm Arctic Front AdvanceIn patients with an indication for permanent anticoagulation either with direct oral anticoagulants or warfarin, the procedure was performed with a previous interruption of anticoagulant therapy 24–48 hours before the procedure or when the international normalized ratio ranged from 2–3.®; Abbott, Chicago, IL, USA), and right jugular vein access with a 7-Fr sheath for a decapolar  or duo-decapolar  catheter placed in the coronary sinus. Immediately after the venous punctures, the patient was anticoagulated with sodium heparin at an initial bolus of 100 IU/kg with an activated clotting time target of 200–350 seconds during the procedure.All procedures were performed under general anesthesia. Vascular punctures were performed using ultrasound guidance. Vein accesses included the right femoral vein for 6-French (Fr) and 8-Fr sheaths, the left femoral vein with an 11-Fr sheath for ICE  was advanced. Using a 71-cm transseptal needle  and under fluoroscopic and direct ICE vision, transseptal catheterization was performed. Afterward, the 0.032-in guidewire was exchanged for a 260-cm-long high-support coated hydrophilic guidewire , and a FlexCath Advance™ 15-F steerable sheath (Medtronic) was then placed in the left atrium.Through the 8-Fr sheath, a 0.032-in guidewire was advanced, over which a transseptal introducer sheath (Swartz® circular mapping catheter (Medtronic) were advanced into the left atrium and subsequently into every PV ostium. The Achieve® catheter was advanced into every vein and the cryoballoon  was insufflated over it, with iodinated contrast subsequently injected through the distal tip of the catheter to test the complete sealing of the PV before proceeding with cryoablation during 240 seconds. In cases of incomplete sealing, a new position of the Achieve® catheter in another vein branch was adopted and iodinated contrast was injected again until an acceptable seal between the vein and cryoballoon was achieved. An absence or dissociation of PV potentials was confirmed. In cases with persistent non-dissociated potentials, a second lesion was created. Additional lesions were performed at the discretion of the main operator. During the right PVI, simultaneous phrenic nerve stimulation was carried out to detect its lesion during ablation. Simultaneously, basal and minimal capnography values during each insufflation were recorded. On the right side, a record was taken before starting phrenic nerve stimulation.Guided by ICE and fluoroscopy and using CT imaging of the left atrium as an anatomic reference, the cryoballoon and the Achieve2 is measured in the exhaled gases using infrared light to generate a capnography curve that is used to supervise organ perfusion due to the direct proportional relationship between etCO2 and cardiac output. Likewise, etCO2 can be a marker of a ventilation–perfusion mismatch created during PV occlusion while the cryoballoon is inflated during cryoablation.Side-stream capnography is routinely performed in all patients who undergo major cardiovascular procedures under general anesthesia at our institution. Capnometry of etCO® anesthesia machine  with a corresponding full-size patient monitor (etCO2 monitor included) was used in each procedure. Patients were ventilated under the assisted-controlled ventilation mode (either with volume or pressure) through endotracheal intubation. A tidal volume of 6–8 mL/kg was programed. The respiratory rate was adjusted to maintain an etCO2 of 28–38 mmHg. Hemodynamic variables  were intervened to keep them as stable as possible in order to avoid or minimize any variations in etCO2 . If needed, a noradrenaline infusion or cardiac stimulation (through a decapolar or duo-decapolar catheter) was used. The etCO2 was registered in the pre-designed form before, during, and after each cryoablation session.Orotracheal intubation was performed following the standard institutional protocol. A Datex-Ohmeda Aestiva 52) value was calculated from the difference between the baseline capnography values in every patient and the minimum capnography during ablation for every application in each vein. To our knowledge, there are no reports in the literature that describe a cutoff of ΔetCO2 that correlates with vein occlusion guided by contrast media injection and fluoroscopy.The capnography delta  values or median with interquartile range (IQR) values, while categorical variables were given as absolute numbers and percentages. Comparisons between groups were performed using Student’s 2 that predicts PV occlusion compared to the standard of reference, which, in this study , was the vein occlusion appreciation of the operator. The diagnostic accuracy of the values of ΔetCO2 was assessed during receiver operating curve (ROC) analysis, presented with an area under the ROC curve (AUC) with 95% confidence intervals (CIs). Additional diagnostic parameters, such as sensitivity, specificity, and likelihood ratio (LR), were also determined. Data were not imputed, and missing capnography values were excluded from the analysis. Sample size was not calculated as all patients who underwent cryoablation were included. Statistical analyses were completed in SPSS Statistics version 20 .The main outcome was to determine a cutoff for ΔetCO1 55.6% had 2a (mild) symptoms. One hundred thirty-eight cryoablation applications were performed. Among these, 108 of them occluded the PV as confirmed by contrast media injection and fluoroscopy. The average age of the population was 51.5 years . Twelve patients (66.7%) were men. The main comorbidity found was dyslipidemia in 8 patients (44.4%). None of them had a history of coronary heart disease or previous cardiac surgery. Half of the patients were previously treated with apixaban (22.2%) or rivaroxaban (27.8%). Six had no previous anti-thrombotic management. Every patient was on an anti-arrhythmic, mainly metoprolol tartrate (61.1%). Their median left ventricular ejection fraction as documented by echocardiography was 60%. The left atrium area and volume average were 18.6 cm2  and 26.5 cm3 (21–34 cm3), respectively. In 6 patients, an accessory vein was observed during the left atrial CT scan. One patient presented with cardiac tamponade requiring percutaneous drainage (this complication probably occurred during manipulation of quadripolar catheter for stimulation of the phrenic nerve). The other procedures were performed without any complications .A total of 18 subjects with symptomatic paroxysmal AF were included. Eight of them had associated cavo-tricuspid isthmus–dependent flutter. According to the modified European Heart Rhythm Association classification,Table 2. Also, values classified by the side of the veins are shown. There were no significant differences in either baseline, minimal, or delta capnography when classified by right- or left-side veins . Considering both sides, the overall ROC analysis showed an AUC of 86%  . Further analysis proved that a ΔetCO2 of >3.5 mmHg had the best accuracy cutoff to predict PV occlusion during the cryoballoon ablation procedure, with a sensitivity of 80%, a specificity of 86.7%, a positive LR of 6, a positive predictive value of 96%, and a negative predictive value (NPV) of 54%.Baseline and minimal capnography measures are described in . Left-side veins showed a better diagnostic performance with an AUC of 87%  . Detailed information about diagnostics for left-side veins with a ΔetCO2 cutoff of 3.5 is reported . Right-side veins had a lower AUC compared to the left-side veins at 77%  . However, the difference was not statistically significant . For right-side veins, the best cutoff was also established with a ΔetCO2 of 3.5 mmHg . An exploratory analysis using other capnographic variables, such as the initial capnography and final capnography values, was carried out. However, none of them showed an adequate diagnostic accuracy compared to ΔetCO2.Additional accuracy diagnostic parameters were assessed by every side vein to look for differences depending on the left or right side based on the differential cardiac output by side or the effect exerted by stimulation of the phrenic nerve . The mean diameter was 11.87 mm. In those cases, we attempted to isolate the PV with the same balloon. The response in terms of the drop in capnography was attenuated as a consequence of less flow interrupted. However, given that we did not analyze the drop in capnography for each vein, it is not possible to precisely know the effect of occlusion for each accessory vein.There were 6 patients with accessory PVs The main finding of this exploratory diagnostic registry is the confirmation that, during a cryoballoon PVI procedure, a drop in the capnography value successfully predicted complete occlusion of the PVs. In fact, our data show that a drop of >3.5 mmHg predicts occlusion of the PV with a sensitivity of 80% and a specificity of 86.7%. There was no significant difference between the right- and left-side veins.16 described and quantified the capnographic changes during cryoballoon PVI. They determined the utility of end-tidal CO2 as an indicator of PV occlusion during ablation. In their study, 80 PVs were acutely isolated with a cryoballoon. The sensitivity for PV occlusion was 90% for the right superior PV, 70% for the left superior PV, and 50% for both the right and left inferior PVs. These findings showed a lower sensitivity compared to ours. One possible explanation is the greater proportion of gas exchange occurring in the upper lobes during general anesthesia. Depending on the ventilatory technique chosen, the lower lobes may remain under-ventilated. They originally used this method as a primary tool in order to avoid or minimize contrast agent administration, in particular for patients with renal dysfunction or pre-diagnosed contrast agent allergies. They preferred to use this method as a complementary tool to assess the maintenance of the occlusion during PVI. This method was found to be very useful in obese patients in whom visualization of contrast media is difficult due to a reduction of fluoroscopy penetration across a larger body mass. Also, they mentioned the utility of this method during repositioning of the balloon for repeated ablation applications. Additionally, they demonstrated that there was no influence of the rhythm (AF or sinus rhythm) on this physiological finding.In 2015, Hoyt and Lim10 performed a retrospective examination of observational data from 15 patients who underwent a cryoballoon PVI procedure. They reviewed and recorded the capnography readings during those procedures and the parallel cryoballoon catheter performance parameters  during each cryoablation shot. They found a direct correlation between nadir temperature and the capnography nadir of etCO2 (P = .0001 for the Pearson correlation). Interestingly, in their study, most etCO2 nadirs were typically achieved within 60 seconds of PV-to-balloon occlusion.In 2016, Sircy et al.17 evaluated 30 subjects who underwent cryoballoon PVI for a drug-refractory symptomatic paroxysmal AF. They monitored and used etCO2 to evaluate the occlusion of each PV. Subjects were monitored during long-term follow-up for the recurrence of AF. Strikingly, they excluded patients with a clinical history of diabetes or dysautonomia . They used the same protocol for etCO2 sampling previously described by Hoyt and Lim.16 The primary efficacy endpoint was the 1-year outcome of the cryoballoon ablation procedure. The secondary endpoint was to determine whether any etCO2 level could be used to differentiate between subjects who had recurrent AF and subjects who maintained a normal sinus rhythm. As in our study, they calculated the ΔetCO2  at each PV measured before each cryoablation. They followed all patients for 1 year after the index cryoablation. Of the 30 subjects, 5 had a recurrence of atrial arrhythmia (>30 seconds), meaning a long-term success rate of 83.3%. The main finding of this study was a significant difference in ΔetCO2 between the sinus rhythm and recurrent AF patients . However, a definitive “cutoff” value useful for predicting the persistence of a sinus rhythm at 1 year was not found due to the large overlap in values between the groups. Another interesting finding of their study was that failures to achieve a change in etCO2 in 60 seconds could be used with other parameters to terminate an unsuccessful freeze attempt.In 2018, Pickett et al.2. Therefore, there is a possibility of bias toward occlusion determination by the standard method when a significant drop in etCO2 was observed. Another important limitation is the low NPV obtained. It must be recognized that this is an exploratory registry with few patients. Its primary purpose was to test a concept and estimate a cutoff point for ΔetCO2. A larger study is needed to get more accurate specificity and sensibility values. In this protocol, PV tomography was used to characterize the anatomy of each PV. As a consequence, use of a 3-dimensional mapping system was not necessary. Finally, every procedure was performed under general anesthesia to favor the patient’s comfort, allow the use of an esophageal thermometer, and stabilize ventilatory parameters. The general anesthesia protocol was standardized to eliminate variations in the respiratory pattern as a potential cause of variations in capnography measurements. We do not know whether carrying out the procedure under sedation allows us to appreciate the changes in capnography. To avoid phrenic stimulation from affecting ΔetCO2, each right PV was occluded, and we waited for a drop in capnography to make a measurement. Then, cooling was started with simultaneous phrenic stimulation. Practicing the procedure under deep sedation could be an alternative to explore in the near future to verify if the same changes in capnography can be measured.The most important limitation of our registry is that, in some cases, the main operator, who was responsible for determining the PV occlusion by the conventional method (the standard for comparison), was not blinded to the value of etCO2) able to predict the occlusion of each PV with an acceptable level of sensitivity and specificity for each PV, with independence from the PV side. The main advantage of this tool was the reductions in contrast media and fluoroscopy use to determine PV occlusion. In contrast with other techniques (such as intracardiac echo), this measure can be performed in each PV no matter whether there are anatomic or positional variations. Another advantage of this technique is that no additional devices are needed (such as a direct measure of PV pressure during the occlusion), and it can be practiced routinely without increasing the risks for patients or the cost.To the best of our knowledge, this is the first prospective registry that has achieved an accurate determination of a capnography cutoff value (ΔetCO2 (ΔetCO2) of ≥3.5 mmHg in capnography during inflation of the cryoballoon in each of the PVs directly correlates with PV balloon occlusion. The measurement of ΔetCO2 constitutes a very simple, reliable, and universally available method for monitoring the PV occlusion during PVI by cryoballoon that allows for the use of less contrast media and fluoroscopy.In conclusion, a drop in etCO2 and freedom from atrial arrhythmia at 1-year follow-up.A prospective study in which the main operator is blinded to the capnography results is necessary in order to avoid the previously described risk of bias. Additionally, it would be very useful to know whether there is a direct correlation between ΔetCO


The differentiation of solitary intra-axial hematomas from hemorrhagic neoplasms based on their magnetic resonance imaging (MRI) features is challenging. The treatment and prognosis for these two disease entities are vastly different and distinction between them is often based on MRI findings alone. The aim of this study was to describe the 1.5 tesla MRI features of canine intra-axial hematomas and correlate these findings with the evolution of hemorrhages described in human brains. Retrospective evaluation of patient details, clinical signs, and MRI findings of dogs with intra-axial hematomas that were histopathologically confirmed or determined via repeat MRI study and/or resolution of neurological signs. Ten dogs met the inclusion criteria. All 10 hematoma lesions were determined to be 2–7 days in age. On MRI, all 10 hemorrhagic lesions were comprised of two distinct regions; a relatively thin T1-weighted (T1W), T2-weighted (T2W) and gradient echo (GRE) hypointense (9/10) peripheral border region and a large central region that was heterogenous but predominantly T1W, T2W and GRE hyperintense (8/10). The peripheral border region was complete in its integrity in all 10 cases on T2W and GRE sequences. Contrast enhancement was present in (6/10) hematoma lesions and was always peripheral in nature with no evidence of central enhancement associated with any of the lesions. An intra-axial hematoma should be suspected in solitary hemorrhagic space occupying lesions that have a complete hypointense peripheral rim, elicit a peripheral contrast enhancement pattern, and display the expected temporal pattern of hematoma evolution. The smorrhage –6. Consent echo T* and/or The MRI appearance of intracranial hemorrhage can be complex and has different signal intensity patterns, which depend on several factors including: (a) intrinsic factors  ; (b) bioAngiostrongylus vasorum infection ; (b) the MRI study confirmed a solitary hemorrhagic space-occupying intra-axial lesion, and subsequently; (c) there was histopathological confirmation of a benign hematoma , or a follow-up MRI that indicated resolution or marked improvement of the previous lesion with only supportive treatment, and/or the patients clinical signs resolved with only supportive treatment. In cases where diagnosis of an intra-axial hematoma relied upon clinical resolution alone, a sufficient follow-up period was defined as 3 months after first presentation. Patients were excluded if the MRI study or medical data was incomplete, if they had received cytotoxic therapy , if they had a suspected or known history of trauma, if they underwent repeat MRI and this revealed a worsening of the severity of the intracranial lesion or the lesion remained unchanged, and if insufficient clinical follow-up was available.In this retrospective multicenter descriptive study, medical records of three different referral institutions were reviewed to identify dogs that had intra-axial hematomas on MRI. Ethical approval was granted by the Ethical Review Committee of the University of Nottingham School of Veterinary Medicine and Science. Patient inclusion criteria were set as: (a) a complete brain MRI study was performed  and clinical/neurological findings at re-examination.MRI protocols and sequences varied between institutions, but all MRI examinations were completed with dogs under general anesthesia, using high-field-strength magnets: 1.5 Tesla . Anesthetic protocols were varied and adapted to individual patients by the attending anesthetist. The MRI studies were reviewed by three observers independently [two ECVDI certified veterinary radiologists (AH and IC) and a ECVDI resident (JW)], followed by collective consensual evaluation. All available follow-up MRI studies were also evaluated. Reviewers were aware that all dogs had intra-axial hematomas. The following features were assessed on image evaluation:The location of the lesion, and the distribution of the lesion within either the gray or white matter.Lesion margination was characterized subjectively as well or poorly circumscribed.Lesion shape was defined as spherical, ellipsoidal, or irregular.* or SWI.The signal intensity of the lesion in each sequence was evaluated both at the lesions periphery and at its center and graded as hypointense, isointense, hyperintense, or mixed intensity, relative to cerebral gray matter on T1W, T2W, and T2If there were distinct regional differences in the signal intensity between the periphery of a lesion and its center, then the diameter of the peripheral region was measured at its widest point.The degree of contrast enhancement was subjectively categorized . Furthermore, the pattern of enhancement was also noted .Perilesional edema was graded as absent, mild (present in the area immediately adjacent to the lesion), moderate (extending beyond the area immediately adjacent to the lesion) or marked (involving the entire hemisphere). The amount of edema was determined by the extent of abnormal hyperintensity on T2W images, which was T1W iso to hypointense, non-contrast enhancing, and exceeded the boundary of the hematoma.Any mass effect  was subjectively graded as mild, moderate, or severe.Finally each reviewer estimated the age of each lesion  based on its signal intensity pattern and how this correlated to temporal hemorrhage evolution according to previously published data in dogs  Table 1Table 1.All MRI examinations were assessed using a PACS workstation DICOM viewer . All measurements were performed collectively during group evaluation.Ten dogs met the inclusion criteria. These included four Crossbreeds, two Staffordshire Bull Terriers, a Dachshund, a Border Collie, a Jack Russell Terrier, and a Yorkshire Terrier. Of these, five were neutered females and five were neutered males. The age of the dogs varied between 7 and 13 years (median age 9.8 years) and the weight varied between 5.4 and 25.6 kg (median weight 15.72 kg).The presenting neurological signs for each patient are summarized in A.vasorum. One patient was diagnosed with systemic hypertension on presentation (Case 9) via repetitive non-invasive indirect estimations of systolic blood pressure using a doppler devices. The hypertension was considered idiopathic in nature due to concurrent normal hematology, serum biochemistry, and urinalysis results. Aside from this patient, an underlying etiology responsible for the intracranial hemorrhage was not identified in the remaining cases. Records of blood pressure were not available in the remaining cases. Medical treatment prescribed to the patients included: prednisolone (5/10), fenbendazole (3/10), levetiracetam (4/10), and dexamethasone (2/10). All treatments were discontinued in all patients within 3 months of the initial presentation.Hematology and serum biochemistry results were available for all 10 patients. Hematologic abnormalities included mild neutrophilia (2/10), increased hemoglobin concentration (1/10) and mild anemia (1/10) and in all other cases the hematology results were normal. The only biochemical abnormalities identified were increased alanine aminotransferase (2/10), increased alkaline phosphatase (1/10), and hyperglycemia (1/10). All patients had clotting profiles within normal limits. All patients tested negative for Histopathological confirmation of the diagnosis of a benign intra-axial hematoma was achieved in (2/10) of the patients. A diagnosis of a benign hematoma was made in (4/10) patients based on the results of a follow-up MRI study, which was also accompanied by neurological improvement. In the remaining four patients diagnosis was met by clinical follow-up alone, all these dogs were reported to have shown marked neurological improvement within 1 month after first presentation. On subsequent re-evaluations after 3 months all dogs were considered neurologically normal.In all 10 dogs the hematoma lesion was located within the forebrain and in 9/10 the lesion was localized to the white matter. The most frequently involved forebrain substructure was the frontal lobe , followed by the parietal lobe , the thalamus , and then single cases of occipital and temporal lobe involvement . All 10 Contrast enhancement was evident in (6/10) hematomas and absent in 4/10) cases. When present the enhancement was always present at the periphery of the hematoma , 4. Enha/10 casesFluid-fluid level lesions were evident within the central portions of the hematomas in Case 3 and 4. In Case 3 this feature manifested as the dependent and ventral region being T2W hyperintense and T1W hypointense, and the dorsal region being T2W hypointense and T1W isointense . In CaseFour out of the ten dogs had repeat MRI studies; case 2, case 4, case 8 and case 10 at 207, 32, 46, and 83 days following their initial MRI study, respectively . On evaluation of these follow-up studies, it was observed that in all four cases the lesions had markedly reduced in volume, were hypointense in all sequences, and both perilesional edema and contrast enhancement were absent .*W hypointense peripheral border region that is also T1W hypointense but thinner, and a large predominantly T2W hyperintense central region. Additionally, intra-axial hematomas were shown to have mild to moderate peripheral contrast enhancement with no central enhancement. Furthermore, hematomas were frequently associated with mild to moderate mass effect and mild to moderate perilesional edema. These findings largely reflect the MRI characteristics of intra-axial hematomas observed in humans.This study demonstrates that in the period of 2–7 days post hemorrhage, intra-axial hematomas most commonly appear as ordered dichotomous lesions on MRI images consisting of a complete T2W and T2The fact that all hematoma lesions in this study demonstrated a uniform regional duality consisting of a complete thin peripheral region and a large central region is in direct agreement with human studies , 29. ThiWhen considering the predominant blood breakdown products within the hematoma lesion it is important to bear in mind the peripheral to central nature of hematoma maturation. The “oldest” or most advanced breakdown products will be present at the periphery of the lesion. When considering the likely constituents of the peripheral border in the hematoma lesions in our study, this factor was considered alongside the hypointense signal of this region in all sequences and the pattern of signal intensity within the central region that showed loss of the magnetic susceptibility effect (indicating the presence of extracellular methemoglobin). Consequently, the peripheral border had to be ferritin and hemosiderin and not a less advanced product like deoxyhemoglobin that would have also satisfied the signal intensity pattern seen.* weighted hypointense peripheral region noted in all 10 cases. This is in agreement with previous human literature which also found that the presence of a complete peripheral hypointense rim in a hemorrhagic lesion was associated with benign intra-axial hematomas  rather than the 7–14 day period (late subacute phase) following the hemorrhage . CompartTwo hematomas had regions where a fluid-fluid interface was evident. This feature in intracranial hemorrhage has been associated with coagulopathic disorders . HoweverThe underlying etiology for the development of the intracranial hematoma was not established in 9/10 cases. This is not uncommon with a previous study observing that 32 of 43 dogs (74%) with a solitary intracranial hematoma had no concurrent underlying medical condition and the underlying etiology could not be established . The undThe principal limitation of this study is the small number of cases where gold-standard histopathological confirmation of a hematoma was available. Nonetheless, resolution of the clinical and neurological signs, long-term clinical follow-up, and a repeat MRI study (where available) for the remaining cases provides a reasonable assertion that these lesions were attributable to simple hematomas rather than hemorrhagic malignancies. The subjective and qualitative nature of assessment of multiple factors on the MRI studies  may have also led to inaccuracies. However, the individual and collective group assessments of the MRI findings may have mitigated these to a degree. Additionally, the study was limited by the low numbers present in the sample population, but this reflects the uncommon occurrence and/or identification of solitary benign intra-axial hematomas in the clinical setting. Serial monitoring of the hematoma lesions with MRI over time would have been rewarding to document the characteristics of more chronic phases of hematoma evolution, but this was not viable in the clinical setting. Finally, it must always be kept in mind that the imaging characteristics of hemorrhage are linked to magnetic field strength with higher magnetic field strengths enhancing susceptibility effects and related signal loss. Consequently, this changes the appearance of hematomas at 1.5 T relative to lower magnetic field strengths because more profound hypointensity will be shown in the presence of deoxyhaemoglobin, hemosiderin, or other compartmentalized paramagnetic material .In conclusion, this study suggests that a benign intra-axial hematoma should be considered as a primary differential when a solitary intra-axial hemorrhagic lesion with a complete thin hypointense peripheral rim, a variable intensity central region, and a peripheral enhancement pattern is identified on MRI. Accurately differentiating between benign intra-axial hematomas and hemorrhagic tumors using MRI is challenging and can be stressful for all those involved in the clinical decision-making process, particularly given that euthanasia or an expensive treatment could be chosen if a malignancy is suspected. This is further compounded by the fact that a definitive diagnosis is seldom achieved for such lesions antemortem. In this scenario, depending on the patient's clinical status and severity of the neurological signs, the authors propose that it would be prudent to monitor the patient for further improvement or deterioration combined with follow-up advanced imaging, if feasible. Further studies could also be conducted evaluating the potential inverse MRI characteristics of solitary intra-axial hemorrhagic neoplasms in dogs.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Ethical Review Committee of the University of Nottingham School of Veterinary Medicine and Science. Written informed consent was obtained from the owners for the participation of their animals in this study.IC conceived of the presented idea. JW, IC, and AH performed the image evaluation. JW wrote the manuscript. All authors contributed cases and discussed the results and contributed to the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bovine viral diarrhea virus (BVDV) infection results in a wide variety of clinical manifestations and is a pathogen that is able to cause huge economic losses in the cattle industry worldwide. It is important to identify cattle that are persistently infected (PI) by BVDV within the herd as early as possible because PI animals are the main reservoir of the virus. In contrast, cattle who are acutely infected (AI) with BVDV show various clinical signs, but most cattle show either mild symptoms or are asymptomatic. In general, AI and PI animals can be distinguished by repeat testing within an interval of at least 21 days. However, we found a rare case of a BVDV2-infected AI animal with long-term viral presence, making it indistinguishable from PI through two tests within an interval of 21 days. As a result, we diagnosed one infected animal as AI after 35 days from the initial sample collection via multiple analyses. Our findings recommend performing an additional test using samples that have been collected after 14–21 days from the second sample collection in cases where it is difficult to accurately differentiate an AI diagnosis from a PI diagnosis after only two tests. Additionally, our analysis exhibits that monitoring the number of copies of viruses with similar genomes in the sera by means of quantitative real-time RT-PCR through several sample collections periods might be useful to distinguish AI from PI. Furthermore, our data suggest that the AI animals with a long-term viral presence who show test results similar to those of PI animals might be the result of a coincidental combination of various factors that are present in cattle fields. These findings provide useful information that can be used to improve the diagnosis of BVDV in the field. Pestivirus of the family Flaviviridae and are one of important causative pathogens among cattle disorders and that have a worldwide economic impact . Th. Th30]. The partial genomes of the 5′-untranslated region (UTR) and the E2 region were amplified using the SuperScript III One-step RT-PCR System with Platinum Taq DNA Polymerase  and primer sets that were previously reported  31,32].,32.31,32Upon initial sample collection, the Ct values  that were positive for BVDV were detected in the sera from six of seven calves (nos. A1–A3 and B2–B4) by RT-qPCR . In addiDuring the the second sample collection period, which occurred approximately 21 days after the initial sample collection, sera were collected from seven calves. Ct values  that were positive for BVDV were obtained in the sera from four calves . In addition, ncp-type BVDVs and cp-type BVDV were isolated in the sera from four calves  and in the serum from one calf (no. A3), respectively. Moreover, the analysis of the AgELISA results showed S-N values (3.276–3.702) that were positive for BVDV in the sera from four calves . Furthermore, the sera from seven calves showed titers (negative to 32 and negative to 1024) against BVDV1 and BVDV2 in the virus neutralization test, respectively. Of them, the sera from three calves  showed relatively higher titers (512 to 1024) against BVDV2 than those from the remaining four calves.During the third sample collection period, which took place approximately 35 days after the initial sample collection period, serum was collected from one calf (no. A1). A Ct value (35.2: GE copies 3) that was positive for BVDV was detected in the serum. In addition, no BVDV was isolated in the serum. Moreover, the analysis of the AgELISA results showed an S-N value (1.030) that was positive for BVDV in the serum. Furthermore, the serum showed titers (4 and 4) against BVDV1 and BVDV2 in the virus neutralization test, respectively.In addition, the BVDVs that were detected in these animals were classified as belonging to BVDV2 via BVDV genotyping by multiplex RT-qPCR .Collectively, these findings showed that the three calves  who obtained positive results in the RT-qPCR, virus isolation, AgELISA tests and no seroconversion through two tests occurring within 21 days of each other were determined to be PI animals with BVDV2. On the other hand, three calves  obtained negative results in the RT-qPCR, virus isolation, and AgELISA tests and had undergone seroconversion through two tests occurring within 21 days of each other were identified as being AI animals with BVDV2. Regarding the last calf (no. A1), who demonstrated positive results for the RT-qPCR, virus isolation, AgELISA tests and no seroconversion at both the initial and second sample collection period was identified as a PI animal. However, the negative virus isolation and seroconversion results that were detected by virus neutralization test during the third sample collection period were mismatched to the PI animal criteria . ConsequThe BVDVs that were detected from five  of seven calves were classified into BVDV2 the category via the phylogenetic analysis for the 5′-UTR and E2 genomic sequences  . The 5′-Sera from 143 milking cows (24 to 106 ages in months) in the farm A and from 53 milking cows (24 to 119 ages in months) in the farm B showed median titers of 8 and 4 and 32 and 512 against BVDV1 and BVDV2 in the virus neutralization test, respectively .Our study identified a rare field case of an AI animal with the long-term presence of BVDV2 in serum, resulting in the animal being indistinguishable from the PI animals, even after two tests conducted 21 days apart. Eventually, our study demonstrated that 35 days and multiple sample collection periods were need before we could diagnose that one calf (no. A1) as an AI animal with BVDV2.During the initial and second sample collection periods, all of analyses were suggested three calves  were PI animals, as they were shown to have positive Ct values as demonstrated by RT-qPCR, ncp-type virus isolation, high S-N values, and no seroconversion of the BVDV2 antibody. In contrast, all of the analyses that were based on the two sample collection periods from three other caves calves  indicated that they were AI not PI animals, as they were shown to have negative Ct values as demonstrated by RT-qPCR, no virus isolation, low S-N values, and seroconversion of the BVDV2 antibody. All of the analyses using paired samples that had been collected from the last one calf (no. A1) suggested that the calf was a PI animal, as it was shown to have positive Ct values as demonstrated by RT-qPCR, ncp-type virus isolation, high S-N values, and no seroconversion of the BVDV2 antibody. Specifically, the Ct value (28.1: GE copies 280) in the serum from the calf at second sample collection period was matched to the threshold value  of that of a putative BVDV PI animal, but it was more increased from the Ct value  that was found in the serum at the initial sample collection period. Our previous study suggested that it is important to conduct tests using various analyses throughout multiple sample collection periods in order to accurately diagnose BVDV . TherefoCalves that are under one month of age do not have a fully functional adaptive immune system ,36,37. TIn general, the PI calf is the most important reservoir of BVDV due to the shed viruses that are present in various secretions that an animal is exposed to over the course of its life ,41. On fThe most important point to consider when trying to control BVDV infection is to find PI animals early and to eliminate them from the herd ,42. ThisIn conclusion, our analyses for seven calves via multiple tests identified four AI calves and three PI calves who were infected with BVDV2. One AI calf could not be excluded as a possible PI after retesting 21 days after the initial sample collection period based on the OIE Terrestrial manual. However, the calf that was unable to be distinguished as being AI or PI after a third test was conducted 35 days after the initial sample collection period. Our data suggest that the prolongation of AI presenting similarly to PI might be the result of the three following factors: the immature and delayed adaptive immune response of a newborn calf, passive immunity from colostrum, and long-term virus exposure from another PI calf. The data presented herein provide useful information that can be used to help distinguish AI from PI. Further information should be accumulated through multiple analyses of more field cases focusing on BVDV outbreaks in order to contribute to better criteria for a more accurate diagnoses of BVDV in the future.


This cross-sectional study uses surveillance data to assess temporal trends in suicide attempts among children admitted to a pediatric emergency department in Paris, France, in the decade before and during the COVID-19 pandemic. We conducted a cross-sectional study of surveillance data collected over the past 10 years at the Robert Debré Hospital in Paris, France, which is one of the largest pediatric centers in Europe.4 Deaths by suicide were not included in the analysis because those data were not available in real time.This cross-sectional study included all children aged 15 years or younger who attempted suicide and were admitted to the pediatric emergency department of Robert Debré Hospital between January 2010 and April 2021. The study comprised 830 children, with a mean (SD) age of 13.5 (1.5) years and a 1:4 ratio of boys to girls. A suicide attempt was defined as a nonfatal self-directed potentially injurious behavior with any intent to die as a result of the behavior.5 The number of suicide attempts observed at time t was decomposed as (1) a seasonal component accounting for the variations observed during the year, (2) a long-term trend, and (3) a remainder. In the absence of atypical events, the remainder should be centered and uncorrelated and should have a constant variance.For this study, we conducted a robust seasonal-trend decomposition using locally estimated scatterplot smoothing analysis on the bimestrial time series of all suicide attempts in the 2010 to 2021 surveillance data.STROBE) reporting guideline. The independent ethics committee of the Assistance Publique–Hôpitaux de Paris Health Data Warehouse approved the study protocol. Informed consent was not required, in accordance with French law regarding the retrospective use of monocentric anonymized routine data.This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (P = .003), with a coefficient of −0.079 .The deseasonalized time series results showed that the number of suicide attempts among children decreased from 12.2 at the lowest level (July to August) in 2019, to 7.8 during the first lockdown period (March to April) in 2020 in France −36%; ; Table. 4 Our analysis further suggests a dramatic increase in suicide attempts among children in late 2020 and early 2021 after the start of the COVID-19 pandemic in France.Since 2010, the incidence of suicide attempts among children has increased worldwide.6 Many factors may have contributed to this acceleration, such as children’s specific sensitivity to mitigation measures, deterioration of family health and economic conditions, increased screen time and social media dependence, or bereavement. Interestingly, we also observed a decrease in suicide attempts in the early months of the pandemic during the March 2020 lockdown period in France, which may have resulted from not only increased parental supervision but also difficulties in accessing urgent care.Similar results in the dynamics of suicide, suicide attempts, or suicide ideations among children have been reported recently.Our study had some limitations in that it was monocentric, subject to representativeness bias, and also underpowered to stratify patients by sex. Only patients aged 15 years or younger were included, which limits the comparison of our findings with similar works that include patients aged 16 to 21 years. Finally, our careful monitoring of potential changes in children’s mental health since the pandemic onset may also have led to an overestimation of the outcomes we reported.In conclusion, our findings suggest that the COVID-19 pandemic is associated with profound changes in the dynamics of suicide attempts among children. There is a need for rapid deployment of evidence-based prevention and intervention strategies to address factors influencing suicide attempts among children during and likely after the pandemic.