Background: This study was conducted to determine the prevalence of headache in coronavirus disease 2019 (COVID-19) and to assess its association as a predictor for COVID-19. This study also aimed to discuss the possible pathogenesis of headache in COVID-19.Methods: Available articles from PubMed, Scopus, and Web of Science were searched as of September 2nd, 2020. Data on characteristics of the study, headache and COVID-19 were extracted following the PRISMA guidelines. Biases were assessed using the Newcastle-Ottawa scale. The cumulative prevalence of headache was calculated for the general population (i.e. adults and children). The pooled odd ratio (OR) with 95% confidence intervals (95%CI) was calculated using the Z test to assess the association between headache and the presence of COVID-19 cases.Results: We included 104,751 COVID-19 cases from 78 eligible studies to calculate the global prevalence of headache in COVID-19 and 17 studies were included to calculate the association of headache and COVID-19. The cumulative prevalence of headache in COVID-19 was 25.2% . Headache was found to be more prevalent, approximately by two-fold, in COVID-19 patients than in non-COVID-19 patients , OR: 1.73; 95% CI: 1.94, 2.5 with p=0.04.Conclusion: Headache is common among COVID-19 patients and seems to be more common in COVID-19 patients compared to those with the non-COVID-19 viral infection. No definitive mechanisms on how headache  emerges in COVID-19 patients but several possible hypotheses have been proposed. However, extensive studies are warranted to elucidate the mechanisms.PROSPERO registration:CRD42020210332 (28/09/2020) Coronavirinae family and Betacoronavirus subfamily together with severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle Eastern respiratory syndrome coronavirus (MERS-CoV)1. The virus is primarily transmitted from person-to-person through droplets from symptomatic and pre-symptomatic patients, and is likely to also be transmitted by asymptomatic individuals6. Currently, no effective vaccines or pharmaceutical agents against SARS-CoV-2 are available but some progressions have been made to produce vaccines and drugs against the disease10.The current coronavirus disease 2019 (COVID-19) pandemic has caused a global crisis for both the health and economic sectors. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is a member of the11 with complications such as hypoxemia, acute respiratory distress syndrome (ARDS), arrhythmia, shock, acute cardiac injury, and acute kidney injury14, most SARS-CoV-2 infections are asymptomatic or have mild symptoms16. The common clinical symptoms of COVID-19 include fever, dry cough, dyspnea, chest pain, fatigue and myalgia18. In some cases, other neurological manifestations such as headache, dizziness, seizure, taste and smell impairment were also reported21. Headache is one of the symptoms that is also reported in various viral infections such as dengue and chikungunya that are common in the tropical regions23 and therefore may not be specific for COVID-19. In addition, the prevalence of headache in COVID-19 patients varies across studies24. A study found that the prevalence of headache was 17.4% (94/540) in Hubei province, the epicenter of the outbreak, and 14.1% (111/788) among patients outside the epicenter21. Another study in European countries found that the headache was reported in more than 40% of 417 COVID-19 patients19. Furthermore, the association of headache with the presence of COVID-19 is unknown. This systematic review was undertaken to provide robust evidence on the prevalence of headache in COVID-19 patients globally and its association with COVID-19 cases. Information described in this study might help clinicians to decide whether headache could be used as one of the basic symptoms to be included in diagnosing SARS-CoV-2 infection, especially those in the front line with limited resources.Although up to 20.3% of hospitalized patients require admission to the intensive care unit (ICU)25. The protocol of this study was registered with PROSPERO, an international database of prospectively registered systematic reviews at the University of York, on 28th September 2020 (CRD42020210332).This systematic review was conducted as recommended by the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelinesArticles reporting headache as a symptom of COVID-19 cases were included. COVID-19 cases should be diagnosed with RT-PCR test using either nasopharyngeal and oropharyngeal swab, bronchoalveolar lavage or cerebrospinal fluid (CSF). All cross-sectional and cohort studies that included COVID-19 cases randomly selected from the population were considered eligible while case reports and case series including all editorials, reviews, and commentaries were excluded. Case-control studies with pre-allocated number of patients with headache and non-headache were excluded. Studies that were conducted in specific populations only such as in pregnancy, children, cancer patients and other groups were excluded. Only articles written in English were included in this study.PubMed,Scopus, andWeb of Science as of September 2nd, 2020). The search criteria were as follows. Pubmed ([Title] "SARS-CoV-2" OR "COVID-19" OR "Wuhan coronavirus" OR "Wuhan virus" OR "novel coronavirus" OR "nCoV" OR "severe acute respiratory syndrome coronavirus 2" OR "coronavirus disease 2019 virus" OR "2019-nCoV" OR "2019 novel coronavirus" OR "severe acute respiratory syndrome coronavirus 2" OR "coronavirus" OR "coronaviruses" OR "SARS 2" OR "2019-nCoV acute respiratory disease" OR "novel coronavirus pneumonia"OR "COVID") AND ([All] “Headache”). Scopus ([Title] "SARS-CoV-2" OR "COVID-19" OR "Wuhan coronavirus" OR "Wuhan virus" OR "novel coronavirus" OR "nCoV" OR "severe acute respiratory syndrome coronavirus 2" OR "coronavirus disease 2019 virus" OR "2019-nCoV" OR "2019 novel coronavirus" OR "severe acute respiratory syndrome coronavirus 2" OR "coronavirus" OR "coronaviruses" OR "SARS 2" OR "2019-nCoV acute respiratory disease" OR "novel coronavirus pneumonia"OR "COVID") AND ([All] “Headache”). Web of Science ([Title] "SARS-CoV-2" OR "COVID-19" OR "Wuhan coronavirus" OR "Wuhan virus" OR "novel coronavirus" OR "nCoV" OR "severe acute respiratory syndrome coronavirus 2" OR "coronavirus disease 2019 virus" OR "2019-nCoV" OR "2019 novel coronavirus" OR "severe acute respiratory syndrome coronavirus 2" OR "coronavirus" OR "coronaviruses" OR "SARS 2" OR "2019-nCoV acute respiratory disease" OR "novel coronavirus pneumonia"OR "COVID") AND ([All] “Headache”). English as language limitation was imposed in the searches. Only peer-reviewed articles were included. Data were extracted both from the articles and the supplementary materials. Reference lists from the eligible articles were retrieved for further relevant studies.To identify potential articles for analysis, systematic searches were conducted using three bibliographical databases  and duplicate records between databases were removed. Retrieved articles were initially screened based on title and abstract to identify possible eligible studies. The full texts of potentially eligible articles were then reviewed. The screening and review processes were conducted by two authors (MF and JKF). After reviewing the full texts, the eligibility of each study was decided. Any discrepancies between the two authors were solved by consulting with another investigator (HH).The following data were extracted from eligible articles: study characteristics , headache characteristics , COVID-19 characteristics .This study received no external funding.The primary outcomes of this systematic review were: a) the prevalence of headache in COVID-19 cases; and b) the association between headache and COVID-19 cases compared to other viral infections.The cumulative prevalence rate of headache was calculated for COVID-19 cases in the general population. The prevalence was calculated as the number of COVID-19 cases with headache divided by the total number of COVID-19 cases with and without headache, expressed as a percentage (%). Pooled odd ratios (OR) and 95% confidence intervals (95% CI) were calculated to assess the association of headache and COVID-19 compared to non-COVID-19 cases .26. This scale evaluates 9 criteria of the study including the sample selection (4 items), group comparison (1 item), and the outcome (3 items). The scores range between 0 to 9 in which classified into three groups: low (≤ 4), moderate (between 5–6), and high quality study (≥ 7).To reduce sample selection bias, a critical assessment was specifically conducted in terms of setting of study and diagnosis of COVID-19. The quality of eligible studies was assessed using critical appraisals based on the Newcastle-Ottawa scale (NOS)Review Manager version 5.327. The cumulative pooled OR and 95%CI was presented in a forest plot.The association between headache and the presence of COVID-19 was assessed by the calculation of a pooled OR and 95%CI using the Z test . Prior to analysis, gathered data from studies were evaluated for heterogeneity and potential publication bias. Heterogeneity among studies was assessed using the Q test. Initial analysis found that the data had heterogeneity (p<0.10) and therefore a random effect model was employed. Egger’s test and a funnel plot were used to assess the reporting or publication bias . The data were analyzed usingThe literature searches yielded 732 articles, of which 229 were excluded as duplicates between databases. Following a screening process of the titles and abstracts of the remaining 503 articles, an additional 253 articles were excluded due to irrelevant studies leaving 250 articles . The ful31, diabetic patients32, pediatrics33, pregnant women34, cancer patients35, children and young adults40, patient undergone surgery41, critical patients42, women undergone delivery43, patients with mild-moderate COVID-1946, patients with gastrointestinal symptoms47, patients with severe headache only48, and patients with anosmia only49. In total, 78 studies were included to calculate the prevalence of headache in COVID-19 and all studies were published in 2020. The studies were conducted in Brazil50, China96, Egypt97, France101, Germany102, India104, Italy111, Japan113, Jordan114, Somalia115, South Korea117, Spain119, Turkey121, and the US126. Two studies were cross-sectional122, five were prospective cohort studies121 and the remaining 71 studies were retrospective studies.To calculate the prevalence of headache in COVID-19, full-text assessment resulted in the exclusion of 123 articles for the following reasons: case control studies (n=2), case report studies (n=69), case series (n=29), duplicated dataset (n=1), and conducted in specific population only (n=23). The targeted population studies were conducted among healthcare workers127, Belgium29, Brazil50, China70, France99, Hongkong128, Israel129, Italy130, Germany131, Netherlands31, Turkey120, and the US133. Out of the studies, ten were case-control132, four were cross-sectional133, and two were prospective cohort studies129.To calculate the association between headache and COVID-19, the full-text assessment yielded 16 eligible studies. The rest of the references had been excluded for these reasons: (a) the studies were case reports or case-series (n=98); (b) the full-text did not include data of outcome of interest (n=84); and (c) low quality of study (n=3) . The inc99. Another study which involved 46 patients reported that 40 (86%) had tension-type pain and 6 (14%) had migraine-like headache107. Data from 18 studies indicated that 72.17% (236/327) of headaches were reported in mild-moderate COVID-19 cases117. The prevalence of headache in severe COVID-19 cases from 15 studies was 27.83% (86/309)117.Our systematic review included 78 studies consisting of 104,751 COVID-19 patients and headache was reported in 26,464 patients with a cumulative prevalence of 25.26%. The list of the studies and the prevalence of headache of each study is presented in132 while seven studies reported no association134 , were analyzed to determine the association between headache and COVID-19. Of these studies, an association between headache and the occurrence of COVID-19 was observed in 9 studiesion134 . Our cum23. However, a study described that headache is one of the main neurological symptoms of coronavirus infection including SARS-CoV-2135. The global prevalence of headache in our systematic review is more than 25% out of 104,751 COVID-19 cases. This result was almost double that of the previously reported prevalence from studies in China early in the pandemic that ranged from 6.5–13.1%88. This suggests that headache is prevalent in SARS-CoV-2 infection and therefore could potentially be used as one of the indicators to diagnose COVID-19 cases. In this analysis we did not analyze the prevalence of headache based on COVID-19 severity, the existence of COVID-19 co-morbidity (such as diabetes and hypertension) and based on demographic characteristics such as gender due to scarcity of the available data. Therefore, whenever enough data are available, such sub-analyses are critical to be conducted. As a non-specific symptom, headache might present not only in COVID-19 cases but also in other viral diseases, therefore, headache might not raise suspicion of SARS-CoV-2 infection136. A study in COVID-19 patients with pre-existing primary headache disorders revealed that the headache during COVID-19 had an unusual presentation with 42% (44/104) reporting a recent onset of headaches, 49% (51/104) had a change in headache pattern, and 39% (39/104) reported the worst headache they had ever had119. These results suggest that new onset headache and changes of headache pattern should be carefully explored as this might be able to differentiate patients with COVID-19 from those without.Our study also highlights that headache was significantly more prevalent in COVID-19 patients, 2.2-fold, than suspected non-COVID-19 viral infection . A study found that only around 11% of MERS patients reported they suffered from headaches140. No fixed mechanisms have been reported on how these headaches emerge in COVID-19 patients. However, it has been proposed that the activation of trigeminal nerve ending in the periphery followed by the sensitization of various sites in the brain is one of the main pathomechanism of headache in these patients138.We explored the available literature to broaden our knowledge of the pathogenesis of headache in COVID-19. In general, three main primary headaches are observed in COVID-19 patients i.e. migraine, cluster headache and tension-type headache143. The release of those peptides from the fibers could be due to either electrical, chemical or mechanical induction emerged from three branches of the trigeminal nerve i.e. ophthalmic, maxillary and mandibular branches143. However, because of its wider area of innervation in the meninges and cranial blood vessels, ophthalmic branch seems to play more of a role in stimulating nociceptive processes in meningeal structures than the other two branches144.A headache attack is initiated by the release of several vasoactive neuropeptides such as glutamate, calcitonin gene-related peptide (CGRP), substance P and pituitary adenylate cyclase-activating polypeptide (PACAP), from nociceptive sensory fibers  innervating blood vessels located in the meninges and other cranial structures leading to vasodilation, degranulation of mast cells and plasma protein extravasation in those vascular structures141. Although cerebral and meningeal vasodilation is not the sole cause of headache145, most studies have agreed on the critical role of blood vessel dilation in the emergence of headaches.Next, any physiological events, such as vasodilation, that have occurred in the meningeal and large cerebral blood vessels will become a stimulus which is sent to the trigeminal ganglion (TG) where other nociceptive information from other afferent trigeminal branches are also converging143. These transmissions are then projected to the diencephalon structures, including the thalamus and hypothalamus, via the second-order neurons146. The third-order neurons are subsequently responsible for transmitting the information from diencephalic systems to various cortical areas associated with motoric, somatosensory, auditory, retrosplenial and visual functions143, leading to the manifestation of headache pain and other related symptoms  via first-order neuronsymptoms .147. When the transmission reaches TCC structures, CGRP and substance P may act to induce the release of glutamate and reduce gamma aminobutyric acid (GABA) production148. It has been proposed that during a headache attack, the level of glutamate in the TCC increases, while GABA release is decreased148. This condition could result in the increase of nociceptive neurons excitability149. Moreover, low level of serotonin in trigeminal nerve might also be involved in migraine pathophysiology as the release of this neurotransmitter has been linked to the inhibition of CGRP in trigeminal nerves151.During these transmission processes, the release of neuropeptides, especially CGRP, is limited only in the meninges and in the central terminals of trigeminal afferents137. This theory is hypothesized according to a study, confirming that trigeminal ganglia possess an angiotensinergic activity152. Thus, the viral attack would hypothetically disturb the activity of the renin-angiotensin-aldosterone system (RAAS), which may increase the level of CGRP153. Although the invasion of SARS-CoV-2 into the olfactory nerve ending seems to be the main route156, the action of the virus on the trigeminal nerve must not be overlooked, as suggested by Perlmanet al. (1989). They demonstrated that the trigeminal nerve, in addition to the olfactory nerve, was a route used by neurotropic murine coronavirus to invade the central nervous system (CNS)157. The hypothesis of trigeminal nerve attack by SARS-CoV-2 is also supported by the fact that olfactory mucosa is innervated by the trigeminal nerve159 suggesting the invasion of the olfactory mucosa by SARS-CoV-2 may also induce trigeminal nerve injury.Several mechanisms have been postulated to explain how trigeminovascular system is activated in COVID-19. Firstly, direct invasion of the virus may activate the peripheral trigeminal system160. Once the virus gains access to the nucleus in the cell body through the microtubule-organizing center (MTOC), viral replication is initiated161. Finally, viral progenies may spread to other areas of the body, including the CNS, via anterograde axonal transport assisted by the kinesin motor protein family160.Following entry into the trigeminal nerve, SARS-CoV-2 may hijack the transneuronal transport system to direct the virus to enter the nucleus via a retrograde axonal transport mechanism. This transport occurs by the involvement of cytoskeletal motor proteins called dynein supported by cofactor dynactin that function to move substances, such as endosomes and vesicles, including hijacking viruses, on microtubule towards the cell body162. Furthermore, the movement of SARS-CoV-2 via retrograde axonal transport is also hypothesized as it has been reported that the envelope protein of SARS-CoV could subvert dynein function either directly or indirectly163. The role of dynein in the retrograde axonal movement of several viruses, such as herpesviruses, West Nile virus, rabies, and influenza virus, upon their penetration in the neuronal plasma membrane has also been reported169.A study suggested a transneuronal transport system used by coronavirus after investigating the neuroinvasiveness of HCoV-OC43 in mice137. Cytokine storm has attracted substantial interest from researchers and clinicians as this unwanted condition is strongly suggested to be related to the increased mortality in SARS-CoV-2-infected patients171. It is hypothesized that the headache suffered by COVID-19 patients at the later stage of this infection is induced by the cytokine storm173. This notion has been supported by the fact that proinflammatory cytokines, such as IL-1, IL-6 and TNF-α, have been linked to the activation of the trigeminovascular system, which is responsible for the emergence and development of headache through the modulation of CGRP177.Secondly, SARS-CoV-2 may also invade the trigeminal nerve by indirect mechanisms. Cytokine storm and vasculopathy mechanisms are also proposed to explain the activation of trigeminal nerve upon SARS-CoV-2 infection179. It is known that ACE2 is associated with several protective mechanisms within the body, such as vasodilation180 and antinociception181. ACE2 also diminishes excessive free radical production which prevents oxidative stress182. The utilization of this receptor by the virus may decrease its activities, leading to the disturbance of vascular function. The perivascular trigeminal nerve may in turn be affected resulting in the COVID-19-related headache137. More studies are required to improve our understanding on the role of the ACE2 receptor in headache pathophysiology.Moreover, the presence of angiotensin-converting enzyme 2 (ACE2) receptor on the endothelial cells makes the blood vessels vulnerable to invasion by SARS-CoV-2et al. (2020). They proposed that gas exchange disturbance in alveolar tissues triggered by the viruses would induce hypoxia, which in turn leads to ischemia183. Ischemia itself has been known to have a strong relation with headache incidents184 that could be induced by exaggerating the production of free radicals.Another hypothesis by which SARS-CoV-2 could induce headache is offered by Abboud139. It is hypothesized that headaches occurring in COVID-19 patients might be the result of the same mechanisms as observed in influenza A and influenza B infections, which could be related to the activity of cytokines187. A recent report on dengue-related headache suggested that the headache could be pulsating and either affect the entire brain, only frontal, or orbital area, which may resemble primary headaches reported in COVID-19 patients188. Therefore, although we found that headache is more frequent in COVID-19 patients than those of non-COVID-19 patients, diagnosis of COVID-19 should not be based on the presence of a headache.In regards to the headache characteristics presented by COVID-19 patients compared to the headache induced by other viral infections, no apparent differences could be observed. Headache in COVID-19 could be worsened by physical or head movement, felt in either the entire head  or unilaterally  and the pain is typically pressing or tighteningIn conclusion, headache is a common symptom in COVID-19 cases. Some mechanisms have been proposed as to the mechanism for headaches in COVID-19 such as the activation of the trigeminovascular system by either direct action of the virus or indirect mechanisms induced by cytokine storm, vasculopathy, or ischemia induced by gas exchange disturbance in COVID-19 patients. Extensive efforts must be carried out to provide definitive answers about COVID-19-related headaches. Detailed investigations on the mechanisms by which SARS-CoV-2 attacks the CNS and thus generates headaches are important to improve our understanding on the pathophysiology of COVID-19, and therefore influences possible pharmacological intervention decisions.All data underlying the results are available as part of the article and no additional source data are required.https://doi.org/10.6084/m9.figshare.13166783.v1189Figshare: PRISMA checklist for ‘Global prevalence and pathogenesis of headache in COVID-19: A systematic review and meta-analysis’,Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however, I have significant reservations, as outlined above.Are the rationale for, and objectives of, the Systematic Review clearly stated?PartlyIs the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are sufficient details of the methods and analysis provided to allow replication by others?NoAre the conclusions drawn adequately supported by the results presented in the review?PartlyReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for modifying your paper and clearly addressing my comments. In my opinion, the manuscript is acceptable. CheersAre the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?YesAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Health Policy and Management, an expert in systematic review and meta-analysis methodology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In the study selection and/or quality appraisal stages are the authors faced with discrepancies? Please state how resolved these potential discrepancies.Was it not possible to analyze the subgroups? For example, based on age groups or gender.Please add some details about the NOS checklist and also add the result of the quality appraisal to the manuscript.Thank you for considering a great area of interest related to COVID-19. This study aimed to investigate the prevalence and pathogenesis of headache in COVID-19 patients, globally. The methodology is completely rigorous and the results are interesting. I have a few minor comments to the authors as follow:Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?YesAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Health Policy and Management, an expert in systematic review and meta-analysis methodology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Dear Dr Morteza Arab-Zozani,  Thank you for your comments. 1. We have provide the approach how to solve in case there was discrepancies between two authors during evaluation of the studies.  2. We were unable to do sub-group analysis based on age and gender due to available of data.  We have added this as one of our limitation: We have added this in our manuscript: "In this analysis we did not analyze the prevalence of headache based on COVID-19 severity, the existence of COVID-19 co-morbidity (such as diabetes and hypertension) and based on demographic characteristics such as gender due to scarcity of the available data. Therefore, whenever enough data are available, such sub-analyses are critical to be conducted." 3. We have provide more detail information of Newcastle-Ottawa scale (NOS) - What items that were assessed and how to score as well how the studies classified. We also have added the NOS scores briefly to all studies included in the study.  Thank you Authors seem not considered potential important bias while choosing headaches to be analysed. While among patients with fever (mainly), headache is in the same pathophysiology response due to prostaglandin release. Not to include other symptoms, so it is not merely a headache as a symptom. How about the existence of co-morbidity such as Hypertension (uncontrolled hypertension)? It is not clear how to justify data with such condition concomitantly. In the results section, what does this statement mean? "Headache was found to be more prevalent, approximately by two-fold, in COVID-19 patients than in non-COVID-19 patients with symptoms of other respiratory viral infections, OR: 1.73; 95% CI: 1.94, 2.5 with p=0.04." What kind of non-covid-19 patients with symptoms of other respiratory viral infections? This is a confusing statement. As a clinician taking care of Covid-19 patients, the importance of this article is low.Are the rationale for, and objectives of, the Systematic Review clearly stated?PartlyIs the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are sufficient details of the methods and analysis provided to allow replication by others?NoAre the conclusions drawn adequately supported by the results presented in the review?PartlyReviewer Expertise:infectious diseaseI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Dear Dr Erni J. Nelwan We agreed that our study have limitation since we did not able to analysed the data based on existence of COVID-19 co-morbidity. This is mainly because the available of data. The data was scarce.  We have added this in our manuscript: "In this analysis we did not analyze the prevalence of headache based on COVID-19 severity, the existence of COVID-19 co-morbidity (such as diabetes and hypertension) and based on demographic characteristics such as gender due to scarcity of the available data. Therefore, whenever enough data are available, such sub-analyses are critical to be conducted." We have provided a clear explanation about the non-COVID-19 in Method section as requestioned. Not-COVID-19 in our study refers to other respiratory viral infections such as rhinovirus, influenza, parainfluenza and respiratory syncytial virus. We added: "non-COVID-19 cases " in text.  We also also agreed that the pathogenesis of headache is complex including the production of prostaglandin while fever. Therefore in our study we compared the occurrence of headache in COVID-19 and other non-COVID-19 respiratory infection as control. This to reduce the effect of fever since in both COVID-19 and non-COVID-19 cases the fever are present. Due to this complexity we did not claim any diagnostic for direct clinical practice.


Several strategies have demonstrated the ability to improve the survival of patients with both metastatic and nonmetastatic prostate cancer. The old backbone of androgen-deprivation monotherapy has been disrupted in the hormone-sensitive setting, and several options have been introduced for the management of the castration-resistant disease. However, no optimal sequencing is still defined, and few randomized comparisons are currently available to identify the approach that maximizes the long-term benefit for these patients. This comprehensive review aims at resuming the current evidence on this topic to help physicians during the treatment choice for patients with advanced prostate cancer.PTEN).The treatment landscape of advanced prostate cancer has completely changed during the last decades. Chemotherapy , androgen-receptor signaling inhibitors (ARSi) , and radium-223 have revolutionized the management of metastatic castration-resistant prostate cancer (mCRPC). Lutetium-177–PSMA-617 is also going to become another treatment option for these patients. In addition, docetaxel, abiraterone acetate, apalutamide, enzalutamide, and radiotherapy to primary tumor have demonstrated the ability to significantly prolong the survival of patients with metastatic hormone-sensitive prostate cancer (mHSPC). Finally, apalutamide, enzalutamide, and darolutamide have recently provided impactful data in patients with nonmetastatic castration-resistant disease (nmCRPC). However, which is the best treatment sequence for patients with advanced prostate cancer? This comprehensive review aims at discussing the available literature data to identify the optimal sequencing approaches in patients with prostate cancer at different disease stages. Our work also highlights the potential impact of predictive biomarkers in treatment sequencing and exploring the role of specific agents  in biomarker-selected populations of patients with prostate cancer (i.e., those harboring alterations in DNA damage and response genes or  The treatment landscape of advanced prostate cancer has completely changed in recent years. Several treatment options have demonstrated the ability to improve the overall survival (OS) of patients with metastatic hormone-sensitive prostate cancer (mHSPC) and metastatic castration resistant prostate cancer (mCRPC) when added to the androgen-deprivation therapy (ADT). These treatments include chemotherapy with docetaxel and cabazitaxel, androgen-receptor signaling inhibitors (ARSi), radium-223, and radiotherapy to primary tumor. Phase 3 trials have also shown that the addition of ARSi to ADT improves the outcomes of patients with nonmetastatic castration resistant prostate cancer (nmCRPC). However, no standardized approach to correctly sequence all of these treatment options is still defined. Few randomized trials have compared these different strategies, and the majority of information is provided by retrospective series and secondary analyses. This comprehensive review aims at providing the most convincing evidence on the best sequencing of agents in different settings of advanced prostate cancer and to discuss current data that support the use of specific biomarkers during the treatment choice.Long-term ADT has been the treatment of choice in the mHSPC setting for decades, with an estimated median OS of about 3.5 years in contemporary series ,2. In reThe addition of docetaxel to ADT demonstrated an OS gain ranging from 10.4 to 16 months in the CHAARTED and STAMPEDE trials, respectively . The OS Although these trials have introduced new active options for the treatment of mHSPC, no direct head-to-head comparisons are currently available. Cross-trials comparisons appear to be inappropriate given that the median OS observed in the control arms varies from 36.5 months of the LATITUDE trial to 52.2 months of the TITAN trial. In addition, the majority of retrospective analyses or indirect comparisons are subjected to the bias of different trials’ design, population, treatment duration, and follow-up.In the only indirect, patient-level comparison available to date, the outcomes from 566 patients treated in the STAMPEDE trial who were contemporarily randomized to abiraterone acetate and docetaxel were assessed. Although abiraterone was associated with longer failure-free and progression-free survival (PFS), there was no difference in terms of OS between abiraterone- and docetaxel-treated patients  .p = 0.033) [p = 0.827) [p = 0.0098) [Clinical variables, such as disease volume or patients’ risk, have been proposed to select the appropriate treatment choice for patients with mHSPC. In the CHAARTED study, a prospective stratification of high- versus low-volume disease  was included in the trial. Docetaxel was found to significantly improve OS in patients with high-volume disease  but not in those with low metastatic burden  . Further 0.0098) , and thePatients with nmCRPC show biochemical progression on ADT, with baseline PSA ≥ 2 ng/mL, and no evidence of metastatic disease on conventional imaging (bone scan and computed tomography or magnetic resonance). Before 2018, no standard of care was established for these patients. After initial studies with zoledronic acid in patients with nmCRPC were stopped due to the lack of events , subsequSubtle differences in trial design, such as the inclusion of patients with lymph node disease in SPARTAN but not in PROSPER or ARAMIS, may limit cross-trial comparisons of efficacy. Although tolerability seemed improved in the ARAMIS trial, where rates of discontinuation due to AEs were not different to placebo (8.9% vs. 8.7%) in contrast to SPARTAN (14.9% vs. 7.3%) or PROSPER (17% vs. 9%) trials, the different reporting of AEs can also limit conclusions on toxicity . Of noteDocetaxel , abiraterone acetate , and enzalutamide  have all shown a significant survival benefit as first-line therapies for mCRPC and are considered standard options in initial therapy ,17,38 T. The curNo formal randomized comparison between chemotherapy and ARSi is currently available in the first-line setting of mCRPC. The marked difference in median OS observed in the control arms of the TAX-327 (16.5 months), COU-AA-302 (30.3 months), and PREVAIL (31 months) trials suggests that different patient populations were investigated and cross-trial efficacy comparisons are inappropriate. In a large, real-world, observational study, patients treated with first-line ARSi experienced longer times to progression than those treated with docetaxel, but there was no difference in terms of OS . Additio2 dose [Docetaxel remains the first-line taxane of choice in mCRPC based on the results of the FIRSTANA trial, where no difference in survival was observed when comparing first-line docetaxel with cabazitaxel, although cabazitaxel seemed to be better tolerated than docetaxel at the 20 mg/m2 dose . Since tTo date, no recommendation based on efficacy can be made for the selection of a first-line ARSi. Indirect comparisons of phase 3 trials have not reported statistically significant differences in OS between abiraterone and enzalutamide both pre- and post-chemotherapy for mCRPC , althougp = 0.039) [p = 0.083), whereas there was no difference in terms of radiographic response rate or confirmed PSA decline ≥ 50%.The choice of an optimal treatment for mCRPC patients who have received prior treatment with docetaxel for mHSPC remains unclear a. Data f= 0.039) . PatientChemotherapy appears to be a reasonable option for the first-line mCRPC treatment of eligible patients who have previously received ARSi in the mHSPC setting b and in p < 0.0001) were observed in patients treated with the abiraterone -> enzalutamide sequence, with no difference in OS .Cross-resistance between different ARSi is likely, and the sequence including two sequential ARSi is often discouraged. Although analyses from the SPARTAN trial in nmCRPC, where up to 80% of patients received abiraterone at progression, reported a benefit in PFS2 for patients in the apalutamide -> abiraterone over the placebo -> abiraterone sequence, most of the benefit was driven by the superior PFS of apalutamide over placebo in first-line nmCRPC, and the outcome comparisons in patients that received second-line therapy are lacking. Data from the control arm of the PLATO trial, in which patients received abiraterone acetate after first-line enzalutamide for mCRPC, are quite discouraging, with a median time to PSA progression of only 2.8 months and a PSA response ≥ 50% observed in 2% of patients . A phaseMethodological issues arise when interpreting retrospective sequencing studies in mCRPC since, frequently, only results of patients treated in sequence are presented. The entire population of patients who start a first-line treatment should be analyzed to determine the best first-line approach in order to avoid a selection bias. The outcomes of patients with substantial benefit from first-line therapy and of those with aggressive disease that die while on first-line therapy, neither of which receive second-line treatment, can significantly affect the final results.Cabazitaxel , abiraterone acetate , enzalutamide (AFFIRM), and radium-223  have demonstrated a significant improvement in OS after treatment with docetaxel in an mCRPC setting ,23,24,51Taken together, the data suggest the activity of agents in second-line is lower than in first line. The PSA response rates observed with enzalutamide in post-docetaxel mCRPC were lower than those observed in chemo-naïve mCRPC 78% vs. 54%) ,52. Simi% ,52. SiThe choice of therapy in patients that have received both an ARSi and docetaxel has been established in the phase III CARD trial, where cabazitaxel proved to be superior to a second ARSi . In thisRadium-223 is an intravenous alpha-emitting radiotherapeutic drug that mimics calcium and binds to bone mineral hydroxyapatite in areas of high bone turnover. In the phase III ALSYMPCA trial, six cycles of radium-223 at 50 kBq/kg prolonged OS (HR 0.70 95% CI 0.58–0.83) and delayed time to first symptomatic skeletal event (SSE) compared to placebo (HR 0.66 95% CI 0.54–0.77) in mCRPC patients with symptomatic bone metastases  . Of noteIn the Expanded Access Program, the safety and activity of radium-223 was examined in a single-arm cohort of patients, including those with asymptomatic disease, and the combination of radium-223 with abiraterone or enzalutamide was allowed . Patientp < 0.0001) and fewer grade 3 or 4 adverse events (33% vs. 53%).Lutetium-177-prostate-specific membrane antigen (PSMA)-617 (LuPSMA) is an investigational radioligand therapy that has been investigated for patients with mCRPC . LuPSMA The results of the phase 3 VISION study involving patients with mCRPC treated with LuPSMA were recently presented at the ASCO Congress 2021   have emerged as potential options for the prevention of SRE among men with prostate cancer. Zoledronic acid and denosumab have demonstrated the ability to reduce the risk of skeletal-related events (SRE)—including asymptomatic fractures—and time to first SRE in men with mCRPC ,72. Of nIn an attempt to maximize benefits, a number of combinations of agents with seemingly non-overlapping mechanisms of action have been studied in advanced prostate cancer . Combinap = 0.14), and this result should be interpreted with caution. Similar data were observed in the post-hoc analysis of the TITAN trial of apalutamide in mHSPC [In the ENZAMET trial, the use of enzalutamide in combination with docetaxel was associated with significant improvement in clinical PFS (HR 0.48 95% CI 0.37–0.62), but the hazard ratio was suggestive for no OS benefit . Of note, no evidence of heterogeneity of effect according to docetaxel use was found  ; data onIn the mCRPC setting, two phase III trials evaluated the combination of abiraterone with the antiandrogens enzalutamide (ALLIANCE A031201) and apalutamide  compared with ARSi alone as first-line mCRPC treatment. Both abiraterone plus enzalutamide (HR: 0.70 95% CI 0.67–0.72) and abiraterone plus apalutamide  showed a significant benefit in terms of radiographic PFS over ARSi monotherapy but no OS benefit ,80. The BRCA1/2, have been clinically validated as biomarkers for selecting patients who are sensitive to poly ADP-ribose polymerase (PARP) inhibition. Pembrolizumab has received tissue-agnostic approval by the U.S. Food and Drug Administration (FDA) for patients with microsatellite instability or mismatch repair-deficient prostate cancer. In addition, the AKT inhibitor ipatasertib has demonstrated significant activity in patients with PTEN loss. Many of these biomarker-driven treatments are going to be implemented in routine clinical practice. However, to what extent these treatments will affect the sequencing and response of other therapies is largely unknown and will be the object of investigation in the future. Several biomolecular alterations, including alterations in tumor driving genes, have been observed in patients with prostate cancer. Some of these molecular alterations could be explored as predictive biomarkers for planning treatment to early identify primary resistance, avoiding useless toxicity to patients. In some cases, these alterations involve inherited or spontaneously acquired gene mutations in the germline. More frequently, alterations are acquired at the somatic level during the oncogenesis and/or cancer progression, or they could arise or be enriched as a result of the selective pressure induced by treatments. Examples of molecular alterations associated with the mechanisms of treatment resistance that could be helpful in castration-resistant disease to select the appropriate therapy include androgen receptor (AR) amplification, mutation, or splice variants. Other resistance mechanisms bypass AR by exploiting alternative signaling and metabolic pathways . Table 2BRCA2 gene alterations are the most common DDR event both in the somatic- and germline [Alterations in DDR genes have recently become a field of major interest in prostate cancer research, given their potential prognostic and predictive implications ,89. DDR germline ,93.BRCA2 mutations have been associated with aggressive disease and poor clinical outcomes [BRCA2 alterations has negative prognostic significance. Additionally, a significant interaction between germinal BRCA2 status and treatment type (ARSi versus taxane therapy) has been observed, suggesting that BRCA2 might be a valid biomarker during the selection of the first-line treatment choice in patients with mCRPC [BRCA2 alterations as a predictive biomarker for the selection of ARSi or taxanes as first-line of therapy [Germline outcomes ,95. The th mCRPC . The BRC therapy .BRCA1 or BRCA2. Several case series and retrospective studies suggest that DDR-deficient prostate cancer patients might benefit from this therapeutic approach, and many clinical trials are ongoing to assess the role of platinum-based chemotherapy in patients with DDR defects [Platinum-based chemotherapy represents one of the first fields of investigation in patients with prostate cancer harboring DDR defects. Platinum generates DNA crosslinks that cannot be easily repaired when the homologous recombination repair (HRR) pathway is impaired, leading to cell death. This strategy has proven successful in treating breast and ovarian cancers with alterations in  defects .BRCA1, BRCA2, or ATM, establishing the first validated biomarker in patients with prostate cancer.Practice-changing data came from trials including patients with DDR defects treated with PARP inhibitors. The phase III PROFOUND study has recently established the predictive value of certain DDR genes defects in patients with mCRPC whose disease had progressed during previous treatment with enzalutamide, abiraterone, or both ,83. PatiBRCA1/2 alterations who had progressed after one to two lines of ARSi and one taxane-based chemotherapy for mCRPC, with complete response rates and a confirmed PSA response rate of 43.5% and 54.8%, respectively [The subgroup analysis of PFS and OS favored olaparib irrespective of prior taxane use . The useectively .BRCA alterations are those who derive the greatest benefit from olaparib, whereas those with ATM alterations showed unclear PFS  and OS benefit  [BRCA1/2 germline and somatic pathogenic mutations were associated with similar benefit from olaparib; greater benefit was observed in patients with homozygous BRCA deletion. Biallelic, but not mono-allelic, PALB2 deleterious alterations were associated with clinical benefit. In addition, the loss of ATM protein by immunohistochemistry was associated with a better outcome. Of note, the loss of RAD51 foci, a functional biomarker of HRR function, was primarily found in tumors with biallelic BRCA1/2 and PALB2 alterations, and the authors have suggested that the RAD51 assay could help identify less-common genomic variants impacting HRR function that sensitize to PARP inhibition.Current evidence suggests that different DDR alterations could provide different sensitivity to PARP inhibitors. In the PROFOUND trial, the gene subgroup analysis suggested that patients with 53–1.75) . In the 53–1.75) , BRCA1/2BRCA1/2 mutations, in biallelic versus monoallelic mutations, and in homozygous deletions versus other deleterious mutations. In addition, the efficacy of rucaparib was greater in patients with BRCA2- versus BRCA1-altered mCRPC, as assessed by PSA50 response rates, overall response rates, and median radiographic PFS estimates. This apparent discrepancy in PARP inhibitor sensitivity between patients with BRCA1- and BRCA2-mutated mCRPC seems to be a class effect of PARP inhibitors in prostate cancer [BRCA1- versus BRCA2-altered mCRPC in a cohort of 123 BRCA1/2-altered mCRPC patients receiving the PARP inhibitor, and this differential activity was not explained by mutation origin (germline vs. somatic) or allelic status  [BRCA1/2 alterations, 33% in PALB2 and 12% in ATM, whereas the complete response rates were 76% in BRCA1/2, 50% in PALB2, and 28% in ATM. The phase II GALAHAD trial is assessing niraparib in patients with mCRPC and biallelic DDR defects with disease progression on taxane and ARSi [BRCA carriers, with durable responses, particularly in biallelic BRCA mutation carriers.In the TRITON2 trial, PSA response rates were greater in patients with germline versus somatic e cancer . Taza anallelic) . The phaallelic) . The oveand ARSi . At the BRCA1/2 alterations. The clinical use of these agents is dependent on local regulatory approval; for example, the U.S. FDA approved olaparib for men with deleterious or suspected deleterious germline or somatic HRR gene-mutated mCRPC who have progressed on ARSi. In contrast, the EMA have restricted its use to patients with germline or somatic BRCA1/2 mutations.We could conclude that olaparib and other PARP-inhibitors as monotherapy showed significant benefit in patients with pretreated mCRPC and alterations in DDR, especially in those with Ongoing studies are assessing the role of these agents in combination with ARSi at earlier stages of mCRPC, given the strict relationship between PARP1 activity and AR function. It is also hypothesized that the co-blockade of PARP1 and AR using could be active regardless of DDR deficiency status. A phase II trial of olaparib in combination with abiraterone in post-docetaxel mCRPC showed a significant improvement in terms of radiographic PFS with the combination compared to abiraterone alone . The ongAR CNV in plasma DNA are associated with a worse outcome in patients with mCRPC treated with ARSi [AR gain in plasma DNA is also associated with a worse outcome in docetaxel-treated mCRPC patients, but AR-gained patients seem to derive greater benefit from treatment with taxanes than with ARSi [Several studies support the notion that alterations in the AR pathway represent an important driver of resistance in the context of mCRPC. AR aberrations including point mutations, copy number variations (CNV), structural variations, and alternatively spliced forms of AR are frequent among mCRPC patients, particularly after the use of ARSi . An analith ARSi . AR gainith ARSi ,126.The androgen-receptor variant 7 (AR-V7) has been proposed to predict for poor response to treatment with ARSi, such as abiraterone acetate or enzalutamide. Antonarakis and colleagues firstly showed that the detection of this AR variant in circulating tumor cells (CTCs) was associated with treatment resistance to ARSi . InteresAKT phosphatase PTEN, with hyper-activation of the oncogenic PI3K/AKT signaling [AKT inhibitor ipatasertib plus abiraterone vs. abiraterone alone in patients with mCRPC after docetaxel chemotherapy [PTEN-loss tumors. Based on these data, the phase III IPATential150 trial assessed the efficacy ipatasertib in combination with abiraterone compared to abiraterone alone for the first-line treatment of patients with mCRPC [PTEN-loss-by-immunohistochemistry population and in the intention-to-treat population. Of 1101 patients enrolled in this study, 521 (47%) harbored PTEN loss. In patients with PTEN loss, the combination arm with ipatasertib achieved significantly superior radiographic PFS  and antitumor activity compared to the placebo arm. However, the improvement of radiographic PFS in the intention-to-treat (ITT) population was not statistically significant. The subgroup analysis of the IPATential150 trial suggests that patients with PTEN loss previously treated with taxanes may not benefit from the addition of ipatasertib to abiraterone (HR 1.0 95% CI 0.58–1.74). However, given the limited number of patients, this observation should be interpreted with caution. A biomarkers analysis of the IPATential150 trial also showed that patients with PTEN loss and with genomic alterations in PIK3CA/AKT1/PTEN by next generation sequencing had a larger magnitude of radiographic PFS benefit with ipatasertib than patients with no detectable alterations [PI3K/AKT pathway alterations. The ProCAID phase 2 trial assessed the efficacy of docetaxel combined with pan-AKT inhibitor capivasertib compared to docetaxel alone in patients with mCRPC. The primary endpoint of PFS was not met, irrespective of the biomarker status for the PI3K/AKT/PTEN signaling pathway. However, OS (secondary endpoint) was longer in patients who received the combination compared with chemotherapy alone, and prospective validation studies are required to identify patients most likely to benefit from capivasertib [About a half of patients with mCRPC show a loss of the ignaling . These pignaling . The phaotherapy . The radth mCRPC ,137. Theerations . These rvasertib .The PAM50 is a well-known gene expression classifier that categorizes breast cancer into luminal A, luminal B, HER2, and basal subtypes. Zhao and colleagues applied this classifier to subtype prostate cancer samples into luminal A, luminal B, and basal subtypes . The autp = 0.051) compared to the basal subtype  [p = 0.0008) compared to the luminal subtype  [Regardless of basal/luminal subtype, >50% of patients enrolled in the phase III SPARTAN trial  achieved ≥90% reduction in PSA with apalutamide. However, PSA decline was deepest and most rapid in patients with the luminal subtype. Similarly, the OS improvement with apalutamide seemed to be more marked in patients with the luminal subtype  . Convers = 0.33) .It is unclear whether statistical inadequacy or the distinct setting (mHSPC vs. mCRPC) might explain these discordant results. Of note, an increased proportion of patients with the basal subtype was found in SPARTAN compared to TITAN (66% vs 50%). However, biomarkers analyses were performed in archival primary tumors, including patients that received these treatments in later stages during castration-resistance. The molecular characteristics of metastatic sites might differ from those of primary tumors; therefore, caution should be used when interpreting these analyses. Overall, these data suggest that luminal versus basal classification might be useful to selecting patients who are expected to derive the greatest benefit from ARSi and docetaxel. However, prospective biomarker-driven studies are needed to determine the real potential predictive impact of this classification.RB1 and PTEN, TP53 mutations, and the amplification of MYCN and AURKA are common events in NEPC and AVPC [Aggressive-variant prostate cancer (AVPC) refers to AR-independent anaplastic forms of prostate cancer that are characterized by a rapidly progressive disease, weak response to therapies, and poor prognosis . Many ofand AVPC ,143. AVPand AVPC ,144,145.AURKA inhibitor alisertib in patients with metastatic NEPC [N-myc and Aurora-A overactivity showed exceptional responses, including the complete resolution of liver metastases and prolonged stable disease. Many trials are currently ongoing in patients with AVPC and NEPC to test the activity of immunotherapy, PARP inhibitors, and EZH2 inhibitors in these patients [NEPC generally shows a high response rate, generally of short duration, to platinum-based chemotherapy . These ptic NEPC . Althougpatients .For patients with AVPC  there is no consensus for the optimal first-line treatment. At the Advanced Prostate Cancer Consensus Conference (APCCC) 2019, 75% of panelists voted to add docetaxel to ADT, 16% voted to add platinum-based combination therapy, and 9% voted to add an ARSi. Finally, the potential effect of a first-line platinum-based chemotherapy on the efficacy of subsequent treatments such as PARP inhibitors, docetaxel, or ARSi is largely unknown and requires further studies.BRCA or ATM aberrations included in this trial showed response to pembrolizumab, compared to 4/124 (3%) of those without alterations in DDR. The data also suggest that a proportion of patients with CDK12 deficiency may respond favorably to anti-PD-1 checkpoint inhibitors [SPOP mutations have been suggested to predict for response to abiraterone acetate [RB1 aberrations increase in prevalence after treatment-selective pressure [RB1 alterations showed worse clinical outcomes and worse progression-free survival [RB1 and TP53 are associated with shorter time on treatment with abiraterone or enzalutamide [TP53, PTEN, and RB1, may drive more aggressive disease and an increased risk of relapse [Given its tissue-agnostic approval by the FDA, patients with microsatellite instability or mismatch repair-deficient prostate cancer tumors might benefit from treatment with pembrolizumab . In the hibitors ,151. SPO acetate . RB1 abepressure ; patientsurvival . A studylutamide . Another relapse .BRCA1, BRCA2, or ATM. These data show the important limits of tumor tissue analysis. An increase in the sequencing success rate or the implementation of liquid biopsy approaches are necessary to enlarge the number of patients who could benefit from biomarker-driven treatments. It has been shown that ctDNA can sufficiently identify all driver DNA alterations found in matched metastatic tissue in the majority of patients with mCRPC [TP53, BRCA2, PTEN, RB1, and AR all have clinical relevance in mCRPC, but these alterations are not always possible to identify in samples with low ctDNA% [Of 4425 patients initially enrolled in the PROFOUND trial, 4047 patients had tumor tissue available for testing. Among these, 2792 (69%) were successfully sequenced, and only 162 patients  were found to harbor germline or somatic alterations in these th mCRPC . Data frth mCRPC . In metath mCRPC . The ctDw ctDNA% . Importaw ctDNA% . This obGiven that the majority of pivotal trials in mHSPC and mCRPC have been conducted using standard imaging for staging, limited evidence is available regarding the potential predictive role of PET tracers during the staging and re-staging of patients with prostate cancer. Several PET-derived parameters might be of value for the prognostic stratification of patients with mCRPC before systemic therapy . In addiIn recent years, new standards of care have been established for the treatment of advanced prostate cancer. However, few randomized trials have investigated which might be the best sequencing approach among these options in order to maximize the benefit and prolong the survival of patients. The choice of first-line treatment for mCRPC is complicated by the introduction of treatment options at the earlier stages of mHSPC and nmCRPC. The CARD trial has defined that chemotherapy with cabazitaxel is the best choice for patients with mCRPC who have already received docetaxel and are progressing during an ARSi. The ARSi -> ARSi approach is commonly discouraged given the potential development of cross-resistance. The advent of precision treatments, such as PARP- and AKT-inhibitors, and the pending approval of LuPSMA are going to further complicate this complex scenario. In the future, randomized trials are warranted to identify the optimal sequencing strategy and to improve the outcomes of patients with both hormone-sensitive and castration-resistant disease.


To raise awareness about preventive measures in COVID-19 pandemic, even though fully vaccinated. Although recent trials showed high efficacy of vaccines in preventing symptomatic infections, there are some individuals experiencing symptomatic SARS-CoV-2 infection. In this case report, a fully vaccinated young dental practitioner experienced symptomatic SARS-CoV-2 infection 55 days postvaccination with BNT162b2 Pfizer vaccine with evident ageusia. Diagnostic swabs were performed and used for viral genome sequencing. The patient fully recovered 15 days after diagnosis. Loss of smell and taste, together with nasal congestion were the main reported symptoms. The use of personal protective equipment prevented spread of infection in patients and co-workers. With the increase of people being fully vaccinated, it is still necessary to follow infection preventive protocols by correctly applying personal protective equipment. Although high efficacy has been proved, some individuals may still be vulnerable to symptomatic infection and new guidelines and markers should be adopted and investigated to find out patients for whom vaccination may not determine full immunization. After the SARS-CoV-2 pandemic, the hope of the whole world is placed in the effectiveness of vaccines that should ensure immunity to the entire population. For this reason, the FDA authorized early the new mRNA-based vaccines. Their short-term efficacy has been comparable with that of other common vaccines in phase 3 trials and a reduced symptomatic SARS-CoV-2 infection by over 90% was observed in healthy participants, after vaccination ,2. mRNA Sometimes the immune system of some subjects does not respond effectively to the vaccination, especially in some categories of patients, such as the immunocompromised. One study, based on solid organ transplant (SOT) recipients, showed reduced humoral response to mRNA vaccines .However, COVID-19 disease after full-dose mRNA vaccination can involve healthy subjects too. A recent study reported eight symptomatic SARS-CoV-2 infections occurring in fully vaccinated health care workers  .We reported, to our knowledge, the first case of an Italian fully vaccinated dental practitioner in whom symptomatic infection emerged. This unique case was described by the patient himself. Transmission of SARS-CoV-2 virus has been implicated in other published reports in fully vaccinated individuals and by publishing several case reports would be useful for understanding COVID-19 disease in vaccinated people, improving prognostication, patients’ selection and general public health policies.A 28-year-old male dentist received the first inoculation of BNT162b2 Pfizer vaccine on 23 January 2021, and the second dose on 15 February . The patAs reported by the patient, on the first dose no adverse events occurred and it was reported only a pain in the arm, where the vaccine was inoculated. Similar symptoms were reported after getting the second dose, without signs of fever, fatigue or more serious events. He spent the second week of 5–11 April 2021, at home, where he met his parents and relatives who had already recovered from COVID-19 from November 2020. Then he moved back to his workplace, at the Dental Clinic of Foggia University. On 11 April, he noted changes in his voice, nasal congestion and symptoms similar to seasonal allergy. On 20 April, he received a call from his aunt stating her positivity to SARS-CoV-2. On 21 April, he was referred to the Infectious Disease and Control Department of the University where he underwent to SARS-CoV-2 antigen test first, and later to RT-PCR test on nasopharyngeal swab. Both tests were positive for new coronavirus infection.AFIAS COVID-19 Ag  was used for the first screening. AFIAS COVID-19 Ag is an automatic fluorescence Immunoassay (FIA) for the qualitative/semiquantitative ) detection of novel coronavirus in human nasopharyngeal swab specimens .Subsequently, viral RNA was extracted within 2 h from the sample collection using the STARMag 96×4 Universal Cartridge kit with the Microlab NIMBUS IVD instrument according to the manufacturer’s instructions . Amplification and detection of four target genes N, E, S and RNA-dependent RNA-polymerase was performed using the commercially available kit Allplex SARS-CoV-2 Assay  with the CFX96TM instrument . Results interpretation was performed with the Seegene Viewer software .https://clades.nextstrain.org/, accessed on 21 April 2021) was used to assign clade and call mutations. The obtained genome sequence was deposited at the GISAID global repository  with the EPI_ISL_1890572 Accession ID. According to the currently accepted classification criteria, the viral genome belonged to the VOC Alpha 202012/01 GRY (B.1.1.7) and was characterized by the presence of several known relevant alterations in the S protein .Sample was therefore used for viral genome sequencing. In brief, total RNA extraction was performed starting from 400 µL of the swab transport medium using a Viral Nucleic Acid Extraction Kit (Low inhibition cartridge 202) with the MagCore workstation. Target enrichment and library preparation for next-generation sequencing was performed using the CleanPlex SARS-CoV-2 Research and Surveillance Panel . Sequencing was performed on a MiSeq Illumina System with a MiSeq v2 reagent kit (300 cycles). Downstream analysis was performed using the SOPHiA DDM software pipeline . Nextclade software  lasting around one hour together with changes in voice and nasal congestion, loss of taste and smell. Paracetamol was administered to control the fever.On 17 April, patient had already reported to have lost salt taste and nasal congestion, days before testing positive on 21 April. In the following days, nasal congestion decreased, together with recovery of taste and smell. Finally, he was tested negative 4 May, after 14 days of quarantine as recommended by Italian ministerial guidelines. He reported feeling fully recovered even a few days before the last test. Medical and working staff at the dental clinic tested negative even if there was a close contact with the positive patient.Unfortunately, we did not have data about immune response after vaccination.Recently, in this scenario, healthy vaccinated subjects report positive tests and symptoms of SARS-CoV-2 infection ,8, despiAlthough more severe susceptibility emerged in patients with obesity, metabolic syndrome, or type 2 diabetes ,11, alteRecent studies showed that immunization can be less potent against the beta variant (B.1.351) for a limited protection of neutralizing antibodies created by natural contact with the virus or vaccination ,13.In a group of 417 volunteers inoculated with the second dose of BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccine, Hacisuleyman et al. identified two women with infection . Genome Indeed, both the patients presented with clinically mild symptoms . MoreoveA recent case report showed the presence of COVID-19 symptoms 40 days postvaccination . Based oNowadays, we are still living in an uncertain scenario, in which it is necessary to assess the role of variants in determining postvaccination infection and how the host immune-response may impact in the development of the disease. So, in order to deeply understand these mechanisms, it could be useful, whenever it is possible, to perform viral DNA sequencing and testing for immune-related markers in patients with breakthrough infections.This case outlines some useful considerations; firstly, the classic variant of SARS-CoV-2 infected a healthy subject, 55 days postvaccination, pointing out that breakthrough infection is possible after vaccination in healthy subjects with common variants; in addition, moreover, it is necessary to continue observing all the prescriptions recently indicated in the literature in order to avoid new contagion for all healthy workers also after vaccination, since, as seen, the vaccination does not ensure complete immunity in 100% of cases. In this case report, the correct use of personal protective equipment by the dental staff prevented the spread of infection between patients and workers. Personal protective equipment included the use of filtering facepiece 2 (FFP2) and working spaces were arranged to keep a distance of at least 2 m between workers in the office. Each room was also provided with portable air cleaners. In the last instance, we are lacking standardized means to predict a guaranteed immunity status after vaccination. Future research should focus on the development of affordable means for monitoring a population’s immunity postvaccination response in order to adopt preventive measures and understand new mechanisms beyond this particular disease.


Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is a serious liver disease with pathogenesis remaining unclear. This study aims to investigate the association between testosterone levels, stage , severity scores, and clinical outcomes of HBV-ACLF.This single-center observational study involved 160 male patients with HBV-ACLF, 151 chronic hepatitis B patients without liver failure (CHB) and 106 healthy controls (HC). Morning blood samples were collected and androgen levels analyzed by chemi-bioluminescent immunoassay. Time to death or liver transplantation within 90 days comprised the primary composite outcome.P = 0.030).Serum levels of total testosterone (TT), free testosterone index (FTI), dehydroepiandrosterone sulfate and cortisol were significantly lower among HBV-ACLF than CHB and HC, while androstenedione was higher. Low TT, sex hormone binding globulin and FTI were associated with increased stage  and severity scores (Model for End-stage Liver Disease and Chinese Group on the Study of Severe Hepatitis B-ACLF scores). Low TT (< 142.39 ng/dL) was a risk factor for both the composite outcome and for death alone within 90 days. Multivariate analysis revealed TT to be an independent predictor for the composite outcome (hazard ratio 2.57, 95% CI 1.09–6.02; Low serum testosterone is common among male patients with HBV-ACLF and predictive of increased severity and worse outcome of the disease and may play an important role in the progression of HBV-ACLF. Acute-on-chronic liver failure (ACLF) is a complex syndrome, characterized by acute and severe liver injury in the context of pre-existing chronic liver disease . Among ATestosterone, the most important androgen in males, plays a critical role in the metabolism of proteins, carbohydrates, and fat; and is believed to have suppressive effects on the immune system . And itsConsidering the dynamic transformation between the bound and free forms of testosterone, interactions between steroid hormones and the potential influences of sex, age, body mass index (BMI), and other clinical factors, we sought to compare the circulating serum levels of total testosterone (TT), free testosterone index (FTI), sex hormone binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and androstenedione (AND) in men with HBV-ACLF with those of age- and BMI-matched chronic hepatitis B patients and healthy controls without liver disease. Secondarily, we evaluated whether the five androgens and SHBG are associated with the severity and outcome of HBV-ACLF in cases. We hypothesized that low testosterone is related to increased severity of disease and risk of death or liver transplantation among men with HBV-ACLF. Deeper understanding of the role and functions of testosterone levels in HBV-ACLF may contribute to further studies to identify contributors to severity and mortality of liver disease and determine if application of testosterone therapy improves patient outcomes.This is a single-center, prospective observational study. The study were performed in accordance with the ethical guidelines of the 2013 Declaration of Helsinki and all its protocols were approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine . Written informed consent was obtained from all the participants and for dead participants from their legal representatives prior to enrollment in the study.We consecutively recruited 229 patients with HBV-ACLF who underwent treatment with an artificial liver support system at the First Affiliated Hospital of Zhejiang University College of Medicine during the 14-month period between December 2018 and January 2020 by invitation. Of these, 69 were excluded from analysis . During the study period, we recruited 151 age- and BMI-matched males with chronic hepatitis B but without liver failure from sample bank of major diseases in Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province as disease controls and 106 matched healthy controls without liver disease from the physical examination center. Exclusion criteria were: other diagnosed chronic liver disease ; hepatocellular carcinoma; previous transplant; hypogonadism; the presence of testicular, prostatic, or pituitary tumor; and use of hormone therapy  or anticoagulation therapy such as warfarin.The diagnostic criteria for HBV-ACLF followed the criteria of the Chinese Group on the Study of Severe Hepatitis B (COSSH) : acute hcreatinine [mg/dL]) + (3.8 × LogETbil [mg/dL]) + (11.2 × LogEINR) + 6.4 and COSSH-ACLF [Clinical data was obtained from medical records relating to essential information , comorbidities , precipitating events, laboratory indexes , and complications . The Model for End-stage Liver Disease (MELD)  score waMorning fasting blood samples were drawn from all subjects prior to treatment with the artificial liver support system and from controls at the time of recruitment. We analyzed the androgens using chemi-bioluminescent immunoassay. Serum TT, SHBG and DHEAS were measured on an Architect i4000 analyzer , cortisol was measured on an ADVIA Centaur XP  and androstenedione was measured on an Immulite 2000XPi . The FTI was calculated for each participant as (TT × 10)/SHBG . The norP < 0.05 was considered statistically significant.All continuous variables are expressed as median, interquartile range , categorical data are presented as percentage and frequency. TT, FTI and SHBG levels were natural log-transformed and other androgen levels were square root-transformed. In univariate statistical comparisons, the Mann–Whitney non-parametric U test was used for continuous variables and the Kruskal–Wallis test to compare more than two groups. Categorical data were evaluated using a chi-squared test or Fisher’s exact test, as appropriate. Generalized linear models were used to predict an increase in stage of HBV-ACLF, ascites, and hepatic encephalopathy with decreasing or increasing androgen level. The relationships between androgen level and severity scores were examined in subjects with HBV-ACLF through multiple linear regression analysis. Kaplan–Meier estimation was used to evaluate the survival rates without transplant of groups with different testosterone levels. The log-rank test was used to compare mortality rates in terms of the composite outcome and in terms of death alone between groups with different testosterone levels. When analyzing mortality rates, subjects who underwent liver transplant were excluded. With regards to HBV-ACLF prognosis, the Cox proportional hazards model was fitted with a forward stepwise selection method (p-in: 0.05 and p-out: 0.10) to identify risk factors associated with the composite outcome of death or transplantation. All statistical analyses were carried out using SPSS version 24.0 . A value of A total of 160 males aged 45 (36–56) years with HBV-ACLF were enrolled for analysis in the present study. Of these, 32 were categorized as having early stage liver failure, 61 as middle stage, and 67 as end stage. The most frequent potential precipitating event was hepatitis B relapse (58.1%), followed by bacterial infection (18.8%), hepatotoxic drugs (3.7%), superimposed HEV infection (1.3%), gastrointestinal hemorrhage (1.3%), more than one hepatic insult (5.6%) and unknown cause (11.2%). All patients received antiviral therapy upon diagnosis of hepatitis B and patients with HBV-ACLF underwent plasma exchange every 1 or 2 days until improvement or the primary outcome. All 160 subjects completed follow-up. The in-hospital 90-day mortality rate was 34.4% (21 subjects underwent liver transplantation and 34 died).Table P < 0.001).Lower levels of TT, FTI, and SHBG and higher levels of androstenedione were associated with increased risk of advanced severity stage Table . For eveThe clinical characteristics and outcomes of subgroups after stratification by TT are presented in Table P < 0.001) compared with normal TT. After adjustment for age, BMI, SHBG, free testosterone index, cortisol, and androstenedione (Model 2), the significant association between TT and the composite outcome remained. Model 3 showed that among the clinical variables significantly associated with the outcome in univariate analysis, only TT, INR and urea nitrogen levels were independent factors predicting composite outcome rates. As Fig. P < 0.001 by log-rank test; Fig. P = 0.018). Analyzing death alone, low TT was associated with a significantly higher risk of 90-day mortality than normal TT .After dividing patients into survivor and death/liver-transplant groups according to the 90-day outcome, we found TT, SHBG, FTI to be lower and cortisol, androstenedione higher in the death/liver-transplantation group Fig. . Univarine Model , the signe Model , the signe Model , the sigThe researches on association between testosterone level and severity of HBV-ACLF in males are limited. In the present study, we confirmed that men with HBV-ACLF had lower levels of TT, FTI, DHEAS and cortisol and higher levels of androstenedione compared with age- and BMI-matched healthy controls and those with chronic hepatitis B. In secondary analysis among HBV-ACLF cases, lower levels of TT, FTI, and SHBG and higher levels of androstenedione are strongly associated with higher stages of HBV-ACLF and increased clinical severity of HBV-ACLF according to complications  and multiple severity scores (MELD and COSSH-ACLF scores). Low TT is associated with an increased risk of death or the need for liver transplantation independently, but the association between FTI and this risk is weaker.This is, to the best of our knowledge, the first prospective study to survey the associations between low serum TT and disease stage, severity, and outcome in men with HBV-ACLF. To reduce any influence of treatment, we only recruited patients who were planned to undergo treatment with the artificial liver support system, which may help to reduce the mortality rate of ACLF . Our resThe precise mechanism of low testosterone levels in men with HBV-ACLF is complex and involves various biological actions. Testosterone is mainly secreted by Leydig cells and regulated by hypothalamic-pituitary–testicular axis in males. Testosterone deficiency can develop as a consequence of the direct damage of the testis caused by HBV. Early in 1990, HBV DNA / RNA signals were found in testis of acute HBV infections and decompensated but not compensated CHB males , indicatAs the most important endogenous anabolic steroid, testosterone deficiency may conceivably contribute to increased risk of advanced severity and poor outcome in patients with HBV-ACLF. A double-blind placebo-controlled trial  demonstrMost of all, excessive immune response triggered by HBV exacerbation is considered to be the driver of HBV-ACLF and testosterone may play a major role in it. The liver was recently demonstrated to be an androgen-sensitive organ because it expresses androgen receptors, and HBV has been suggested to be a sex-hormone-responsive virus . ResearcThis study has some limitations which should be acknowledged. First, it is a single-center study which analyzed data of 160 subjects, with 55 cases of the composite outcome. Further studies involving larger cohorts recruited from multiple centers would provide information about the generalizability of these results to other populations. Second, we recruited male subjects, but changes in testosterone levels may also occur in females and affect female patients with HBV-ACLF. However, due to the rarity of HBV-ACLF in women, studies involving such subjects will require longer-term or multi-center collaborations. Finally, our observational study cannot determine the causality between low testosterone and increased death or need for transplantation among patients with HBV-ACLF. Extended longitudinal studies and prospective interventional trials may help to elucidate the underlying mechanisms of this relationship.In summary, we demonstrate the high prevalence of decreased serum testosterone levels among male patients with HBV-ACLF and show that low testosterone levels are independently associated with severity and outcomes of HBV-ACLF. Our results indicate low total testosterone level to be a marker of disease progression and poor prognosis in male patients with HBV-ACLF and testosterone supplementation may be a potential treatment to reduce the high mortality rate of male patients with HBV-ACLF, but the safety of the therapy remains to be proved in future.


Timely data from official sources regarding the impact of the COVID-19 pandemic on people who use prescription and illegal opioids is lacking. We conducted a large-scale, natural language processing (NLP) analysis of conversations on opioid-related drug forums to better understand concerns among people who use opioids.key themes: (i) prescription and/or illegal opioid use; (ii) substance use disorder treatment access and care; and (iii) withdrawal. Phrases that were unmappable to any particular theme were discarded. We computed the frequencies of substance and theme mentions, and quantified their volumes over time. We compared changes in post volumes by key themes and substances between pre-COVID-19 (1/1/2019—2/29/2020) and COVID-19 (3/1/2020—11/30/2020) periods.In this retrospective observational study, we analyzed posts from 14 opioid-related forums on the social network Reddit. We applied NLP to identify frequently mentioned substances and phrases, and grouped the phrases manually based on their contents into three broad Seventy-seven thousand six hundred fifty-two and 119,168 posts were collected for the pre-COVID-19 and COVID-19 periods, respectively. By theme, posts about treatment and access to care increased by 300%, from 0.631 to 2.526 per 1000 posts between the pre-COVID-19 and COVID-19 periods. Conversations about withdrawal increased by 812% between the same periods . Posts about drug use did not increase . By substance, among medications for opioid use disorder, methadone had the largest increase in conversations . Among other medications, posts about diphenhydramine exhibited the largest increase .Conversations on opioid-related forums among people who use opioids revealed increased concerns about treatment and access to care along with withdrawal following the emergence of COVID-19. Greater attention to social media data may help inform timely responses to the needs of people who use opioids during COVID-19.The online version contains supplementary material available at 10.1186/s13011-022-00442-w. As of June 2021, the novel coronavirus disease (COVID-19) pandemic has claimed the lives of over 590,000 individuals in the US and 3.8 million globally .Persons with substance use disorder (SUD) are thought to be directly at a higher risk for COVID-19 and poor outcomes because of higher rates of immunocompromised status and other underlying comorbidities . IndirecHowever, little is known about immediate concerns among communities of people who use prescription and illegal opioids, and how those concerns have evolved during the COVID-19 pandemic. Our goal was to harness large-scale, publicly-available conversation data from posts made on Reddit, which houses the largest online opioid-related drug forums, to better understand how communities of people who use opioids have been impacted by the pandemic. We use natural language processing (NLP) and a mixed-methods design to study trends in topics prior to and since the emergence of the COVID-19 pandemic. Improved, timely awareness of issues affecting people who use opioids during the pandemic can help local, state, and federal organizations better respond to the needs of these communities in the moment.pre-COVID-19 period: January 1, 2019–February 29, 2020.COVID-19 period: March 1, 2020–November 30, 2020. Reddit, one of the largest social networks and the largest online forum with over 52 million daily users, hosts active community forums on a range of substance-use related topics including, but not limited to, opioid use and has been extensively used for similar research . We idenThe included subreddits span topics ranging from opioid use disorder (OUD) recovery strategies to personal experiences with opioids. Consistent with standard convention in NLP research, posts were initially preprocessed by converting text to lowercase, reducing words to their stems , and removing stopwords  using the Python Natural Language Toolkit.2Frequency distributions of words/phrases 1–3 tokens long for each study period.Term frequency inverse document frequency (TF-IDF) values, including monthly and aggregate TF-IDF values for both periods. TF-IDF calculations assign weights to a given word/phrase by frequency of appearance in a data subset relative to posts from the remaining dataset. We employed a customized TF-IDF method where “TF” denotes the term frequency for a given period  and “IDF” denotes “the inverse of the term frequency” from other periods .Next, potential phrases of interest were identified quantitatively by calculating:Subject matter experts in substance use (CMJ and SAS) then manually reviewed the phrases of interest, combined with their spelling and phrasing variants, to identify the top 50 unique terms relevant to substance use . These wThe subject matter experts grouped the selected terms into three broad themes by primary content discussed: 1) prescription and illegal opioid use, 2) SUD treatment and access to care, and 3) withdrawal. Relative frequencies were then calculated by month for each theme using the total monthly number of posts from the 14 included subreddits as the denominator. We also plotted trends in the frequency of the term ‘COVID’. Since this term was understandably absent before the pandemic, its rise following the outbreak served as a suitable comparison for other substance-related themes explored. Qualitative review of posts by theme was conducted to validate our automatic findings and further explore the nature of conversations.We also studied changes in the mentions of specific substances in the two periods. Drugs of interest were identified using the aforementioned quantitative and qualitative approaches (Appendices-C-D). We combined brand, generic, and street names for substances into single items, where necessary. Since drug names are often misspelled or expressed using non-standard expressions on social media, we used an automatic variant generator to include commonly-used lexical variants. We then computed the relative frequencies of these substances in the pre-COVID-19 and COVID-19 periods to explore trends. All analyses were conducted in Python (Version 3.8).Posts were collected and analyzed across the 14 opioid-related subreddits, with 77,652 and 119,168 posts during the pre-COVID-19 and COVID-19 periods, respectively. Figure Table The most [take-homes] one can get at [my opioid treatment program] is 1 week. I asked if they would have to make special exceptions because of the [COVID-19] crisis. I was told that nobody has to do anything for us… I understand that take home medications could kill kids, pets, etc.[.] if someone is irresponsible but I think they should let up a little bit.”. Other posts highlighted changes opioid treatment programs had made since the onset of the pandemic: “… I usually have 2 week [take-homes], I'm getting 28 days now [because] of COVID19. My clinic has ONLY been testing unstable patients, new patients, or ones with recently failed [urinalyses]. But all of it is up to the individual clinics or corporate owners of the clinic.”. Additional examples are available in Qualitative assessment of posts that contained treatment and access to care related content revealed that the majority of posts were from individuals currently in recovery. A large volume of conversations focused on take-home access to methadone. For example, one post (abbreviated for clarity and anonymity) stated: … “This study used large-scale real-world conversation data to explore the concerns of people who use opioids after the emergence of the COVID-19 pandemic. Our mixed-methods approach reveals that conversations about treatment and access to care increased markedly during COVID-19, with a focus on methadone access and increased discussion of over-the-counter medications  and other substances .. Leading sources of information on drug use traditionally include large-scale surveys such as the National Survey on Drug Use and Health and data derived from toxicology testing of overdose deaths [There is increased demand for timely data to guide public health prevention activities as health professionals have raised several potential concerns facing SUD treatment and overdose prevention arising from the COVID-19 pandemic , 12. Leae deaths . Unfortue deaths , 15, ourManual examination of posts verified that the NLP analysis findings are indeed informative. We found large increases in conversations about treatment and access to care from March to November 2020. For methadone, a considerable amount of the increase concerned regional policies about medications for opioid use disorder access. In March 2020, the Substance Abuse and Mental Health Services Administration released guidance allowing opioid treatment programs to provide take-home doses of methadone and buprenorphine for established, stable patients. Under access to care, discussions surrounding methadone increased the most relative to other medications for opioid use disorders. Interestingly, while policy changes attempted to facilitate access to medications for opioid use disorder during COVID-19, access to methadone was under stricter regulation than buprenorphine; for example, clinicians at opioid treatment programs could initiate buprenorphine remotely for new patients without an in-person examination unlike methadone initiation which still required in-person examinations. Additionally, in office-based settings, clinicians with a Drug Addiction Treatment Act waiver to prescribe buprenorphine for OUD were provided emergency authority to initiate new patients remotely without in-person examinations.Despite considerable speculation about how COVID-19 is shifting substance-use markets and patterns, we found that conversations among people who use opioids were overwhelmingly focused on treatment relative to conversations about procuring and using illegal substances. This underscores the interest in medications for opioid use disorder, despite the myriad challenges that face the medications for opioid use disorder maintenance, especially during the pandemic. We note that our analysis did reveal an increase in conversations about fentanyl during the COVID-19 period when overdose deaths involving synthetic opioids such as illegally made fentanyl accelerated .Non-opioid substances showed marked increases in conversations within the opioid subreddits, the largest of which were seen for over-the-counter  or prescription  medications used to treat symptoms, including those associated with withdrawal . Of noteThis work has some limitations. Although the data sources employed represent large-scale data from the largest drug forums online, we recognize that such data are a convenience sample and may not fully represent concerns of populations who do not use these platforms. Additionally, stay-at-home orders could have resulted in conversations about the studied themes shifting from in-person to online channels. Thus, results from these efforts should be used to help generate early hypotheses and insights that can be further assessed using traditional health and substance use surveillance systems. Furthermore, this study uses qualitative review of posts; future work should evaluate the accuracy of fully-automated, unsupervised machine learning methods to further accelerate the timeliness of analyses and insights.Nevertheless, our findings show that treatment and access to care continue to be major themes discussed by people who used opioids during the COVID-19 pandemic. Rapidly identifying and addressing concerns of these communities through novel data and analytic methodologies are crucial. Removing barriers to care, including through the expansion of telehealth and allowance of take-home doses of medications for opioid use disorder from opioid treatment programs, are important to support overdose prevention activities and long-term recovery as part of the overdose public health emergency response.Our analysis demonstrates the utility of social media for studying SUD, particularly at times when significant social changes occur, such as the spread of the COVID-19 pandemic. There is still considerable uncertainty about how COVID-19 will keep impacting the lives of people. COVID-19 resulted in substantial increases in chatter related to treatment and access, and withdrawal. We found that this was because people suffering from SUD had major concerns about maintaining their treatment once stay-at-home orders were issued. Active surveillance using social media data can help track emerging concerns and problems as the world prepares for post-COVID-19 life. Currently, despite the usefulness of social media for SUD research and surveillance, they have been largely under-utilized. We believe that caregivers and policy makers should put greater attention to social media data, as they may help inform timely responses to the needs of people who use opioids during COVID-19.Additional file 1. A.TF-IDF equation. B.Top 50 selected terms and phrases. C. Drug names and misspellings. D. Medication names and grouping. E. Sample posts.