The aim of this study was to investigate the effect of possible late effects of cancer treatment  and of two job resources (autonomy and supportive leadership style) on future burnout complaints, among employees living 2–10 years beyond breast cancer diagnosis.N = 287). These data were part of a longitudinal study among Dutch speaking workers with a cancer diagnosis 2–10 years ago. All complaints and job resources were self-reported. Longitudinal multivariate regression analyses were executed, controlling for years since diagnosis, living with cancer (recurrence or metastasis), and other chronic or severe diseases. Mediation by baseline burnout complaints was considered.Data at T1 (baseline questionnaire) and at T2 (9 months later) were collected in 2018 and 2019 (A higher level of fatigue and cognitive complaints at baseline (T1) resulted in higher future burnout complaints (at T2), with partial mediation by baseline burnout complaints. No effect of physical complaints at T1 was observed. Higher levels of autonomy or a supportive leadership style resulted in lower burnout complaints, with full mediation by baseline burnout complaints. Buffering was observed by autonomy in the relationship of cognitive complaints with future burnout complaints. No moderation was observed by supportive leadership.The level of burnout complaints among employees 2–10 years beyond breast cancer diagnosis may be an effect of fatigue or cognitive complaints, and awareness of this effect is necessary. Interventions to stimulate supportive leadership and autonomy are advisable, the latter especially in the case of cognitive complaints. Burnout complaints receive a lot of attention, both in the media, within companies and organizations, among occupational physicians and within the area of work and organizational psychology. The number of research studies into burnout complaints has increased enormously in recent decades . The conTo the question how burnout complaints can be prevented or reduced, several answers have been given by means of research based on the well-established Job-Demands Resources (JD-R) model . Within The World Health Organization recently defined burnout as an occupational phenomenon, characterized by exhaustion, mental distance and decreased personal effectiveness . These cWorkers beyond cancer diagnosis may experience late effects of cancer treatments, like physical complaints , fatigueIt is conceivable that the late effects of cancer treatments aggravate the job demands, as the effort it takes to perform certain tasks may increase. Furthermore, professionals  that guide this subpopulation of workers have mentioned that it may be unclear if and how the late effects of cancer treatments and burnout complaints are related to each other . This imTherefore, the aim of this study is to investigate (1) if and to what extent possible late effects of cancer treatments  affect the level of future burnout complaints, and (2) to identify a possible direct effect of autonomy or a supportive leadership style on future burnout complaints, as well as buffering of the presumed association of late effects with future burnout complaints, among female salaried employees diagnosed with breast cancer 2–10 years ago.Possible late effects of cancer treatments can last for more than decades or even develop many years after treatment . As prevN = 1,009) reported perceived health to predict a decrease in job burnout and the predicted effect of perceived health on job burnout to be significantly larger than the effect of burnout on health  , as wellAll in all, a higher level of physical complaints, fatigue or cognitive complaints is expected to have an increasing effect on future burnout complaints beyond the first 2 years after breast cancer diagnosis among employees. Therefore, our first hypothesis (H1) is as follows:H1: A higher level of physical complaints, fatigue, or cognitive complaints at T1 (baseline) is associated with higher burnout complaints at T2.Various job resources that are important for the work ability of workers who experience late effects after cancer treatments have emerged in qualitative studies , as wellFirst, a lack of autonomy is reported to be correlated with burnout risk . FurtherAll in all, although research data among workers more than 2 years beyond breast cancer diagnosis are lacking, a higher level of autonomy or a supportive leadership style are expected to be associated with lower burnout complaints among employees 2–10 years beyond breast cancer diagnosis. Hence, our second hypothesis (H2) is as follows:H2: Higher levels of autonomy or of supportive leadership style at T1 (baseline) are associated with lower burnout complaints at T2.No quantitative studies among workers beyond cancer diagnosis concerning buffering effects by autonomy or by a supportive leadership style of an association of physical complaints, fatigue or cognitive complaints with burnout complaints are available. However, qualitative studies indicate that these job resources are experienced as relevant factors when workers are confronted with late effects of cancer treatments . FurtherH3: The relationships between physical complaints, fatigue, or cognitive complaints (late effects) at T1 and burnout complaints at T2 are moderated by autonomy or by a supportive leadership style at T1 such that when the job resource is high, the burnout complaints are lower than when the job resource is low .In addition, three covariates are included in the analyses, namely years since diagnosis, living with cancer (recurrent or metastasis), and the presence of comorbid chronic conditions, as these factors may influence the relationships mentioned because of differences in physical and mental burden. The level of burnout complaints at baseline (T1) will be studied as a possible mediator, as it is expected that burnout complaints will have associations with individual factors as well .All in all, the present study is quite unique in focusing on burnout complaints among workers beyond cancer diagnosis and the first to investigate the influence of late effects after cancer treatments on future burnout complaints. It is important for professionals to know whether late effects contribute to a possible diagnosis of burnout complaints and whether autonomy or a supportive leadership style require additional attention among workers confronted with late effects of cancer treatment in preventing burnout complaints.N = 287) were used in the present study. Various methods and channels were used to inform  participants about the study and the first (online) questionnaire such as social media, a short video clip and a website . An invitation (and a reminder 1 month later) for the second questionnaire was sent out by e-mail to the participants of the first questionnaire. Before the online questionnaire started, the respondent gave informed consent. The Research Ethics Committee (cETO) of the Open Universiteit in the Netherlands assessed the ethical acceptability of the study and agreed with the study design and method (reference cETO: U2018/03891/MQF).A survey study was carried out (between June 2018 and December 2019) among workers 2 – 10 years beyond cancer diagnosis. Data from workers beyond breast cancer diagnosis and with exclusively salaried employment at T1 (baseline) and at T2  physical complaints, fatigue, cognitive complaints at T1, (2) burnout complaints at T1 and at T2, (3) autonomy and supportive leadership style at T1, and (4) years since diagnosis, living with cancer (recurrence or metastasis), and other chronic or severe diseases at T2.Physical complaints were measured by a question about complaints caused by ten possible conditions . These conditions were neuropathy, hormonal complaints, hot flushes, bone decalcification, heart complaints (due to treatment), scar adhesions, joint pain, lymph edema, lung problems, and bowel and/or bladder problems. For the level of physical complaints, the total score (0–20) is used. Fatigue was measured by the subjective fatigue subscale  of the Checklist Individual Strength (CIS) , 1999. TThe general version of the Utrecht Burnout Scale (UBOS-A) was used to measure burnout complaints . The UBOAutonomy and supportive leadership style, were measured using one of the scales by The year of diagnosis was collected in the first questionnaire, from which the number of years after cancer diagnosis was determined. In the second questionnaire, the respondents indicated whether they were living with cancer (recurrence or metastasis) and whether they had other chronic or severe diseases.The numbers of respondents with a breast cancer diagnosis and exclusively salaried employment at T1 and at T2 was 287.®/Apple Mac®.The data were analyzed using SPSS software, version 25  for WindowsN = 287) and for the dropouts between T1 and T2 (N = 174). Descriptives were demographics , years since diagnosis (at T1), living with cancer (recurrence or metastasis), and other chronic diseases (at T1 and at T2), late effects  at T1, burnout complaints (at T1 and at T2), autonomy and supportive leadership style (at T1).Descriptives were analyzed for the study sample at T2 , and other chronic or severe diseases at T2. Missing data were not imputed, and this could result in lower numbers of respondents to be used in analyses. Physical complaints, fatigue, cognitive complaints, autonomy, and supportive leadership style were centered at their means. The hypotheses were tested by regression analyses in six steps; first one step for each of the three covariates, then the late effects together in the fourth step, the two job resources in the fifth step, and the interaction terms to test possible buffering by the job resources of the association of late effects and burnout complaints at T2 in the sixth step. If this regression showed that the late effects significantly predicted burnout complaints at T2, possible mediation by baseline burnout complaints at T1 was analyzed.To be able to establish if the variable burnout complaints at T1 (baseline) is functioning as a mediator, three assumptions should be met . First, N = 287) was 49.7 years (SD 7.72) and the mean number of years since diagnosis was 4.5 years (SD 2.39). At T2 6% (N = 17) was living with cancer (recurrence or metastasis), and 44% (N = 125) had other chronic or severe diseases. Dropouts between T1 and T2 (N = 174) were younger and reported a higher level of cognitive complaints. See At T1 the mean age of the study sample (p < 0.001). A paired sample test resulted in no significant difference between burnout complaints at T1 and burnout complaints at T2. The three late effects showed low mutual correlations, and the same was observed for the job resources. The late effects were correlated positively (p < 0.01) both with burnout complaints at T1, and with burnout complaints at T2. The two job resources were correlated negatively (p < 0.01) both with burnout complaints at T1, and with burnout complaints at T2. See Burnout complaints at T1 were strongly correlated with burnout complaints at T2 . Years since diagnosis and living with cancer (recurrence or metastasis) and were not associated with burnout complaints at T2. See One of the three covariates showed a significant association with burnout complaints at T2, namely other chronic or severe diseases , however, a higher level of fatigue and a higher level of cognitive complaints did . See p < 0.05 for fatigue and β = 0.121, p < 0.05 for cognitive complaints. As the associations were significant to a lesser extent, this means the mediation by burnout complaints at T1 was partial. See Thereafter hypothesis H1 was tested in step 4 of the multivariate regression analyses, with physical complaints, fatigue, and cognitive complaints at T1 as possible predictors of burnout complaints at T2. A higher degree of the number of physical complaints did not predict higher burnout complaints at T2 . See p = 0.223 for autonomy and β = –0.060, p = 0.179 for supportive leadership style. As the associations were not significant anymore, this means burnout complaints at T1 fully mediated the association between the job resources and burnout complaints at T2. See Hypothesis H2 was tested in step 5 of the above-described multivariate regression analyses, with the job resources (autonomy and supportive leadership style) at T1 as possible predictors, and burnout complaints at T2 as dependent variable. Our hypothesis was confirmed. A higher level of autonomy or a supportive leadership style predicted lower burnout complaints at T2 . Furthermore, there was one other case of moderation  by autonomy of the association of physical complaints with burnout complaints at T2 . In the analysis of H3, no mediation by burnout complaints at T1 was observed as the regression analysis with burnout complaints at T1 as dependent variable did not show any significant result for the interaction terms. See Hypotheses H3 concerned the expected buffering of the job resources (autonomy and supportive leadership style) at T1 of the relationships between physical complaints, fatigue, or cognitive complaints at T1 and burnout complaints at T2. Buffering was observed by autonomy of the association of cognitive complaints at T1 with burnout complaints at T2  among a population of employees with a breast cancer diagnosis 2–10 years ago is remarkable. Burnout complaints appeared to be quite stable in time, and further analyses showed that baseline burnout complaints partially mediated the relation between fatigue and cognitive complaints at T1, and burnout complaints at T2. This causal relationship between fatigue and cognitive complaints on one side and future burnout complaints on the other side among employees more than 2 years beyond breast cancer diagnosis is not reported before. Studies among other populations did report negative cross-sectional associations of self-reported fatigue  or of coIt was already mentioned that it may be difficult to distinguish burnout complaints from the late effects as a result of cancer treatments . This poAs expected, the level of autonomy and supportive leadership style did predict lower future burnout complaints in the present longitudinal study, however, this effect changed into non-significant when controlled for burnout complaints at baseline, as burnout complaints at baseline have a fully mediating effect. Cross-sectional studies among this population also reported positive associations with burnout complaints for these job resources . These rPhysical complaints did not affect the level of burnout complaints in the current analyses, which is unexpected as we took note of some other studies where this was the case . AgainstOf course, we have to take into account that this study result may be influenced by certain limitations in the design of this study. An important limitation is the possibility that the experienced complaints are not (completely) caused by the cancer treatments, but that other causes also play a role. Only in studies with very large cohorts that are followed for many years is it possible to also make measurements before an occurring cancer diagnoses and to compare these measurements on the same factors in the years after the cancer diagnosis and treatment. Furthermore, hindering late effects may cause stress. Elevated levels of stress affecting burnout measures are reported among individuals experiencing short-term stress . MoreoveTo summarize, the present study indicates that fatigue and cognitive complaints increase future burnout complaints among employees 2–10 years beyond breast cancer diagnosis, which is a novel finding. Furthermore, a supportive leadership style and autonomy are regarded as important, the latter especially if cognitive complaints are at higher levels. It is important to also investigate other job resources (like job crafting of coaching) for their positive effect among these workers. In other words, it is very important to conduct more studies into effective job resources specifically to protect workers beyond cancer diagnosis at risk of higher burnout complaints in the future.Finally, it is important for managers and human resource management within companies and organizations and for other professionals guiding these employees, to realize which late effects of cancer treatments may present an extra risk to show and develop higher levels of the self-reported burnout complaints and to know what can be done in the workplace to reduce or even prevent increasing burnout complaints. Therefore, it is important to always be alert if it is known that an employee has been treated for cancer and therefore possibly experiences late effects of the treatments, in order to include this information in the choice of interventions. Possibly, for employees who are known to have undergone cancer treatments, an additional focus on the treatment of possible fatigue or cognitive complaints from the psycho-oncological perspective may be considered, apart from the attention for factors in the work situation.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Research Ethics Committee (cETO) of the Open Universiteit in the Netherlands who assessed the ethical acceptability of the study and agreed with the study design and method (reference cETO: U2018/03891/MQF). The participants provided their written informed consent to participate in this study.IB, WV, and TV developed the study design. IB was responsible for the data collection. IB prepared the data analysis with support from TV and WV. IB wrote the first draft of the manuscript and the later drafts of the manuscript were adjusted by all authors in collaboration. All the authors read and approved the submitted version.TV was employed by the company Loyalis Knowledge & Consult. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Monkeypox is an emerging infectious disease, which has a clinical presentation similar to smallpox. In the two past decades, Central Africa has seen an increase in the frequency of cases, with many monkeypox virus (MPXV) isolates detected in the Democratic Republic of Congo (DRC) and the Central African Republic (CAR). To date, no complete MPXV viral genome has been published from the human cases identified in the CAR. The objective of this study was to sequence the full genome of 10 MPXV isolates collected during the CAR epidemics between 2001 and 2018 in order to determine their phylogenetic relationships among MPXV lineages previously described in Central Africa and West Africa. Our phylogenetic results indicate that the 10 CAR isolates belong to three lineages closely related to those found in DRC. The phylogenetic pattern shows that all of them emerged in the rainforest block of the Congo Basin. Since most human index cases in CAR occurred at the northern edge of western and eastern rainforests, transmissions from wild animals living in the rainforest is the most probable hypothesis. In addition, molecular dating estimates suggest that periods of intense political instability resulting in population movements within the country often associated also with increased poverty may have led to more frequent contact with host wild animals. The CAR socio-economic situation, armed conflicts and ecological disturbances will likely incite populations to interact more and more with wild animals and thus increase the risk of zoonotic spillover. Orthopoxvirus in the family Poxviridae1. The disease has a clinical presentation similar to smallpox, with additional symptoms including adenopathy and maculopapular rash, especially on the palms of the hands and the soles of the feet2. Although there is no specific treatment or vaccine for monkeypox, cross-immunity with smallpox vaccination may offer some protection in human populations3. Nonetheless, the monkeypox lethality rate varies from 1 to 10%4. Transmission of MPXV in humans occurs either through direct contact with infected animals or through contact with body fluids or respiratory droplets of an infected person, resulting in secondary transmission. Although monkeypox is a zoonotic disease, the animal reservoir has not yet been identified. MPXV has been isolated twice from wild animals: in 1985 from a symptomatic Thomas’s rope squirrel (Funisciurus anerythrus) caught in the Democratic Republic of the Congo (DRC)5 and in 2012 from a sooty mangabey (Cercocebus atys) in Ivory Coast6. More recently, MPXV was sequenced from the feces of chimpanzees in Ivory Coast7. Serological studies have suggested, however, that several rodent species are potential reservoirs8.Monkeypox, an emerging and neglected infectious disease, is caused by the monkeypox virus (MPXV), which belongs to the genus 11. In 2018, monkeypox human cases were also reported in western Cameroon 12, near the border with the Nigeria. The sequence of the ATI gene (639 bp) was obtained from one of these cases. Given the close phylogenetic relationship identified between the Cameroonian (for the 2018 strain) and Nigerian isolates, the close geographical proximity of the affected regions and the absence of human cases in Cameroon since 1989, the origin of this isolate is likely an import from Nigeria (West Africa). However, as also suggested by the authors, the hypothesis of a natural circulation within Cameroon cannot be completely ruled out12. Monkeypox cases caused by the West African clade of MPXV have recently been identified outside of the African continent14. Three human cases were diagnosed in the United Kingdom (UK), the first of which involved a person traveling through Paris from Nigeria, and the second resulting in a nosocomial transmission to a health worker in the UK15. Additional cases have recently been imported into Israel and Singapore from Nigeria17.In the past two decades, Congo Basin MPXVs have primarily affected two Central African countries, the DRC and the Central African Republic (CAR), at a growing case rate Many MPXV genomes have been sequenced from previous outbreaks in the DRC and Nigeria, but no genomic data is currently available for MPXV detected over the past two decades in the CAR. In Central Africa, the Congo Basin is a large block of tropical rainforests covering, from West to East, Equatorial Guinea, Gabon, southern Cameroon, the southern and northern parts the Republic of the Congo (R. Congo), the northern half of the DRC, and several southern regions of the CAR  were collected by Institut Pasteur Bangui (IPB) staff, and stored in a refrigerated compartment during the transportation to the IPB. The tools used for the notification of suspected cases, the collection of biological samples (scabs and/or pus) to investigate the etiological cause, and the registration of epidemiological data have been standardized and validated by the CAR authorities. As soon as a case was reported, a team was commissioned to go into the outbreak area to carry out field investigations and interview officials from the local health care teams. Outbreak investigations consisted of the identification of human monkeypox cases and their contacts to establish the chain of transmission, including suspicious human contacts with wild animals. Moreover, they also looked at patient consultation records, health records, and hospitalization forms to supplement the epidemiological data. The term micro-outbreak that we used in our article corresponds to us to outbreak with less that ten human cases.All suspected cases of monkeypox in the CAR were investigated according to a well-established procedure validated by both the CAR Ministry of Health and the World Health Organization (WHO)19. The mice were monitored daily for a week until mortality or morbidity was observed in at least one mouse. Then, the brains of the dead mice were harvested and suspended in a 1 × PBS solution (pH = 7.4). For all samples, whether harvested from mouse brains, pus or scabs . Finally, high-throughput sequencing was performed using a Miseq benchtop sequencer (Illumina) with 300 cycles to generate 2–18 million pairs of 150 nucleotides.The libraries were prepared using the SureSelect XT library preparation kit for the Illumina sequencing kit. The SureSelect biotinylated probes for targeted enrichment were custom designed and are specific to a panel of known complete MPXV genomes . DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and at least 200 ng of DNA (suspended in 50 μl) was fragmented using a Covaris M220 ultrasonicator . This fragmented DNA was processed for final repair, addition of adapters and purified using Agencourt AMPure XP beads  according to the manufacturer’s instructions. The libraries were amplified by PCR with DNA polymerase (Herculase II) provided by Agilent Technologies and captured with 1 μl biotinylated probes. The hybridization step lasted 16 h at a constant temperature of 60 °C. The captured libraries were then amplified by PCR (16 cycles). After purification with AMPure XP beads , an enrichment step, identical to the initial capture step, was performed according to the same protocol as described above27.The quality of raw reads was initially assessed and filtered using CLC workbench 10.0.1, and the trimmed reads were mapped against all MPXV references extracted from GenBank using the following stringent parameters: 95% of the whole read had to align with at least 95% identity. Mapped reads were then assembled de novo using SPAdes v3.10. To evaluate the accuracy of the assembly, reads were mapped back  to the resulting contigs. All genomes were annotated using VAPid28. Cowpox virus  and horsepox virus  sequences were used as outgroups. Both extremities of the preliminary alignment containing missing data, as well as many insertions or deletions (indels), they were trimmed and the length of the final alignment was 204,963 bp. The best-fit nucleotide substitution model, i.e., GTR+G, was selected using the Akaike information criterion (AIC) in jModelTest 2.1.1030. The Bayesian tree was reconstructed with MrBayes v3.2.7 and the posterior probabilities (PP) were computed with four independent Markov chains for ten million generations with a sampling every 1000 generations and a burn-in of 25%. IQ-TREE31 was used to calculate bootstrap proportions (BP) using the maximum likelihood method and 1000 replicates.The 10 MPXV genomes specially sequenced in this study were aligned with the 56 MPXV genomes available in NCBI using MAFFT v7.471 (2020/Jul/3)32. BEAST v1.10.433 was used for Bayesian MCMC analysis to estimate temporal nodes. As recommended in Patrono et al., we selected the simplest model: strict clock and constant population size with a HKY substitution model and four Gamma categories7. An MCMC model was run with 50,000,000 generations and a burn-in of 25,000 sampled trees. All other parameters were by default. BEAST executions were completed checking chain convergence and sufficient sampling of the posterior space (ESS > 200) with Tracer v1.7.1. The final chronogram was generated using TreeAnnotator v1.10.433.Molecular estimations of divergence times were carried out on an alignment of the complete MPXV genomes found in the Central Africa clade. Estimations were based on the same methodology as described in “34. All multiple sequence alignments were then edited by Aliview35 to analyse synonymous and non-synonymous substitutions and alterations (insertion and/or deletion) in MPXV protein-coding genes.The characterization of the polymorphism in ORFs was carried out based on the genomic annotations of the sequence of the MPXV isolate from Zaire isolated in 1996 (NC_003310). At first, all coding sequences (CDS) were extracted from the reference sequence and used to build a BLAST reference database. We then used BLASTN to compare all the genomic sequences in our Central Africa dataset with our reference database to identify homologs. Homologous genes were grouped by family, and codons aligned using translatorx_vLocal.pl scriptComité Ethique et Scientifique; University of Bangui). Moreover, oral and written informed consent was obtained from all included patients. All the experiments carried on mice were done in accordance with relevant guidelines and regulations on the use of laboratory animals including the ARRIVE guidelines. All the experiments were done in accordance with relevant guidelines and regulations. The authorization number UB/FACSS/IPB/CES/20 provided by the CES includes both investigations of human suspected Monkeypox cases and experiments carried on mice.The investigation of all suspected cases of monkeypox occurring in the CAR was approved by the local ethics committee  in Lidjombo (Sangha Mbaéré prefecture) in the extreme southwestern region of the country, where the father of a patient reported hunting a duiker and a monkey that both showed skin lesions37. The next four cases were identified in a Bantu family in August 2001 in the city of Bangassou (Mbomou prefecture)4. In June 2010, two cases were confirmed in two young Pygmy children who had hunted and eaten a wild rodent in the city of Mongoumba (Lobaye prefecture)10. Between December 2015 and February 2016, 10 suspected cases were reported in Bangassou (Mbomou prefecture). The index cases were two brothers in Madigui/Bakouma (Mbomou prefecture)11, who also infected their mother and their younger brother. A second cluster was also identified in Bangassou, following nosocomial transmission at the Bangassou health care center from the Madigui/Bakouma index case, with a secondary intrafamilial transmission to the family of the nurse. Another case was reported at the same period in Bakouma (Mbomou prefecture)11, but epidemiological investigations suggested that it was not linked to the previous outbreak. In 2017, two new micro-outbreaks were reported. The first was in February 2017 in the city of Bao (Mbomou prefecture) where at least two cases were confirmed, and the second was in April in the city of Mbaïki (Lobaye prefecture) with a single confirmed case38. Finally, between March and April 2018, six cases were detected in the town of Ippy (Ouaka prefecture), followed by three other cases in the town of Rafaï (Mbomou prefecture)24. Epidemiological investigations suggested that one of the cases in Rafaï (18c) may be related to the Ippy case (15c). The Ippy case involved a street vendor who visited his girlfriend in Rafaï (18c). Among all the MPXV cases reported in CAR since 1984, we selected 10 isolates from nine micro-outbreaks or isolated cases that occurred between 2001 and 2018 in eight cities, located in four prefectures of the country Except for the two libraries from the A1 and A2 samples that were prepared from DNA extracted from neonatal mouse brain biopsies, all other libraries were prepared from DNA extracted from scab or pus samples. Threshold cycle (Ct) values determined using quantitative PCR ranged from 18 to 35 Ct for DNA extracted from new born mouse brain biopsies and scabs, respectively. On average, the enrichment protocol provided Ct values between 15 and 18 for the final libraries. The greatest improvements in Ct values (− 16 Ct on average) were obtained with DNA extracted from primary samples, whereas for DNA extracted from the MPXV isolate, the decrease in Ct values was smaller (− 3 Ct on average) (data not shown). Finally, the ten whole-genome sequences were assembled de novo. Then, the raw reads were mapped back to these draft sequences. On average, 98% of the reads were successfully mapped back to these draft sequences. Moreover, this mapping showed that the proportion of MPXV reads ranged from 50% to more than 90% of the total number of raw reads  between 2016 and 2018 . It appears as the sister-group of a cluster of MPXV isolates collected in the DRC in 1979  and 1985 , as well as one MPXV found in South-Sudan in 2005 (KC257459) , we extracted, using sequence similarities, 191 homologous CDS for each CAR genomic sequence. These CDS were multiple-aligned to detect variations between the CAR sequences, as well as those belonging to MPXV Congo Basin group II. At least one SNP was detected in 47 different ORFs, even though three of these ORFs were identical because they were located in the inverted terminal regions (ITR) of the genome. In 70.2% (33/47) of the cases, a single SNP was identified in an ORF. However, in 21.3% (10/47), 6.4% (3/47) and 2.1% (1/47) of the other cases, two, three or four SNPs were found in the same ORF, respectively Table . MoreoveIn parallel to the search for these SNPs, a total of 10 alterations, indels of one or more amino acids, were found in nine ORFs. These indels were specific to certain isolates or sequence groups Table . Of the This study is the first to describe the genomic data for MPXVs isolated from human cases in the CAR.–6  substitutions per site per year. Our estimate is slightly lower than that obtained by Patrono et al. on MPXV sequences belonging to the West Africa clade (1.93 × 10–6)7 or for the variola virus (8.5 × 10–6)39. However our estimate remains within the expected range for double-stranded DNA viruses40. As expected, the MPXV sequences belonging to the Central Africa clade generally differ from each other by only single nucleotides. There were also some indels, but most of these mutations were located in non-coding regions where tandem repeats have been described previously. There is a single exception, with one insertion in the gene that codes for an interleukin receptor (IL-1β). In fact, this gene has already been identified as one of the less conserved genes between the Central and West Africa clades, which may help to explain the difference in virulence observed between isolates of these two clades41. The IL-1β gene produces a protein that prevents cytokine from binding to the IL-1 receptor43. IL-1, and one of its forms (IL-1β) in particular, is involved in the inflammatory response during infection by binding to IL-1 receptors that activate several signaling pathways45. In addition, experiments on the vaccinia virus have shown that the IL-1β binding protein inhibits the immune response by affecting the proliferation of murine B and T lymphocytes; mice infected with a deletion mutant are not as ill as those infected with the wild isolate43. Our analysis shows that several sequences belonging to the Central Africa clade, including the Zaire-1979 and CAR-15c/18c sequences, have an ORF that codes for a truncated protein (210 aa instead of 326 aa in the other isolates). The consequences of this alteration on protein function and the virulence of the virus are not known. Therefore, despite a retrospective analysis of the two CAR cases related to this isolate, we could not conclude as to the potential impact of this alteration on the clinical outcome of the disease. However, patient 18c had more than 180 lesions, which corresponds to a severe clinical outcome46. Unfortunately, this rash severity score cannot be compared with the other cases, either in patient 15c or in the nine other patients in this study.First of all, our analysis showed that the 10 CAR MPXV genomes have the typical organization observed in other orthopoxviruses: there is a conserved central region and ITRs with tandem repeats at both extremities. As expected, all ORFs—except four that are located in the ITRs—were present in the central region of the genome. Moreover, the mean genome-wide substitution rate for sequences belonging to the Central Africa clade was estimated at 1.06 × 1047.The distribution of MPXVs examined in this study Fig.  shows thOur data show that many MPXV isolates detected from individual cases or micro-outbreaks identified in the Mbomou prefecture were epidemiologically unrelated, although several of them belong to the same CAR1 lineage, i.e. the isolates from Bao (A4), Rafaï (38c) and Bangassou (B2) collected between 2016 and 2018. Moreover, these isolates might have diverged from each other between 1999 and 2004, whereas the isolates from Bangassou (A1) and Mongoumba (A2) separated around 2001. This period coincides with a period of intense political instability in the CAR, occurring between 1992 and 2005, when a military takeover in 2001 led to a large number of population movements within the CAR or across borders to neighboring countries. These movements associated with increased poverty may have resulted in more frequent contacts with infected animals, perhaps due to increased activities such as hunting or bushmeat trade.9. Over the last two decades, an increasing number of cases have been reported, particularly in the CAR9. Improvements in disease surveillance have certainly contributed to such increase in incidence, but not exclusively50. Other possible explanations include the cessation of routine smallpox vaccination campaigns, which ended between 1978 and 1980. As a result, there was a drop in cross-protection against Orthopoxvirus species in these populations52. Other MPXV exposure factors include the generally difficult living conditions, either due to poverty or recurrent wars or civil unrest, which tend to force people to move over long distances, and for some to flee to rainforests. Deforestation has also led to ecosystem changes, leading to local extinction, colonization by invasive species/alien species, and may also foster the enzootic circulation of MPXV53. All of these changes may have led to an increase in repeated contact with wild animals potentially infected by MPXV, particularly in children and young male adults3. In Nigeria or Sierra Leone, human cases more likely correspond to indigenous zoonotic spillover than to importation from a neighboring country 54. Although three modes of transmission  were identified during the small outbreak that occurred in Bangassou in 2016 11, human-to-human transmission in CAR is generally confined to a single family55. It rarely spreads over long distances, with the exception of the street vendor from Ippy who probably infected his girlfriend in the town of Rafaï, several hundred kilometers away. Unlike the outbreak in Nigeria where several cases related to it have been reported outside the country, no human case related to a MPXV isolate from the CAR has been reported outside the country to date.To date, MPXV has been reported in human populations in only four West African countries  and six Central African countries 56. In addition, molecular dating estimates suggest that periods of intense political instability resulting in population movements within or outside the country often associated also with increased poverty may have led to more frequent contact with host wild animals and to monkeypox human cases. To limit transmission and prevent the spread of MPXV to the human population8, new studies must seek to better characterize all CAR isolates that remain unsequenced and all animal hosts still unidentified to date, to evaluate ethological and ecological risk factors involved in human infections.In conclusion, our phylogenetic results indicate that the 10 CAR isolates belong to three lineages closely related to those found in DRC. Furthermore, the phylogenetic pattern shows that all of them emerged in the rainforest block of the Congo Basin. Since most human index cases in CAR occurred at the northern edge of western and eastern rainforests, transmissions from wild animals living in the rainforest is the most probable hypothesis. Ecological disturbances through land-use changes, deforestation and geographical expansion of human activities will probably lead populations to interact more and more with wild forest mammals and thus increase the risk of zoonotic spilloverSupplementary Table S1.Supplementary Table S2.


Text message  smoking cessation interventions have demonstrated efficacy in high-income countries but are less well studied in low- and middle-income countries, including Vietnam.The goal of the research is to assess the feasibility, acceptability, and preliminary efficacy of a fully automated bidirectional SMS cessation intervention adapted for Vietnamese smokers.The study was conducted in 3 phases. In phase 1, we adapted the SMS library from US-based SMS cessation programs . The adaptation process consisted of 7 focus groups with 58 smokers to provide data on culturally relevant patterns of tobacco use and assess message preferences. In phase 2, we conducted a single-arm pilot test of the SMS intervention with 40 smokers followed by in-depth interviews with 10 participants to inform additional changes to the SMS library. In phase 3, we conducted a 2-arm pilot randomized controlled trial (RCT) with 100 smokers. Participants received either the SMS program  or weekly text assessment on smoking status . The 6-week SMS program consisted of a 2-week prequit period and a 4-week postquit period. Participants received 2 to 4 automated messages per day. The main outcomes were engagement and acceptability which were assessed at 6 weeks (end of intervention). We assessed biochemically confirmed smoking abstinence at 6 weeks and 12 weeks. Postintervention in-depth interviews explored user experiences among a random sample of 16 participants in the intervention arm.P=.01), but the effect was not significant at 12 weeks . In-depth interviews conducted after the RCT suggested additional modifications to enhance the program including tailoring the timing of messages, adding more opportunities to interact with the program, and placing a greater emphasis on messages that described the harms of smoking.Participants in both arms reported high levels of engagement and acceptability. Participants reported using the program for an average of 36.4 (SD 3.4) days for the intervention arm and 36.0 (SD 3.9) days for the control arm. Four of the 50 participants in the intervention arm (8%) reset the quit date and 19 (38%) texted the keyword TIPS. The majority of participants in both arms reported that they always or usually read the text messages. Compared to the control arm, a higher proportion of participants in the intervention arm reported being satisfied with the program (98% [49/50] vs 82% [41/50]). Biochemically verified abstinence was higher in the intervention arm at 6 weeks (20% [10/50] vs 2% [1/50]; The study supported the feasibility and acceptability of an SMS program adapted for Vietnamese smokers. Future studies need to assess whether, with additional modifications, the program is associated with prolonged abstinence.ClinicalTrials.gov NCT03219541; https://clinicaltrials.gov/ct2/show/NCT03219541 Of the world’s 1.1 billion smokers, 80% live in low- and middle-income countries (LMICs) . PromotiVietnam, an LMIC, has one of the highest smoking rates in the world . AccordiTo continue to meet goals for decreasing smoking prevalence globally, effective cessation interventions must be easily accessible, adapted to local languages and cultural contexts, and scalable. Mobile technology  that uses text messaging or short message service (SMS) meets these criteria by creating a relatively low-cost platform for wide dissemination of tailored tobacco cessation interventions. A growing literature indicates that automated, bidirectional SMS cessation programs can be effective in increasing smoking cessation compared to minimal or no smoking cessation support -12. HoweThis study was conducted to address the gap in the literature by assessing the feasibility, acceptability, and preliminary efficacy of an SMS intervention for tobacco users in Vietnam. The study also provided an important opportunity to describe methods for adapting text message interventions found efficacious in high-income countries to different sociocultural contexts and forms of tobacco use.Despite the large number of SMS studies conducted in high-income countries, few studies have compared the efficacy of combining SMS programs with additional cessation support to SMS alone, and findings have been mixed . For exaThe study was conducted in 3 phases. In the first phase, we conducted 7 focus groups (n=58 participants) to adapt text messages from SmokefreeTXT, a freely available public resource . We suppWe partnered with a community health center in Hanoi to recruit participants for each phase of the study. Community health collaborators, who are similar to community health workers and are assigned to work with the community health centers, were trained to disseminate study information through community outreach activities. During their routine outreach, they assessed smoking status of community members and shared study information with current tobacco users. If interested these individuals were asked for permission to share their contact information with research staff. Research staff then contacted potential participants to provide additional details, obtain consent, and enroll them in the study. This study was approved by the institutional review boards of New York University Grossman School of Medicine and Institute of Social and Medical Studies in Vietnam.The content of efficacious SMS interventions is based on a combination of several theoretical frameworks including social cognitive theory, transtheoretical model, and cognitive behavioral theory ,19-23. TThe final message library was developed through an iterative process that included first translating messages from the English language SMS libraries into Vietnamese with some initial changes to align the content to the Vietnamese context. For example, strategies for coping with nicotine cravings were edited to include practices that were relevant to Vietnamese smokers. We then conducted focus groups to assess message preferences; elicit suggestions to guide further adaptations; and assess reasons for and barriers to quitting, smoking triggers, and participants’ social networks and their influence on smoking behavior.Focus groups included quantitative assessment of message preference followed by group discussions. Participants were asked to rate 46 text messages on a 1 to 4 scale . Ratings were summarized while the focus groups elicited more details about smoking patterns and past quit attempts, reasons for quitting, and barriers to quitting. We then discussed a sample of the messages that were rated across the response scale options to gain additional insights about what types of messages were preferred and elicit suggestions for improving the messages.Focus groups were moderated by two researchers and were audiorecorded, transcribed, and translated into English. Qualitative data analyses were conducted using NVivo 12 . Using an inductive analytic approach, two research team members independently read a subset of transcripts (2-3) to identify preliminary themes, relevant patterns, and clustered concepts and generate questions ,27. UsinBased on the findings from focus groups, the messages were further adapted. For example, compared to the original SMS programs, greater emphasis was placed on the impact of smoking on the family’s health and the dangers of smoking. Other content that was added to align with Vietnamese culture and specific tobacco use patterns included the health hazards of waterpipe use, which is still commonly used in Vietnam. Findings from focus groups also pointed to a need to develop additional messages that encouraged smokers to identify people in their network who could support their quit attempt and offered suggestions on how to refuse an offer of cigarettes or decline to smoke when others are smoking, a common scenario in a country with high male smoking rates .After finalizing the first draft of the SMS library, we enrolled 40 participants in a single-arm pilot test. The participants received automated bidirectional text messages for 6 weeks. At the end of the pilot test, two researchers conducted in-depth interviews with a random sample of 10 participants. The interviews explored 5 areas:Overall perceptions about the program Perceptions about specific program features such as the opportunity to type in the keyword TIPS to obtain additional advice on how to deal with cravingsPerceptions about specific message themes . We read some text messages that participants received during the intervention and asked what they liked and didn’t like about the messages, as well as their suggestions Perceptions about other program characteristics including the number and timing of text messages and length of the programSuggestions for improving the SMS program.Similar to the focus groups, the in-depth interview was guided by our integrated conceptual framework. Two researchers moderated and audiorecorded the interviews. Interviews were transcribed and translated into English. Two researchers used the same approach used to analyze the focus group data. Findings from the single-arm pilot test demonstrated the feasibility of retaining participants in the 6-week SMS cessation intervention and informed additional modifications to the content of the message library. These included a greater emphasis on harms of smoking versus benefits of quitting and adding more messages that offered concrete advice about coping with cravings rather than vague messages meant to motivate smokers .We conducted the pilot RCT  between The final message library consisted of 188 text messages. Messages were designed to increase knowledge about smoking and motivation to quit ; change outcome expectancies (benefits of quitting); and offer cognitive and behavioral strategies such as refusal skills to assist smokers in maintaining the quit attempt. Behavioral strategies encouraged self-efficacy for quitting and encouraged smokers to obtain social support from family and friends.The intervention consisted of a 2-week prequit period and a 4-week postquit period. Participants received 2 or 3 messages per day during the 2-week prequit period, 4 on the quit date, 3 or 4 per day during the first 2 weeks after the quit date, and 2 or 3 messages per day during the subsequent postquit period. Prequit messages encouraged smokers to track their smoking behavior and identify triggers, reinforced reasons for quitting, elicited smokers’ reasons for quitting, and provided advice on obtaining social support as they neared their quit date. Postquit messages were oriented toward relapse prevention and maintaining motivation and included the themes described above. In addition to programmed outgoing messages, participants could send the keyword TIPS to the program to trigger on-demand messages for additional support.Starting from the quit date, participants received a weekly bidirectional text message to assess smoking status as follows: “Are you smoke-free? Reply: Yes or No.” Those who responded yes continued to receive postquit messages. Those who answered no received a message asking if they preferred to set a new quit date. Those who responded no continued to receive postquit messages, and those answering yes received a call from the research assistant to obtain a new quit date and reset the quit date in the SMS program which returned to the prequit protocol. In addition, participants received bidirectional text messages that assessed their level of craving  on days 1, 3, 5, 8, 15, and 25. A high or medium craving response triggered an automated message offering TIPS from the SMS program. Participants could opt out of the SMS program at any time by texting STOP. At the start of the program, participants were made aware that they had the option to text STOP at any time during the trial to discontinue receiving messages.Participants in the control arm received one text assessment message per week at a fixed time in the evening during the 6-week intervention period: “Are you smoke-free? Reply: Yes or No.” The control condition was consistent with previous SMS cessation trials that included minimum exposure for participants in the control group ,28.We conducted surveys at baseline and at 6 weeks (end of treatment) and 12 weeks. The baseline survey captured sociodemographic information such as gender, age, education, and household income level; text messaging habits; and smoking behavior, including CPD and waterpipe sessions per day. Participants were dichotomized as dual users if they reported waterpipe use on some day or every day or cigarette-only smokers if they responded not at all to the waterpipe use question.Measures of feasibility included reach  and survey assessment response rates. We also tracked if participants experienced technical problems.Two measures of program engagement were assessed using the 6-week survey: (1) the number of weeks that participants reported using the program  and (2) self-reported frequency of reading the messages . Two additional measures included the proportion of participants who responded to the bidirectional text message assessments with mean number of times they responded and the proportion who texted the keywords  with mean number of times they texted the keyword.Program acceptability was assessed at 6 weeks by asking participants to rate their overall satisfaction with the SMS program (for intervention arm) or the weekly text assessment , perceived number of messages (too many/just right/too few), and their agreement with statements such as “The text messages helped me quit smoking” using a 4-point Likert scale from “strongly disagree” to “strongly agree.”Smoking abstinence was assessed at 6 weeks and 12 weeks. Abstinence was defined as self-reported no smoking in the past 7 days confirmed with a carbon monoxide of 10 ppm or less . Quit atWe conducted in-depth interviews with 16 randomly selected participants from the intervention arm. Using a semistructured guide similar to the single-arm pilot test, two researchers moderated and audiorecorded the interviews to obtain more in-depth information about what they liked and didn’t like about the program and elicit recommendations for improving the SMS program. Interviews were transcribed and translated into English.t tests. We used descriptive statistics to summarize program acceptability and engagement results. All tests of statistical significance were 2-tailed, and P<.05 was considered significant. The process for qualitative analyses was the same across the 3 aims and described above.We analyzed quantitative data using the R statistical computing environment  . SociodeOn average, participants were aged 38.9 (SD 8.2) years . A totalAlmost all of those screened were eligible and 99.0% (100/101 eligible participants) enrolled in the study. All participants completed the 6-week and 12-week follow-up surveys. There were no technical issues reported by participants or the SMS vendor.The mean number of days that participants reported using the program was 36.4 (SD 3.4), out of a total of 42 days, in the intervention arm and 36.0 (SD 3.9) for the control arm . None ofAll but one participant in the intervention arm reported being satisfied or very satisfied with the program overall and none reported being very unsatisfied . In contP=.01; 12 week: 12% [6/50] vs 6% [3/50]; P=.49), although the difference was not significant at 12 weeks , and recommendations for enhancing the program. Almost all of the participants liked the SMS program and described it as helpful, primarily because it offered encouragement and enhanced motivation and served as a reminder to stay on track.The messages motivated me and reminded me not to smoke.#15, 39 years, maleI felt like they [text messages] made me determined to quit.#16, 43 years, femaleMessages that included craving management strategies and addressed the harmful effects of smoking were described as particularly useful. A participant noted that he “learned many things…about the ways to overcome cravings” . Another explained that messages about the negative consequences of tobacco were “like a warning, helping us understand the danger of smoking and benefits of quitting. So we became conscious and then decided to quit” .Reactions to messages that suggested strategies for refusing cigarettes in social situations were mostly positive. One participant noted that those messages “provided the most simple and effective way to refuse invitations to smoke” . However, a few participants suggested rewording these in ways that were more consistent with how they communicate with friends and family.Many of the participants preferred a more tailored approach in terms of message timing.You should send text messages to the relevant time frame of each individual.#6, 34 years, maleOne participant wanted the messages to be sent when he had the cravings.You could send the messages at those time.#6, 34 years, maleParticipants did have the option to proactively text the keyword TIPS to generate messages during those difficult times, but few routinely used the option.Participants suggested several additional modifications to enhance the program. Additional content changes including adding more text messages about the health consequences of smoking.I want to receive more text messages on the risks of smoking so my determination in quitting may be stronger.#8, 50 years, maleParticipants expressed a strong interest in a more interactive approach.Sometimes I wanted to interact with the person who sent the messages, but I could not do that. The interaction was limited to the responses of yes or no.#6, 34 years, maleyears, male) rather than using the keyword. A few participants suggested adding telephone interactions with a counselor. Last, the majority of smokers suggested extending the intervention duration.Similarly, another suggested allowing users to “ask [text] my own questions” . A review of studies that analyzed the impact of emphasizing benefits (gain framed) versus costs (loss framed) found a small advantage of gain versus loss framed messages, but findings were mixed, and these data are based on studies in high income countries . In the The control arm’s high level of engagement and satisfaction with the program that only included weekly text assessments was an unexpected finding. This may reflect the lack of prior experiences with any smoking cessation services among Vietnamese smokers. Vietnam has not widely disseminated WHO guidelines for integrating routine tobacco use screening and brief advice into the primary care health system , and altThese results should be interpreted as preliminary given the small sample size of this pilot study, as well as the relative brevity of our program as compared to some of the existing SMS cessation interventions ,20,32,33Finally, the review by Krishnan et al  also sugOur study has limitations. First, participants were recruited from two urban wards in one city. Thus, the findings may not be generalizable to all smokers in Vietnam. In addition, we excluded waterpipe-only smokers. Additional research is needed to explore the value of tailoring mHealth programs to specific types of tobacco users. Second, we did not conduct qualitative interviews with the control arm, which could have provided additional insights into their experience and reasons for the high levels of engagement and acceptability of weekly text assessment. Third, while the study sample was small, a strength of the study was the high retention rates, with all participants completing both follow-up assessments. Last, our study sample included only 2 women, which is consistent with the low smoking rate among Vietnamese women (1.1%) as smoking is less acceptable among women ,40. ThisDespite these limitations, this study contributes to the small body of literature on mobile phone smoking cessation treatment carried out in LMICs. The data supported the feasibility and acceptability of a culturally adapted SMS cessation treatment program and demonstrated short-term efficacy in promoting abstinence among Vietnamese smokers. While ongoing and future research continues to grow the evidence for effective mHealth cessation programs in a given context, LMICs are beginning to adopt and scale these programs . Therefo


Although there have been many published reports on fatigue and pain in patients with chronic obstructive pulmonary disease (COPD), it is considered that these symptoms are seldom, if ever, asked about during consultations in Japanese clinical practice. To bridge this gap between the literature and daily clinical experience, the authors attempted to gain a better understanding of fatigue and pain in Japanese subjects with COPD. The Brief Fatigue Inventory (BFI) to analyse and quantify the degree of fatigue, the revised Short–Form McGill Pain Questionnaire 2 (SF-MPQ-2) for measuring pain and the Kihon Checklist to judge whether a participant is frail and elderly were administered to 89 subjects with stable COPD. The median BFI and SF-MPQ-2 Total scores were 1.00 [IQR: 0.11–2.78] and 0.00 [IQR: 0.00–0.27], respectively. They were all skewed toward the milder end of the respective scales. A floor effect was noted in around a quarter on the BFI and over half on the SF-MPQ-2. The BFI scores were significantly different between groups regarding frailty determined by the Kihon Checklist but not between groups classified by the severity of airflow limitation. Compared to the literature, neither fatigue nor pain are considered to be frequent, important problems in a real-world Japanese clinical setting, especially among subjects with mild to moderate COPD. In addition, our results might suggest that fatigue is more closely related to frailty than COPD. Breathlessness is undoubtedly believed to be one of the most important perceptions experienced in subjects with chronic obstructive pulmonary disease (COPD). This is likely followed by coughing, as well as sputum production, symptoms which are described in most of the clinical practice guidelines . AntoniuPrevalence rates for pain have also been reported to be surprisingly high in subjects with COPD over the last decade ,15,16,17When interviewing a candidate suspected of COPD, we often ask about breathlessness, cough, sputum and wheezing but not fatigue or pain. We are aware of only a small number of chest physicians in Japan who are of the opinion that these symptoms should be asked about in the consulting room. Although many published reports have studied fatigue and pain, it is unclear whether they should be checked during every examination. The aim of this study was to bridge the gap between the literature and daily clinical experience by gaining a better understanding of fatigue and pain in subjects with COPD. COPD is occasionally considered to be an accelerated aging disease since it is well known that aging of the lungs and COPD have many similarities and are sometimes difficult to distinguish from each other . The fre1) to forced vital capacity (FVC) of under 0.7. All participants gave written informed consent.We recruited 89 consecutive patients with stable COPD who attended the outpatient clinic at the Department of Respiratory Medicine of the National Center for Geriatrics and Gerontology (NCGG) from August 2018 to August 2020. The inclusion criteria were: (1) age more than 50 years; (2) smoking history exceeding 10 pack-years; (3) chronic fixed airflow limitation; (4) regular clinic attendance for more than half a year to avoid any changes induced by new medical interventions; (5) no uncontrolled co-morbidities and (6) no variation in treatment in the preceding four weeks. Chronic fixed airflow limitation was defined as a maximal ratio of forced expiratory volume in 1 s (FEVCO) measured by the single-breath technique as reported by the American Thoracic Society and European Respiratory Society Task Force in 2005  and 0.00 [IQR: 0.00–0.27], respectively. They were all skewed toward the milder end of the respective scales. A floor effect was noted in around a quarter on the BFI and over half on the SF-MPQ-2. The BFI scores were significantly different between groups regarding frailty determined by the Kihon Checklist, but not between groups classified by the severity of airflow limitation. Compared to the literature, neither fatigue nor pain are considered to be frequent important problems in a real-world Japanese clinical setting, especially among subjects with mild to moderate COPD. In addition, our results might suggest that fatigue is more closely related to frailty than COPD.


BRCA2 mutation.The impact of various breast-cancer treatments on patients with a BRCA2 mutation has not been studied. We sought to estimate the impact of bilateral oophorectomy and other treatments on breast cancer-specific survival among patients with a germline We identified 664 women with stage I–III breast cancer and a BRCA2 mutation by combining five different datasets (retrospective and prospective). Subjects were followed for 7.2 years from diagnosis to death from breast cancer. Tumour characteristics and cancer treatments were patient-reported and derived from medical records. Predictors of survival were determined using Cox proportional hazard models, adjusted for other treatments and for prognostic features.p = 0.55)). The 10-year breast-cancer survival for women who had a bilateral oophorectomy was 89.1% and for women who did not have an oophorectomy was 59.0% . The adjusted hazard ratio for chemotherapy was 0.83 .The 10-year breast-cancer survival for ER-positive patients was 78.9% and for ER-negative patients was 82.3% (adjusted HR = 1.23 (95% CI, 0.62–2.45, BRCA2 mutation, positive ER status does not predict superior survival. Oophorectomy is associated with a reduced risk of death from breast cancer and should be considered in the treatment plan.For women with breast cancer and a germline  Evidence is emerging that knowledge of inherited mutations in predisposing genes, such as BRCA1 and BRCA2, is also important.BRCA1 mutation carriers3 with hazard ratios ranging from 0.4 to 0.6. It is important to confirm that oophorectomy is helpful in the treatment of breast cancer in patients with a BRCA2 mutation. Many of the patients are premenopausal, and oophorectomy will curtail fertility and induce surgical menopause, which increases risks for osteoporosis, cardiovascular disease and possibly cognitive dysfunction.8 In general, hormone-replacement therapy is not advised for women with ER-positive breast cancer. It is not likely that a randomised clinical trial will be conducted in BRCA2 mutation carriers; therefore, we must rely on large, well-conducted observational studies. Our primary goal was to assess the efficacy of various treatments in reducing breast-cancer mortality in the BRCA2 patient population. We combined data from five different databases and conducted a multicentre historical cohort study of women with breast cancer and a BRCA2 mutation, resident in North America, Europe and Australia, to identify the impact of various treatments  on survival.A beneficial effect of oophorectomy on breast-cancer survival has been seen in BRCA2 mutation. We excluded patients with known metastatic disease at diagnosis. Age at diagnosis was restricted to 25 and 70 years and year of diagnosis was between 1990 and 2019. Patients were eligible if they had genetic testing within 5 years of the diagnosis of breast cancer  and were found to be BRCA2-positive. Patients were also eligible if they developed breast cancer after genetic testing . Patients with a previous diagnosis of cancer in the same or contralateral breast or cancer at another site were excluded. We wished to study the impact of oophorectomy after diagnosis, and therefore patients who had a bilateral oophorectomy prior to their breast-cancer diagnosis were excluded. The indication for oophorectomy was not recorded.Patients were eligible for the study if they had a diagnosis of stage I–III invasive breast cancer that was pathologically confirmed and carried a germline BRCA2 mutation carriers from 67 participating centres in Canada and the United States who were enrolled in a multicentre, longitudinal cohort study. All subjects sought testing for BRCA1 and BRCA2 mutations because of a personal or family history of breast and/or ovarian cancer. Mutation detection was performed using a range of techniques, but all nucleotide sequences were confirmed by direct sequencing of DNA. The study was approved by the institutional ethics review boards of the respective host institutions and all study subjects provided written informed consent. All subjects completed a baseline research questionnaire at the time of a clinic appointment or at their home at a later date. Subjects were eligible if they were cancer-free at the time of study enrolments (the baseline questionnaire) and reported a new diagnosis of invasive breast cancer in one of the follow-up questionnaires. They were also eligible if they had been diagnosed with breast cancer in the 5-year period prior to genetic testing. The questionnaires requested detailed information on family and personal history of cancer, reproductive and medical histories as well as medication use. Follow-up questionnaires are completed every 2 years thereafter to update exposure information and to capture incident disease and deaths from all causes. Information on diagnoses and treatment of breast cancer was collected from the questionnaires and from review of medical records. Hormone-receptor status of the tumour and other pathologic features were abstracted from the pathology report and/or medical record review. Cause and date of death was obtained by the collaborating investigator at each of the participating sites. This was determined by patient record, by correspondence with the treating physician or by next of kin.Eligible study subjects included female BRCA2 mutation is present in approximately 2% of the familial breast-cancer cases in Poland.9 Since 1996, the team has collected blood samples for DNA extraction from unselected women with newly diagnosed breast cancer in various clinical centres across Poland. From 1996 to 2001, this was done primarily for young breast-cancer patients (age of diagnosis <50), and from 2002 onwards, included breast cancer patients diagnosed at all ages. In total, DNA samples from approximately 13 000 breast-cancer patients were collected between 1996 and 2014. The patients were tested for the presence of two founder mutations in BRCA2. The patients in the study were those who were found to be positive for one of the two mutations. The mean time from diagnosis to blood testing was 1.4 years. Patients had no prior diagnosis of breast cancer or another cancer. Information on clinical presentation and treatments received was retrieved from the medical records. Vital status and date of death was retrieved by linkage to the Vital Statistics Database of the Polish Ministry of Administration and Internal Affairs. Cause of death was determined by medical record review and interview with the treating physician.A 10 They have been systematically followed up with a mailed questionnaire at 3-yearly intervals as part of the kConFab Follow-Up Study.11 Study participants are predominantly from families with multiple cases of breast and/or ovarian cancer and were recruited from one of 16 Familial Cancer Centres across Australia and New Zealand. At the time of enrolment, blood was collected for BRCA1 and BRCA2 mutation analysis. The baseline and 3-yearly follow-up questionnaires ask about demographics, cancer risk factors, cancer diagnoses, surgeries (treatment and prophylactic) and cancer treatments.12 Breast cancers are verified by obtaining pathology reports and medical records. Vital status and cause of death was obtained by relative report or from the death registry. The kConFab Follow-up Study includes 508 women with a BRCA2 germline mutation with a mean of 11.3 years of follow-up; 278 of these were affected with Stage I–III invasive breast cancer either before baseline or during follow-up. Patients were included if there was no prior history of cancer, if they were between 25 and 70 years of age and if less than 2 years had elapsed between the date of breast cancer and the date of genetic testing.Between 1997 and 2008, more than 6000 women enrolled in the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab).BRCA1 and BRCA2 mutation carriers initiated in Manchester in 1999. This includes all carriers of pathogenic mutations in BRCA2 identified through genetic testing in the Manchester region. The regional testing laboratory opened in 1996. Data include all treatment details relating to breast and ovarian cancer and the associated pathology. Vital status is determined through NHS systems.The source of patients was a clinical database of BRCA2 mutation carriers treated at the outpatient clinic of the San Gerardo hospital in Milan between 2007 and 2019. Data were retrieved for women who had identified a mutation prior to 2018. All patients participated in an ongoing cohort study and all indicated their willingness to be followed. Each of the 57 patients enrolled in the study was contacted via email or by phone, and details about the pathological examination of breast cancer were recorded. Details about treatment received were collected from the patients and from review of clinic notes and hospital records.The source of patients was the clinical database of BRCA2. For each case, the medical record was reviewed, and information on age of diagnosis, tumour size (cm) and nodal status (positive/negative) was obtained. Information was also obtained from the questionnaires (patient- reported outcomes). Grade, oestrogen-receptor status (positive/negative), progesterone-receptor status (positive/negative) and HER2 status (positive/negative) was recorded as assigned by the pathologist associated with the hospital of treatment. Chemotherapy, tamoxifen, aromatase inhibitors and radiotherapy were recorded as yes/no. Most studies did not routinely record the specific chemotherapy formulation or the timing of administration (neoadjuvant or adjuvant) or use of ovarian-suppression therapy. Oophorectomy was recorded (yes/no) as well as the date of oophorectomy. In general, most operations are bilateral salpingo-oophorectomy (TAH-BSO). Hysterectomy is done in some centres but not all. Oophorectomies that were done for the treatment of ovarian cancer, metastatic breast cancer or other cancer were considered as no oophorectomy. We recorded the date of contralateral mastectomy; some were done as initial surgical treatment and some were done at a later date. We recorded the dates of all contralateral breast cancers.The nature of patient selection and the testing process varied by centre (see above), but all patients carried a confirmed pathogenic mutation in Survival analysis was conducted using Cox proportional hazard models; subjects were followed from the date of diagnosis  until age 80, the date of completion of the last questionnaire or date last known alive, ovarian cancer, pancreatic cancer, death from breast cancer or death from another cause. Women for whom the cause of death was missing were censored as death from another cause. To adjust for potential survivorship bias that might result because genetic testing was done after diagnosis, follow-up was left-truncated at the date of genetic testing. We estimated the hazard ratio for breast-cancer-specific death associated with radiotherapy (yes/no), chemotherapy (yes/no), tamoxifen/AI use and oophorectomy. Oophorectomy and contralateral mastectomy were treated as time-dependent covariates. The covariates included tumour size , nodal status (negative/positive), ER status (+/−), PR status (+/−/missing), HER2 (+/−/missing) and tumour grade (I/II vs. III). The analysis was repeated restricting the study set to ER-positive breast-cancer patients. The analysis was also repeated for subgroups according to age at diagnosis .A secondary matched analysis was done specifically to study the effect of oophorectomy on survival. In this study, each woman who had an oophorectomy was matched to a woman who had not. Pairs were generated, matched on date of birth (within 4 years), year of genetic test (within 2 years) and country. Further, to be matched to an exposed woman, the unexposed women had to have an age of the last follow-up older than age at the date of oophorectomy in the matched exposed woman. The unexposed control had an age at diagnosis younger than that of the exposed woman. Both subjects were followed for death from the date of oophorectomy in the case. The date of oophorectomy in the exposed woman was used as the date of the initial follow-up for the control. The hazard ratio generated for the matched pairs was adjusted for tumour size, nodal status and ER status using a Cox proportional hazard model.We also assessed time from diagnosis to oophorectomy as a predictor of mortality. The average time elapsed from diagnosis to oophorectomy was 3.2 years. In total, 82 of the oophorectomies were done within the first year following diagnosis. We assessed the hazard ratio in the Cox model for three subgroups of patients based on time from breast surgery to oophorectomy , compared to women who never had an oophorectomy. We also conducted an analysis comparing women who had an oophorectomy in the first year with all other women (no oophorectomy and later oophorectomy).Summary information for the 664 patients is presented in Table The majority of the patients with ER-positive breast cancer received endocrine therapy: tamoxifen alone  an AI alone  or both . When women with missing data were excluded, 419 (71.8%) of all patients received chemotherapy (neoadjuvant or adjuvant) and 248 (67.0%) received both endocrine therapy and chemotherapy. About 46.2% had a contralateral mastectomy and 56.8% had a bilateral oophorectomy after diagnosis. The proportion of women who had an oophorectomy was similar for women with ER-positive (58.7%) and ER-negative breast cancer (55.1%).Overall, there were 112 deaths in the cohort; of these, 91 were from breast cancer and nine were from causes other than breast cancer (Supplementary Table p = 0.31). The adjusted hazard ratio for tamoxifen (vs. no endocrine therapy) was 1.57 . The adjusted hazard ratio for AI alone versus no endocrine therapy was 1.23 . Figure Among the 455 ER-positive patients, the 10-year survival for those who had taken any endocrine therapy was 78.8% and for those who had taken no endocrine therapy was 82.8% . The adjusted hazard ratio for any endocrine therapy (tamoxifen or AI) versus no endocrine therapy was 1.48 . Among the 455 ER-positive women, the adjusted hazard ratio for chemotherapy was 1.05 , and among the 107 ER-negative women, the hazard ratio for chemotherapy was 0.48 .Among all 664 patients, the adjusted hazard ratio for chemotherapy (yes/no) was 0.83 .About 307 of the patients had a contralateral mastectomy; the prevalence ranged from 16.1% in Poland to 67.1% in Australia. The mean interval from initial to contralateral surgery was 2.0 years (range 0–20 years). The adjusted hazard ratio for breast-cancer death associated with contralateral mastectomy (time-dependent) was 0.71 .Among the 664 women, the adjusted hazard ratio for radiotherapy (yes/no) on breast-cancer mortality was 0.81 . About 82 oophorectomies were done within a year of diagnosis and 295 were done within 1 or more years after diagnosis.The overall 10-year survival rate was 78.6% for all 625 women in the cohort. The 10-year survival rate was 89.7% for women who had an oophorectomy and was 59.0% for those who did not have an oophorectomy Fig. . The 10-p = 0.0009) for breast-cancer-specific death and was 0.44  for all-cause death.Among all patients, the adjusted hazard ratio for oophorectomy (time-dependent) was 0.45  and for women over 50 at diagnosis it was 0.33 .Among women under age 50 at diagnosis, the adjusted hazard ratio for oophorectomy and breast-cancer-specific death was 0.51  for ER-negative patients and was 0.48  for ER-positive patients.The adjusted hazard ratio for oophorectomy was 0.28  for women with small node-negative breast cancers (<2 cm) and was 0.48  for women with breast cancer that had node-positive breast cancer or greater than 2 cm.The adjusted hazard ratio for oophorectomy was 0.32  for those who had chemotherapy and was 0.25  for those who did not have chemotherapy.The adjusted hazard ratio for oophorectomy was 0.55  compared to those who never had an oophorectomy.The adjusted hazard ratio for those who had an oophorectomy in the first year after diagnosis was 0.56  compared to all others (those who never had an oophorectomy and those who had an oophorectomy at a later time).The adjusted hazard ratio for those who had an oophorectomy in the first year after diagnosis was 0.77   Fig. .BRCA2 patient population. We observed a decrease in the proportion of cancers that were ER-positive with age of diagnosis (rather than the expected increase) and observed that ER-positive cancers were more likely to be node-positive than ER-negative cancers. These associations have also been seen in an earlier study from our group13 and in the Nordic BRCA2 mutation carrier study.14 Moreover, positive ER status was not predictive of better survival, compared to negative ER status.The goal of our study was to assess the associations of various treatments in reducing breast-cancer mortality in the p = 0.0009). The results were similar for the analysis of the 149 matched pairs . Surprisingly, the association with oophorectomy was present in women with ER-negative breast cancer, but there were only 107 ER-negative patients and the association did not reach statistical significance. It is important that possible sources of bias be considered. Oophorectomy might occur years after the diagnosis; therefore, it is important that we corrected for immortal time bias by including oophorectomy in survival models as a time-dependent covariate. We also adjusted for potential confounders, including tumour size, nodal status and contralateral mastectomy. We excluded women who had known metastatic disease at diagnosis. It is also possible that some women developed distant recurrence early in the follow-up period and that these women did not have an oophorectomy for this reason. This could bias the result towards longer survival for the oophorectomy group. However, among the women with stage I breast cancer, the hazard ratio associated with oophorectomy was 0.32  and it is unlikely for these women that the choice of oophorectomy or not was based on prognosis.Of the four treatments studied, oophorectomy appeared to be the most effective. The adjusted hazard ratio for breast-cancer-specific death associated with oophorectomy was 0.45  would be done in the first year of treatment. In this subgroup, the association was still significant but when we compared women with an oophorectomy in the first year with all other women , the association was attenuated. Further, to ensure that women with and without an oophorectomy were as similar as possible, we confirmed our results in a separate matched analysis, matched on date of birth (within 4 years), year of genetic test (within 2 years) and country of residence. A protective association of oophorectomy on breast-cancer mortality was also seen in our earlier studies of BRCA1-associated breast cancer.BRCA2 carriers.15 We did not distinguish between neoadjuvant and adjuvant chemotherapy, and we did not have the dates of distant recurrences. In cases where neoadjuvant chemotherapy was given, the size and nodal status was based on clinical parameters and pre-treatment imaging. Genetic testing was done for some women after diagnosis, and therefore we did a left-censored survival analysis from the date of genetic testing.There are other limitations to the study. Missing values for several prognostic factors might limit our ability to adjust for potential confounding. We had limited data on HER2 status although HER2 positivity is rare among BRCA2 mutation carriers was similar to that observed in studies of non-carriers, and there is no indication from the data presented here that the decision to prescribe chemotherapy should depend on BRCA2 carrier status.The effect of chemotherapy in p = 0.31). This could be because those treated with endocrine therapy had more aggressive cancers than those who did not get treated, but the association was adjusted for age and conventional prognostic factors. A similar lack of the effect of tamoxifen was seen in an earlier study by Goodwin et al.16 It is not clear if tamoxifen or aromatase inhibitors are effective treatments for ER-positive breast cancer in BRCA2 mutation carriers. The mean follow-up in this cohort was 7.2 years and this may be insufficient to characterise the effect of a given treatment in ER-positive breast cancers. Nevertheless, it is surprising that oophorectomy appeared to be effective in this patient cohort, which did not show a beneficial effect of anti-hormonal therapy.We saw little evidence for the benefit of tamoxifen or an aromatase inhibitor in ER-positive patients in the current study , but this approach has not been studied in mutation carriers.Increasingly, premenopausal women with breast cancer are offered ovarian suppression with a gonadotropin-releasing hormone agonist, particularly if they wish to maintain fertility, and regardless of their mutation status. We did not evaluate the effect of medical ovarian suppression on breast cancer survival in the BRCA2 and BRCA1 carriers.The majority of patients in the study were stage II or III (85%) and few were aware of their mutation status at the time of diagnosis. It is hoped that as genetic testing becomes more widespread, a greater proportion of patients will be diagnosed in MRI surveillance programmes and this will result in more stage I breast cancers. It will be important to revisit predictors of survival for women with MRI-detected breast cancer and for women with stage I cancers in both BRCA2 carriers with breast cancer and should be considered in the treatment plan. Oophorectomy is also of value in preventing the second primary ovarian cancer. Further studies on the benefits of oophorectomy and of other endocrine therapies in this group of women are warranted.In conclusion, we found that oophorectomy is associated with a reduced risk of death from breast cancer in Supplementary Tables