Patients co-infected with hepatitis B virus (HBV) and the human immunodeficiency virus (HIV) are at risk of developing hepatocellular carcinoma (HCC). In sub-Saharan Africa, the overlap between high HIV and HBV prevalence may increase the incidence of HCC. This study investigated the impact of HBV/HIV co-infection on age at presentation and survival of HCC.Ethical approval was obtained to recruit, following informed written consent, patients diagnosed with HCC at oncology units at four South African hospitals. Between December 2012 and August 2015, patients newly diagnosed with HCC were recruited and provided demographic and clinical data and blood specimens. Patients were tested for HBV, hepatitis C virus (HCV) and HIV. Survival data was available for a subset of patients.p = 0.04). A greater proportion of HBV/HIV co-infected cases were female than HBV mono-infected . In addition, HBV/HIV co-infected females presented at a younger mean age (36.8 years) than HBV mono-infected women (50.5 years) (p = 0.09). Median survival was 82 days among the HIV-infected HCC patients compared to 181 days among those without HIV (p = 0.15).Of 107 HCC cases, 83 (78%) were male. Median age was 46 years (range 18 to 90 years), 68/106 (64%) were HBsAg-positive, and 22/100 (22%) were HIV infected. Among HBV surface antigen (HBsAg)-positive HCC cases, 18/66 (27%) were HIV-infected compared to 3/34 (9%) among those that were HBsAg-negative . Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide . Sub-SahHCC in African populations appears to affect men more commonly than women . PatientCHB has an intermediate to high prevalence (5–7%) among adults in southern SSA , althougLittle is known about the natural history of HCC in the HBV/HIV co-infected patient. In fact, the question of whether HIV increases the risk of HCC in patients with CHB still remains largely unanswered. Two studies from resource rich settings suggest that HIV may hasten the evolution of HBV-related HCC, resulting in an earlier age of presentation in the HIV co-infected patient , 19. TheBetween December 2012 and August 2015, patients who were newly diagnosed with primary HCC were prospectively recruited into the study from oncology departments at four university teaching hospitals in South Africa: Tygerberg Hospital and Groote Schuur Hospital in the Western Cape Province and Chris Hani-Baragwanath Academic Hospital and Charlotte Maxeke Johannesburg Academic Hospital in Gauteng Province. The diagnosis of HCC was based on radiological examination by contrast-enhanced computed tomography, abdominal ultrasound scan and/or magnetic resonance imaging and clinical findings. An experienced oncologist reviewed all patients. A subset of 25 cases also had histologic diagnosis of HCC. Serum alpha-fetoprotein (AFP) was measured, but levels < 400 μg/L did not exclude diagnosis of HCC. Following consent patients were interviewed using a standardized questionnaire, had a physical examination and provided blood samples. Clinical laboratory data were extracted from clinical records. A sub-group of the patients recruited from the Western Cape, underwent follow up for survival analysis. This was achieved by means of following up cases via the South African Department of Home Affairs to determine date of death (where applicable) using the national identification number.All participants were recruited following written informed consent according to the Declaration of Helsinki 2008. Ethical approval for the study was obtained from the health research ethics committees at Universities of Stellenbosch, Cape Town and Witwatersrand.Blood specimens were collected in EDTA anticoagulant and SST tubes with silica clot activator upon enrolment into the study. The tubes were centrifuged to separate plasma or serum and stored at − 80 °C until they were used for serologic and molecular testing. The HBsAg test was performed using the DiaSorin Murex HBsAg Version 3 immunoassay kit . HBeAg and antibodies to HBeAg (anti-HBe) were tested using the ETI-EBK PLUS and ETI-AB-EBK PLUS (DiaSorin), respectively. Total antibodies against HBV core antigen (anti-HBc) were measured using the Murex Anti-HBc  immunoassay kit (DiaSorin). Antibodies to HCV and HIV were tested on the Abbott Architect i2000SR immunoanalyzer . HBV DNA quantification and genotyping were performed as previously described on HBsAg-positive samples , 23. HBVp < 0.05.All data entry and storage were performed in Epi-Info 7  and statistical analysis was carried out in Statistica version 12 . Non-parametric data or small-sample data were described using medians and interquartile ranges and compared using the Mann-Whitney test where there were only two groups or the Kruskal-Wallis test in cases of three or more groups. Data with normal distribution were described using means and 95% confidence intervals. Comparisons were performed using the unpaired t-test where data were normally distributed. Categorical data were described using proportions and analysed using the chi-square test or Fisher’s exact test, depending on the number of observations. Survival after diagnosis of HCC was evaluated using the Kaplan-Meier method, and comparisons between patients infected and uninfected with HIV were performed with the log-rank test. Graphs were constructed using GraphPad Prism version 5 . All hypothesis testing was done at 95% confidence intervals and results were regarded as significant if A total of 107 HCC patients were recruited between December 2012 and August 2015. The mean age was 46 years (95% CI: 43–49), range 18–90 years. The majority were male 78% (83/107) and 55% (59/107) were Black African. Twenty two percent (22/100) of cases were HIV infected, of whom 91% (20/22) had evidence of current or past HBV infection. There was insufficient specimen for HIV testing for seven cases. Table The median serum AFP was 6176 μg/L . Of 98 cases, 69 (70%) had serum AFP greater than 400 μg/L. The majority of cases were diagnosed using imaging modalities in combination with clinical laboratory data. Contrast-enhanced computed tomography (CT) scan was done in 77 of 107. Liver histological examination was done on 31/100 of cases on whom information was provided (data was missing on seven cases).p = 0.16. Among the 34 HBsAg negative samples, 17  were positive for anti-HBc, indicating HBV exposure. Male HBsAg positive patients were younger  than male HBsAg negative patients , (p = 0.07). However, the HBsAg-positive female patients were similar in age  to the HBsAg negative females .Of 106 serum samples tested (one had insufficient volume for testing), 68  were seropositive for HBsAg, and 85/103  were anti-HBc positive. The mean age of the HBV-infected cases was 44 years (95% CI: 41–47) compared to 49 years (95% CI: 43–54) among those HBV-uninfected, p = 0.29.Of the 68 HBsAg seropositive patients, 65 were tested for HBeAg. There was insufficient serum in three cases. The overall prevalence of HBeAg among the HBsAg positive cases was 20/65 (31%). Five of 11 female patients  were HBeAg-positive, while only 15 of 54 male patients  were HBeAg positive, Table p = 0.07. The mean age of HIV-infected female patients was 35.8 years (95% CI: 34.1–40.0), 13 years younger than the mean age of HIV-uninfected females, 48.5 years (95% CI: 37.4–59.6), p = 0.08. The mean age of HIV-infected male HCC cases was 42.0 years (95% CI: 36.6–47.4), the mean age of HIV-uninfected males was 45.9 years (95% CI: 42.2–49.8) p = 0.4.The mean age of HIV-infected patients at HCC diagnosis was 39.7 years (95% CI: 36.0–43.5); that of uninfected patients was 46.4 years (95% CI: 42.8–50.0), p = 0.06). The two patients who were HIV infected and had no evidence of HBV infection, both had histological confirmation of the diagnosis. One of the two HIV-infected patients had a history of alcoholic liver disease as a risk factor.Eighteen of the HIV-infected (81.8%) and 47 (60.3%) of the uninfected patients had active HBV infection (HBsAg seropositive)  of the mono-infected patients had HBV genotype A. HCC cases infected with HBV genotype A had a median age of 36 years (IQR: 31–48) compared to 53 years (IQR: 50–58) in those infected with HBV genotype D, p = 0.01. Of the ten anti-HCV seropositive HCC cases, 9 (90%) were male. Among the ten anti-HCV positive cases, three were HBV/HCV co-infected while one case had triple infection with HCV, HBV and HIV. Of eight anti-HCV positive cases, 5 (63%) had detectable HCV RNA with a median of 6.27 log10 copies/ml (IQR: 5.67 log10–6.42 log10). Samples from four HCV-infected HCC cases were successfully sequenced - two cases were infected with HCV genotype 5a while one case each had genotype 1a and the other genotype 1b.Antibodies to HCV were detected in 10 of 101 cases . There was insufficient sample for testing in six cases. The mean age of the anti-HCV positive subjects was 57.5 years (95% CI: 45.8–69.2), while that of the anti-HCV negative cases was 12 years younger, 44.6 years (95% CI: 41.8–48.0) p = 0.15. The survival curves of the HCC cases with and without HIV infection are shown in Fig. Of the 22 HIV-infected HCC patients, 15 (68.2%) died during follow up, while of the 78 patients without HIV, 44 (56.4%) died during follow-up. The Kaplan-Meier survival curves of the two groups were not significantly different, Primary liver cancer accounts for 600,000 deaths each year . HCC is The prevalence of HIV among HCC patients in this study was 22% (95% CI: 14–30), higher than the prevalence of HIV in the South African adult population, estimated at 14.4% among females and 9.9% among males from the 2012 South African National HIV Prevalence, Incidence and Behaviour Survey Report . MoreoveThe patients with HBV infection were younger at HCC diagnosis than those that were infected with HCV in this study. This finding is consistent with reports that HCV-associated HCC tends to peak in the sixth decade of life and HBV-associated HCC in the fourth decade . The difWe found that patients who were infected with HBV genotype A presented at a significantly younger age than those with genotype D. That finding is consistent with that of Kew et al., who described a mean age difference of almost 7 years . The freFifty-five percent of HCC patients in this study were Black Africans who are thought to be at higher risk of HCC than other racial groups due to greater exposure to factors predisposing to HCC, such as dietary iron overload and HBV and HIV infection , 33. CirOur findings raise questions regarding implementation of current hepatitis B treatment and HCC screening guidelines in South Africa. Patients with viral hepatitis have access to tertiary level treatment and surveillance programmes but without wider access to screening for HBV infection, it is not possible to implement them. Unfortunately, many HBV infections are only diagnosed once they present with HCC. Few of our HBV-mono-infected patients had been screened and treated for CHB before their HCC diagnosis. As a result, many patients have radiological findings of multiple or large liver lesions at presentation. Only 30% of cases in this current study had histologic diagnosis of HCC. This has implications with regard to understanding the true burden of HCC with South Africa whose national cancer registry is pathology-based. As a result, there is an underestimation of the burden of HCC and this impacts on the allocation of resources towards the health burden.Among these HCC patients in this study, survival was dismal regardless of HBV or HIV status. Although the HIV-infected HCC patients had a median survival of 82 days, compared to 181 days among those without HIV infection, the difference was not statistically significant. Nonetheless, the poor survival of HIV-infected patients compared to HIV-uninfected patients argues against the probability that the difference in age at diagnosis between HIV-infected and uninfected HCC patients is due to lead-time bias. More intense surveillance of HIV-infected than uninfected patients would be expected to lead to earlier HCC diagnosis with better prognosis and survival which was not observed in this study.An important limitation of this study is recruitment bias; because we recruited HCC cases only from oncology units of state-owned tertiary hospitals, we may have missed patients presenting with HCC at primary health care facilities and therefore unable to recruit all cases. In the setting of a strong health care system, such patients might be diagnosed earlier and survive longer. However, anecdotal evidence suggests that even if patients presented to the primary care facilities with early-stage HCC, those facilities are not equipped or staffed with appropriately trained individuals to provide early detection and effective treatment. It is more likely that our recruitment approach missed patients with HCC who did not survive long enough  to be seen at the recruiting sites for this study. It is unlikely that HIV and HBV infection would be less prevalent among such patients than among those in this cohort.Our findings suggest that HIV infection may cause early development of HCC in HBV-infected patients, particularly in females, and that many patients who present late with HCC have underlying undiagnosed and/or untreated HBV infection. Estimates suggest that only 5% of patients infected with viral hepatitis are aware of their infection status prior to HCC diagnosis . Most ofOur results highlight the need to address the problem of hepatitis B within sub-Saharan Africa. The majority of childhood-acquired HBV infections become chronic, and CHB is the greatest risk factor for HCC in sub-Saharan Africa. HBV birth dose vaccination is the cornerstone to the HBV prevention strategy and should be available to all infants, particularly those born in high prevalence countries. The South African Ministry of Health takes the position that it is unnecessary to screen HIV-infected individuals for HBV because the current first line ART that includes tenofovir and lamivudine is active against both HIV and HBV. However, the hypothesis that ART is sufficient to prevent the evolution of HCC in HIV-infected patients needs further study. In order to identify those most at risk of HCC, there is a need to screen HIV-infected individuals for HBV, to treat those who test positive for HBV, and to vaccinate those who are not immune. In addition, better screening and management strategies for HCC should be developed for sub-Saharan African populations, where young people, especially those infected with HIV are at risk of this rapidly fatal malignancy.


Chronic lung disease of prematurity (CLD), also called bronchopulmonary dysplasia, is a major consequence of preterm birth, but the role of the microbiome in its development remains unclear. Therefore, we assessed the progression of the bacterial community in ventilated preterm infants over time in the upper and lower airways, and assessed the gut–lung axis by comparing bacterial communities in the upper and lower airways with stool findings. Finally, we assessed whether the bacterial communities were associated with lung inflammation to suggest dysbiosis.We serially sampled multiple anatomical sites including the upper airway , lower airways  and the gut (stool) of ventilated preterm-born infants. Bacterial DNA load was measured in all samples and sequenced using the V3–V4 region of the 16S rRNA gene.From 1102  samples from 55 preterm infants, 352 (32%) amplified suitably for 16S RNA gene sequencing. Bacterial load was low at birth and quickly increased with time, but was associated with predominant operational taxonomic units (OTUs) in all sample types. There was dissimilarity in bacterial communities between the upper and lower airways and the gut, with a separate dysbiotic inflammatory process occurring in the lower airways of infants. Individual OTUs were associated with increased inflammatory markers.Ureaplasma spp., may decrease the development of CLD in preterm-born infants.Taken together, these findings suggest that targeted treatment of the predominant organisms, including those not routinely treated, such as  Respiratory colonisation was acquired after birth and associated with a pro-inflammatory response, suggesting an infectious process was present in babies at risk of developing chronic lung disease of prematurity (CLD), thus providing a target to reduce CLDhttp://bit.ly/31C27iX The six studies included [e.g. one reported progression from Proteobacteria to Firmicutes, but another showed reverse progression. To provide a more comprehensive assessment of the bacterial community in preterm infants who are at risk of developing CLD, we 1) serially studied multiple anatomical sites, including obtaining tracheal aspirate fluid (TAF) and bronchoalveolar lavage (BAL) fluid from ventilated preterm infants to assess the tracheal and lower airways, respectively; and nasopharyngeal aspirates (NPA) and stool samples to assess the progression of the bacterial community in ventilated preterm infants over time in the upper and lower airways; 2) assessed the microbial relationship between the stool and upper/lower airways, i.e. the gut–lung axis, for which there are no studies thus far in term or preterm-born infants; 3) assessed the relationship between the bacterial communities and clinical factors including mode of delivery, sex, clinical site of sampling and effect of antibiotics; and 4) measured proinflammatory cytokines interleukin (IL)-6 and -8 in all sample types and related the findings to the bacterial communities observed.Chronic lung disease of prematurity (CLD), also called bronchopulmonary dysplasia, is a major consequence of preterm birth being influenced by many early life factors, including delivery at an early stage of lung development, need for respiratory support and supplemental oxygen therapy . We have pathway , 3. Furts of CLD , 5, withs of CLD . In the s of CLD  or possis of CLD . A recens of CLD  investigincluded –15 showesupplementary material. TAF, BAL and NPA samples were collected daily during the first week then twice weekly or until extubation, whichever occurred earlier. Stool samples were collected weekly until 28 days of life. Ethical approval (ref 14/WA/0190) was obtained from the Wales Research Ethics Committee 2 and written informed consent was obtained from the parents.Preterm infants of ≤32 weeks' gestation were recruited from two tertiary regional neonatal intensive care units in the United Kingdom. TAF and NPA were collected at the time of routine endotracheal tube (ETT) suctioning. BAL samples were collected as per our previous studies  and accosupplementary material. Total bacterial load was quantified using a quantitative PCR assay based on published protocols [DNA extraction, bacterial load and 16S sequencing are described in more detail in the rotocols  amplifyirotocols .www.r-project.org) using Phyloseq v1.20.0 [https://cran.r-project.org/web/packages/vegan/index.html). STAMP v2.13 [Mothur v1.39.5  was used v1.20.0  and VegaMP v2.13  was usedversus 25.9 weeks, p=0.07). More patent ductus arteriosus were identified at NBT, since they screen for this condition. As expected with recruitment of only ventilated preterm infants, most infants developed CLD defined using the National Institute of Child Health and Human Development definitions [1102 samples were collected from 55 preterm infants born at ≤32 weeks' gestation  during the first 28 days of age including 539 NPA, 276 TAF, 89 BAL and 198 stool samples. The demographics are shown in −1 for TAF and NPA, 0.1 ng·µL−1 for BAL and 0.5 ng·µL−1 for stool). Bacterial load peaked at day 4–7 for BAL and TAF samples and at days 8–14 for NPA and stool samples. Interestingly, a similar pattern of increase was observed for the TAF and BAL samples as for the stool and NPA samples (352 (32%) of the 1102 samples were successfully sequenced for the 16S rRNA gene with the greatest success rate for stool (73%) samples and least for TAF (17%) samples . Bacteri samples .After low-quality reads and chimeric sequences were removed, the mean (range) reads were 109 457 (8665–426 696) for TAF samples; 44 548 (27–177 875) for BAL samples; 60 295 (5951–222 539) for NPA samples; and 59 329 (1–481 757) for stool samples, with 0, 2, 0 and 39 samples removed, respectively, for reads of <1000 per sample. The maximum reads for negative controls were 7558, 588, 580 and 206, respectively, with one TAF sample discarded due to co-clustering with a negative control sample.supplementary figure S1, were Proteobacteria and Firmicutes; Tenericutes were also present, particularly in TAF and BAL samples, and to a lesser extent in NPA samples; Bacteroidetes were noted in stool samples, with lesser presence in NPA, TAF and BAL samples. The pattern of phyla in TAF and BAL samples over the first month of life remained static, with a mean relative abundance ∼40–50% of Proteobacteria, while Proteobacteria in NPA and stool samples increased from a mean relative abundance of 15% in NPA and 30% in stool in the first week of life to 46% and 55%, respectively, by the third/fourth week life (supplementary figure S2). Most samples contained a single dominant phylum (>50% of reads from a single phylum). At genus level, Staphylococcus, as shown in Klebsiella, Escherichia, unclassified Enterobacteriaceae, Enterococcus and Serratia predominating in 35%, and the Tenericutes Ureaplasma and Mycoplasma for 5.2% of all samples. While Proteobacteria increased and Firmicutes decreased with age, especially in NPA  and stool samples, there was no obvious pattern of change at genus level, although dominance of the aforementioned genera was evident. Both at genus and OTU level, a dominant genus/OTU was present in most samples, with 31 (91%) out of 34 BAL samples, 43 (92%) out of 47 TAF samples, 122 (97%) out of 126 NPA samples and 130 (90%) out of 145 stool samples having >50% of their reads for a single dominant OTU, as shown in The majority of phyla in all samples, as shown in supplementary figure S4).Interestingly, each infant had their own individual bacterial colonisation pattern which differed between individual infants and between anatomical sites  and c. Sversus TAF; BAL versus NPA; BAL versus stool; TAF versus NPA; TAF versus stool; and NPA versus stool .When bacterial communities were examined between the four sample types by PERMANOVA, all differed significantly from each other, as shown in Acinetobacter (p=0.04) and marginally with Pseudomonas (p=0.06)); Mycoplasma (p=0.04) in TAF samples; and Serratia (p=0.01) in NPA from infants delivered vaginally. For infants delivered by caesarean section, Staphylococcus were increased in NPA (p=0.02), and marginally in TAF samples (p=0.056) (supplementary figure S2a). However, sex did not influence TAF or NPA bacterial communities (supplementary figure S2b). A significant difference for bacterial community structure was noted between the recruitment centres (p<0.05) (supplementary figure S2c). This may be partially explained by the outbreak of Acinetobacter at one site, resulting in greater Acinetobacter observed at that recruitment centre.Delivery mode influences early colonisation across many anatomical sites in term newborn infants and in the limited data for preterm infants in stool samples , 25, 26.supplementary figure S3). Samples taken when not on antibiotics were significantly more likely to be successfully sequenced for the 16S rRNA gene than samples taken while on antibiotics . Antibiotics did not affect α-diversity in successfully sequenced TAF and NPA samples, suggesting that they globally suppressed bacterial growth rather than suppressing specific species. Nevertheless, antibiotics were associated with increased proportion of Tenericutes (p=0.03), probably due to Mycoplasma, which were marginally increased (p=0.059) in TAF samples compared to the no-antibiotic group.At both recruiting centres the initial prophylactic antibiotic regimen of benzylpenicillin and gentamicin was altered according to subsequent microbiological assessments. 219 (79%) out of 276 TAF and 335 (62%) out of 539 NPA samples were taken while the infants were receiving antibiotic therapy (versus unsuccessful sequencing samples: IL-6 1222 (534–3224) pg·mL−1versus 539 (217–1377) pg·mL−1; and IL-8 26 383 (3460–51 740) pg·mL−1versus 9031 (2535–20 511) pg·mL−1; and BAL IL-6 3629 (2029–8538) pg·mL−1versus 1213 (485–2459) pg·mL−1; and IL-8 80 689 (12 303–196 477) pg·mL−1versus 9520 (3060–18 572) pg·mL−1 . Next, we tracked the dynamic changes in individual infants, as shown in −1 observed in 19 infants, 13 (65%) episodes were associated with presence of bacterial DNA within 24 h of the peak cytokine concentration. Both IL-6 and IL-8 showed peak concentration at 7 days of age as we have shown previously in infants developing CLD [Acinetobacter, unclassified Enterobacteriaceae and Mollicutes including Mycoplasma and Ureaplasma were strongly associated with the greatest increases in IL-6 and IL-8 in BAL, being several-fold greater than in samples which could not be sequenced (Since a single OTU predominated (>50% of reads) in most of the successfully sequenced samples, potentially those samples with greater initial biomass, we measured IL-6 and IL-8 in all samples to evaluate whether successful bacterial DNA detection in samples was associated with an inflammatory response. IL-6 and IL-8 were largely undetectable in the NPA and faecal fluid extracts, but were noted in considerable quantities in the BAL and TAF samples if they could be sequenced, as shown in -6 1222 54–3224 pgping CLD   were satisfactorily amplified for bacterial sequencing. Bacterial load was low in all samples during the first 3 days of life, but peaked at days 4–7 of life in BAL and TAF samples, and at days 8–14 in stool and NPA samples. Bacterial acquisition is most likely to be from the mother with vaginally associated microbes including Tenericutes predominating in vaginally delivered infants, and Ureaplasma and Mycoplasma were mainly confined to the lower airway samples with only some presence in NPA samples, but were almost absent from stool samples. In contrast, Acinetobacter was present in stool and in NPA samples, but not in lower airway samples, reflecting the outbreak in that unit. Almost all samples had a predominant OTU, with >90% of samples having a predominant OTU of >50% of reads with Staphylococcus, Mycoplasma and Ureaplasma predominating especially in the lower airways. Interestingly, all infants had their own bacterial pattern with little overlap between the sample types, suggesting an active infective episodic process. Significant differences were present in the community structure of the samples from each anatomical site as was shown by the PERMANOVA analysis. α-diversity indices estimating richness showed greater number of genera present in BAL and TAF than in NPA or stool samples, but the differences disappeared at OTU level suggesting greater richness at OTU level for NPA and stool samples. Since measures of evenness (Shannon diversity index) did not show a difference between the sample sites, the data suggest that the degree of dominance of a single OTU was no different between anatomical sites. Differences between the NPA and BAL/TAF samples may be partly explained by the presence of an uncuffed endotracheal tube which partially separates the upper and lower airways. Obtaining lower airways samples from nonventilated preterm or term newborn infants is not possible, thus limited data exist for the microbial evolution of the lower airways [At phylum level, the NPA and stool samples changed similarly over time with an increase in Proteobacteria, which remained relatively static in the BAL/TAF samples. The Tenericutes,  airways .Staphylococcus in NPA (p=0.022) and marginally in TAF (p=0.056) in those who were delivered by caesarean section and increased Mycoplasma (p=0.039) and Acinetobacter (p=0.042) in TAF samples and Serratia (p=0.008), Ureaplasma (p=0.024) and Corynebacterium (p=0.028) in NPA samples from vaginally delivered infants. Sex did not affect the bacterial colonisation, but the centre at which the sample was obtained did influence the results. These results may be partly explained by the outbreak of Acinetobacter in one unit, but the differences between the two centres remained in sensitivity analyses when the infants colonised with Acinetobacter were removed, thus suggesting that the local environment or local personnel appear to influence colonisation in addition to the maternal milieu. Antibiotics, as expected, decreased bacterial load, with fewer samples being suitable for bacterial gene sequencing. However, the diversity and number of OTUs were similar when the babies were on or off antibiotics. Presence of Tenericutes was increased in TAF samples (p=0.03) and a trend towards Tenericutes increased in NPA samples (p=0.059) in the antibiotic group.As previously observed for term-born infants, mode of delivery was associated with increased proportion of Most interestingly, we noted several-fold differences in IL-6 and IL-8 concentrations in BAL and TAF when bacterial sequencing was possible against when it was not. Our data suggest that an infective dysbiotic environment is occurring in the lungs of babies who are at risk of developing CLD. Low biomass samples may have lower cytokine concentrations due to dilution caused by variation in volume of samples returned. However, as per our previous observations, the collated data for IL-6 and IL-8 showed a peak concentration of both cytokines at ∼7 days of age , 3, 16. Taken together, these data suggest that both the bacterial community and pulmonary inflammation are potential targets with antimicrobials such as azithromycin, which has both anti-infective and anti-inflammatory activities to decrease rates of CLD .There are several limitations to this study, most notably the presence of an endotracheal tube to support the infant's respiratory system. Most importantly, this precludes the acquisition of samples from “healthy” preterm- and term-born infants which is not ethically possible. The presence of an endotracheal tube may physically separate the lower and upper airways, but, despite prolonged ventilation in many infants, we noted separate processes in the upper and lower airways and in the gastrointestinal tract. The strength of the study is the large number of samples from multiple anatomical sites obtained longitudinally, making this by far the largest study of preterm infants. Furthermore, we have related the findings between recruitment centres and with the inflammatory processes occurring in the lungs of infants at risk of developing CLD.Ureaplasma and Mycoplasma may predominate and potentially provide a therapeutic target to decrease the development of CLD in preterm-born infants [In summary, bacterial load was low at birth, but quickly increases and microbes are most likely to be acquired from the mother. There was dissimilarity between the upper and lower airways and the gut in bacterial community structures with a separate dysbiotic inflammatory process occurring in the lower airways of infants at increased risk of developing CLD. Additionally, the pulmonary inflammatory processes were occurring despite the infants being on antibiotics suggesting that microbes not susceptible to routinely used antibiotics such as  infants .10.1183/13993003.01909-2019.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.ERJ-01909-2019.SUPPLEMENTSupplementary material 10.1183/13993003.01909-2019.Shareable1This one-page PDF can be shared freely online.ERJ-01909-2019.ShareableShareable PDF


Objective. To assess the long-term test–retest reliability of measures of muscle power and lower body functional performance in healthy, nonexercising, older women. Methods. Data were derived from a nonexercising control group  years; height = 159.6 (7.7) cm; body mass = 69.5 (12.7) kg; BMI = 27.3 (4.8)) of a randomized controlled trial of muscle power training in older women. Participants underwent lower extremity muscle power (Biodex) and functional testing  at week 0 (baseline), 9, and 15. Results. For the upper leg, intraclass correlation coefficients (ICCs) were very high for knee extension power (0.90–0.97) and high to very high for knee flexion power (0.83–0.96). For lower-leg power, ICCs were high to very high for plantar flexion and dorsiflexion (0.83–0.96). ICCs for functional performance were moderate to very high (0.64–0.93). Coefficient of variation of the typical error (CVTE) was <10.5% for knee extension/flexion power, 9.9–20.0% for plantar flexion/dorsiflexion power, and 1.9–14.9% for functional performance. Knee extension power and stair climb power demonstrated the highest reliability for muscle power and function, respectively. Mean values did not change over time, with the exception of the chair stands (p < 0.05); however, these changes were not considered clinically meaningful. Conclusions. The current study provides evidence supporting the long-term reliability of performance assessments of muscle power and lower body functional capacity over a period of up to 15 weeks in healthy, older women.Evaluation of the long-term reliability of muscle power and functional performance tests in older, healthy adults is warranted since determining whether performance is consistent over longer durations is more relevant for intervention studies.  Functional status is associated with hospitalization, health decline, and death in older adults –3. SinceSince resistance training is one of the primary recommended treatments for attenuating age-related functional decline  and imprData for this study come from the nonexercising control group of a randomized controlled trial . Older women were recruited in the Greater Toronto Area through flyers, e-mail lists, and electronic newsletters. Inclusion criteria consisted of women, ≥ 65 years old, untrained (< 1 hour/week of structured moderate/high intensity exercise and not currently participating in resistance training), and who provided medical clearance from their physician to participate in exercise. Exclusion criteria consisted of osteoporosis, diabetes, uncontrolled hypertension, or a new/unstable condition diagnosed in the last six months. The University of Toronto's Research Ethics Board approved the study (Protocol #27773) which complies with the Declaration of Helsinki. The reliability analysis included 18 women (mean age = 73.3 (3.4) years, mean height = 159.6 (7.7) cm, mean body mass = 69.5 (12.7) kg, and mean BMI = 27.3 (4.8)).https://wnarifin.github.io/ssc/ssicc.html) and methods from Walter et al.  and the 400 MW  were significant.For balance, almost all participants received maximal scores (4/4) at all time points (data not shown). There were no significant differences in the means of the functional performance tests across time points (iability . Stair cThe main findings include the following: (1) isotonic muscle power demonstrated high to very high reliability over a nine- and fifteen-week duration with knee extension power being the most stable measure; (2) all of the functional tests studied demonstrated reliability that ranged from moderate to high, with stair climb power exhibiting the highest reliability over durations that are commonly used in training studies for older adults ; (3) staTE of 14 and 12%, respectively. The current study did not determine one-week test-retest results; however, in the shorter test-retest period (nine weeks), PF power ICC was 0.96 , and DF power ICC was 0.92 . Therefore, the very high test-retest reliability of lower-leg isotonic power previously demonstrated over one week is maintained over nine weeks in a healthy older population.The current study utilized the isotonic mode of the Biodex dynamometer to evaluate muscle power and hence used a less common velocity-dependent power measure where movement velocity is measured and the load used can be quantified . The curTE were <15% over 15 weeks. In a 2013 systematic review of the properties of physical performance measures in community-dwelling older adults, the SPPB was recommended for use since it was found to have good intrarater reliability  and validity [TE  and the 30 sCS (increase in chair stands: 1.6 reps). These average changes are less than the estimated minimally important changes found in the literature for clinical populations, which were 1.7 seconds for the 5 CS  and 3.3 The inter/intrarater analyses produced ICCs that were high to extremely high, supporting the use of video-recorded functional performance assessment in older adults. Our findings are in agreement with other studies in clinical populations that have investigated the reliability of assessing functional performance remotely using video technology versus face-to-face testing , 35. ForThe current study's participants represent high-functioning, community-dwelling older women (baseline SPPB median score = 12 units), and interpretation of results might not apply to clinical populations or frail older women, which is a limitation. In addition, the change in the mean for the chair stand tests (5 CS and 30 sCS) does impact the interpretation of the stability of these tests over 15 weeks in healthy, older women. The increase in chair stand performance, while not clinically meaningful, does reduce the long-term reliability of this measure and suggests a potential learning effect.Lower extremity muscle power and functional tests were found to be reliable in older, healthy women over 15 weeks, given a standardized testing environment and administration, minimal familiarization in study design, and multiple raters. These measures were stable over time and can be used to detect changes in response to interventions in older, healthy women. Future research should examine the long-term reliability of functional test performance in healthy, older men, as well as clinical populations.


Acute pyelonephritis (APN) with obstructive uropathy often causes sepsis. Recently, sepsis was redefined using the sequential organ failure assessment (SOFA) score, based on the new Sepsis-3 criteria. We investigated predictors for sepsis using this new definition in patients with obstructive APN associated with upper urinary tract calculi.We retrospectively evaluated patients who were admitted to our hospital for treatment of obstructive APN associated with upper urinary tract calculi. Blood and urine samples were collected before treatment of obstructive APN. Treatment included adequate antimicrobial therapy and emergency drainage to decompress the renal collecting system. We diagnosed sepsis using the new Sepsis-3 definition. We assessed predictors for sepsis by multivariate logistic regression analysis.p = 0.001 and p = 0.016, respectively). Procalcitonin (PCT) and presepsin (PSEP) levels were significantly higher in the sepsis group than in the non-sepsis group . Multivariate analysis showed that PCT elevation  and PSEP elevation  were independent predictors for sepsis.Sixty-one patients were included in this study. Overall, all patients underwent emergency drainage, and 11 (18.0%) patients showed sepsis. There were no significant differences in performance status or comorbidities between sepsis and non-sepsis groups. Platelet count and serum albumin level were significantly lower in the sepsis group than in the non-sepsis group (Elevation of PCT and PSEP levels before treatment might predict the development of sepsis in patients with obstructive APN. Sepsis is a major cause of morbidity and mortality among hospitalized patients, and its reported incidence is increasing . Until rUrosepsis is commonly caused by obstructive disease of the urinary tract, such as urinary stones, stenosis, and tumor, and affects 20–30% of all septic patients . A Japan2) at baseline were also excluded from the final cohort to minimize the impact of kidney function on PCT and/or PSEP values. The severity of acute kidney injury (AKI) was graded according to the AKI network (AKIN) criteria [This retrospective study was based on a medical chart review of patients who were admitted to our institution for the treatment of obstructive APN with upper urinary tract calculi between September 2015 and February 2018. The definition of APN and the regimen of antimicrobial treatment that we used were described in our previous report [criteria . MidstreSepsis was defined based on the Sepsis-3 criteria. Briefly, patients with suspected infection were evaluated using the quick SOFA (qSOFA) . Patients with positive for qSOFA and ≥ 2 SOFA score points were diagnosed with sepsis . Performp < 0.05 were considered to be statistically significant. Statistical analyses were conducted using SPSS software .The variables were compared among groups using the Mann–Whitney U-test and Kruskal-Wallis test. The independence of categorical data was estimated by the chi-squared test or Fisher’s exact test. Continuous variables  level, platelet count, serum albumin level, PCT level, and PSEP level) were used to divide the patients into two groups by receiver operating characteristic curve analysis. Independent predictors of sepsis were determined using logistic regression analysis. Differences with This study included 61 patients. Median patient age was 64 years, 24 patients were male and 37 patients were female. In total, five patients (8.2%) exhibited poor performance status (2–4). Of the 61 patients, there were 16, eight, six, three, and two with diabetes mellitus, cardiovascular diseases, neurologic diseases, immunocompromised status, and urinary tract abnormalities, respectively. Median serum creatinine was 1.22 mg/dL. Overall, 41 patients (67.2%) showed a large shift in leukocyte count . Twenty and 19 patients exhibited reduced platelet count  and reduced serum albumin level (< 3.1 g/dL), respectively. Median PCT and PSEP levels were 1.49 ng/mL and 445 pg/mL, respectively. PCT and PSEP levels increased significantly with the severity of AKI (Table Most stones were located in the ureter (90.2%). The median longest and shortest diameters of stones were 8.0 mm and 5.0 mm, respectively. Overall, 22 patients (36.1%) had high-grade hydronephrosis. Fifty-six (91.8%) and five patients (8.2%) underwent indwelling ureteral stenting and nephrostomy, respectively, to achieve decompression of the collecting system. The median duration from initial treatment to decompression of the collecting system was 1.0 days.p = 0.209). Performance status and underlying diseases did not significantly differ between sepsis and non-sepsis groups. With respect to laboratory data before treatment, platelet count and serum albumin level were significantly lower in the sepsis group than in the non-sepsis group . However, inflammatory markers (leukocyte count and CRP level) and serum creatinine did not significantly differ between sepsis and non-sepsis groups . PCT and PSEP levels were significantly higher in the sepsis group than in the non-sepsis group . There were no significant differences in stone characteristics between the sepsis and non-sepsis groups  were positive for sepsis, using the Sepsis-3 criteria  [The present study showed that elevated PCT and PSEP levels before treatment might be independent predictors for sepsis, based on the Sepsis-3 criteria, in patients with obstructive APN. Obstructive APN can be lethal, and a recent survey revealed a mortality rate of 2.3% . Based o< 0.001) . The limA recent meta-analysis showed that PCT and PSEP levels had moderate diagnostic value for sepsis . HoweverPCT is a prohormone of calcitonin; under normal conditions, it is only produced in C-cells of the thyroid gland. During infection, PCT can be produced by several cell types and many organs in response to pro-inflammatory cytokines . Because it shows an early increase during infection, PCT is a useful biomarker for infection . PSEP is2) were excluded from analysis. PSEP levels inversely correlate with GFR, and PSEP levels in patients with GFR < 30 mL/min/1.73 m2 were reportedly significantly higher than those in patients with GFR ≥ 30 mL/min/1.73 m2 [2 demonstrated PSEP levels of 1150 pg/mL, 889 pg/mL, and 3620 pg/mL, respectively; all were negative for sepsis. Second, levels of PCT and PSEP could be influenced by acute kidney injury (AKI). Recently, a retrospective study in patients with AKI showed that different thresholds of PSEP and PCT levels might be useful markers of bacterial infections [There were several limitations in this study. First, patients with severe chronic kidney disease (GFR < 30 mL/min/1.73 m/1.73 m2 . The evafections . HoweverElevated PCT and PSEP levels before treatment might predict the development of sepsis in patients with obstructive APN.


Autonomous cortisol secretion (ACS) is a condition with ACTH-independent cortisol overproduction from adrenal incidentalomas (AI) or adrenal hyperplasia. The hypercortisolism is often mild, and most patients lack typical clinical features of overt Cushing’s syndrome (CS). ACS is not well defined and diagnostic tests lack validation.Retrospective study of 165 patients with AI evaluated clinically and by assay of morning plasma ACTH, late-night saliva cortisol, serum DHEA sulphate (DHEAS), 24-h urine-free cortisol, and cortisol after dexamethasone suppression.n = 165) were diagnosed as non-functioning incidentalomas (NFI) (n = 82) or ACS (n = 83) according to current European guidelines. Late-night saliva cortisol discriminated poorly between NFI and ACS, showing a high rate of false-positive (23/63) and false-negative (38/69) results. The conventional low-dose dexamethasone suppression test (LDDST) did not improve the diagnostic specificity, compared with the 1 mg overnight DST. Receiver operating characteristic curve analysis of DHEAS in the two cohorts demonstrated an area under the curve of 0.76 (P < 0.01) with a sensitivity for ACS of 58% and a specificity of 80% using the recommended cutoff at 1.04 µmol/L (40 µg/dL).Patients with AI (We here demonstrate in a large retrospective cohort of incidentaloma patients, that neither DHEAS, late-night saliva cortisol nor 24-h urine free cortisol are useful to discriminate between non-functioning adrenal incidentalomas and ACS. The conventional LDDST do not add further information compared with the 1 mg overnight DST. Alternative biomarkers are needed to improve the diagnostic workup of ACS. Autonomous cortisol secretion (ACS) is usually caused by adrenal adenomas or hyperplasia and is frequently diagnosed in patients with adrenal incidentalomas (AI). AI are adrenal masses discovered on imaging undertaken for other reasons than suspicion of adrenal disease and are found in about 3% of people at 50 years of age, with the prevalence increasing to up to 10% in elderly above 70 years of age .Due to large inter- and intraindividual biological variation of cortisol secretion, most patients with ACS shows serum cortisol levels in the normal range; however, the diurnal and ultradian variation of cortisol secretion is dysregulated. Also, these patients lack the classical phenotype of overt Cushing syndrome (CS). Formerly known as subclinical CS, ACS has a much higher prevalence than overt CS, affecting up to 2% of the adult population . DespiteThe most recent international guidelines recommend the 1 mg overnight dexamethasone suppression test (DST) to screen for ACS . Plasma Saliva cortisol (sa-cortisol) drawn at late night is commonly used in the diagnostics of overt CS, but is not found useful to reveal low graded cortisol overproduction. Sa-cortisol and the 48-h conventional low-dose (0.5 mg four times daily) dexamethasone suppression test (LDDST) are not well validated in the setting of ACS, and none of them are recommended in the ESE/ENS@T guidelines . Twenty-How the various diagnostic tests perform in the diagnosis of AI have been evaluated mostly in small insufficiently powered studies. Here, we checked the performance of these tests in an adequately sized patient cohort in a ‘real-life’ clinical setting.n = 3), primary aldosteronism (n = 1) and adrenocortical carcinomas (n = 1) were excluded. The study cohort consisted of the remaining patients (n = 165), and categorised as having either non-functioning incidentalomas  or ACS (n = 83). The ESE guidelines recognise three categories of patients based on post DST cortisol levels. S-cortisol below 50 nmol/l is named NFI, between 50 and 139 nmol/L is named possible ACS and 140 nmol/L or above is ACS. We have defined ACS here as a s-cortisol value above 50 nmol/L after the 1 mg overnight DST (positive test). This because we have excluded the most common reasons for a false DST, namely patients with low dexamethasone bioavailability < 3.3 nmol/L (n = 5)  curve was made for DHEAS and sa-cortisol to evaluate the diagnostic performance, and for DHEAS also to evaluate the proposed cutoff of 1.04 µmol/L (40 µg/dL) in the diagnostics of ACS  and continuous data as median (range). Since the data was not normally distributed, non-parametric statistics were applied. A significance level of 0.05% was chosen. The Mann–Whitney s of ACS .The study was approved by the local ethics committee (REK) and all participants signed an informed consent form (REK no: 2011/1810 and 2014/2170).One hundred and sixty-five patients were included in the study; 82 had NFI, and 83 had ACS . OverallP < 0.05). The median s-cortisol after DST was 84 nmol/L (range 51–486) in the ACS group, and 31 nmol/L (range 12–50) in the NFI group.The results of the diagnostic tests performed in the two groups of patients are summarised in P < 0.05) even though both medians were below the cutoff for a normal test (2.8 nmol/L). Thirty-eight of sixty-nine patients in the ACS group and 40/63 in the NFI group had normal saliva samples. However, in patients who delivered two saliva samples, 16/55 in the ACS group and 4/51 with NFI had one positive and one negative measurement (P < 0.05), and there were not possible to derive a better cutoff level than 2.8 nmol/L, suitable to differentiate between ACS and NFI.In the ACS group, we had valid saliva data for 69/83 patients, 14/69 patients had one saliva sample performed and 55/69 patients had two. In the NFI group, 63/82 patients had valid saliva data, one saliva sample was obtained in 12/63 patients and 51/63 patients delivered two samples. The median sa-cortisol level was higher in the ACS than in the NFI group (1.75 nmol/L (0.4–45) vs 0.9 nmol/L (0.1–8.2); surement . In the surement . There wP < 0.001) in the NFI group. A proposed cutoff for DHEAS in the diagnostics of ACS is 1.04 µmol/L (40 µg/dL), using the exact same method as our laboratory (P < 0.05). Applied to our cohort, the cutoff of 1.04 µmol/L (40 µg/dL) yields a sensitivity of 58% and a specificity 80% for diagnosing ACS. In the ACS group, we had valid DHEAS data for 58/83 patients, and in the NFI group 54/82 patients. The median value of DHEAS was 0.95 µmol/L (range <0.4–3.8 µmol/L) in the ACS group, compared to 1.85 µmol/L . Calculating a ROC curve for the use of DHEAS-ratio in the diagnostics of ACS gave an AUC of 0.69 .Dennedy et al. have suggested to calculate a ratio derived by dividing DHEAS by the lower limit of the sex- and age-specific reference range for the analysis. We used the recommended lower limits shown in P = 0.12) or reviewed as two separate groups P < 0.001).The median level of s-cortisol after the LDDST in the ACS group was 89 nmol/L 51–517, . A scattn = 49/58) or twice (n = 29/58) in the cohort of ACS. Seven patients had one or more positive test results. Two patients tested positive in both samplings, while four tested positive in one sample. One delivered a single positive urine. The two patients with two positive UFC were from the category of patients with post-DST-cortisol above 140 nmol/L.UFC was assayed once  or mortality data after surgery. Furthermore, many patients with ACS are quite old, an age group expected to have physiologically low DHEAS levels.DHEAS as a sulphate conjugate is excreted through the kidneys, and higher levels may be expected in patients with kidney failure. We found no difference between s-creatinine in the two groups, and there were no correlation between s-creatinine and DHEAS in the whole cohort, or when each patients group were reviewed separately. Thus, it is unlikely that our findings are confounded by kidney function.The activity of enzymes involved in the sulphation of DHEA to DHEAS may vary between individuals and increase variability; thus DHEA could perhaps be a substitute. However, assay of DHEA with its shorter half-life and circadian variation is not straight forward.Furthermore, the manufacturer does not state any known interference issue and we are not aware of any such reports, other than heterophilic autoantibodies, concerning the Immulite 2000 DHEAS analysis. This may be expected because DHEAS are present in blood at levels orders of magnitude higher than other steroid hormones.et al. found that the circadian cortisol rhythm was not impaired using serial sa-cortisol samples and calculating AUCs. The cortisol exposure was only increased in the morning in patients with ACS  in the diagnosis of overt CS . Howeverwith ACS . This coThe LDDST has mainly been used in a further assessment of patients with positive overnight DST, when there are discrepancies between the overnight DST and other diagnostic tests. After excluding patients with low dexamethasone bioavailability, we found a very strong correlation between the overnight DST and LDDST. The LDDST was not able to exclude ACS in any patients diagnosed by DST. Thus, we found no support for the continued use of LDDST in the diagnostics of ACS.The UFC-test has been considered the gold standard in the diagnostics of overt CS since the 1970s , but havDespite the fact that we here report findings in a sizeable cohort of subjects, the retrospective nature of the study is an important limitation. Furthermore, a significant amount of missing data for both DHEAS, sa-cortisol and UFC and p-ACTH is an important shortcoming. Also, two salivary cortisol samples were only available for 55/83 ACS and 51/82 NFI patients – which given the well-recognised limitations of a single salivary cortisol measurements, is a weakness worth mentioning.In conclusion, we recognise that the conventional approach using spot measurements, dynamic testing and daily hormone excretion are far from suitable to accurately diagnose ACS. DHEAS was not found to be an adequate replacement for p-ACTH in the diagnostics of ACS. Late-night sa-cortisol was not useful to distinguish between ACS and NFI, and the LDDST did not increase the diagnostic specificity compared to the 1 mg overnight DST. The UFC had insufficient sensitivity for detecting patients with ACS. Thus, for the time being, morning ACTH and the 1 mg overnight DST are the tests of choice.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This work was supported by a grant from Stiftelsen Kristina Gerhard Jebsen and The regional Health Authorities of Western Norway.


Bone scintigraphy (BS) of disseminated skeletal metastasis is sometimes misinterpreted as normal. The use of computer-assisted diagnosis (CAD) may resolve this problem. We investigated the performance of a CAD system, BONENAVI, in the diagnosis of disseminated skeletal metastasis.Cases of disseminated skeletal metastasis were selected from a BS log. These patients’ BSs were analyzed by BONENAVI to obtain an artificial neural network (ANN) and bone scan index (BSI). Clinical features  were compared with the BONENAVI (ANN and BSI) results. The BS findings  and ANN or BSI were evaluated. Then, negative ANN patients were presented.Fifty-four patients were diagnosed as having disseminated skeletal metastasis. Regarding the primary cancers, 12 had prostate cancer, 16 gastric cancers, 16 breast cancers, and 10 miscellaneous cancers. Total sensitivity of ANN (≥ 0.5) was 76% (41/54). ANN values correlated with the BS type among clinical features. Diffuse increased axial skeleton uptake was mostly correlated with ANN of the BS findings.The BONENAVI CAD system was partially helpful in diagnosing disseminated skeletal metastasis, but the sensitivity of BONENAVI was not sufficient and underestimated the disseminated skeletal metastasis. Further improvement of this CAD system is necessary to improve the detectability of disseminated skeletal metastasis. The diagnosis of disseminated skeletal metastasis by bone scintigraphy (BS) is usually easily established when typical findings are present. However, the BS pattern is easily misinterpreted as normal unless there is an absence of renal uptake, or the presence of diminished activity in the bones of the appendicular skeleton, and when a high ratio of bone to soft tissue activity is recognized , 2.Until recently, BS was interpreted visually. However, considering the need for an appropriate quantitative approach for BS, computer-assisted diagnostic (CAD) software for BS was developed and evaluated –5. A JapOur clinical questions are how the BONENAVI performed in patients with disseminated skeletal metastasis, and what factors, if any, caused the failures  in the diagnosis of the skeletal metastasis by BONENAVI in disseminated skeletal metastasis patients.Therefore, we tried to clarify the sensitivity of BONENAVI II in patients with disseminated skeletal metastasis. We also tried to determine the clinical features or BS features that cause mismatches in the skeletal involvement and negative CAD analysis.We selected patients with disseminated skeletal metastasis from the BS log from January 2013 to August 2019 at the cancer institute hospital, Tokyo, Japan. From the log, 63 patients were listed. These patients were diagnosed on BS to have definite, suspected or not-ruled-out disseminated skeletal metastasis. We reviewed the BS and other clinical data, including computed tomography (CT), magnetic resonance imaging (MRI) and/or fluoro-deoxy glucose positron emission tomography with CT (FDG-PET/CT). Fifty-four patients were confirmed to have disseminated skeletal metastasis; that is, skeletal metastatic lesions had spread to nearly the entire axial skeleton, equivalent to the extent of the disease (EOD) = IV . These 5The diagnosis of disseminated skeletal metastasis was established clinically; that is, no skeletal metastatic lesion was diagnosed histologically. The diagnosis of disseminated skeletal metastasis was established and confirmed using at least two diagnostic modalities .Primary cancer sites were variable: 12 patients had prostate cancer, 16 gastric cancers, 15 breast cancers, and 10 miscellaneous cancers .This retrospective study was approved by the local institutional review board.99mTc-MDP. After obtaining anterior and posterior whole-body scans, local images, the pelvic axial view, were also acquired. Whole-body (anterior and posterior) images were taken using E-CAM (Canon), Infinia (GE) or Intevo (Siemens) using 15–20-cm /min scan speed with a 256 × 1024 matrix size. After taking whole-body images, static studies were conducted using 6–8 million counts/view with a 512 × 512 matrix size.BS was performed about 3 h (range 2–4 h) after injection of 740 MBq of BS findings were reviewed, and the presence of the characteristics of disseminated skeletal metastasis based on each BS clinical report was recorded. BS findings of disseminated skeletal metastasis were characterized by intense uptake throughout the skeleton; faint or absent renal uptake; patchiness (inhomogeneity) of uptake, particularly in the ribs; and the presence of diminished activity in the appendicular skeleton , 2. ThesBased on the above findings, BS visual interpretations were classified into (a) definite BS type: definite super bone scan; diffuse increased axial skeleton RN uptake irrespective of other findings, (b) suspected BS type: suspected disseminated bone metastasis ; weak or no diffuse axial skeleton RN uptake and inhomogeneity of RN uptake, irrespective of other findings, (c) not-ruled-out BS type: not-ruled-out disseminated skeletal metastasis; other than (a) or (b) above; weak or no diffuse axial skeleton uptake, partial or no inhomogeneity, and increased proximal extremity uptake or absent or weak renal uptake.BONENAVI analysis was performed as previously reported . BrieflyCT scans were obtained with a 2-mm thickness at 5-mm intervals.The CT morphological classification of skeletal metastasis included the categories osteoblastic, osteolytic, mixed osteoblastic and osteolytic, and intertrabecular (invisible on CT). This classification was independently performed by two radiologist-nuclear physicians, and discordant cases were decided through discussion. The diagnostic confirmation of disseminated skeletal metastasis was made by the agreement of two physicians using at least two imaging modalities .We observed the relationships among the types of primary cancer, CT types, and BS types.We investigated the relationship between BONENAVI: ANN ≥ 0.5 and the types of primary cancer, CT types or BS types.We analyzed the relationship between BONENAVI  and clinical features; the types of primary cancer, CT types or BS types by one-way analysis of variance (one-way ANOVA).To clarify the BS factors that were related to BONENAVI , the relationship of ANN values and BS findings  was investigated by one-way ANOVA. Using BONENAVI: ANN ≥ 0.9 as a dependent factor, multivariate logistic analysis was also employed to determine which factor was related to a high ANN value. We initially used ANN ≥ 0.5; however, there was no patient whose diffuse axial skeleton uptake was below 0.5 (ANN), and analysis was not properly performed. Therefore, we selected ANN ≥ 0.9 as the dependent factor instead of ANN ≥ 0.5.We also observed the relationship between BSI values and BS findings by one-way ANOVA.We added the list of patients with various factors who showed negative ANN (ANN < 0.5) despite the presence of disseminated skeletal metastasis.p < 0.05 was considered to indicate statistical significance. For one-way ANOVA, Levene’s test for homogeneity of variance was performed first. When the homogeneity of variance was assumed, a t test was followed by Tukey’ test. When the homogeneity was not assumed, Welch’s test was followed by the Games–Howell test. For contingency table analysis, if more than 20% of cell boxes was expected to number < 5, Fisher’s exact test was used. Pearson’s Chi-square test was applied to the others.Statistical analysis software  was used. A value of The multivariate logistic regression analysis was carried out using ANN ≥ 0.9 as a dependent factor both by forward and backward stepwise analysis using a likelihood ratio.Totally 54 patients were diagnosed clinically to have disseminated skeletal metastasis. Primary cancer sites were variable: 12 patients had prostate cancer, 16 gastric cancers, 15 breast cancers, and 10 miscellaneous cancers .Twenty-five CT patterns were osteoblastic type, 19 mixed type, and 10 intertrabecular type. Twenty-eight BS types were of definite type, 16 were of suspected type, and 10 were of not-ruled-out type. The relationships among these factors are summarized in Table Disseminated skeletal metastasis from prostate cancer tended to have a high percentage of CT osteoblastic type (11/12) and BS definite type (10/12).Disseminated skeletal metastasis from gastric cancer showed the next highest ratios as CT osteoblastic type (9/16) and BS definite type (10/16).Breast cancer showed the lowest ratios among the three most frequent cancers as CT osteoblastic type (3/16) and BS definite type (6/16).In all patients, CT osteoblastic  and mixed  types showed similar ratios to the definite BS type, and the CT intertrabecular pattern showed a low ratio to the definite BS type . There were no patients with osteolytic type who showed disseminated skeletal metastasis.Figures Figures The sensitivities of the ANN value (ANN ≥ 0.5) are shown in Table Regarding the types of primary cancer, patients with prostate cancer showed 100% (12/12) sensitivity, those with gastric cancer and breast cancer 75% (12/16) each, and those with miscellaneous cancers 50% (5/10).Regarding CT type, osteoblastic type showed 72% (18/25) sensitivity, mixed type 89% (17/19), and intertrabecular type 60% (6/10). The intertrabecular type could not be found on CT scan (CT negative), and therefore, CT sensitivity was calculated as 81% (44/54).With BS types, definite BS type showed 100% (28/28), suspected BS type 56% (9/16) and not-ruled-out BS type 40% (4/10). Because we recruited the disseminated skeletal metastasis patients based on visual BS results , we could not calculate the sensitivity of the visual BS method.Relation to BONENAVI  and clinical features .To clarify the relationships of primary cancer types, CT types, and BS types to ANN value, one-way ANOVA was performed. The results are shown in Table p = 0.019); however, there were no significant differences among ANN values for each type of cancer.The whole analysis between ANN values and primary cancer types showed a statistically significant difference , and there was a significant difference between definite type and suspected type (p = 0.01), and between definite type and not-ruled-out type (p = 0.028).Statistically significant difference was shown in BS types (p = 0.000) and BS patterns (p = 0.000) were statistically significant; however, CT types (p = 0.247) were not significant. Inter-group sub-analysis showed that there was a statistically significant difference between the BSI of prostate cancer and gastric cancer patients, and breast cancer and miscellaneous cancers patients. BSI values of prostate cancer and gastric cancer patients were not significantly different, and neither was there a significant difference in the BSI values between breast cancer and miscellaneous cancers.About BSI values, kinds of cancer .p = 0.001) and absent kidney uptake (p = 0.000) were significant factors. However, inhomogeneity of uptake (p = 0.45) and proximal extremity relatively increased uptake (p = 0.135) were not significant factors.To investigate how BS findings are related to ANN value, one-way ANOVA was performed with ANN value as a dependent factor and BS findings as independent factors. The results are summarized in Table We further analyzed the relation of ANN and BS findings using a multivariate logistic regression analysis. We initially used ANN ≥ 0.5; however, no patient with diffuse skeleton uptake was below 0.5 (ANN < 0.5), and the analysis was not properly performed. Therefore, we selected ANN ≥ 0.9 as the dependent factor.p = 0.001), as shown in Table This multivariate analysis showed diffuse increased skeleton uptake to be the only significant factor , all BS factors showed statistically significant differences, but diffuse increased skeleton uptake and absent kidney sign showed strong correlations with the BSI values. On sub-group analysis, BSI values of diffuse increased skeleton uptake patients showed a significant difference compared with those of others . BSI values of patients showing absent kidney sign were statistically significantly high compared with those of others .p = 0.027, Fisher exact test). Patients with active cancer therapy (chemotherapy and hormone therapy) statistically significantly frequently showed negative ANN results in the BONENAVI analysis.Table The diagnosis of disseminated skeletal metastasis by BS is sometimes easily misinterpreted as normal unless the absence of renal uptake, the presence of diminished activity in the bones of the appendicular skeleton, and a high ratio of bone to soft tissue activity are recognized , 2. FiguWe aimed to evaluate the clinical application of the BONENAVI system on BS to resolve this problem. As shown in Table Regarding CT interpretation, intertrabecular CT type means undetectable on CT study. Therefore, the sensitivity of CT for disseminated skeletal metastasis was 81% (44/54). Furthermore, in intertrabecular type (CT negative) patients, BONENAVI detected skeletal metastasis in 60% (6/10). These figures are promising that in some patients with BS difficult  and CT negative  showed positive ANN values but were not satisfactory. Total sensitivity of ANN in disseminated skeletal metastasis (76%) was lower than the reported sensitivity for all cancers. Nakajima et al. reported that the sensitivity of ANN for all cancers was 91% [According to our classification, our clinical interpretation was that patients with suspected BS could be diagnosed as having bone metastasis, but whether or not disseminated spread was present was not certain. A false-negative example of such a patient is shown in Fig. The ANN value correlated with the BS types rather than the primary cancer or CT types Tables , 3. AmonTo improve the sensitivity of BONENAVI, factors other than diffuse increased skeleton uptake, such as inhomogeneity of uptake, might be considered or included to reach the artificial intelligence of BONENAVI.Chemotherapy and radiotherapy may influence the bone seeking RN uptake to bone. Radiation usually causes deceased bone seeking RN to bone with well demarcated margins limited to the boundaries of radiation areas . The intA serious limitation of this retrospective study was that we started this study from the BS log. We aimed to investigate the performance of BONENAVI in disseminated skeletal metastasis, and we regarded a BS study to be essential in this study. However, there is a possibility that BS was not performed (or not ordered) in all patients with disseminated skeletal metastasis. We recorded the BS log when any suspicious or not-ruled-out findings in BS study were identified. However, there is a possibility that patients with normal BS finding might have had disseminated skeletal metastasis. Therefore, we could not calculate the sensitivity of visual BS interpretation. We established a diagnosis of disseminated skeletal metastasis when at least two imaging modalities were consistent with the diagnosis. However, the sensitivities and specificities of each modality are not 100%, that is, currently there is no gold standard to diagnose disseminated skeletal metastasis.Another drawback was that the diagnosis of disseminated skeletal metastasis was performed clinically; that is, no histopathological confirmation was carried out.The BONENAVI CAD system was partially helpful in diagnosing disseminated skeletal metastasis, but the sensitivity of BONENAVI was not sufficient (76%) and underestimated disseminated skeletal metastasis. BONENAVI results may underestimate the disseminated skeletal metastasis by the present version.


Incomplete aneurysmal occlusion is a common feature of immediate posttreatment angiography. The safety and outcomes of acutely ruptured intracranial aneurysms (RIAs) with incomplete occlusion after stent-assisted coiling (SAC) and no-stent coiling (NSC) have not been well clarified. Progressive occlusion of stents can promote the complete occlusion of intracranial aneurysms (IAs), but it remains to be determined if progressive occlusion in acutely RIAs with incomplete occlusion after coiling may be enhanced by protective stenting. This study aimed to evaluate the safety and outcomes of those aneurysms after SAC and NSC; And to discover whether the stents can promote progressive aneurysm occlusion in such lesions or not.We reviewed 199 patients with acutely RIAs underwent endovascular coiling and developed incomplete occlusion in the past seven years. The patients’ clinical and imaging information were recorded and analyzed. Univariate and multivariate analyses were performed to determine the association of recurrence rate with potential risk factors.P = 0.009) and smaller dome-to-neck ratio  than in NSC group. There was no significant difference between the two groups in total procedure-related complications rate , procedure-related mortality  and modified Rankin Scale (mRS) score at 6-month follow-up (P > 0.05). However, SAC group had significantly higher ischemic complications rate  and complete occlusion rate , and lower recurrence rate  than NSC group based on 6-month follow-up angiograms. Additionally, Multivariable analysis showed NSC was an independent risk factor for aneurysm recurrence .SAC group had wider aneurysms neck (3.471 mm vs 2.830 mm, Acutely RIAs with incomplete occlusion after SAC is associated with higher complications rate and mortality, but has an acceptable safety profile and similar clinical outcome compared to NSC, as well as gives patients superior angiography outcome by progressive occlusion of stents. Despite advances achieved in endovascular techniques, incomplete occlusion is a common feature of immediate posttreatment angiographic studies . Yang etFortunately, several studies had revealed the capability of stent assistance to augment the progression from incomplete occlusion (MRRC II or III) to complete occlusion (MRRC I) , 5–7. OtFor acutely RIAs, it remains to be determined if progressive occlusion in such lesions after coiling may be enhanced by protective stenting. The safety and outcomes of acutely RIAs with incomplete occlusion after SAC and NSC have also not been well clarified. Thus, we performed this retrospective study to compare the safety and outcomes of those aneurysms after SAC and NSC, and to elucidate progressive aneurysm occlusion of stents in such lesions.According to the agreement approved by the Institutional Review Board. Two independent investigators  retrospectively reviewed the angiography and clinical data of 199 patients treated with endovascular coiling with/without stent for acutely RIAs with incomplete occlusion during 7 years between January 2012 and May 2019.Ruptured saccular aneurysms treated with SAC or NSC;Acutely RIA, which was defined as an aneurysm treated no more than 28 days after the initial rupture;Incomplete occlusion aneurysms were defined as MRRC II or III based on immediately postoperative angiograms.Inclusion criteria for this study were:Aneurysms other than saccular—that is dissecting, fusiform, traumatic, and blood blister-like;Aneurysms treated with “special” treatment strategies—that is, staged stent placement and stent placement alone;Aneurysms treated with clipping or assisted coiling with balloon or flow diverters;Patients with multiple aneurysms, the ruptured one cannot be certainly identified;Patients with multiple aneurysms and treated both the ruptured and unruptured one.Exclusion criteria for this study were:Treatment decisions of clipping or coiling concerning patient comorbidities, clinical status, surgical feasibility, and grade of hemorrhage were made in consensus among the neurosurgeons and neuro-interventionalists. Stent placement was determined according to the aneurysm feature and the intraoperative requirement when the endovascular coiling was determined as the treatment plan for patients. All operations were performed under general anesthesia. A 6-Fr or 8-Fr guiding catheter was positioned in the internal carotid artery via the common femoral artery for anterior circulation aneurysms or in the vertebral artery for posterior circulation aneurysms. Types of stents used in this procedure included the Leo stent , the Enterprise stent , and the LVIS stent . Stents were chosen according to the surgeon’s preference concerning anatomical variations.A femoral artery catheter was inserted in a standard fashion, and the femoral artery catheter performed the systemic heparinization for all patients. Glycoprotein IIb/IIIa inhibitor  was used to prevent thrombosis when the stents were opened. The loading dose of the first intravenous infusion was 0.3 mg (intravenous infusion time was 3 min), and then injected intravenously at 0.3 mg per hour by a trace syringe pump. If acute thrombosis occurred during the procedure, additional doses of tirofiban would be used. If there was no thrombosis or hemorrhagic events happened during the process, tirofiban will be discontinued after the operation finished. The postoperative antiplatelet regimen has been aspirin 100 mg daily for 6 months and clopidogrel 75 mg daily for 4 weeks. Moreover, the first 3 days after surgery, the low-molecular-weight heparin  is used in combination with antiplatelet agents.occluded, defined as no contrast material filling into the aneurysm sac and neck;improved, defined as decreased contrast material filling into the aneurysm sac or neck;stable, defined as unchanged contrast material filling into the aneurysm sac or neck;recurrent, defined as increased contrast material filling into the aneurysm sac.The safety was measured by total procedure-related complications, mortality, mRS score at discharge, and the 6-month follow-up. Based on the mRS score at the 6-month follow-up, a favorable clinical outcome was defined as mRS score 0–2 and an unfavorable outcome as mRS score 3–5. Immediately postoperative angiograms were used to analyze in terms of the degree of IAs obliteration by using the MRRC . The angiography follow-up results obtained with digital subtraction angiography (DSA) or three-dimensional contrast-enhanced magnetic resonance angiography (3D CE-MRA). In patients undergoing 3D CE-MRA at 6 months, the findings with not-stable aneurysms (any change) would be confirmed by DSA. And these results were classified into 4 categories when compared with the immediate embolization degree:The same two physicians who evaluated the immediate postoperative angiograms also evaluated the angiography follow-up outcome.2 or Fisher exact test was used for categorical variables. Univariate analysis and multivariate analysis were performed to determine the association of recurrence rate with potential risk factors. The univariate analysis cutoff for inclusion in the multivariate analysis was P < 0.05. The level of statistical significance was set at P < 0.05.Statistical analysis was performed by using SPSS version 22.0 software (SPSS. Chicago) by two authors. Data are presented as the mean and range for continuous variables, and frequency and percentage for categorical variables. Mann-Whitney U tests were used for non–normally distributed but continuous variables. PearsonχP > 0.05). However, there were a significantly wider aneurysm neck and smaller dome-to-neck ratio in the SAC group (P < 0.05). Additionally, the SAC group had a significantly higher incidence in internal carotid aneurysms (ICA) than the NSC group (P = 0.034).Of the 199 patients, 101 patients were treated with SAC, and 98 patients were treated with NSC. The patient’s and aneurysm’s baseline characteristics were summarized in Tables P = 0.195) and mortality , but with no statistical difference. Ischemic events  and intraoperative thrombosis  were prominent in the SAC group. Hemorrhagic events  and intraoperative rupture (IOR) rates  between the two groups were comparable. The postoperative thrombosis (5.9% vs 3.1%), postoperative hemorrhage (3.0% vs 0%) and aneurysm rebleeding (5.9% vs 1.0%) in SAC group were slightly higher than in NSC group, but with no statistical difference (P > 0.05). The detailed data were shown in Table The SAC group had a higher rate than the NSC group in total procedure-related complications , the data were shown in Tables We also performed a comparative analysis of complications in early (before 72 h) and late (day 3–28) stage of SAC and NSC group. And there was no difference in different operation timing of the two groups P > 0.0, the datP = 0.170). 9 (4.5%) patients died of the two groups. Among these death cases, 7 patients died of IOR (5 in SAC and 2 in NSC), 1 patient died of intraoperative thrombosis which led to massive cerebral infarctions and cerebral hernia, and 1 patient died of postoperative cerebral hemorrhage combined with multiple cerebral infarctions. Clinical evaluation was performed for all 190 surviving patients at discharge based on mRS score, 164 (82.4%) of whom got the favorable clinical outcome (mRS score of 0–2), while the other 26 were dependent (mRS score of 3–5). All 179 patients underwent clinical follow-up after discharge at postoperative 6 months, 156 (87.2%) patients got mRS score 0–2. All 7 patients with aneurysm rebleeding didn’t get admission again and lose further follow-up data , but without statistical difference (P = 0.020). And the recurrence rate of SAC and NSC group was 15.6% vs. 28.1%, still with an obvious statistical difference (P = 0.042)  treated with SAC and 89 (90.8%) patients treated with NSC got follow-up 6-month. The follow-up angiograms showed that 102 aneurysms (57.0%) were occluded, 19 (10.6%) were improved, 20 (11.2%) were stable, and 39 (21.8%) were recurrent. The complete occlusion rate of SAC and NSC group was 65.6 and 48.3%, respectively, with a significantly statistical difference (P = 0.018) was an independent risk factor for aneurysm recurrence  and stent. The balloon-assisted coiling (BAC) provides a contemporary scaffold effect that prevents coil protrusion and is associated with an improved occlusion rate after endovascular therapy in previous studies –19. WangAcutely RIAs treated with SAC is still controversial because of concerns about the complexity of the procedure, thrombotic risk, and antiplatelet drugs , 23. TheIn the SAC group of this study, the incidence of ischemic and hemorrhagic complication was 21.8% 22/101), 14.8% (15/101), respectively. It showed a higher incidence than previous systematic reviews. Nishido et al. /101, 14. reportedComplications of the stent group in this study was mainly caused by intraoperative thrombosis  and IOR 11.9%, 12/101). The number one reason of high intraoperative thrombosis events probably was related with the method of antiplatelet administration. Ryu et al.  conclude/101. TheIOR is a severe complication of coiling and is associated with high rates of morbidity and mortality. According to the review of the Cerebral Aneurysm Re-rupture After Treatment (CARAT) study, the coiling-related IOR occurred in 5% of patients, and IOR was associated with 64% rate of periprocedural death and disability . RecentlAdditionally, some studies even reported that ruptured aneurysm was an independent predictor of complications , 37, 42.P = 0.081), 83.3% vs. 89.9% (P = 0.244) respectively in this study. Zhang et al. [There was no statistical difference in mortality and rebleeding rate between the two groups in this study, and are consistent with previous studies –29, 43. g et al.  reportedg et al. , 19, 27.The use of stent usually brings future superior angiography outcome for patients, even in incomplete occlusion aneurysms , 22, 27.Although we conducted some objective and reliable results, and still with some limitations. The major limitation of this study is that this is a retrospective study with no randomization; the patient’s selection of SAC group didn’t establish uniform inclusion and exclusion criteria, so selection bias was inevitable. Additionally, aneurysm characteristics between the SAC group and the NSC group were the slight differences, especially the differences in aneurysm neck and dome-to-neck ratio. Finally, the BAC was not thoroughly considered in deciding the treatment plan because the BAC technique is still developing in our institute and we didn’t have much experience, so the complications of SAC group may overestimate by this study due to patients who are suitable for BAC are selected into the SAC group. Another limitation is that this study had no long-term follow-up results and small sample size.The overall procedure-related complications rate and mortality in the SAC group were higher than in the NSC group, but it is still comparable. Moreover, the favorable clinical outcome rate of stents placement was similar to those in no-stent group. These results suggest that acutely RIAs with incomplete occlusion treated by SAC were safe for patients. As well as we performed a comparative analysis of complications in early (before 72 h) and late (day 3–28) stage of SAC and NSC group, and show that the SAC is as safe early as well as late after aneurysm rupture. The follow-up angiography outcome elucidated that stent placement could increase aneurysm occlusion rate, reduce recurrence rate for those patients. And the progressive occlusion of stent deployment may not be entirely compensated by the progressive regrowth of aneurysms and the use of DAPT.


Prunus japonica is an ornamental and medicinal plant that is widely cultivated. The complete chloroplast genome of P. japonica was sequenced using Illumina Hiseq X Ten platform. The chloroplast genome was 158,080 bp in length, containing two short inverted repeat (IRa and IRb) regions of 26,385 bp, which was separated by a large single copy (LSC) region of 86,270 bp and a small single copy (SSC) region of 19,040 bp. The GC content of the whole chloroplast genome was 36.8%. The chloroplast DNA of P. japonica comprised 112 distinct genes, including 78 protein-coding genes, 4 ribosomal RNA genes and 30 transfer RNA genes. Phylogenetic analysis indicated that all species of Prunus formed a monophyletic group, P. japonica was closely related to P. hulimis. Prunus japonica Thunb., also called Korean bush cherry, is a shrub species in the genus Prunus of family Rosaceae, which is widely cultivated for ornamental and medicinal use. Its native range extends from Northeast and East China to Korea . Voucher specimen was stored at the herbarium of Institute of Chinese Materia Medica (CMMI), China Academy of Chinese Medical Sciences with the specimen voucher number is 341124LY0988. Total genomic DNA was isolated from fresh leaves using a DNeasy Plant Mini Kit  according to the manufacturer’s instructions. And the sequencing library was constructed and quantified following the methods introduced by Dong et al.  as the reference. The selected contigs were assembled using Sequencher 4.10. Gene annotation of P. japonica was performed using DOGMA annotation  regions of 26,385 bp, which was separated by a large single copy (LSC) region of 86,270 bp and a small single copy (SSC) region of 19,040 bp. The GC content of the whole chloroplast genome was 36.8%. The cpDNA of C. formosana comprised 112 distinct genes, including 78 protein-coding genes, 4 ribosomal RNA genes and 30 transfer RNA genes. In these genes, 19 were duplicated in the IR regions and 19 genes contained one or two introns.17 harbored a single intron, and two (ycf3、clpP) contained double introns.The circular cpDNA of P. japonica based on Seventy-eight common coding genes which extracted from Twenty-five chloroplast genome sequences, including two outgroup samples and 22 samples of Rosaceae from the GenBank. All the sequences were aligned using MAFFT (Katoh et al. Prunus were strongly supported as a monophyletic tree (P. japonica was closely related to P. hulimis. The complete chloroplast genome reported in this study will be a valuable resource for future studies on genetic diversity, taxonomy, and phylogeny of family Rosaceae.A phylogenetic tree was constructed to confirm the location of tic tree  and P. j


Since the skin appears affected in up to 80% of cases of acute GVHD (aGVHD), its prognosis and diagnosis are essential for the correct management of these patients. Plasma concentration of elafin, an elastase inhibitor produced by keratinocytes, has been described elevated at the diagnosis of skin GVHD, correlated with the grade of GVHD, and associated with an increased risk of death. In this study we explored elafin plasma levels in the largest series reported of T cell–replete haplo-HSCT with PTCy. Plasma samples drawn from 87 patients at days +15 and +30 were analyzed (“discovery cohort”). Elafin levels at days +15 were no associated with chronic GVHD, non-relapse mortality, relapse, therapy-resistant GVHD, or overall survival. In our series, elafin levels at day +30 were not associated with post-transplant complications. On the other hand, elafin plasma levels at day +15 were higher in patients with severe skin aGVHD . Of note, patients with higher elafin plasma levels at day +15 presented a higher incidence of stage III-IV skin aGVHD . These results were confirmed  in an independent group of patients (n = 62), i.e. the “validation cohort.” These data suggest that measurement of elafin in patients undergoing haplo-HSCT with PTCy might be useful for an early identification of those patients who are at higher risk of suffering severe skin aGVHD and thus, improve their treatment and prognosis.Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has shown favorable results in the treatment of hematological malignancies. Despite the use of post-transplant cyclophosphamide (PTCy), graft  Despite setting , 3. The  setting . The dia setting . Thus, i setting –9. One o setting .et al. analyzed plasma samples from 492 patients who received HSCT derived from bone marrow and described increased elafin plasma concentrations at the onset of skin aGVHD and its correlation with aGVHD severity. In the multivariate analysis, elafin levels also predicted non relapse mortality (NRM) and overall survival (OS) independently of the area of the skin rash , is an epithelial protein that is secreted by keratinocytes in response to IL-1 and TNFα. It is overexpressed in inflamed epidermis and absent in normal skin. Paczesny kin rash . These rkin rash . Similarkin rash , 12. Mos  in all cases  was defined as the time from transplantation to disease relapse or progression, re-transplantation due to graft failure, or death from any cause, whichever occurred first. OS was defined as the time from transplantation to death from any cause. aGVHD and chronic GVHD (cGVHD) were scored according to established criteria , 15. SteSamples from the 87 patients included in the “discovery cohort” were collected at days +15 and/or +30 after transplantation. All patients had at least one sample from one of the two timepoints. Elafin plasma levels were available on day +15 in 70 patients and on day +30 in 75 patients. Results for both plasma samples were available in 58 patients. Additionally, samples at day +15 were collected from 62 patients from the “validation cohort.” Plasma was obtained from blood samples by refrigerated (4°C) centrifugation at 2,000 rpm for 30 min in the 2–6 h following extraction. Samples were aliquoted without additives into cryovials and stored at −80°C. Elafin was detected using ELISA according to the manufacturer’s instructions . Samples (diluted 1/50) and standards were run in duplicate and absorbance was measured using the VICTOR2 D fluorometer™ (multilabel plate reader).Numerical and categorical variables were expressed as median (range) and frequency (percentage), respectively. The Mann-Whitney test was used to compare differences between two independent variables. The determination of the best cut-off for elafin levels to stratify patients was derived from receiver operating characteristic (ROC) curves.Competing risks were death, relapse, and DLI before +180 for aGVHD.Univariate analyses were done using the log-rank test for EFS, and OS and Gray’s test for cumulative incidence. For the subanalysis carried out to study the relationship between elafin levels on day +30 and the appearance of aGVHD, those patients who had presented GVHD before day +30 were censored. OS and EFS were calculated using the Kaplan-Meier method. Statistical analyses were performed using SPSS v18 for Windows . Cumulative incidence (CI) rates were calculated using the statistical package R ver. 3.3.2.Baseline characteristics of 87 patients from the “discovery cohort” who underwent haplo-HSCT with PTCy are detailed in + cells (data not shown).No association was found between median elafin levels and clinical variables such as age, sex, stem cell source, donor sex, hematological malignancy, disease status at transplant, HSCT–associated comorbidity, previous transplant, conditioning regimen intensity, and number of infused CD34We correlated median elafin levels at days +15 and +30 with post-transplant complications , with statistically significant differences in those with severe skin involvement . No association was found for the other variables analyzed . Both cohorts of patients were homogeneous except for the pretransplant disease status, the proportion of patients who had previously undergone autologous transplantation, the stem cell source, and the amount of PB CD34Patients with elafin levels higher than 20,373 pg/ml at day +15 presented a significantly higher incidence of stage III-IV skin aGVHD . In addition, all patients with stage III-IV skin aGVHD had elafin levels on day +15 greater than 20,373 pg/ml  and we have performed the measurement of elafin levels on day +15 in an independent “validation cohort.” Although the latter confirmed our results, the use of biomarkers in routine clinical practice should be validated in a larger cohort in a prospective multicenter study.Even with the need for further research, our work supports the fact that elafin is a valid plasma biomarker in the setting of haplo-HSCT with PTCy. Elafin could have a predictive role for the development of severe skin aGVHD. The results reported here could provide the basis for future clinical trials to analyze, in a controlled way, the optimal modulation of the immunosuppressive treatment in this particular transplant setting.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Comité de Ética de la Investigación con Medicamentos (CEIm) del Hosp. G.U. Gregorio Marañón. The patients/participants provided their written informed consent to participate in this study.Conception and design: LS, CM-L, IB. Data collection: PM, ND, EL, JA. Data analysis: LS, MK, DC, MC-L, JG, JD-M, CM-L, IB. Manuscript drafting: LS, CM-L, IB. Critical manuscript revision: all authors. All authors contributed to the article and approved the submitted version.The present study was partially supported by the Ministry of Economy and Competitiveness ISCIII-FIS (grants PI17/01880 and PI20/00521), co-financed by ERDF (FEDER) funds from the European Commission, “A way of making Europe,” as well as grants from the Asociación Madrileña de Hematología y Hemoterapia (AMHH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.