At 3 months after initiation of faricimab therapy, a dry macula  was achieved in 82% of the eyes. Complete regression of polypoidal lesions was observed in 52% of eyes with PCV. Subfoveal choroidal thickness also decreased significantly at 1, 2, and 3 months compared to baseline (p < 0.0001). Although retinal pigment epithelium tears developed in two eyes, there were no other ocular or systemic complications observed during the 3 months of loading therapy. In conclusion, loading therapy using faricimab resulted in improved visual acuity and retinal morphology in Japanese patients with wAMD without particular safety issues.This multicenter study aimed to assess the short-term effectiveness and safety of faricimab in treatment-naïve patients with wet age-related macular degeneration (wAMD) in Japan. We retrospectively reviewed 63 eyes of 61 patients with wAMD, including types 1, 2, and 3 macular neovascularization as well as polypoidal choroidal vasculopathy (PCV). Patients received three consecutive monthly intravitreal injections of faricimab as loading therapy. Over these 3 months, visual acuity improved gradually compared to baseline. Moreover, the central foveal thickness decreased significantly at 1, 2, and 3 months compared to baseline ( In the pathologic phase of macular neovascularisation (MNV) in wet AMD (wAMD), angiopoietin-2 (Ang-2) expression increases in the vascular endothelium; this competitively inhibits angiopoietin-1 (Ang-1) from binding tyrosine kinase immunoglobulin-like receptors (Tie 2) on the surface of vascular endothelial cells, thus preventing Ang-1/Tie 2 signalling and resulting in capillary inflammation or pericyte loss4. In patients with wAMD, Ang-2 expression is upregulated in the retina and vitreous5.Worldwide, age-related macular degeneration (AMD) is a leading cause of central vision loss in the elderly population7. Intensive research has been conducted on optimal treatment protocols based on these drugs for wAMD8. In 2022, the first humanized, bispecific IgG monoclonal antibody, faricimab, which inhibits both VEGF-A and Ang-2, was approved after two phase 3 trials (TENAYA and LUCERNE). It demonstrated the visual benefits of treatment with faricimab given at up to 16-week intervals in patients with wAMD9. This study aimed to evaluate the safety and efficacy of faricimab treatment for patients with wAMD in real-world settings.Vascular endothelial growth factor (VEGF) inhibitors are the main treatment option for patients with wAMD; there are several commercially available anti-VEGF agents, including bevacizumab (off-label), ranibizumab, aflibercept, and brolucizumab as shown in Fig. p < 0.0001) as shown in Fig. The background characteristics of the participating patients with wAMD are presented in Table 10. Matsumoto et al. reported similar results in patients with type 1 MNV11. However, Fukuda et al. reported that visual function significantly recovered 3 months after treatment with aflibercept in eyes with PCV12, but no significant recovery was achieved at 3 months by treatment with brolucizumab.This Japanese multicentre study demonstrated visual and anatomical improvement as well as the general safety of loading therapy with faricimab in untreated eyes with wAMD. BCVA improved 2 months after the first faricimab treatment, which further improved at 3 months. The loading therapy of faricimab gradually induced these improvements in patients with wAMD. In comparison, we reported that the short-term BCVA significantly improved 1 month after the first brolucizumab injection in patients with wAMD, except for those with PCV10. Another study reported dry macula rates of 47.2%, 86.1%, and 94.4% at 1, 2, and 3 months, respectively, after treatment with brolucizumab11 in type 1 MNV. Among patients with PCV treated with aflibercept, the SRF-free rates were 44.7%, 76.3%, and 84.2% at 1, 2, and 3 months after aflibercept treatment, respectively, while the corresponding values after brolucizumab treatment were 64.3%, 85.7%, and 100%12. Overall, the improvement in visual function and retinal anatomy after treatment with faricimab during loading therapy was comparable to that after treatment with aflibercept. In our cohort, faricimab allowed complete regression of polypoidal lesions in 50% of patients with PCV. In our previous study, the rate of complete regression of polypoidal lesions in eyes treated with aflibercept was 47.8%13, which is consistent with other reports15. In eyes treated with brolucizumab, the regression rate has been reported to be 73.9–78.9%16. Thus, faricimab and aflibercept have similar effects on the regression of polypoidal lesions.Moreover, 35%, 69%, and 82% of patient eyes in our study attained a dry macula at 1, 2, and 3 months, respectively, and polypoidal lesions completely disappeared in 50% of the patients with PCV. We previously reported that dry macular rates 3 months after brolucizumab treatment were 91%, 88%, and 100% for type 1 and/or type 2 MNV, PCV, and type 3 MNV, respectively17. Ang-1–Tie 2 receptors are expressed in the choroid5, where they maintain the stability of choroidal vessels18. As faricimab affects these receptors, it could also affect choroidal vessels. Accordingly, there was a decrease in SCT at 1, 2, and 3 months after initiation of faricimab treatment . In our previous study, the SCT decreased by 91.3%, 89.8%, and 86.5% at 1, 2, and 3 months, respectively, after treatment with aflibercept in PCV cases19. Related results regarding the effects of aflibercept 21 on SCT have been reported. Moreover, in our previous study, SCT in eyes with wAMD decreased by 90.6%, 86.6%, and 84.7% at 1, 2, and 3 months, respectively, after treatment with brolucizumab22. In other studies, SCT in eyes with type 1 MNV or PCV decreased by 83.3–89.4%, 82.5–86.0%, and 84.1–84.4% at 1, 2, and 3 months, respectively, after treatment with brolucizumab12. Faricimab might have a slightly lower impact on the choroid than brolucizumab.Furthermore, the loading treatment significantly reduced the SCT in the affected eyes. VEGF is secreted from the basement membrane of the RPE to maintain choroidal homeostasis9. This trend requires careful monitoring in patients with a large PED, in which active type 1 MNV spreads through sub-RPE fluid23.There were two cases of RPE tears: one developed 1 month after the first faricimab treatment in an eye with type 1 and type 2 MNV, while the other developed at 3 months in an eye with PCV. By the end of the loading therapy, the bilateral visual acuity was maintained. In a study conducted by TENAYA and LUCERNE, RPE tears after treatment with faricimab developed in five eyes (1.3%) and three eyes (0.9%), respectively24; however, reducing Ang-2 can reduce inflammation, as was shown in a mouse model of myocardial infarction 25. It is possible that the anti-inflammatory effects of Ang-2 inhibition contributed to the low rate of ocular inflammation.In the 3 months after initiation of faricimab treatment, no cases with ocular inflammation were observed, even though in the previous phase 3 trials (TENAYA and LUCERNE), 1.3% and 1.5% ocular adverse events were reported in each, respectively, including 0.3% and 0.6% vitritis, 0.5% and 0.6% iritis, and 0.5% and 0.6% uveitis, respectively. Ang-2 may be essential for constructing normal vasculature in response to physiological events The limitations of this study include its retrospective nature and short follow-up period. Thus, further studies are required to verify the long-term efficacy and safety of faricimab in patients with wAMD.In conclusion, a loading therapy of faricimab effectively improved visual acuity and retinal morphology in eyes with wAMD. Ophthalmologists should carefully monitor safety during follow-up.26, PCV27, and type 3 MNV26. PCV was diagnosed based on the presence of polypoidal lesions on ICGA27. The diagnosis criteria for type 3 MNV were retinal–retinal anastomosis on early-phase FA or ICGA and a hot spot on late-phase ICGA28. We determined the BCVA using the early treatment diabetic retinopathy study (ETDRS) visual acuity chart at Nihon University as well as the Landolt C chart at the other four institutions. BCVA was converted to the logarithm of the minimal angle of resolution (logMAR) units for outcome analyses. On the OCT images, the macula was considered dry if the subretinal and intraretinal fluids were completely resolved. CFT was measured from the superior border of the RPE to the border of the inner retinal layer at the foveal centre. Additionally, SCT was measured as the vertical distance between the hyperreflective line corresponding to Bruch’s membrane under the RPE and the inner scleral boundary at the foveal centre using the caliper function of the OCT .This study included 63 untreated eyes of 61 patients with neovascular AMD, who visited the Fukushima Medical University Hospital; Kyorin University Hospital; Nihon University Hospital; University of the Ryukyus Hospital; and Tokyo Women’s Medical University Hospital (Japan AMD Research Consortium: JARC) between June 2022 and August 2022. The study protocol was approved by the Institutional Review Board of Fukushima University, Kyorin University Hospital, Nihon University Hospital, Ryukyus Hospital and Tokyo Women’s Medical University Hospital as a retrospective study. All study protocols adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all the patients included in this study. We included untreated patients with wAMD aged ≥ 45 years. We excluded patients with myopia of > − 6 dioptres, a history of uveitis, or a history of vitrectomy. One patient (1 eye) did not return for personal reasons. Accordingly, we included 62 eyes from 60 patients who received faricimab at 1-month intervals for 3 consecutive months, with assessments at baseline and then after 1 and 2 months. The outcomes included visual acuity, fluid resolution rate, CFT, SCT, changes in PED, and polypoidal lesion regression rate in patients with PCV after 3 months. Fluorescein angiography (FA) and indocyanine green angiography (ICGA) were performed using a confocal scanning laser ophthalmoscope  to determine the subtypes of neovascular AMD, including type 1 MNV, type 2 MNVPED was defined as one or more detached areas, the same size as the optic disc, in the macula on FA; its height was also recorded for comparison with previous measurements. The height of PED from the inner layer of Bruch’s membrane to the top of the RPE was measured using OCT. Changes in polypoidal lesions were recorded as complete regression, partial regression, or increase relative to baseline levels as measured using ICGA. Exclusion criteria were massive submacular haemorrhages extending beyond the equator.p < 0.05. GraphPad Prism version 9  was used for the statistical analyses.Data are presented as the mean ± standard deviation (SD). The Wilcoxon signed-rank test was used to assess changes in visual acuity. One-way analysis of variance was used to compare CRT and SCT before and after treatment. Statistical significance was set at


In IVD embryos, fewer errors were found at blastocyst stage compared to cleavage (4-cell) stage . One androgenetic and two parthenogenetic embryos were also identified. Triploidy was the most common error in IVD embryos (15.8%), but only observed at cleavage, not blastocyst stage, followed by whole chromosome aneuploidy (9.9%). In IVP blastocysts, 32.8% were parthenogenetic, 25.0% (hypo-)triploid, 12.5% aneuploid, and 9.4% haploid. Parthenogenetic blastocysts arose from just three out of ten sows, suggesting a possible donor effect. The high incidence of chromosomal abnormalities in general, but in IVP embryos in particular, suggests an explanation for the low success of porcine IVP. The approaches described provide a means of monitoring technical improvements and suggest future application of PGT-A might improve embryo transfer success.Preimplantation genetic testing for aneuploidy (PGT-A) is widespread, but controversial, in humans and improves pregnancy and live birth rates in cattle. In pigs, it presents a possible solution to improve in vitro embryo production (IVP), however, the incidence and origin of chromosomal errors remains under-explored. To address this, we used single nucleotide polymorphism (SNP)-based PGT-A algorithms in 101 in vivo-derived (IVD) and 64 IVP porcine embryos. More errors were observed in IVP vs. IVD blastocysts (79.7% vs. 13.6%  Chromosomal errors in embryos are a major cause of implantation failure, spontaneous abortions, and congenital birth defects in humans. Preimplantation genetic testing for aneuploidy (PGT-A) is frequently  used in IVF embryos as a fertility treatment to identify chromosomal abnormalities and select euploid embryos prior to transfer ,2. In caPrior studies on cattle, sheep, horse, and pig suggested that there is a higher incidence of aneuploidy (extra or missing whole chromosomes) in IVP compared to IVD embryos ,12,13,14n = 123) were collected, and in vitro blastocysts (n = 68) were produced between March 2020 and August 2021. Oocytes and embryos were collected from sows originating from Nucleus farms that were routinely slaughtered because of their breeding value, not because of sub/infertility. A tissue sample from each sow was stored at −80 °C for genotyping for identification. The DNA extraction from the ovaries was performed by BioBank, Hamar and genotyping was performed at CIGENE, Norwegian University of Life Sciences, Ås, using the Illumina GeneSeek custom 25K SNP chip .In vivo-derived embryos  and embryos were collected from the uterine tracts by flushing the top part of the horn. Briefly, 30 mL of prewarmed TL-HEPES-PVA at 37 °C was inserted with a syringe and needle at the side of the oviduct and approximately 40 cm of the uterine horn was flushed, after which the medium was collected in a 50 mL tube. This was repeated twice to collect most embryos. The number of embryos and embryo stages were recorded and the number of corpora lutea were counted on the ovaries to calculate the collection rate .2 in air. Subsequently, COCs were matured for another 24 h in POM without hormones and dbcAMP. Oocytes from the different sows were not pooled, but matured, fertilized, and cultured per sow in groups of 25–35 per well of a Nunc® four-well multidish containing 500 mL pre-equilibrated medium. Fertilization was performed with cryopreserved sperm from one Landrace boar and straws originated from the same ejaculate. Each 2.5 mL straw was thawed at 50 °C for 50 s and diluted in 40 mL Tri-X-cell  at room temperature (RT). Sperm cells were washed and selected at RT using Percoll® density gradient centrifugation by layering 2 mL of 45% Percoll on top of 2 mL 90% Percoll. Finally, 1 mL of semen was carefully placed on top followed by centrifugation at 700× g for 20 min. Supernatant was removed by aspiration, the pellet was resuspended in 4 mL PGM without BSA and centrifuged at 500× g for 5 min. The pellet was then resuspended in 150–200 µL PGM without BSA. Sperm motility and concentration were measured by computer assisted sperm analysis (CASA) using a Sperm Class Analyzer® version 6.1 , equipped with a phase contrast Eclipse Ci-S/Ci-L microscope  and Basler digital camera . Spermatozoa were diluted to 5 × 105 progressively motile sperm cells/mL in 300 µL pre-equilibrated PGM with BSA. Groups of 25–35 COCs were co-incubated at 3.0 × 104 progressively motile sperm cells/mL. After 2 h of co-incubation, oocytes were transferred to a new well with 500 µL PGM medium to remove an excess of sperm cells. After a total of 4 h fertilization, presumptive zygotes were denuded of cumulus cells by vortexing for 1 min in 2 mL PXM in a 15 mL tube. The presumptive zygotes were washed twice in PXM medium and once in PZM-5 before culture in 500 µL PZM-5 under 400 µL mineral oil  at 38.9 °C in an humified atmosphere containing 6% CO2 and 7% O2. At day 4 of culture (fertilization day 0), PZM-5 medium was replaced with 250 µL new equilibrated PZM media. Embryos were cultured until day 5 or 6 when they reached blastocyst stage.In vitro embryo production was carried out as previously described . PorcineAll embryos were incubated with 0.5% pronase in phosphate buffered saline (PBS) to remove the zona pellucida and DNA of any attached cumulus and/or sperm cells. In vivo-derived embryos were incubated for approximately 45–60 s, and in vitro-produced blastocysts for 30–45 s. Embryos were individually washed twice in PBS and once in REPLI-g Advanced Single Cell Storage buffer before they were individually stored in 3 µL storage buffer at −80 °C until further analysis. Samples were transported on dry ice to CIGENE, Norwegian University of Life Sciences, Ås, where the embryos were subjected to DNA extraction and whole genome amplification (WGA) using the REPLI-g Advanced DNA Single Cell Kit  following the manufacturer’s instructions. Briefly, samples were denatured in 3 µL Denaturation Buffer by incubation at RT for 10 min, after which 3 µL Stop Solution was added. Samples were then kept on ice and 40 µL REPLI-g sc Master mix was added to 9 µL of denatured DNA after which the samples were amplified at 30 °C for 2 h, followed by inactivation of REPLI-g sc DNA Polymerase by incubation at 65 °C for 3 min. Amplified DNA was genotyped the same day using the Illumina GeneSeek custom 25K SNP chip. Only samples with a call rate ≥ 80% were included in the further study. After filtering, 101 IVD embryos and 64 IVP blastocysts were analyzed.Based on the SNP genotypes, the chromosome analysis was conducted at the University of Kent, UK. Computing and PGT-A diagnosis were performed using a combination of three PGT-A algorithms per embryo : signal p ≤ 0.05 was considered statistically significant. Figures were plotted using GraphPad Prism version 9.0 .The results are presented as percentages with their 95% confidence intervals (CI) for proportions unless stated otherwise. Proportions between different groups were analyzed by Fisher’s exact test and The data presented in this study are not publicly available due to being part of the intellectual property of Norsvin SA and Topigs Norsvin.n = 32/101) in IVD embryos. Fewer chromosomal errors were indicated at the blastocyst stage compared to the 4-cell stage , 35.0% in 6–12 cells (n = 7/20), 35.3% in morulae (n = 12/34) and 13.6% in blastocysts (n = 3/22), respectively. The different chromosome abnormality classes found among the embryos in all development stages are shown in n = 16/101) and embryos with aneuploidy   was observed. This last class includes embryos showing monosomy, trisomy, and/or UPD. Triploid embryos were only observed at the early embryo developmental stages and not at the blastocyst stage . A high incidence of parthenogenetic  and (hypo)triploid blastocysts  was indicated followed by 12.5% of the blastocysts having aneuploidy  (n = 8/64) and 9.4% being haploid (n = 6/64) (n = 4/9) and 57.1% (n = 4/7) of the embryos were parthenogenetic from sow 6 and 10, respectively. Furthermore, euploid blastocysts were obtained from 6 out of 10 sows, while only blastocysts with errors were produced from the other 4 sows.In IVP blastocysts, only 20.3% were euploid ( = 6/64) . Parthen = 6/64) . From son = 51/64) as compared to IVD blastocysts  (p < 0.001).The results indicate that IVP blastocysts have a higher incidence of errors , while errors from paternal origin where prevalent in IVD embryos  (p < 0.01). Furthermore, 2.0% (n = 2/101) and 4.7% (n = 3/64) of the errors originated from the embryo in IVD and IVP embryos, respectively. Tri- and hypotriploidy in both IVD and IVP embryos originated with a higher frequency from the paternal side , which is most likely related to polyspermy, compared to the maternal side  which is related to meiotic non-disjunction in the oocyte. All monosomies in both IVD and IVP embryos were of maternal origin. One case of uniparental disomy was identified in an IVP blastocyst on chromosome 18, which had a paternal origin. For trisomies, it was assessed whether the errors originated from meiosis I, meiosis II, or during embryonic development (mitotic error) (n = 6/12) and IVP (n = 4/8) embryos). In IVD embryos, most trisomies originated from errors during paternal meiosis I , while no paternal trisomies were observed in the IVP embryos. The androgenetic in vivo embryo arose from a dispermic event, in which two sperm cells fertilized an empty oocyte.In total, 36 errors were observed in IVD embryos and 57 in IVP embryos . Errors c error) . Most trn = 14) and IVP (n = 14) embryos. Chromosome 18 showed a higher incidence of errors (n = 3/14) in IVD embryos, as well as a higher frequency of trisomies (n = 3/12). Only chromosomes 2 and 14 presented monosomies, accounting for one each of the total of two monosomies.The incidence of errors by chromosome are shown in n = 4/14), showing a similar incidence for monosomy and trisomy. This was followed by chromosomes 13 and 15 (both n = 3/14). Chromosome 13 presented more trisomies, whereas chromosome 15 presented more monosomies. Interestingly, the only UPD found in this dataset belonged to an IVP blastocyst on chromosome 18.Meanwhile, in IVP blastocysts, chromosome 14 was the most affected  but not in IVP embryos . Female and male embryos were affected by chromosomal abnormalities at similar rates, with 30.4% vs. 26.8% in IVD embryos and 78.9% vs. 50.0% (p = 0.09) in IVP blastocysts  and IVP embryos   . FurtherThe results presented suggest that there is a high prevalence of chromosomal errors in porcine  embryos, which potentially can be one of the reasons behind the inconsistency of IVP in establishing blastocyst rates. Although it might be argued that a high prevalence in multiparous animals is expected, the disparity between IVP and IVD embryos suggests that technical improvements in the IVP process need to be made before it mimics the in vivo situation and the benefits of porcine IVP can be fully realized. We establish proof of principle for the use of SNP-based data, applying algorithms that can monitor the progress of the improvement of the IVP process, and ultimately apply PGT-A to select chromosomally abnormal embryos as has been established in cattle . HoweverWe found an error incidence of 13.6% in IVD and 79.7% in IVP blastocysts, suggesting that the application of in vitro technologies might induce these errors. Previous separate studies in pig embryos show an incidence of 14.3–36.7% in in vivo ,24 and 3Our results indicated a lower incidence of chromosomal errors in IVD blastocysts (13.6%) compared to 4-cell stage embryos (40%). This is in line with the suggestion that chromosome abnormality is mostly responsible for early embryo mortality ,10,27. GSurprisingly, two parthenogenetic  and one androgenetic embryo  were classified in the IVD embryos. To the best of our knowledge, this is the first time that parthenogenetic and androgenetic embryos have been characterized in in vivo-derived porcine embryos. The overall error incidence in the present study was higher compared to results from others who reported 14.3% aneuploidy in pig embryos collected 3 days after insemination  and onlyThe study also raises the question of whether the high incidence of aneuploidy in in vivo pig  embryos is the natural state of affairs. Early embryo loss has been described to be 30–40% in pigs and cattle . MoreoveA high incidence of parthenogenetic blastocysts 32.8%) was indicated within our IVP system, which was presented with recombination throughout the chromosome (observed by Karyomapping), except one case that was presented without any recombination. Parthenogenesis was not induced in this study by electrical or chemical stimuli and further research into the cause is therefore of interest as creating parthenotes is undesirable for animal breeding. The parthenogenetic embryos arose from just 3 out of 10 sows in two of the three IVP rounds while cryopreserved sperm from the same boar and ejaculate was used during fertilization, which suggests a possible donor effect. Spontaneous parthenogenetic activation has also been observed in bovine IVP, with 4/61 embryos being parthenogenetic .8% was i,34. NormFurthermore, we observed a high incidence of triploid embryos 25.0%) in the IVP blastocysts. In porcine IVP, polyspermy has been stated to be a challenge and is shown to be affected by individual boar differences, breed differences, and sperm–oocyte ratio ,37,38. W.0% in thWe observed that errors with a paternal origin were more prevalent in IVD embryos, while maternal errors predominated in IVP embryos. This might be explained by the fact that 32.8% of the IVP blastocysts were parthenogenetic. Furthermore, a higher incidence of tri- and hypotriploidy were originating from the sire in both IVD and IVP embryos, which indicate polyspermic fertilization ,43 as diOur results showed a high incidence of errors on chromosomes 13, 14, and 15 in the IVP blastocysts. In pigs, chromosome 13–18 are acrocentric, which means that the centromere is located near the end of the chromosome. Acrocentric chromosomes seem to be more at risk for non-disjunction events due to the crossing over during the prophase I of meiosis and later during prophase II, as they can suffer premature separation of sister chromatids or reverse segregation ,45. PrevOverall, PGT-A discovered a high incidence of aneuploidy and triploidy in IVD and IVP embryos, suggesting that the future application of this technology might improve embryo transfer success in the pig.


Despite the preserved LVEF, patients with acromegaly are characterized by subclinical systolic dysfunction i.e., abnormal global longitudinal strain (GLS) assessed by speckle tracking echocardiography (STE). The effect of acromegaly treatment on LV systolic function assessed by STE, has not been evaluated so far.Thirty-two naïve acromegalic patients without detectable heart disease were enrolled in a prospective, single-center study. 2D-Echocardiography and STE were performed at diagnosis, 3&6 months on preoperative somatostatin receptor ligand (SRL) treatment and 3 months after transsphenoidal surgery (TSS).2 in males and 94g/m2 in females. Most patients (78.1%) had increased LAVi and the median value was 41.8mL/m2. At baseline 50% of patients, mostly men (62.5% vs. 37.5%) had GLS values higher than -20%. There was a positive correlation between baseline GLS and BMI r=0.446 (p=0.011) and BSA r=0.411 (p=0.019). The median GLS significantly improved after 3 months of SRL treatment compared to baseline: -20.4% vs. -20.0% (p=0.045). The median GLS was lower in patients with surgical remission compared to patients with elevated GH&IGF-1 levels: -22.5% vs. -19.8% (p=0.029). There was a positive correlation between GLS and IGF-1 levels after TSS r=0.570 (p=0.007).Treatment with SRL resulted in reduction in median (IQR) GH&IGF-1 levels after 3 months, from 9.1(3.2-21.9) to 1.8(0.9-5.2) ng/mL (p<0.001) and from 3.2(2.3-4.3) to 1.5(1.1-2.5) xULN (p<0.001), respectively. Biochemical control on SRL was achieved in 25.8% of patients after 6 months and complete surgical remission was achieved in 41.7% of patients. TSS resulted in decrease in median (IQR) IGF-1 compared to IGF-1 levels on SRL treatment: from 1.5(1.2-2.5) to 1.3(1.0-1.6) xULN (p=0.003). Females had lower IGF-1 levels at baseline, on SRL and after TSS compared to males. The median end diastolic and end systolic left ventricle volumes were normal. Almost half of the patients (46.9%) had increased LVMi, however the median value of LVMi was normal in both sex groups: 99g/mThe greatest beneficial effect of acromegaly treatment on LV systolic function is visible already after 3 months of preoperative SRL treatment, especially in women. Patients with surgical remission have better GLS compared to patients with persistent acromegaly. Acromegaly is a systemic disease that affects multiple organs including the cardiovascular system . Recent Acromegaly can cause a typical cardiomyopathy characterized by mainly left ventricular hypertrophy (LVH) that is associated with mildly impaired diastolic function, which can progress to systolic dysfunction . HoweverThis was a prospective, single-center study. The study group consisted of 35 consecutive newly diagnosed acromegaly patients admitted to the Department of Endocrinology at the Center of Postgraduate Medical Education in Warsaw, Poland, from January 2018 to July 2020. Only adult patients (age ≥ 18 years) were enrolled to this study. Acromegaly was diagnosed based on typical clinical features associated with elevated serum IGF-1 levels for age and sex and lack of GH suppression below 0.4 ng/mL during a 75 g oral glucose tolerance test (OGTT), and positive pituitary magnetic resonance (MRI) findings.After the diagnosis of acromegaly, each patient received a preoperative treatment with the first-generation somatostatin receptor ligand (SRL) (lanreotide autogel 120 mg every 4 weeks s.c.) while waiting for transsphenoidal pituitary surgery (TSS). Biochemical response to the medical therapy was assessed after a 6-month treatment period for 31 patients. Good response to first-generation SRLs was defined as fasting GH ≤1 ng/mL and IGF-1 ≤ the upper limit of normal (ULN) for age and sex on treatment with a maximum dose of SRL.Surgical remission was defined as IGF-1 concentration below the ULN for sex and age-matched groups and suppression of GH on OGTT below 0.4 ng/mL assessed 3 months after TSS. Out of 32 enrolled patients 24 underwent the TSS (75%) during the study after preoperative treatment with SRL.All patients were operated on by one neurosurgeon experienced in pituitary surgeries from the Department of Neurosurgery, Military Institute of Medicine in Warsaw, Poland.Each patient underwent echocardiography at the time of diagnosis of acromegaly, 3 and 6 months after the initiation of treatment with SRL and 3 months after TSS. Patients with a known coronary artery disease, with a history of myocardial infarction or myocarditis, with impairment of LV systolic function (EF < 54% in women and < 52% in men), and moderate to severe valvular disease were not included in this study.From the initial number of 35 patients with newly diagnosed acromegaly enrolled to this study, 32 patients were included to the final analysis. Three patients were excluded: 1 with a history of myocardial infarction, 1 with multivessel coronary artery disease and concomitant ventricular tachycardias and 1 with heart failure and reduced EF. Additionally, 28 out of 32 enrolled patients underwent carotid Doppler ultrasonography at the diagnosis of acromegaly.The project received the approval of the Bioethics Committee of the Center of Postgraduate Medical Education in Warsaw, Poland.2), body surface area (BSA) was calculated according to the DuBois formula, and systolic and diastolic blood pressures were measured in millimeters of mercury (mmHg). Arterial hypertension was defined as a systolic blood pressure of ≥ 140 mmHg and/or diastolic blood pressure of ≥ 90 mmHg or the treatment of previously diagnosed hypertension. Plasma glucose, glycosylated hemoglobin A1c, total cholesterol, high-density lipoprotein cholesterol, and triglycerides were measured in the morning after a 12-hour fasting period. Low density lipoprotein cholesterol was calculated by the Friedewald formula. Diabetes or prediabetes were diagnosed according to the recent criteria  was calculated using the formula weight (kg) divided by height squared . The GH assay has a sensitivity of 0.05 ng/mL, an intra-assay coefficient of variation (CV) of 1.93% for a GH concentration of 1.18 ng/mL and inter-assay CV of. 3.77% for a GH concentration of 1.11 ng/mL. The intra-assay CV is 4.59% for an IGF-1 concentration of 189.3 ng/mL and inter-assay CV is 4.3% for an IGF-1 concentration of 202.6 ng/mL.Blood samples taken for GH and IGF-1 were analyzed with chemiluminescence immunoassay using the LIAISONEach patient with confirmed acromegaly was referred to the Echocardiography Laboratory of the Department of Cardiology at the Center of Postgraduate Medical Education, Warsaw.Standard two-dimensional transthoracic echocardiographic study and Speckle Tracking Echocardiography were performed at four time points as detailed above.2 for women and ≤ 74mL/m2 for men, were accepted as normal. The cut-off points for indexed left ventricular end-systolic volume (LVESVi) were as follows: ≤24 mL/m2 for women, ≤ 31mL/m2 for men.All studies were performed by the same experienced investigator. Echocardiography exam was done using Vivid 9 device . A sector transducer with a frequency of 3.2 MHz was used. The images were stored digitally for later offline analysis using dedicated software . LV and left atrial (LA) volumes were calculated using the apical four- and two-chamber views. LV volumes were normalized by body surface area (BSA) and the value of left ventricular end diastolic volume (LVEDVi) ≤ 61mL/m2 was considered normal for both men and women.Left atrium volume (LAV) was also indexed to BSA, and the value of LAVi ≤ 34mL/mLV EF was calculated using the biplane Simpson formula and value ≥ 52% for men, and ≥ 54% for women were considered normal. The LV mass (LVM) was calculated using the linear method and was indexed in accordance with BSA to obtain the left ventricular mass index (LVMi) value. This parameter was calculated by using Penncube method according to Devereux’s formula: LVMi (g/m2) = (1.04 × (IVST + LVID + PWT)3 − (LVID)3 − 13.6)/BSA . LVMi ≤ 95 g/m2 in women and ≤ 115 g/m2 in men were accepted as cut-off points for normal.These echocardiographic parameters were measured in accordance with Recommendations for Cardiac Chamber Quantification by Echocardiography in Adults: An Update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging .LV diastolic function was measured using pulse Doppler from the apical 4-chamber view  and using tissue Doppler imaging . To calculate the ratio E/e’ the average value of the septal and lateral mitral annulus velocities was used.E’lat was not measured in 5 patients, and in this group E’ med was used to calculate the E/E’ ratio. E’med value above and equal to 7 and E’lat above and equal to 10 were considered normal.E/E’ ratio of less than 10 was considered the cut-off for normal. The value of the E/A ratio in the range of 0.8 to 2 is considered normal, and values below 0.8 and above 2 were interpreted in the context of other parameters, including age.These pulse and tissue Doppler parameters were evaluated and interpreted in accordance with Recommendations for the Evaluation of Left Ventricular Diastolic Function by Echocardiography: An Update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging .The images for GLS measurement were stored for later offline analysis using dedicated software . We used the automated function imaging (AFI) application to evaluate GLS.The analysis involved ECG-gated digital images in four-, three-, and two-chamber apical views, and a high temporal resolution of 50–60 frames per second was obtained to assure acoustic-marker tracking. The LV walls were divided into six segments in each apical view, and strain values were assessed for each LV segment. Only the images with appropriate tracking in all the myocardial segments were used in the analysis. GLS was the average of the values that were obtained for three apical views. GLS is a negative percentage number and indicates fiber shortening. In accordance with the guidelines, a value of GLS of –20% or lower (i.e. more negative) was taken as normal in our study .Carotid Doppler ultrasound was performed using Vivid 9 device . A linear transducer with a frequency of 9.5 MHz was used (GE 11L). The measurements of the carotid intima media thickness (CIMT) were obtained 5-10 mm from the bifurcation around the posterior wall. To optimize the image zoom was used. Color Doppler and pulse Doppler ultrasonography have also been used. CIMT values ≤0.9 mm were considered normal, and CIMT values ≥ 1.5mm were considered as atherosclerosis plaque. Intermediate values (1.0-1.4mm) in our study were classified as abnormal. This interpretation is in accordance with:1. Recommendations for the Assessment of Carotid Arterial Plaque by Ultrasound for the Characterization of Atherosclerosis and Evaluation of Cardiovascular Risk: From the American Society of Echocardiography .2. Standards of ultrasound examinations of the Polish Ultrasound Society - update. Examination of the intracranial sections of the carotid and vertebral arteries .The data were statistically analyzed using STATISTICA 13 software.Median and interquartile range (IQR) (25% - 75%) were estimated for numerical variables, while counts (n) and percentages (%) of the occurrence of items for categorical variables.Mann-Whitney U test was used to compare numerical variables between two categories of categorical characteristics. Kruskal-Wallis H test was used to compare numerical variables between three categories of categorical characteristics. Fisher exact test was used to compare categorical variables between categories of categorical characteristics. Wilcoxon signed ranks test was used to compare numerical variables between baseline and after 3 and 6 months of SRL treatment, and 3 months after TSS. Scatter plots and Pearson’s correlation coefficient were used to correlate numerical variables between each other. The missing data were omitted in all the analyses. The significance level was assumed at 0.05.The median age of 32 enrolled patients with newly diagnosed acromegaly was 52.5 (IQR 42-60) years and there were 16 (50%) males. The median duration of symptoms until the diagnosis of acromegaly was 10 (IQR 5-10) years. Seventeen patients (53.13%) had hypertension at acromegaly diagnosis and the majority (82.3%) of them were treated either with angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB). Only 18.75% of the patients had normal glucose tolerance, 21.88% had diabetes and 59.38% - prediabetes. Dyslipidemia was present in 37.5% of acromegalic patients and 25% had carotid artery atherosclerosis diagnosed by Doppler ultrasonography. None of the patients with confirmed atherosclerosis had significant stenoses above 50% of the vessel lumen. Seven females (46.7%) were considered to be eugonadal because they had regular menstrual cycles and/or conceived spontaneously and had normal FSH/LH and estradiol concentrations at diagnosis while 9 females (56.3%) were considered hypogonadal either due to menopause or due to gonadotroph deficiency.2, p=0.006 and 2.2 (2.1-2.3) vs. 1.8 (1.7-1.8) m2, p<0.001.There were no statistically significant differences in the occurrence of comorbidities and the duration of symptoms to diagnosis between women and men. Men had statistically significant higher BMI and BSA compared to women: 29.0 (IQR 27.4-30.5) vs. 26.0 (21.8-27.7) kg/mThe median baseline GH concentration was 9.1 (IQR 3.2-21.9) ng/mL, nadir GH on OGTT was 4.9 (IQR 1.9-21.1) ng/mL and IGF-1 concentration was 3.2 (IQR 2.3-4.3) x ULN for sex and age. Men had significantly higher biochemical activity of acromegaly compared to women measured by IGF-1 concentration: 3.9 (IQR 3.0-4.7) x ULN vs. 2.8 (IQR 2.1-3.3) x ULN, p=0.027. The detailed baseline clinical and biochemical characteristics of the studied group are presented in After preoperative SRL treatment the median fasting GH concentration decreased significantly after 3 months since the initiation of SRL from 9.1 (IQR 3.2-21.9) ng/mL to 1.8 (IQR 0.9-5.2) ng/mL, p<0.001. Longer treatment with SRL did not result in further significant decrease in GH concentration (p=0.836). However, a significant further decrease in GH concentration was reached after TSS: from 1.2 (IQR 0.8-2.1) ng/mL after 6 months SRL treatment to 0.9 (IQR 0.3-2.4) ng/mL after TSS, p=0.016 (The baseline median IGF-1 concentration was 3.2 (IQR 2.3-4.3) x ULN and significantly decreased 3 months after SRL initiation to 1.5 (IQR 1.1-2.5) x ULN, p<0.001. Longer treatment with SRL did not lead to further decrease in IGF-1 concentration (p=0.620). Biochemical control after 6 months of SRL treatment was achieved in 8 out of 31 patients (25.81%). A significant decrease in IGF-1 levels compared to IGF-1 levels on SRL treatment was observed 3 months after TSS: from 1.5 (IQR 1.2-2.5) x ULN after 3 months SRL treatment to 1.3 (IQR 1.0-1.6) x ULN after TSS, p=0.003 . Females had significantly lower IGF-1 levels not only before treatment but also after 6 months of SRL and 3 months after TSS compared to males Figure 2The detailed characteristics of biochemical parameters during treatment for each patient is presented in 2 in males and 44.5mL/m2 in females, LVESVi 23.4mL/m2 in males, and 16.7mL/m2 in females.The majority of patients had normal end diastolic and end systolic LV volumes. Only 9.38% of patients had increased end diastolic volume and 12.5% had increased end systolic volume. The median values of these parameters were within normal limits: LVEDVi 56.4mL/m2 in males and 94g/m2 in females.46.88% of patients  had abnormal LVMi, however the median values of LVMi remained normal in both sex groups: 99g/m2. Interestingly, in the group of women this value was higher compared to men (43mL/m2 vs. 38.5mL/m2), but the difference was not statistically significant (p=0.3). More than 50% of the patients had abnormal diastolic function assessed by TDI; the median E’med was 6 cm/s and E’lat was 9 cm/s, these values were mildly lower than normal, and the median E/E’ ratio was 10 and it remained within the ULN. Most patients (68.75%) had normal mitral inflow as assessed by pulse Doppler; the median E/A ratio was 0.9 and it was normal. All patients included in the study had normal EF, the median EF was 64% . The baseline echocardiography parameters are presented in Most patients  had abnormal LAVi and the median was 41.8mL/mAt baseline 50% of newly diagnosed patients with acromegaly had abnormal GLS, i.e., higher than -20%. The majority of patients with abnormal GLS constituted men (62.5% vs. 37.5%). GLS significantly improved after 3 months of SRL treatment comparing to baseline: -20.4% vs. -20.0%, respectively, p=0.045. There was no further change in GLS after 6 months of SRL therapy and no significant change after TSS. The number of patients with abnormal GLS decreased at the end of the study to 43%. The change in GLS during acromegaly treatment is presented in We found a significant positive correlation of baseline GLS with BMI  and BSA . It means that the higher the patient’s BMI and BSA was, the worse their GLS was, on average. We did not find any statistical correlation of baseline GLS with age, disease duration, arterial hypertension, diabetes status or disease activity at baseline (p>0.05) Table 3.There was also a strong positive correlation between GLS and IGF-1 levels after TSS  Table 3.We found a significant negative correlation between baseline GLS and LAVi: r= -0.354, p=0.047 Table 4.2 and 94g/m2, respectively, though almost 47% of patients had LVH. In the studies available in the literature, where abnormal GLS in acromegaly patients was found, the LVMi values were definitely higher than in our group of patients  and that was consistent with subclinical systolic dysfunction . The betpatients , 21.2 vs. 29 kg/m2, p<0.001 and 1.8 m2 vs. 2.2 m2, p<0.001, respectively and a strong positive correlation between these parameters and GLS.Another reasons that could explain the normal median GLS result and normal median LVMi were possibly the good hypertension control and the cardioprotective effect of novel antihypertensive drugs which has been proven in many studies –29. In oWomen with acromegaly, like women in the general population, have better GLS compared to men . SimilarThese results are surprising especially when we realize that women with acromegaly, but also with other cardiovascular diseases have a worse prognosis and shorter life expectancy . A KoreaIn the light of the previous data on healthy subjects and our results, it may be necessary to redefine and differentiate the cut-off points for women and men for the GLS parameter, similarly to the different normal ranges used for other echocardiographic parameters.Pierre et al. wrote that it was necessary, because a woman’s heart differed not only in size but above all, it had a different microstructural architecture .The female heart has a larger EF and beats at a faster rate but generates a smaller cardiac output. It has a lower blood pressure but produces universally larger contractile strains .The sex differences in cardiac form and function are complex, important, and obvious, and can’t be ignored. When we use similar diagnostic criteria for female and male hearts, cardiac diseases in women can be overlooked, diagnosed later and with more severe symptoms than in men. This should be applied also to females with acromegaly to improve their increased cardiovascular mortality compared to males.To the best of our knowledge this is the first prospective study to evaluate the effect of acromegaly treatment on GLS value, not only the effect of SRL treatment but also the effect of TSS. Silva et al. and Bogazzi et al. in prospective studies evaluated patients with acromegaly before and after 12 months of octreotide and 6 months of lanreotide treatment , 39. HowIn the above-mentioned studies, echocardiography and cardiac magnetic resonance imaging (CMRI) were used to assess changes in the patients’ heart. Silva et al. in a group of 40 patients  did not find any clinically relevant differences in cardiac variables after 12 months of acromegaly treatment. Interestingly, only 5% of patients had LV hypertrophy features in CMRI versus 31% in echocardiography . In turnHypersecretion of GH appears to have a direct effect on the heart and is associated with a specific cardiomyopathy that results in structural and functional abnormalities due to interstitial fibrosis and edema. It is well known that GH affects water balance, and that GH excess increases myocardial water content. Gouya et al. showed that increased myocardial T2 relaxation time in CMRI which indicates myocardial edema can be normalized soon after effective treatment (10 days for seven patients) and is significantly correlated with successful reduction of GH and IGF-1 levels . We showThe beneficial effect of acromegaly treatment could explain the discrepancies in GLS in recent studies, where STE was performed in patients who previously underwent TSS and were treated with SRL. Volschan et al. and Gadelha et al. did not find a significant differences in GLS between acromegaly patients and control groups. However, 62.2% of the patients enrolled to the Volschan et al. study were during acromegaly treatment, and 80% of the patients in Gadelha et al. study had a history of TSS and 72% were using medical therapy , 20. In Almost 80% of our patients had an enlarged LA with mild diastolic dysfunction (in about 50% of patients) and with LVH occurring in less than 50% of patients, however the mean LVMi was normal in both male and female groups. It is difficult to explain such changes only by an increase in the afterload. Some similarities can be found with an athlete’s heart in which atrial remodeling is a physiological adaptation to volume overload allowing for greater volume delivery and increased cardiac output .In patients with acromegaly, volume overload may result from the action of GH leading to water and sodium retention . When weThe LA has been incorrectly perceived as a simple transport chamber for many years.Currently, its hemodynamic function is known in three integrated phases:1. reservoir 2. conduit and 3. booster-pump. Similarly to the LV, the Frank-Starling mechanism works in the LA. The increase in blood volume stretches the myocardial fibers, causing the cardiac muscle to contract more forcefully with increase in its mechanical performance .In our study, we showed a negative correlation between LAVi and GLS. It may indicate that the dynamic and interactive relationship between LA and LV in the heart of an acromegaly patient is similar to that of an athlete’s heart. Nevertheless, atrial fiber shortening, and contractility begin to fail with progressive dilation of LA. It means the threshold fiber length has been reached and further enlargement will only result in a decline of atrial function . RegardlTherefore, people with the so-called healthy heart and enlarged LA, like athletes, should be periodically examined using the STE method to evaluate both the LV and the LA  and theyWe expect all our patients to improve or at least maintain their results in the further follow up, and we are particularly interested in the group of patients with abnormal GLS values, whether they will maintain a normal EF after 5 years of follow-up.To our knowledge, this is the first prospective study using GLS as a marker to assess the impact of a comprehensive medical and surgical treatment on LV function in patients with acromegaly, taking into account sex differences. Half of the newly diagnosed patients with acromegaly presented subclinical LV systolic dysfunction assessed by STE. However, the beneficial effect of acromegaly treatment on the LV systolic function was noticeable already after 3 months of SRL therapy, especially in females who had better GLS values compared to males during medical treatment. Surgical remission should always be aimed at, because surgical remission leads to better systolic function assessed by STE in acromegaly patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Bioethics Committee at The Centre of Postgraduate Medical Education, Warsaw, Poland. The patients/participants provided their written informed consent to participate in this study.AP-G and MS-B designed the study, enrolled the patients, analyzed the data, and wrote the manuscript. MS-B and IC-O were involved in managing the patients. AP-G performed all 2D-Echocardiography and STE studies. GZ performed pituitary surgeries. IC-O, WZ, MD, and WK critically revised the manuscript. DR performed the statistical analysis. All authors approved the final version. All authors contributed to the article.


Aggression and negative behaviours are used to be present in individuals with bipolar disorder, who are sensitive to life events. Thus, many studies investigated the emergence of impulsivity and aggression in the developmental process and revealed its relationship with childhood adversities.The aim of this study was to determine the relationship between childhood trauma and aggressive behaviour in euthymic patients with bipolar disorder.It was a cross-sectional descriptive and analytical study involving patients diagnosed with bipolar disorder and followed in the psychiatric department at the University Hospital ofSfax (Tunisia).All subjects completed the Childhood trauma questionnaire (CTQ) and the Buss–Perry Aggression Scale (BPAS). Euthymiawas defined as a score on the Montgomery-Åsberg Depression Rating Scale (MADRS) not higher than 14 and by a score on theYoung Mania Rating Scale (YMRS)not higher than seven.We included 35 patients. Their mean age was 46.69 ± 12.01 years with a sex ratio (M/F) =0.45. Most of them lived in urban areas (91.42%) and had a moderate socioeconomic level(88.57%).The most frequent trauma type was physical neglect with 74.28%, followed by emotional abuse (42.85%), emotional neglect (42.85%), physical abuse (37.14%) and sexual abuse (31.42%).The mean score of CTQ was 58.57 ±9.51. Theaverage total score of BPAS was 82.26 ±14.57.The mean scores of subscales of BPAS were 25.49±4.59 for physical aggression, 13.74±3.51for verbal aggression, 19.14±6.22 for anger and 23.89±5.57 for hostility.A statistically significant and positive correlation was determined between CTQ and BPAS (p=0.011). The score of BPAS was significantly correlated with physical abuse (p=0.003) and physical neglect (p=0.014).The relationship between CTQ and BGHA scores suggests the possibility that childhood trauma may be one determinant of aggression in patients with bipolar disorder. Considering the childhood trauma history in the evaluation of these patients may prevent their aggression and thus their psychosocial functioning.None Declared


Spodoptera frugiperda (Lepidoptera: Noctuidae), is a polyphagous pest highly damaging to maize and other food crops in Africa, particularly in Côte d’Ivoire. Chemical pesticides not only have often proved to be unsuccessful, but cause adverse effects on the environment and human health; therefore, entomopathogenic fungi could represent an alternative biocontrol solution. Against this background, fungi were isolated from soil samples collected in maize fields in three regions of Côte d’Ivoire, by the methods of soil dilution and baiting with Galleria mellonella. The resulting 86 fungal isolates were phenotypically and genetically identified. The pathogenicity of seven isolates of Metarhizium spp., three isolates of Beauveria bassiana and two isolates of Trichoderma sp. was evaluated on fifth instar larvae (L5) of G. mellonella. Larval mortality rates and the median lethal time (LT50) were determined seven days after inoculation for each of these selected isolates. The median lethal concentration (LC50) was determined for a selection of isolates. Beauveria bassiana isolate A214b was the most effective, causing 100% mortality, with an LT50 of 2.64 days and an LC50 of 1.12 × 104 conidia mL−1. Two other promising isolates, A211 and A214a, belonging to B. bassiana, caused 100% mortality with LT50 values of 3.44 and 4.04 days, respectively. Mortality caused by Metarhizium isolates varied from 65.38% to 100%, with Metarhizium anisopliae isolate T331 causing 100% mortality with an LT50 of 3.08 days at an LC50 of 3.33 × 104 conidia mL−1. Trichoderma sp. isolates were the least pathogenic ones. Beauveria bassiana and Metarhizium isolates showed to be virulent against the model Lepidopteran G. mellonella and will be tested on S. frugiperda.The fall armyworm,  Spodoptera frugiperda J.E. Smith) is native to tropical and subtropical regions of America. This pest feeds on the leaves and stems of more than 353 plant species [S. frugiperda has a very high reproductive rate, considering that a female can lay up to 1000 eggs during its life [The fall armyworm or American corn moth  were tested in vitro to determine their efficacy on second instar larvae of S. frugiperda [Trichoderma or Penicillium, which are less studied from an entomopathogenic point of view, but might be considered as well [Chemical control used against this pest proved to be ineffective. In addition, the frequent use of these pesticides causes problems such as the development of resistance in the pest population , the redugiperda . However as well ,14.Galleria mellonella, in a Swiss laboratory, where it is not allowed to import S. frugiperda. Galleria mellonella is easily available and lives long enough to perform such tests. It also seems to be a good insect model for observing the virulence of a microorganism against Lepidoptera [S. frugiperda larvae on maize, in Côte d’Ivoire.Despite the interest aroused by the use of entomopathogenic fungi, very few studies have been carried out in general on these fungi in Western Africa and particularly in Côte d’Ivoire. This study was carried out to generate data and enrich knowledge in this domain. To achieve this, the presence of entomopathogenic fungi in some maize fields in Côte d’Ivoire was assessed. In addition, a pre-screening was carried out on idoptera . The nexv/v). Soil samples were kept in transparent plastic bags with GPS coordinates of the sampling locations . In each locality, 5 fields were prospected and 20 samples of 100 g of soil were taken, using a trowel, from the superficial layer (5–10 cm depth) at the foot of randomly selected maize plants according to the method of Thaochan and Sausa-Ard . To avoiions see . These sG. mellonella as a bait, developed by Zimmermann [Two methods were used for the isolation of fungi, the soil dilution method  and the mmermann  and adapmmermann .−1 to 10−7 starting from a stock solution obtained after dissolving 1 g of soil in 9 mL of distilled water. Then, 100 μL of the dilutions from 10−2 to 10−7 were spread on potato glucose agar  in Petri plates in three replicates before all Petri dishes were sealed and incubated at 25 °C in the laboratory. Two days after inoculation, the fungal colonies that appeared were subcultured separately on new PGA dishes in order to obtain pure cultures.Serial soil dilution suspensions were prepared from 102 Microbox, Deinze, Belgium) and moistened with 1 mL sterile distilled water; five fifth-instar (L5) larvae of G. mellonella were then added in each box. Two control boxes only containing five larvae were also set in parallel. Then, all trays were stored in an incubator Firlabo Bio Concept  at room temperature (25 °C) in the dark. The trays were turned over every day to assure maximum soil–larvae contact. Then, a daily control of the larvae was carried out for three weeks in order to remove the dead larvae from the soil.As for the insect baiting method, the composite soil samples were each subdivided into three subsamples of 50 g. Each subsample was placed into sterile 500 mL plastic boxes , moistened with 1 mL distilled water. Petri dishes were incubated at 25 °C in the dark. The larvae on which fungal sporulation occurred were then removed with sterile forceps and individually deposited on Petri dishes containing PGA amended with 200 mg/L of ampicillin (Carl Roth) 200 mg/L chlortetracycline (Carl Roth), which were then sealed with ParafilmTM and incubated at 25 °C [Dead larvae were disinfected by dipping in 70% ethanol for 10 s, then in 1% sodium hypochlorite for 3 min, followed by rinsing twice in sterile distilled water for 1 min. After disinfection, each larva was placed into a 9 cm diameter Petri dish on a 9 cm diameter cellulose filter . Successfully amplified PCR were purified with the kit Wizard® SV Gel and PCR Clean-Up System  and sent to Microsynth AG  for Sanger Sequencing. Sequences were edited with FinchTV v. 1.4  and identified by searching the NCBI nucleotide database using BLAST .The collected isolates were first sorted and classified by morphotypes; then one or two isolates were selected for species identification and cultured in potato glucose broth  for 4 to 7 days, with gentle shaking. DNA was extracted following a published protocol . The PCR6 conidia mL−1 was prepared by diluting a stock conidial suspension (5 × 108 conidia mL−1) in a solution of 10% phosphate buffered saline  and 0.03% Tween 20 (Carl Roth) in sterile distilled water. Then, 30 L5 larvae of G. mellonella were soaked in 100 mL of the prepared conidial suspension for 10 s. Thirty other larvae representing the controls were also soaked in 100 mL of the diluting solution. For each modality, 10 treated larvae were placed in 500 mL plastic trays (Sac O2 Microbox) and incubated in a climatic chamber  at 25 °C, 60% relative humidity in the dark for 7 days. No food was provided because the L5 stage of G. mellonella is the last larval stage before pupation. Larval mortality was monitored daily. Koch’s postulates were verified by performing re-isolation of the entomopathogenic strains from the dead larvae. For this purpose, remains of G. mellonella were sterilized and incubated according to the baiting method. Three isolates , identified as best performers in the first test, were selected for determination of LC50. At this end, conidia suspensions with concentrations of 5 × 103, 5 × 104, 5 × 105 and 5 × 106 conidia mL−1 were prepared and applied to the larvae as described previously.Conidia were applied to the larvae following the immersion method ,23. A co50) and lethal concentration (LC50) were determined using the log/probit methods of analysis [The percentage of larval mortality was calculated using the formula of Abbott . Data weanalysis .https://www.ncbi.nlm.nih.gov/nuccore/; accessed on 2 April 2022) under the accessions numbers to ON121940 to ON121983 . Larval mortality rates varied from 3.33% to 100%. Beauveria bassiana isolates  and M. anisopliae isolate T331 induced 100% mortality of G. mellonella larvae. Metarhizium anisopliae isolate T35 and Metarhizium sp. isolates  caused 93.33%, 90%; 90%; 86.67%; 80% and 70% mortality, respectively. The mortality rates were 16.33% for the T. longibrachiatum isolate Ko4 and 3.33% for T. asperellum isolate Ou5 .The results obtained showed that all fungi tested in this study except late Ou5 . StatistTrichoderma isolates , B. bassiana isolate A214b quickly developed its pathogenicity, with an LT50 of 2.64 days followed by M. anisopliae isolate T331 and B. bassiana isolate A211. The LT50 values of these latter were respectively 3.08 days and 3.44 days. The LT50 of B. bassiana isolate A214a and Metarhizium sp. isolates T34, T35, T121, T132, T141 and T313 were respectively 4.01, 3.55, 5.70, 3.56, 6.41, 5.20 and 6.61 days. No Trichoderma sp. used in this study yielded a 50% mortality of the G. mellonella population. The LC50 results showed that the rate of mortality increased with the concentration. The lowest mortality percentage (10%) was recorded for larvae inoculated with Metarhizium sp. isolate T34 at a concentration of 5 × 103 conidia mL−1. This rate increased when larvae were exposed to concentrations 5 × 104, 5 × 105 and 5 × 106 conidia mL−1, with mortality rates of 25%, 71%, 42% and 88.57%, respectively. LC50 of 1.12 × 104 and 3.47 × 104 conidia mL−1 were obtained for isolates A214b and T331. Isolate T34 had an LC50 of 2.19 × 105 conidia mL−1  used for the dilution. The difference could also be explained by the sensitivity of G. mellonella to entomopathogenic fungi. This baiting method would therefore be the best way to select for entomopathogenic fungi. According to Meyling [G. mellonella is a very sensitive detection method for selectively isolating entomopathogenic fungi.This study enabled us to isolate a wide range of fungi, regardless of the method used. The method of isolation by dilution made it possible to isolate a greater fungal diversity. The majority of these fungi were saprophytic with the genera and Sera , who isom et al. . Althouga et al. . Our resa et al. , who iso Meyling , who comM. anisopliae and B. bassiana have been effective on G. mellonella larvae. Similar results were also obtained by Saleh et al. [B. bassiana and M. anisopliae. The same rate of mortality (100%) was obtained [G. mellonella larvae inoculated with M. anisopliae isolate ARSEF 5520 at concentration of 3.6 × 106 conidia mL−1. Entomopathogenic fungi B. bassiana and Metarhizium sp. are capable of infecting different species belonging to the order Lepidoptera. Indeed, efficacy tests on second instar larvae of S. frugiperda using the B. bassiana isolate BCMU6 showed a 91.67% mortality rate 12 days after inoculation at a concentration of 108 conidia mL−1 [M. anisopliae and B. bassiana on fourth instar larvae of Spodoptera litura. Mortality rates of 100% were achieved with two B. bassiana isolates  and two M. anisopliae isolates  [Fungi h et al. , who obtobtained  on G. media mL−1 . Similar8, KZ-2) .Trichoderma isolates, which could not be recovered from dead larvae. This could be explained by the fact that Trichoderma sp. isolates would not be responsible for the death of larvae. The shortest LT50 of 2.64 days was obtained with B. bassiana strain A214b. The isolates M. anisopliae T331 and B. bassiana A211 had an LT50 of 3.08 days and 3.44 days, respectively. These results are similar to those of Oreste et al. [B. bassiana AL1 and ALB55. The LT50 and LC50 values showed that some fungi, in particular B. bassiana isolate A214b, M. anisopliae isolate T331 and Metarhizium sp. isolate T34, are highly virulent against G. mellonella larvae. These results could be explained by the viability/concentration of the conidia and the action time of the fungi. According to several authors [In the present study, the verification of Koch’s postulates showed that larval mortality was caused by the strains tested, with the exception e et al. , who obt authors ,36, the This study isolated entomopathogenic fungi from soil samples collected in maize fields in Côte d’Ivoire. The method of isolation by baiting proved to be the most effective.G. mellonella with the exception of two Trichoderma isolates, which caused very low mortality rates. Isolated B. bassiana, M. anisopliae and Metarhizium sp. caused larval mortality rates greater than 50%, and up to 100%. The determination of their LT50 and LC50 has shown that these fungi have insecticidal potential and virulence against G. mellonella. These fungi will therefore be assayed against S. frugiperda in vitro, in greenhouse and field tests, with the hope to soon deliver a biological control agent against the fall armyworm.All the tested fungi were pathogenic to


Enterovirus 71(EV71)-associated hand, foot and mouth disease (HFMD) decreased dramatically in Beijing from 2009 to 2019. This study was to investigate the epidemiological characteristics, evolutionary dynamics, geographic diffusion pathway, and other features of EV71 in Beijing, China.We conducted a retrospective study of EV71-associated HFMD and its causative agent in Beijing, China, from 2009 to 2019. Phylogenetic and phylogeographic methods based on the EV71 genome were used to determine the evolution features, origin, and spatiotemporal dynamics. Positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. Bayesian birth-death skyline model was used to estimate the effective reproductive number (Re).− 3 substitutions per site per year. The genetic diversity of EV71 first increased and peaked in 2012 and then decreased with fluctuations. The time to the most recent common ancestor (TMRCA) of EV71 in Beijing was estimated around 2003 when the EV71 strains were transmitted to Beijing from east China. Beijing played a crucial role in seeding EV71 to central China as well. Two residues  under positive selection were detected from both the VP1 dataset and the P1 dataset. They were embedded within the loop of the VP1 capsid and were exposed externally. Mean Re estimate of EV71 in Beijing was about 1.007.EV71-associated HFMD decreased greatly in Beijing. From 2009 to 2019, EV71 strains prevalent in Beijing shared high homology in each gene segment and evolved with a rate of 4.99*10In recent years, EV71 was not the primary causative agent of HFMD in Beijing. The low Re estimate of EV71 in Beijing implied that strategies for preventing and controlling HFMD were performed effectively. Beijing and east China played a crucial role in disseminating EV71 to other regions in China.The online version contains supplementary material available at 10.1186/s12985-023-02028-9. Hand, foot and mouth disease (HFMD) is a widespread infectious disease characterized by rashes on the hands, feet, mouth, and occasionally buttocks with or without fever. Children under 5 years of age are the most susceptible population. In the past few decades, HFMD has been reported worldwide. HFMD has a significant disease burden in China and attracted considerable attention. From 2009 to 2019, 21.97 million HFMD cases, resulting in 3,561 deaths, were reported to the national surveillance system in China [HFMD is caused by a human enterovirus, among which human enterovirus 71 (EV71), Coxsackievirus A16 (CVA16), and Coxsackievirus A6 (CVA6) were the most prevalent pathogens . IllnessAccording to the VP1 region, EV71 is classified into genotypes A, B, C, D, E, and F. Genotypes B and C are further divided into subgenotypes B1–B5 and C1–C5 –5. SinceIn recent years, the etiology surveillance system in Beijing, and some other provinces in China found that the positive proportion of EV71-associated HFMD decreased dramatically. This phenomenon has attracted considerable attention. However, limited studies were carried out to investigate the molecular epidemiology characteristics of EV71. In this study, we described the epidemiology features and pathogen spectrum of HFMD. We conducted genetic analyses to investigate the phylogenetic characteristics, geographic diffusion pathway, and phylodynamic features of EV71 strains in Beijing from 2009 to 2019.Clinicians and hospitals have been required to report clinical cases of HFMD to National Notifiable Infectious Disease Surveillance System within 24 h of diagnosis since 2008 in China . More thEV, EV71, CVA16, and CVA6 were identified with real-time RT-PCR Kits  according to the manufacturer’s instructions . As previously described, complete nucleotide sequences of EV71 VP1 genes were amplified using specific primers .For EV71 sequences in this study, we reduced the computational load by choosing one closely related sequence that represented sequences with a pairwise distance smaller than 1% collected at the same time. For EV71 strains in mainland China, genome sequences were obtained from the NCBI website (as of 20 June 2021). Sequences with unknown dates or regions or many bases missing from the ORF, containing many N bases, and sequences not EV71 were excluded. Before analysis, we constructed a neighbor-joining phylogenetic tree and selected representative strains from each tree branch. Representative strains should cover more provinces of China and each cluster of the phylogenetic trees. Altogether, three datasets were prepared. Dataset one comprised 86 EV71 genome sequences from Beijing during 2009–2019 . Dataset two included 156 EV71 VP1 sequences obtained in Beijing. Dataset three comprised 353 EV71 VP1 sequences which included 76 in this study and 277 sequences from 29 provinces in China (Supplementary Table 3).Based on TreeTime software, root-to-tip regression analysis was performed to test the temporal signal of the datasets . PhylogeWe performed a Bayesian evolutionary analysis based on 156 EV71 VP1 in Beijing to estimate the rate of evolution, and TMRCA using Markov chain Monte Carlo (MCMC) runs of 200 million generations with BEAST v1.8.4  under a To evaluate phylogenetic correlations between different regions, phylogenetically based Association Index (AI) statistic, Parsimony Score (PS) statistic, and Monophyletic Clade (MC) statistics for discrete-trait were estimated using BaTS v0.9 beta . The AI The MEME method was used to find the key selection pressure sites on the VP1 and P1 coding regions of EV71 in Beijing , 16. TheBayesian birth-death skyline model  implemenThe epidemiology and pathogen distribution of HFMD cases in Beijing from 2009 to 2019 is shown in Table Before 2013, EV71 and CVA16 were the two major pathogens of HFMD. EV71 accounted for 41.6% of HFMD cases and was the most common pathogen associated with HFMD in 2010. Then EV71 positive proportion declined with fluctuations. In 2013, around 26% of samples were EV71 positive. After a rebound in 2014, the positive proportion decreased to 12.3% in 2015. In 2018 and 2019, EV71 was responsible for only 1.6% and 1.1% HFMD, respectively .A total of 86 EV71 genome sequences were obtained in this study. The nucleotide homology of the genomes of all EV71 was 91.6 ~ 99.9%. The nucleotide divergence of overall mean distance of VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C and 3D regions among these strains were 0.043%, 0.049%, 0.054%, 0.051%, 0.052%, 0.066%, 0.049%, 0.060%, 0.089%, 0.042% and 0.051%, respectively (Supplementary Table 4). All 86 EV71 belonged to the C4a genotype. ML phylogenetic trees constructed based on each gene of 86 EV71 genomes showed that VP4, VP2, VP3, and VP1 were located in the same clade with EV71 . At the same time, 2A, 2B, 2C, 3AB, 3C, and 3D were mainly clustered with CVA4 , CVA14 and CVA16 . According to the topological structure of ML phylogenetic trees constructed above, EV71 strains in Beijing were genetically related, shared high homology.According to the phylogenetic characteristics and nucleotide differences of the 3D region, global EV71 is divided into 17 evolutionary lineages (A- Q) . In this− 3  substitutions/site/year. Via the estimated molecular clock, the common ancestor of genogroup C4a in Beijing was dated to around 2003.20 . Maximum clade credibility (MCC) tree constructed using 156 VP1 sequences showed that in around 2006–2007, two clades of VP1 formed on the MCC tree  and parsimony score (PS) value with a significance level < 0.001 showed that EV71 VP1 sequences of partial regions were more phylogenetically clustered by region (Supplementary Table 5). The maximum monophyletic clade (MC) of Beijing (p < 0.01), the central (p < 0.01), south (p < 0.01), and west region (p = 0.04) presented high values, which indicated that the geographic structure of EV71 was significant when the strains were grouped by geographical origin.A past spatial transmission pattern inferred by six regions in mainland China was constructed Fig. A. The reResults inferred through the Markov jump method reflected that the east region dominates the out-migration for EV71, which was followed by Beijing. The state counts of inward migration were much larger than that of outward migration in Beijing, the central, south, west, and north regions of mainland China Fig. B. BeijinThe mixed effects model of evolution (MEME) and single likelihood ancestor counting (SLAC) methods were used to investigate the role of natural selection pressure in EV71 in Beijing (Supplementary Table 6). Individual sites under positive selection were identified. Four key sites  were observed from the VP1 sequence dataset. These four amino acid sites were embedded within the loop of the VP1 capsid and were exposed externally , ranged from 0.923  to 1.185  in Beijing. Re estimates of EV71 experienced a typical phylodynamic characterized by a zigzag fluctuation Fig. . The higEV71 was the leading pathogen responsible for severe HFMD cases, especially death. During 2009–2019, EV71-associated HFMD decreased greatly in Beijing and in many other provinces and cities . For ourGenomic characteristics of EV71 showed that each gene segment shared a high homology among the EV71 sequences in this study, which implied that EV71 prevalent in Beijing shared a common source. 3D region of enterovirus is more susceptible to recombination than any other part . ML phylThe relative genetic diversity of the EV71 VP1 sequences first increased and peaked in 2012, which corresponded with the high positive proportion of EV71-associated HFMD in Beijing. With the non-EV71, non-CVA16 EV became the dominant agent, and EV71 genetic diversity declined with fluctuations from 2013 to 2016. After the EV71 vaccine was introduced in China in 2016, genetic diversity declined again. We speculated that it was the vaccine inoculation that prevented children from infection and as a consequence, reduced genetic diversity.Natural selection pressure analysis showed that the dN/dS value of VP1 and P1 from 2017 to 2019 was smaller than 2009–2016, which implied that both VP1 and P1 had evolved into a relatively stable condition marked by low dN/dS value. Two positive selection sites were obtained from both VP1 and P1 by MEME. VP1-145 is a surface-exposed residue that is located within the DE loop. It has been confirmed that VP1-145 influenced the use of attachment receptors and the three-dimensional structure of the whole virion . AlthougIn this study, the phylogeographic results revealed east China being the primary source sink for the EV71 dispersion all over the country. This is consistent with Zhang’s result . East ChThe epidemic spread of EV71 displayed low Re estimates during the sampling period time, which was close to some previous research , 27, sugThere are limitations to this study. Owing to the severe impact on the surveillance system by the COVID-19 pandemic, the EV71 surveillance result from 2020 to 2022 was not included in this study, which could have provided more information about epidemic characteristics. Not enough EV71 sequences in Beijing or some regions in China of some years might bias the analysis result to a certain extent. Mainly analyzing of VP1 might not be able to clarify the overall epidemic characteristics of EV71, but still help us understand the epidemiological characteristics, phylogenetic features, and bayesian phylodynamics of EV71.In conclusion, EV71 was not the major causative agent of HFMD in Beijing in recent years. EV71 prevalent in Beijing from 2009 to 2019, shared high homology in each gene, and were mainly transmitted from east China around 2003. Beijing played an important role in disseminating EV71 to central China. Low Re estimate of EV71 in Beijing implied strategies of prevention and control of HFMD were performed effectively in Beijing.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8Supplementary Material 9Supplementary Material 10Supplementary Material 11Supplementary Material 12


Introduction: Platelet-rich plasma (PRP) has gained increasing popularity in the orthopedic field. There has been still no consensus on PRP preparation technique, thus providing a variety of final PRP products. Different preparation techniques lead to different compositions of PRP, which include platelet concentration, the number of leukocytes, and their subtypes. Here, we studied those compositions of PRP compared to whole blood samples.5/µL (Group 1), between 2 x 105/µL and 2.99 x 105/µL (Group 2), and greater than 2.99 x 105/µL (Group 3). The primary outcome was reported as the platelet concentration in PRP and the ratio of platelet concentration in PRP compared to baseline whole blood samples. The secondary outcome was reported as the ratios of leukocyte, lymphocyte, and neutrophil in PRP compared to the baseline whole blood samples.Methods: There were 335 participants who met the inclusion and exclusion criteria. Each participant underwent a blood drawing process to prepare PRP for their knee osteoarthritis treatment. We categorized baseline platelet concentration in whole blood samples into three groups: less than 2 x 106/µL (6.3 times higher compared to baseline whole blood samples). The mean platelet concentration of PRP in Group 1, Group 2, and Group 3 was 1.08 x 106/µL, 1.38 x 106/µL, and 1.71 x 106/µL, respectively . The platelet concentration ratio of PRP condition to the baseline whole blood was 6.9, 5.8, and 4.2 in Group 1, Group 2, and Group 3, respectively . The average ratio of leukocytes in PRP to whole blood was 1.5. The average ratio of lymphocytes and neutrophils in PRP to whole blood was 2.0 and 0.5, respectively. Results: The average platelet concentration in PRP was 1.26 x 10Conclusion: Different baseline platelet concentrations in whole blood samples provided significantly different platelet concentrations in PRP. The baseline platelet concentration in whole blood also provided an inverse relation to the fold change of the platelet concentration in PRP. Subtypes of leukocytes changed from neutrophil-predominated in the baseline whole blood samples to lymphocyte-predominated in PRP. Platelet-rich plasma (PRP) is an autologous blood derivative prepared from a centrifugation process that concentrates platelet, removes red blood cells, and provides different leukocytes composition depending on preparation techniques . With thDifferent preparation techniques lead to different components of PRP. To our knowledge, there is still no report on the optimal platelet concentration for PRP . Recent The primary outcome of this current study is to evaluate the components of PRP in terms of platelet concentration and the ratio of platelet concentration in PRP compared to the baseline whole blood samples. We hypothesized that different baseline platelet concentration provides different fold change in platelet concentration in PRP. The secondary outcome is to evaluate the ratios of leukocyte, lymphocyte, and neutrophil in PRP compared to the baseline whole blood samples.A cross-sectional analytic study was conducted, after the Institutional Review Board and the Police General Hospital’s Ethics Committee approval , from June 2020 to October 2021 at the Biotechnological Research and Development Centre, Police General Hospital, Bangkok, Thailand. Knee osteoarthritic patients who were eligible for PRP treatment at our institution’s orthopedic clinic were identified. Inclusion criteria were the following: diagnosis of knee osteoarthritis classified as Kellgren and Lawrence (KL) I-IV and platelet concentration in whole blood > 80,000 cells/µL. Exclusion criteria were the following: history of using non-steroidal anti-inflammatory drugs (NSAIDs) within five days of blood drawn or taking anticoagulant or anti-aggregate drugs. The rationale behind these criteria was to study the hematologic profile of the representative groups that are normally treated with PRP treatment. The hematologic profile may be different from those of healthy participants typical in previous studies.A total of 335 participants met the criteria. All participants were informed about the study protocol and provided with informed consent.PRP preparation protocol A 30-ml peripheral blood sample for a single knee injection  was collected from each participant. A complete blood count was performed at the initial blood draw. The first 20 ml of blood was divided into two centrifuge tubes (red)  Figure  containiHematological analysisPlatelet concentration , total leukocyte count, lymphocyte percentages, and neutrophil percentages from whole blood and PRP were determined using an automated hematology analyzer  immediately after preparation.Statistical analysis The sample size calculation was calculated for a one-way analysis of variance (ANOVA) test. The effect size was 0.4. The α error and β error were 0.05 and 80%, respectively. The total sample size was 159 (53 in each group). Continuous data were elaborated into mean and standard deviation. Discrete data were elaborated into percentages and proportions. We displaced the data to evaluate the distribution by histogram. If the data were normally distributed, the ANOVA test was used to test the difference in the variance. If the data were not normally distributed, Kruskal-Wallis was used to determine the statistically significant difference. The two-tail test was used. We considered a p-value of < 0.008333 significant for the ratio outcome, and a p-value of < 0.05 significant for non-ratio data. We used Stata software, version 16.0 , for statistical analysis.5/µL . We categorized baseline platelet concentration in whole blood samples into three groups according to the classification proposed by Kon et al. [5/µL (Group 1), between 2 x 105/µL and 2.99 x 105/µL (Group 2), and greater than 2.99 x 105/µL (Group 3). There were 141, 188, and six specimens in Group 1, Group 2, and Group 3, respectively. Baseline characteristics  in each group were not statistically significantly different . However, sex in each group was statistically significantly different  with the male predominant in Group 1 and female predominant in Group 2 and Group 3  were female, and 206 participants (61.49%) were male. The ages of participants ranged from 50 years to 93 years with a mean age of 65 years. The average platelet concentration in whole blood samples was 2.08 x 10n et al. : less th5/µL , 10.8 x 105/µL , 13.8 x 105/µL , and 17.1 x 105  for overall samples, Group 1, Group 2, and Group 3, respectively. Platelet concentrations among the subgroups were significantly different  , 6.9 , 5.8 , and 4.2  for overall samples, groups 1, 2, and 3, respectively. There was a statistically significant difference in the ratio of platelet concentration among the three groups  . The average percentage of lymphocytes in PRP was 54.69% . The average ratio of lymphocytes in PRP to whole blood was 2.0 . The average percentage of neutrophils in PRP was 28.9% . The average ratio of neutrophils in PRP to whole blood was 0.5 . We also performed a subgroup analysis of each parameter according to groups. There was no statistically significant difference in each parameter  from knee osteoarthritic patients.We found that different baseline platelet concentration in whole blood samples provides a statistically significant difference in platelet concentration in PRP. We also observed that baseline platelet concentration in whole blood was inversely correlated to the fold change of platelet concentration in PRP products. We proposed that the platelet concentration of PRP products would reach a plateau level to prevent excessively concentrated PRP. This is supported by Weibrich et al. in 2004, who stated that highly concentrated platelets had an inhibitory effect on osteoblast activity when compared to lower concentrations . FurtherLeukocytes originate from myeloid and lymphoid cells. Myeloid stem cells give rise to neutrophils, eosinophils, basophils, and monocytes. Lymphoid stem cells give rise to lymphocytes, which further divide into B cells, T cells, and natural killer cells . It remaWe used LR-PRP. Buffy coat-based PRP preparation in this study protocol provided LR-PRP in which the leukocyte content in PRP increased 1.5 fold from the baseline whole blood. One of the proposed ideas that favor the use of LR-PRP for orthopedics treatment is “macrophage polarization,” according to which peripheral blood mononuclear cells including lymphocytes, natural killer cells, and monocytes transform into macrophages when merging into the tissue. Tissue macrophages then interact reciprocally with activated T&B lymphocytes. Macrophages phenotypically express themselves in two forms: M1 macrophages, which act mainly as pro-inflammatory cells, and M2 macrophages, which act mainly as anti-inflammatory cells. Macrophages can polarize between these two subtypes according to the microenvironment. M1 macrophages usually function in the early phase of healing while M2 macrophages usually function in the late phase of healing. With these functions, macrophages provide a protective immunological role by promoting angiogenesis through growth factors and cytokines. This theory supports that different components of leukocytes in PRP may play an important role in their clinical efficiency . LymphocNeutrophils generally serve as pro-inflammatory mediators and are thought to promote catabolic effects. They release inflammatory cytokines and matrix metalloproteinases, which lead to collagen degradation and fibrosis ,21-23. TOne of the interesting findings in this study is the change in the majority of the composition of leukocytes in PRP products compared to those in whole blood. In baseline whole blood, neutrophils predominate at 59.6%, whereas lymphocytes constitute only 29.4%. In PRP, the neutrophil population decreased to 29% and lymphocytes predominated at 54.7%. The lymphocyte percentage in PRP increased two-fold from baseline whole blood, whereas the neutrophil percentage in PRP decreased 0.5-fold from the baseline whole blood. These findings correlated with many previous studies that found lymphocytes predominated over neutrophils in platelet-rich products ,22,24. T5/µL in the normal population.There are limitations to this study. First, there was no correlation study between the blood component and the clinical outcome of the patient. With further study, researchers may be able to determine the optimal level of platelet, leukocyte, and subtypes of leukocyte concentration in PRP for the treatment efficacy in specific tissues. Future researchers should study the relationship between subtypes of leukocytes and the level of growth factors and cytokine in PRP with clinical effectiveness. Second, PRP in this current study was prepared from one PRP preparation technique. Different PRP preparations may provide different outcomes on their composition. Third, the automated hematology analyzer used in this study did not provide a monocyte profile, which limited the variety of subtypes of leukocytes. Fourth, we were unable to reach the calculated sample size in Group 3 due to the rarity of platelet concentration higher than 2.99 x 10Our study found that different baseline platelet concentrations in whole blood provided significantly different platelet concentrations in PRP. The baseline platelet concentration in whole blood also provided an inverse relation to the fold change of platelet concentration in PRP. We also found that lymphocytes predominated over neutrophils in PRP.


Dear editor,Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory infections (ALRI) associated to hospitalization in children. In 2019, it was estimated that RSV was related to over 100 000 deaths in children aged 0 to 60 months.This observational cohort study was conducted in compliance with institutional guidelines, approved by the Ethics Committee of Universidade Federal de São Paulo (CEP/UNIFESP n. 4.753.851). Data were obtained from the records of the Clinical Virology Laboratory, in Hospital São Paulo.We investigated all children aged 0 to 12 years old hospitalized presenting respiratory symptoms. For every individual, a nasopharyngeal or a tracheal aspirate was collected. A real‐time polymerase chain reaction (RT‐PCR) was performed to detect RSV infection in the samples; primers and probes are described elsewhere.p < 0.0001). The age group 7 to 11 months represented 11.7% (7/60) of infections, followed by 12 to 23 months 11% (8/73). Age group 24 to 59 months had 8.3% (11/135) and children >60 months presented 4.2% (7/168) of RSV infection. Except for the month of August, the detection of RSV cases was possible throughout the year of 2022. In 2023, most cases occurred in April. The distribution of cases per age group and period are in Figure This analysis included 566 hospitalized children from January 2022 to May 2023 aged 0 to 144 months , 261 girls (46.2%) and 305 boys (53.8%). The overall infection rate for RSV was 12.3% (70/566). The age group 0 to 6 months represented 28.5% (37/130) of positive cases, with the highest infection in 2022–2023, as expected , whereas in 2023, 81.5% of cases were identified as RSV‐A. At least one comorbidity was reported by 51.2% (290/566) of the included children, and 27.1% (19/70) among RSV positive cases. Twelve children under 24 months old presented comorbidities 63.2% (12/19).Our findings reflect the atypical circulation of RSV among hospitalized children during the analyzed period. Moreover, we observed important rates of infection in children older than 24 months, resulting in RSV‐related hospitalization in a group of less impact, as demonstrated in another study conducted in the Southern Hemisphere.This present study highlights that it is crucial to keep the surveillance of RSV infections, particularly in children older than 2 years old with comorbidities; this alongside with an interseason RSV testing could provide a better comprehension of the impact of RSV circulation, seasonality, and association with an increase of hospitalization and disease severity.Gabriela Barbosa: Data curation; formal analysis; software; validation; writing—original draft. Ana Helena Perosa: Data curation; formal analysis; investigation; methodology. Nancy Bellei: Conceptualization; funding acquisition; project administration; resources; visualization; writing—review and editing.The authors have no conflicts of interest to declare.https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/irv.13183.The peer review history for this article is available at This study was conducted in compliance with institutional guidelines, approved by the National Commission for Research Ethics (CEP/UNIFESP n. 4.753.851).