Alternaria solani is the main early blight pathogen parasitizing potato. Here, we analyzed genetic and phenotypic diversity in isolates of Alternaria spp. covering all potato production areas in Serbia. We showed that the four species of Alternaria were found in areas with different distributions of the species. The occurrence of Alternaria spp. was studied by analyzing isolates from symptomatic potato leaves during multiyear sampling. In addition to Alternaria solani, we detected three more large-spored species identified as A. linariae (syn. A. tomatophila), A. protenta, and A. grandis that were involved in early blight disease on naturally infected potato leaves in Serbia. Differentiation of species was supported by phylogeny obtained from the DNA sequences of the GAPDH, calmodulin and Rpb2 genes. Our findings present a new perspective into the population structure of large-spored Alternaria species associated with early blight disease. Within the groups of large-spored Alternaria present in Serbia, evidence of A. protenta at high frequency reveals new insight into the contribution of Alternaria species in early blight disease. This work opens new perspectives for early blight management, while the distribution of different species on the same host suggests that the etiology of disease could depend on crop organization and the presence of other Alternaria hosts in close proximity to potato plants.Early blight is an economically important disease of potato worldwide. Understanding which fungal pathogens are the causal agents of early blight and their distribution on the same host is essential to finding the best strategy for the control of this disease. Previous studies have shown that  Early blight is one of the most important fungal diseases of potato and is spread worldwide in all major potato growing areas. Under favorable weather conditions, during warm and wet seasons, it can cause severe yield losses . The patAlternaria species with large conidia have been reported as causal agents of early blight on potato: A. solani, A. grandis, A. protenta, and A. linariae  is known as an important pathogen causing early blight of tomato  gene  gene , RNA polne Rpb2; , transla Alt a 1 , and anoOPA10-2; .Alternaria spp. present on potato leaves. Therefore, the main objectives for the current study were to: (i) study the occurrence and distribution of early blight disease on potato in Serbia, (ii) identify Alternaria spp. causing agents of early blight on potato in the main production areas of Serbia; (iii) to determine the genetic relationship of Serbian Alternaria spp. isolates with strains identified in different parts of the world.Since there is very little knowledge on early blight disease in Serbia, the aim of this study was to gain insight into the distribution of large-spored Alternaria spp., a 3-year survey was conducted from 2016 to 2018. Potato plants with early blight symptoms were collected from large commercial fields and from a small farmer’s garden. Samples were collected in nine different districts of Serbia: North Bačka, South Bačka, Pomoravlje, Moravica, Raška, Jablanica, Zlatibor, Rasina, and City of Belgrade district. Sixty-one fields in 20 different localities were surveyed , and incubated at 23°C. The morphological characteristics of the isolates were studied by taking a 5 mm mycelial plug from the growing colony margin of a 5-day-old culture and subculturing on V8 medium . The cultures were placed under cool white fluorescent light with 12 h/12 h periods of light/dark and incubated on 23°C for 5 days (To identify the presence and distribution of surveyed . Infecter 5 days .p < 0.05) in the statistical software package SPSS version 20.0 .The growing cultures of all isolates were studied with a protocol described by Alt a1 gene to separate the species A. solani and related species (A. grandis and A. protenta) from A. linariae, and with the primer pair OAtF4/OAtR2, which amplified a 483 bp fragment from the calmodulin-encoding gene of A. linariae (A. grandis and A. solani (including A. protenta), PCR identifications of isolates that amplified a 164 bp fragment with the primer pair OasF7/OasR6 were complemented by RFLP of the calmodulin gene that was amplified using the primer pair CALDF1/CALDR1 (HaeII and RsaI (New England Biolabs) according to Ayad et al., (2017a). RFLP patterns were discriminated by the size of the larger fragment, 420 bp for A. solani and 292 bp for A. grandis. To distinguish between A. solani and A. protenta isolates, sequencing of the Rpb2 locus was performed.For molecular identification, 30 mg of dry weight mycelium was collected from PDA plates and used for DNA extraction according to the manufacturer’s instructions of the DNeasy Plant Mini Kit . Polymerase chain reaction (PCR) was used with the specific primers OAsF7/OAsR6 which amplified a 164 bp fragment from the linariae . PCR was1/CALDR1 . The resRpb2) gene, primer pair CALDF1/CALDR1 for the amplification of Calmodulin gene , whereas GAPDH gene amplification was conducted using the PCR conditions described in Berbee et al., (1999). The PCRs were conducted in a Mastercycler Nexus GSX1  in a 25 μl reaction mixture using the following final concentrations or total amounts: 5 ng DNA, 1 × PCR buffer (20 mM Tris/HCl pH 8.4 and 50 mM KCl), 1 μM of each primer, 2.5 mM MgCl2, 0.25 mM of each dNTP, and 1 unit of Taq polymerase . The amplified PCR products were separated by electrophoresis and purified  for Sanger sequencing in both directions . For final identification, the DNA sequences of all isolates were compared with reference sequences of Alternaria species using the BLAST program of NCBI. From DNA sequences of 230 isolates, 37 were selected based on the species and geographic location and as representative strains these sequences were deposited into NCBI GenBank , Rpb2 (772 bp), and calmodulin (778 bp) were concatenated  to conduct a multilocus phylogenetic tree. Analysis is done after the gene sequences are concatenated head to tail to form a gene alignment which is then analyzed to generate the species tree. Manual corrections, alignments, and comparisons of the aligned database were performed using CLUSTALW integrated into MEGA version 11.0 software  was conducted using the best fitting substitution model according to jModeltest 2.1.7  in pot experiments. Potato seed tubers were sown in pots filled with sterilized soil in a glass house at 25°C with an 8 h/16 h photoperiod. One month old plants were used for inoculations. The conidial suspensions prepared from cultures grown on V8 agar at 23°C for 5 days were adjusted to 1 × 10m ImageJ . In the Alternaria pathogens: A. solani, A. protenta, A. grandis, and A. linariae were detected on potatoes in Serbia , Velika Drenova (district Rasina), Bzovik (district Raška) Navalin, and Pržine (district Jablanica). A. protenta was detected in 33% of the isolates in all major potato production areas, except in the localities Velika Drenova (district Rasina), Rudno (district Raška), Pečenjevce (district Jablanica), Krivača, Zablaće, Katići (district Moravica), Drmanovići, and Blato (district Zlatibor). A. grandis and A. linariae were identified in 4 and 3%, respectively. A. grandis was found in Krivača and Zablaće (district Moravica), while A. linariae was detected in Velika Drenova (district Rasina), Dragocvet (district Pomoravlje), and Borča (district City of Belgrade).Throughout a three-year survey 2016–2018), the presence of early blight was confirmed in all surveyed localities and four large-spored n Serbia , 2. In t016–2018,o fields . Most ofAlternaria isolates revealed slight variability in colony morphology. According to A. solani, A. protenta, A. grandis, and A. linariae.Morphological preliminary identification of fungi was performed 7 days after incubation on V-8 medium, and large-spored A. solani isolates produced colonies with conidia dimensions as follows: 190 ± 24 μm in length and 16 ± 2 μm in width with 5–13 transverse and up to three longitudinal septa and branching conidiophores 187.7 ± 4.8 μm with numerous conidiogenous sites and spores. Isolates of A. protenta developed conidia 225 ± 23 μm in length and 17 ± 2 μm in width with 8–13 transverse septa and one longitudinal septum in a few transverse segments. Conidiophores were solitary, reaching 83 μm in primary growth up to 210 μm in the stage of secondary branching (137.7 ± 6.9 μm). A. linariae isolates had solitary conidiophores up to 142 μm long (133.7 ± 7.5 μm), rising straight from the surface with large conidia. The size of the spores was 255 ± 33 μm in length and 18 ± 2 μm in width with 1–12 transverse septa and most commonly only one longitudinal septum in many transverse segments. Isolates identified as A. grandis produced conidia 262 ± 36 μm in length and 16 ± 3 μm in width with 7–12 transverse septa and one longitudinal septum in a few transverse segments. Conidiophores arose as singly, up to 200 μm in length (157.2 ± 9.3 μm).p = 0.002. Parameters for conidial length indicated again that A. solani and A. protenta did not show significant differences in conidial size (p = 0.122).Assumptions for conducting statistical analyses were inspected using Shapiro–Wilk’s normality test, Box’s test of equality of covariance matrices and Levene’s test of equality of error variances. Data showed adequate values for conducting parameter statistical test one-way ANOVA for conidiophores and conidial length but not for conidial width so this category was excluded from the further statistical analyses. A significant difference among the identified groups was found only for conidial length Alternaria isolates, the primer pair OAsF7/OAsR6 amplified the DNA fragment at position 164 bp for 222 isolates , identified four different species distributed in four clusters  employed in jModelTest confessed a general time reversible model with gamma distribution (TIM3 + IG) as the most suitable substitution model. The distance from the end to the end of the concatenated sequences used in this testing was 2,135 bp. Concatenated sequences of three analyzed gene regions, GAPDH, calmodulin, and Rpb2, in supplementary common phylogeny analysis were recognized as a single multilocus dataset for all isolated species. The parameters for the Bayesian analyses were Lset base = (0.2378 0.3131 0.2471) nst = 2 tratio = 1.3022 rates = equal pinvar = 0. For second phylogenetic analyses of GAPDH, calmodulin, and Rpb2 concatenated sequences, maximum likelihood analysis was employed and the T92 model was selected as the best-fit model.Concatenated gene sequences of the After seven days of incubation, typical early blight disease symptoms were observed on inoculated potato plants for all tested isolates with differences in symptom appearance . To confA. solani, A. protenta, A. grandis, and A. linariae isolates resulted in disease symptoms visible one week after inoculation, leaf spots differentiated in color and diameter. Concentric rings with dark color and small diameter were characteristic of isolates of A. grandis and A. linariae. A. solani was the most aggressive, with the largest spots and dark color, whereas A. protenta produced leaf spots light brown to dark in color. No symptoms were observed on plants inoculated with sterile distilled water.During the first week after inoculation, the development of lesions was slow, and spots were small (≤ to 5 mm in diameter). In the second week after inoculation, symptoms appeared as brown to dark spots on leaves similar to those observed in the potato fields . AlthougAlternaria species on potato leaves. The distributions of necrotic leaf area (%) were not similar for all groups, as assessed by visual inspection of a boxplot. The necrotic areas of potato leaves were significantly different between the different species, χ2(3) = 39.142, p = 0.000. Subsequently, pairwise comparisons were performed using A. solani, the most pathogenic species (32.1 ± 6.5), and A. linariae, the weakest pathogen (16.8 ± 4.8) compared to other species; however, A. protenta and A. grandis revealed moderate pathogenicity  with no statistically significant difference in pathogenicity on potato , only A. linariae, as a large-spored Alternaria, was found on potato. A. linariae, the main causal agent of tomato early blight, was found in Serbia as a causative agent of the same disease on potato but only in the fields where tomato was grown next to potato. Interestingly, tomato served as a strong inoculum reservoir of A. linariae, which was able to infect potato when tomato is grown side by side potato. Potato fields in districts Jablanica and North Bačka, which were located only a few kilometers away from the main tomato production areas, were not affected by A. linariae.In this study, we described the first results of a three-year survey on the presence and distribution of  Algeria , Germany Algeria , Brazil  Algeria , Cuba  can be found in the article/ŽI, BI, and GJ contributed to conception and design of the study. DŽ organized the database. JB performed the statistical analysis. ŽI wrote the first draft of the manuscript. JB wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by Ministry of education, science and technological development of Republic of Serbia, Contract no. 451–03-9/2021–14/200010.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The role of hypoalbuminemia and raised C-reactive protein (CRP) levels in predicting critical prognosis has been described extensively in adult literature. However, there are limited studies in pediatrics, particularly neonates. The CRP/albumin (CRP/ALB) ratio is often associated with higher mortality, organ failure and prolonged hospital stay. We hypothesized that the serum CRP/ALB ratio has a prognostic value in predicting surgery and mortality in neonates with necrotizing enterocolitis (NEC).Retrospective review of all neonates with clinical and radiological evidence of non-perforated NEC that were treated in a tertiary-level referral hospital between 2009 and 2018. General patient demographics, laboratory parameters and outcomes were recorded. Receiver operating characteristics analysis was performed to evaluated optimal cut-offs and area under the curve (AUC) with 95% confidence intervals (CI).p < 0.0001] and mortality , respectively.A total of 191 neonates were identified. Of these, 103 (53.9%) were born at ≤ 28 weeks of gestation and 101 (52.9%) had a birth weight of ≤ 1000 g. Eighty-four (44.0%) patients underwent surgical intervention for NEC. The overall survival rate was 161/191 (84.3%). A CRP/ALB ratio of ≥ 3 on day 2 of NEC diagnosis was associated with a statistically significant higher likelihood for surgery [AUC 0.71 (95% CI 0.63–0.79); A CRP/ALB ratio of ≥ 3 on day 2 is indicative of a critical pathway in neonates with radiologically confirmed, non-perforated NEC. This could be used as an additional criterion to guide parental counselling in NEC for surgical intervention and mortality. Necrotizing enterocolitis (NEC) is one of the most common and critical gastrointestinal emergencies in neonates, which predominately affects premature and low birth weight infants in the first weeks of life. Surgical NEC is known to be associated with a significantly higher morbidity and mortality , 2. HoweThe role of reduced serum albumin (ALB) and raised C-reactive protein (CRP) levels in predicting a critical prognosis has been described extensively in adult literature but is very limited in pediatrics. Research in adults has revealed that the CRP/ALB ratio is associated with higher mortality, organ failure and prolonged hospital stay in seriously ill or septic patients on acute medical wards and intensive care units, respectively , 9. MoreA retrospective cohort study of all neonates diagnosed with radiologically confirmed, non-perforated NEC that were consecutively admitted to the specialized neonatal intensive care unit at the Royal London Hospital  from 1 January 2009 to 31 December 2018 was performed. Details of each patient were identified using the BadgerNet platform —a national electronic neonatal database. General patient demographics were collected on gender, pregnancy, gestational age and birth weight. Clinical presentation and examination findings were correlated with abdominal radiographs to ensure the patient met the inclusion criteria. Inclusion criteria were radiological evidence of NEC  and presence of one or more clinical findings . The first day of NEC diagnosis was classified as day 1 of the disease process, and daily measured serum CRP and ALB levels were extracted from the individual electronical or paper medical records. When several blood samples were taken per day, the time of the first set of bloods was used and results from the next nearest tests done at 24 h, 48 h, etc. were recorded. If applicable, date of surgery and mortality data were noted for each patient. Indications for surgery were subsequent evidence of perforation, increasing abdominal distension, erythema, discoloration and tenderness despite maximal medical management and increasing ventilatory/inotropic support. Neonates presenting with radiologically confirmed perforated NEC were excluded from the study, which was formally registered with the clinical effectiveness unit at the Barts Health NHS Trust. As the study was retrospective in nature and therefore did not change the clinical management of patients involved, it was exempt from full IRB review but followed the principles expressed in the Declaration of Helsinki.vs. conservative medical management) as well as outcome were analyzed using Chi-square or Fisher’s exact test, where appropriate. The serum CRP/ALB ratio on day 1, 2 and 3 of NEC diagnosis was determined in each patient. Bivariate and receiver operating characteristics (ROC) analysis was used to assess optimal cut-offs for the CRP/ALB ratio with sensitivity/specificity and to calculate the area under the ROC curves (AUC) with 95% confidence intervals (CI), respectively. A p value of < 0.05 was considered statistically significant.Data was collated into an electronic spreadsheet and statistical analysis was performed using PASW Statistics 18.0 software application . Statistical differences between two cohorts of neonates with NEC . Neonates who died were significantly more likely to be very premature (i.e. ≤ 28 weeks) and extremely low birth weight (i.e. ≤ 1000 g).A total of 191 consecutive neonates with radiologically confirmed, non-perforated NEC were identified during the study period. Of these, 106 (55.5%) were male and 85 (44.5%) were female. Median gestational age was 28.2 weeks (range 23.1–39.0) with a median birth weight of 1113 g (range 490–4140). One hundred and three (53.9%) were born at ≤ 28 weeks of gestation and 101 (52.9%) had a birth weight of ≤ 1000 g. In total, 84 (44.0%) patients eventually underwent surgical intervention for NEC. Indications for surgery were increasing abdominal distension, erythema or discoloration with tenderness despite maximal medical management and increasing ventilatory and/or inotropic support. Six patients had surgical intervention for a subsequent bowel perforation on a median of 3.5 days (range 1–10) after NEC diagnosis. Overall, for all neonates who underwent surgery, this occurred on a median of 3 days (range 1–23) of medical treatment. Except for gender and gestational age, there were no statistical difference between patients that had surgical intervention and those that did not , specificity of 71.3% (95% CI 63.2–78.3) and AUC of 0.66 (0.54–0.77); In this study, neonates with radiologically confirmed, non-perforated NEC who underwent surgery reflected previously well-established findings in NEC research showing correlation between surgery, male gender and gestational age ≤ 28 weeks. Within this group of patients, we have demonstrated that a CRP/ALB ratio of ≥ 2 early in the disease process (i.e. day 1–3) correlates with surgical intervention.CRP is an acute phase protein, which will be raised in conditions that provoke a systemic inflammatory response syndrome (SIRS). This has been noted clinically and CRP is often part of the daily tests to monitor neonates for NEC disease progression . A raisevs. 4.65; p < 0.0001), further indicating that the CRP/ALB ratio at PICU admission is an independent predictor of mortality.The significance of the CRP/ALB ratio has previously been reported in a variety of adult conditions , 9, 17. To date, there has only been one single-center study that analyzed the CRP/ALB ratio in neonates. Yang et al.  investigOur study has a number of limitations. It was retrospective in nature with a limited number of patients and thus dependent on individually written chart records. Therefore, not reported confounding factors could not necessarily be ruled out, which may have caused a potential result bias. Within single-center studies, there is an overall bias towards how NEC assessment and treatment is carried out. In our center, there are several consultant pediatric surgeons, all operating on and managing neonates with NEC. As a result, the decision-making process is different for each surgeon. In particular, the timing of surgery in a neonate who is medically managed for NEC is very subjective, and hence variable. For example, there are some instances when this variability is uniformly minimized, e.g. in neonates with evidence of bowel perforation.To our knowledge, this is the first study analyzing the CRP/ALB ratio specifically in NEC. Our observations showed that a CRP/ALB ratio of ≥ 2 on day 1 has already a reasonably good predictive ability for surgical intervention in neonates with radiologically confirmed, non-perforated NEC. However, a CRP/ALB ratio of ≥ 3 on day 3 had the highest predictive value for surgery in our cohort. This finding may be utilized to triage neonates for transfer to specialist neonatal surgical centers, which would optimize surgical cot utilization. Our study also demonstrated the predicative ability of the CRP/ALB ratio of ≥ 3 on day 2 in view of mortality. This could be used as an additional criterion to guide parental counselling for NEC. Nevertheless, we need to aim for a more standardized decision-making and surgery in NEC. Therefore, we propose a prospective, multicenter study that would enable validation of our results in a larger cohort of neonates with NEC. This would potentially provide a simple, easily accessible and inexpensive tool to provide prediction of surgery and mortality in NEC.


Nucleophosmin‐1 (NPM1) mutations in acute myeloid leukemia (AML) confer a survival advantage in the absence of FLT3‐internal tandem duplication (FLT3‐ITD). Here, we investigated the main predictors of outcome after allogeneic hematopoietic stem cell transplantation . We identified 1572 adult (age ≥ 18 year) patients with NPM1‐mutated AML in first complete remission (CR1:78%) or second complete remission (CR2:22%) who were transplanted from matched sibling donors (30.8%) or unrelated donors (57.4%) between 2007 and 2019 at EBMT participating centers. Median follow‐up for survivors was 23.7 months. FLT3‐ITD was present in 69.3% of patients and 39.2% had detectable minimal/measurable residual disease (MRD) at transplant. In multivariate analysis, relapse incidence (RI) and leukemia‐free survival (LFS) were negatively affected by concomitant FLT3‐ITD mutation , MRD positivity at transplant , and transplant in CR2 , but positively affected by Karnofsky score ≥90 . Overall survival (OS) was also negatively influenced by concomitant FLT3‐ITD , MRD positivity at transplant , and older age , but positively affected by matched sibling donor  and Karnofsky score ≥90 . These results highlight the independent and significant role of FLT3‐ITD, MRD status, and disease status on posttransplant outcomes in patients with NPM1‐mutated AML allowing physicians to identify patients at risk of relapse who may benefit from posttransplant prophylactic interventions. Acute myeloid leukemia (AML) is a heterogenous disease with a highly variable prognosis and high overall mortality whereby the management is influenced by patient characteristics and biological, genetic, and molecular characteristics of the disease. The impact of minimal residual disease (MRD), NPM‐1 mutation status, FLT3‐ITD mutation status, and disease status at transplant has been individually investigated. This study examines the aggregate influence of those factors on outcome post‐transplant in NPM1‐mutated AML.  The entity is thus classified in the favorable risk group as per the European LeukemiaNet (ELN) 2017 classification.NPM1 is present in the setting of FLT3‐ITD at high allelic burden, disease prognosis significantly decreases and patients are referred for allo‐HCT in CR1 irrespective of MRD status.NPM1‐mutated AML and low allelic burden of FLT3‐ITD.Acute myeloid leukemia (AML) is a heterogenous disease with a highly variable prognosis and high overall mortality. The management of AML is guided by patient characteristics including age and performance status as well as biological, genetic, and molecular characteristics of the disease.FLT3‐ITD and NPM1, MRD positivity before or after transplant, and disease status at the time of transplant influence the risk of posttransplant relapse and thus affect outcome.NPM1‐mutated AML with specific emphasis on the individual and aggregate roles of MRD status before transplant, FLT3‐ITD mutation status, and disease status at transplant.Despite the continued progress in transplant techniques, approximately one third of AML patients relapse within 2 years of transplant which prompts the need for a better understanding of factors that influence outcome in this patient population. Current data suggest that the concomitant presence of 22.1This was a retrospective, registry‐based, multicenter analysis.NPM1‐mutated AML, who received a first allo‐HCT between January 2007 and July 2019 and who had FLT3‐ITD mutation status and MRD status at the time of transplant, available in the EBMT registry. Variables collected included recipient and donor age, gender, and cytomegalovirus (CMV) serostatus, FLT3‐ITD mutation status, Karnofsky performance status (KPS) score, disease status, and MRD status at the time of transplant. Transplant‐related factors included year of transplant, conditioning regimen, donor type and degree of mismatch, source of stem cells, graft‐versus‐host disease (GVHD) prophylaxis, in vivo T‐cell depletion, and use of posttransplant cyclophosphamide.Eligibility criteria for this analysis included adult patients (age ≥18 years) diagnosed with normal karyotype, 2.2Myeloablative conditioning (MAC) was defined as a regimen containing either total body irradiation conditioning (TBI) with a dose equal or greater than 8 Gy, a total dose of oral busulfan (Bu) greater than 8 mg/kg, or a total dose of intravenous Bu greater than 6.4 mg/kg. All other regimens were defined as reduced intensity conditioning (RIC).2.3The primary endpoint of our study was assessing the predictive factors of leukemia‐free survival (LFS) post allo‐HCT. Secondary endpoints were overall survival (OS), relapse incidence (RI), non‐relapse mortality (NRM), acute and chronic GVHD, and GVHD‐free, relapse‐free survival (GRFS). LFS was defined as survival without disease relapse or progression whereby patients were censored at the time of last follow‐up. OS was defined as the time until death from any cause and NRM was defined as being alive until death without leukemia relapse. GRFS was defined as being alive with neither grades III–IV acute GVHD, extensive chronic GVHD nor relapse.2.42 statistics for categorical variables and the Mann–Whitney test for continuous variables. Probabilities of OS, LFS, and GRFS were calculated using the Kaplan–Meier method. Cumulative incidence was used to estimate the endpoints of NRM, RI, and acute and chronic GVHD to accommodate for competing risks. To study acute and chronic GVHD, we considered relapse and death to be competing events. A Cox proportional hazards model was used for multivariate regression. All factors associated with one outcome in univariate analysis were included in the Cox model. Results were expressed as a hazard ratio (HR) with a 95% confidence interval (CI). We also performed a univariate analysis separately in CR1 and CR2 patients. All tests were two sided. All analyses were performed using SPSS 24.0  and R version 3.6.2 .Patient, disease, and transplant‐related characteristics were compared using χ33.1FLT3‐ITD mutation including 78.5% of patients transplanted in CR1 and 36.7% of patients transplanted in CR2  who met the inclusion criteria––966 out of an original 2538 were excluded for lack of MRD status pre‐transplant. The majority (78%) of the patients were in CR1, with the remaining 22% in second complete remission (CR2). The KPS was ≥80 and ≥90 in 96.6% and 77.6% of the patients, respectively, at the time of transplant, and most (69.3%) of the patients harbored N = 14, 2.6%), polymerase chain reaction (PCR) , a combination of PCR with either FACS  or next‐generation sequencing (NGS) , and a combination of all three methods  were used to measure MRD. The MRD cutoff ranged from 10−2 to 10−6 depending on the assay used whereby 10−2 was achievable only with NGS which none of the centers solely depended on.Conditioning was MAC in 50.3% of the total study group of patients. Posttransplant cyclophosphamide was used in 13.6%, whereas 61.4% of the patients had in vivo T‐cell depletion. The majority (87%) of patients received peripheral blood stem cells. More than half (57.4%) of the patients were allografted from an unrelated donor, whereas allografts from matched sibling donors and haploidentical donors accounted for the remaining 30.8% and 11.8%, respectively. Most patients had compatible donor/recipient CMV serostatus, whereas 23.7% of the patients were CMV seropositive who had received seronegative allografts. Female to male allografts accounted for 13% of transplants. At the time of transplant, most of the patients (60.8%) were MRD‐negative. Data regarding the means of MRD assessment (Table S1) and corresponding cutoffs (Table S2) were available from 65 centers accounting for 540 patients of whom 224 were MRD‐positive: fluorescent‐activated cell sorting (FACS) . The concomitant presence of FLT3‐ITD , LFS , and OS . Similarly, pretransplant MRD positivity , LFS , OS , and GRFS (Table S4) . The complete set of results of the univariate analysis are included in Tables S4 and S5.Patients in CR1 had significantly better LFS  and OS . Transplantation in CR2 resulted in worse RI (HR 1.36 p = 0.026) and LFS , and FLT3‐ITD mutation negatively affected RI , LFS , OS , GFRS , and incidence of acute GVHD grades II–IV . Similarly, MRD positivity at the time of transplant negatively affected RI , LFS , OS , and GRFS . Conversely, a KPS score ≥90 positively impacted RI , NRM , LFS , OS , and GRFS . Allografts from unrelated and haploidentical donors adversely affected NRM , and OS . In addition, an allograft from an unrelated donor increased the incidence of acute GVHD grades II–IV , whereas one from a haploidentical donor decreased extensive chronic GVHD . A female to male donor resulted in significantly increased acute GVHD grades II–IV  and peripheral blood stem cells negatively impacted the incidence of chronic GVHD  and extensive chronic GVHD  and as such, worsened GRFS . In vivo T‐cell depletion significantly decreased the incidence of acute GVHD grades II–IV and III–IV , and the incidence of chronic GVHD and extensive chronic GVHD , and improved GRFS .In the multivariate analysis Tables  & 3, 4, FLT3‐ITD mutation in patients in CR1 had a similar negative effect on RI , LFS , and OS . Similarly, MRD positivity had a similar negative effect in this subgroup on RI , LFS , OS , and GRFS . In patients in CR2, FLT3‐ITD was also associated with worse LFS , OS , and GRFS  and MRD positivity was associated with higher RI  and lower LFS . When stratified according to FLT3‐ITD mutation status (Table S7), patients harboring the mutation with MRD positivity at transplant had higher acute II–IV  and III–IV  GVHD and RI  with lower LFS , OS , and GRFS . MRD positivity had a similar effect in patients without FLT3‐ITD mutation and was associated with worse RI  and LFS .In the subgroup univariate analysis by disease status at transplant (Table S6), a concomitant 4NPM1‐mutated AML accounts for more than 50% of normal karyotype AML and while the individual impact of MRD, disease status, and FLT3‐ITD has been investigated, little is known about their combined respective contributions in influencing posttransplant outcome.FLT3‐ITD mutation, disease status, and MRD at transplant at transplant in 1572 adult (age ≥18 years) patients known to have NPM1‐mutated normal karyotype AML. We found that a concomitant FLT3‐ITD mutation, MRD positivity before transplant, transplantation in CR2, and KPS <90 increased RI and thus significantly impacted LFS and OS , LFS , OS , and GRFS  , LFS , OS , and GRFS   Table . The sigNPM1‐mutated AML without FLT3‐ITD, who belong to the ELN favorable risk group, are generally referred to allo‐HCT in CR2 or beyond. In our study, transplantation in CR2, as opposed to CR1, resulted in a significant negative impact on RI  and thus, LFS  irrespective of other predictive factors including MRD status and FLT3‐ITD mutational status. These results are in agreement with a previous retrospective study that analyzed 156 patients with NPM1‐mutated AML without FLT3‐ITD, which demonstrated that both CR2 and advanced disease at transplant, negatively affected RI , LFS , and OS .Patients with normal karyotype, NPM1 mutation and FLT3‐ITD low allelic ratio, who also belong to the ELN favorable risk group remains controversial.FLT3 tyrosine kinase inhibitors (TKIs).On the other hand, the indication for allo‐HCT in CR1 in AML patients with FLT3‐ITD to posttransplantation outcome in NPM1‐mutated AML. Given the grave prognosis on OS that relapse imposes in this patient population and the relatively high incidence of relapse, it is rather important to emphasize factors predictive of relapse to identify high‐risk patients who could benefit from personalized treatment approaches such as prophylactic use of FLT3 TKIs or more frequent follow‐up.There is currently, very little is known about the respective contributions of disease status, MRD status, and 5NPM1 or FLT3 MRD. The use of different assays to assess MRD resulted in a wide range of MRD cutoffs utilized by different centers. Furthermore, MRD testing techniques were available for centers as a whole, but data about the techniques used and their results for individual patients were not available, therefore limiting further analysis. The limited MRD technique data, although limiting, are important for solidifying the fact that patients were assessed for MRD according to universally accepted definitions, with the majority of the patients assessed by two modalities. In addition, it would have been valuable to analyze molecular NPM1 MRD which has been shown to be a significant predictor of relapse despite the favorable prognostication that the NPM1 mutation infers.FLT3‐ITD fragment length, and FLT3‐ITD allelic ratio were also unavailable for investigation, further limiting the analysis. This is important since the FLT3‐ITD allelic ratio is vital in allocating ELN risk and aiding treatment decision. Finally, whether patients harboring FLT3‐ITD actually received treatment with FLT3‐inhibitors or whether venetoclax combinations were utilized given their possible promising role in NPM1‐mutated disease was also unavailable.The limitations of this analysis include the retrospective study design, the absence of a unified MRD measurement technique among centers, and the unavailability of molecular MRD assessment, specifically 6NPM1‐mutated AML, FLT3‐ITD mutation status, disease status at transplant, MRD status at transplant, and KPS significantly and independently influence RI and LFS and thus OS. Knowledge of factors that substantially influence posttransplant outcomes allows physicians to undertake, when feasible, an alternative treatment approach to decrease the risk of relapse and thus improve the survival.We demonstrate that in adult patients with normal karyotype The authors declare no potential conflict of interest.the Ministry of Health of Czech Republic under Grant AZV NV18‐03–00277.Dr. Jindra Pavel was supported by R.A.: data curation, investigation, project administration, writing original draft, and editing/reviewing; M.L.: data curation, formal analysis, methodology, and review; E.D., R.N., A.H., G.S., M.S., J.H.B., N.K., E.T., G.C., X.P., H.M., MT.R., P.J., D.B., D.B., H. LW., and A.N: data acquisition, investigation, validation, and review; A.B. and M.M.: conceptualization, investigation, supervision, validation, and review.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.AppendixClick here for additional data file.


Efavirenz (EFV) is commonly used for first‐line antiretroviral therapy in children and adolescents with HIV, but is associated with neuropsychiatric and metabolic side effects. Rilpivirine (RPV) is better tolerated, and switching from EFV to RPV in virologically suppressed adults has been safe and efficacious, but data in adolescents are limited. Our primary objective was to describe the 48‐week immunologic and virologic outcomes in virologically suppressed adolescents switching from EFV‐ to RPV‐based antiretroviral therapy. Secondary objectives included assessment of neuropsychiatric adverse events, quality of life (QOL) and metabolic profiles while on RPV.We conducted an open‐label, single‐arm, multi‐centre study in Thailand in virologically suppressed adolescents aged 12–18 years receiving EFV plus two nucleoside/tide reverse transcriptase inhibitors (NRTIs/NtRTI) for ≥3 months. Participants were switched to an RPV (25 mg) tablet once daily, with the same NRTIs. HIV RNA viral load, CD4 cell count, fasting total cholesterol (TC), triglyceride, glucose, neuropsychiatric adverse events, depression and QOL were assessed over 48 weeks. Data were collected between February 2016 and September 2018.3 (29.9%); 58% were receiving tenofovir‐DF and emtricitabine. At weeks 24 and 48, 96 (94.1%) and 94 (92.2%) participants were virologically suppressed, respectively, with no significant change in CD4 cell counts from baseline. Six (5.9%) participants experienced virologic failure, two of whom had RPV‐associated mutations (K101E and Y181C) and a lamivudine‐associated mutation (M184V/I). There were significant decreases in TC, triglyceride, high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) at weeks 24 and 48 and a significant increase in LDL/HDL ratio at week 48 compared to baseline. No substantial changes in EFV‐related symptoms, depression score or health‐related QOL were observed over time; however, there was significant improvement in performance‐based assessments of executive function at week 24.One hundred and two  adolescents were enrolled. Median age at entry was 15.5 years (IQR 14.4–17.0), median CD4 count was 664 cells/mmA high proportion of adolescents (>92%) remained virologically suppressed up to 48 weeks after switching from EFV to RPV along with no significant change in CD4 cell counts. RPV was well tolerated and associated with improvements in metabolic profiles and executive function. Today, the majority of children living with HIV are surviving into adolescence  and optiEfavirenz (EFV)‐based ART has been the cornerstone of first‐line treatments in adults, adolescents and children older than 3 years in resource‐limited settings since 2016 . HoweverRilpivirine (RPV) is a second‐generation non‐nucleoside reverse transcriptase inhibitor (NNRTI) available in Thailand for free through the national ART program. The efficacy and safety of RPV is well established in adults. Among ART‐naive adults with a baseline RNA viral load (VL) <100,000 copies/ml, the virologic efficacy of RPV was non‐inferior to EFV over 48 weeks, and RPV had a more favourable tolerability profile, with lower rates of CNS toxicity and lipid abnormalities compared to EFV , 8, 9. SOverall, RPV is a potential alternative to standard EFV‐based ART for adolescents struggling with mild CNS side effects, or to help reduce the risk of metabolic side effects associated with long‐term EFV use. Our primary objective was to describe the 48‐week immunologic and virologic outcomes in virologically suppressed adolescents switching from EFV‐ to RPV‐based ART. Assessment of neuropsychiatric adverse events, quality of life (QOL) and metabolic profiles while on RPV were secondary objectives.2We performed an open‐label, single‐arm, multi‐centre study in adolescents with HIV aged 12–18 years old. This study was performed at four sites in Thailand: Siriraj Hospital, Queen Sirikit National Institute of Child Health, HIV‐NAT Chulalongkorn University and Khon Kaen University. Data were collected between 10th February 2016 and 19th September 2018.Prior to screening, caregivers provided written informed consent and adolescents who knew their HIV status provided written assent. Adolescents 12–18 years, with a body weight ≥25 kilograms and receiving ART composed of EFV plus two nucleoside or nucleotide reverse transcriptase inhibitors [N(t)RTI]) for ≥3 months with virological suppression (HIV RNA <50 copies/ml within the last 12 months) were screened for eligibility. We excluded individuals with prior evidence of NNRTI‐associated resistance mutations based on the IAS–USA HIV drug‐resistance mutations list (2019)  [TM 4.0 [At the study entry visit, EFV‐based ART was discontinued and switched to RPV‐based ART. Study participants were advised to take a single RPV 25 mg tablet once daily with a substantial meal to increase drug absorption. This food requirement was emphasized at each study visit. The choice of the NRTIs/NtRTI backbone was according to the standard of care in Thailand and inclusive of drugs and formulations provided for free through the national ART program. Study participants were also advised to report any adverse events  that developed after switching to RPV. Due to the extended inductive effect of EFV on CYP3A4, there were initial questions around the potential for reduced RPV exposures immediately after switching from EFV; thus, at the start of this study, a pharmacokinetic sub‐study was performed in 20 participants. We have previously reported that RPV exposures were adequate in this sub‐cohort immediately after switching from EFV and study‐related dosing was not altered . The plaTM 4.0 , 16 and This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine Siriraj Hospital, Mahidol University, Queen Sirikit National Institute of Child Health Chulalongkorn University and Khon Kaen University.The primary objective was to describe the 48‐week virologic and immunologic outcomes in virologically suppressed adolescents switching from EFV‐ to RPV‐based ART. Under the assumption that all study participants were virologically suppressed at the time of treatment switch, and that 10% would experience virological failure over 48 weeks , a sampl33 (IQR 553–862) (29.9%). Twenty‐seven (26.5%) participants were receiving a single‐tablet regimen of tenofovir‐DF/emtricitabine/EFV (TDF/FTC/EFV) before switching ART. The majority of study participants (58%) were receiving a tenofovir‐DF/emtricitabine (TDF/FTC)‐backbone.A total of 105 adolescents were screened and 102  were enrolled  and Y181C (n = 1). Drug‐resistance mutations detected among study participants with RPV‐associated mutations included: participant #1 NNRTI mutations –– K101E, K103N, Y181C, G190A, N348I and NRTI mutation M184V; participant #2 NNRTI mutation K101E and NRTI mutation M184I. Two study participants experiencing virologic failure also had NRTI mutations. Two other study participants developed the V179D mutation, which is not associated with reduced susceptibility to RPV on its own. Significant decreases in TC, triglyceride, HDL and LDL were observed at weeks 24 and 48 compared to baseline  study participants dropped out, one due to pregnancy (week 24) and another following the advice of their local IRB due to being underweight (week 4). Over the 48 weeks of follow‐up, five participants had detectable VL in the range of 51–202 copies/ml. Of these, four became virologically suppressed after enhanced adherence counselling, while one who had a VL of 202 copies/ml at week 48 was subsequently lost to follow‐up. Overall, 96 (94.1%) and 94 (92.2%) of 102 participants maintained virologic suppression at weeks 24 and 48, respectively. No significant change in CD4 cell counts at weeks 24 and 48 from baseline was observed  participants and 1 (1.0%) participant were slow learner and had intellectual disability evaluated by SPM, respectively. In addition, 41.0%, 54.0% and 68.7% of participants scored lower than average as measured by coding, TMT Part A and Part B, respectively (Supporting Information). At week 24, there was a significant improvement in performance‐based assessments of executive and cognitive function by coding, TMT Part A, and Part B after switching to RPV from EFV . An observational study in Tanzania compared the competence , psychopathology  and cognitive performance (intelligence and working memory) of children with HIV aged 6–12 years receiving an EFV‐based versus non‐EFV‐based regimens, and found that EFV use in children was associated with a mild increase in neuropsychiatric symptoms . The lacWe demonstrated that RPV was well tolerated and was associated with improvement in metabolic profiles, executive and cognitive function, and is a reasonable alternative for adolescents experiencing EFV‐associated side effects. Of note, adolescents must be able to take RPV following a regular schedule with a full meal to assure adequate RPV concentrations, which may limit its usefulness for adolescents with irregular eating schedules. Importantly, our findings support the use of RPV as a part of a long‐acting injectable treatment regimen, which would be an ideal option for adolescents .Our study had several limitations. Firstly, an HIV GRT was not performed prior to initiation of ART. This is consistent with the national antiretroviral treatment guidelines in Thailand. Although GRT could have identified those less likely to respond to RPV, we only enrolled participants who had not previously failed NNRTI‐based ART (based on virologic criteria). Therefore, the presence of NNRTI mutations was expected to be very low and not anticipated to affect treatment outcomes. Secondly, we did not record participant adherence to ART – whether by self‐report or other means (e.g. pill count), which could have been a primary contributor to virologic failure. Thirdly, improvement on repetition of neurocognitive testing could have been due to learning the skills of the test over time. Furthermore, there were many potential psychosocial issues among adolescents living with HIV, which may have influenced the results of the QOL and depression scores while on RPV.3 if the HIV RNA VL is not available. Furthermore, RPV can be used for a treatment switch for those individuals who have had an undetectable VL for 6–12 months and do not have prior NNRTI resistance, or those who cannot tolerate EFV within 2 weeks after initiation [Recently, the World Health Organization revised its recommendations for preferred first‐ and second‐line regimens for adolescents and adults to include dolutegravir (DTG)‐based ART . The Thaitiation . Althougitiation . Until D5Overall, the majority of adolescents on EFV‐based ART with HIV‐VL <50 copies/ml who switched to RPV remained virologically suppressed up to 48 weeks, along with no significant change in CD4 cell counts. Among the few participants with virologic failure, development of RPV‐RAMs (K101E and Y181C) was infrequent. RPV was well tolerated and was associated with improvement in metabolic profiles, executive and cognitive function and is a reasonable alternative for adolescents experiencing EFV‐associated side effects.The authors have no competing interests.Conceptualization: WP, KC and SC. Investigation: WP, WJ, SC, SA, PS, PS, PS and KC. Data analysis: AM. Writing – original draft: WP, SC and AM. Writing – review and editing: KC, WJ, SA, PS, PS and PK. Funding acquisition: WP and KC.This study was supported by grants from amfAR, the Foundation for AIDS Research (TREAT Asia program) and the Faculty of Medicine Siriraj Hospital, Mahidol University (grant number R015836002). Click here for additional data file.


The aim of this study was to observe the effectiveness of botulinum toxin type A (BTA) in the treatment of intermittent exotropia (IXT) in children compared with strabismus surgery.One hundred forty-four children with a clear diagnosis of IXT and an indication for surgery were eligible for inclusion. Subjects were divided into two groups based on parental decision: the BTA injection group (injection group) or the conventional surgery group (surgery group). All cases were followed up for 6 months. The primary outcome was a comparison of the success rate (deviation between − 10 and + 10 PD) between the two groups at 6 months after the initial treatment. Secondary outcomes included change in deviation, visual function, and post-surgical complications.P = 0.13; postoperative deviation − 12.22 ± 10.80 PD vs − 9.17 ± 10.30 PD, P = 0.09). The binocular Visual function, except for near stereoacuity, improved after treatment in both groups, while the fusion recovery rate was higher in the surgical group . Transient complications in the injection group included diplopia, ptosis, and subconjunctival hemorrhage, whereas subconjunctival hemorrhage, conjunctival edema, foreign body sensation, pain, and diplopia were seen in the surgical group. The complications of BTA were relatively mild.Seventy-two patients were enrolled in each group. At 6-month follow-up, there was no significant difference in the success rate between the injection and surgery groups .The study has completed the clinical registration on (The online version contains supplementary material available at 10.1186/s12886-022-02285-2. Intermittent exotropia (IXT) is one of the common eye diseases among children. Surgery has long been considered the treatment of choice for IXT with surgical conditions . However, a number of limitations and complications following surgery have gradually come to light, including scar formation and adhesions, difficulties in subsequent secondary surgery, and compromised surgical outcomes due to the low accuracy of deviation measurements in young children. As a result, minimally invasive approaches to IXT have been explored and shown to have a better safety profile.Clostridium botulinum and was first applied in the treatment of strabismus by Scott [Botulinum toxin type A (BTA) is a neurotoxin synthesized by by Scott  in 1973.by Scott  . The raThe efficacy of BTA injections in the treatment of paralytic strabismus in adults is now widely accepted –6 . In rThis study was a prospective, non-randomized, comparative clinical study. One hundred forty-four children (aged between 3 and 13 years) with IXT attending the strabismus clinic at Beijing Tongren Hospital from April 2018 to October 2019 were eligible for inclusion. Inclusion criteria. (1) IXT patients with indications for surgery ; (2) age < 18 years; (3) ≤ − 50 PD of horizontal deviation. Exclusion criteria: combination of the following (1) other structural ocular pathologies; (2) vertical strabismus, A-V strabismus, dissociated vertical deviation; (3) nystagmus and amblyopia; (4) neurological or developmental abnormalities; and (5) previous history of strabismus surgery. The study complied with the Declaration of Helsinki. Ethics committee approval was obtained from the Institutional Review Board of Beijing Tongren Hospital, Capital Medical University (TRECKY2018–006). All parents or guardians signed an informed consent form, and we obtained informed consent from all pediatric patients.General examination, best corrected visual acuity, and refractive status were performed by a pediatric ophthalmologist. Distance (6 m) and near (33 cm) deviations in primary position were measured using prism and alternate cover test, and deviations that could not be cooperated with were measured by the Krimsky method. Restriction of ductions in horizontal gaze was measured on a 9-point scale (− 4 to + 4), with 0 representing no restriction, − 4 indicating no movement beyond the midline, and + 4 representing extremely strong muscles. Binocular vision at distance fixation, including simultaneous vision, fusion, and distance stereopsis, was examined by synoptophore   guidance without conjunctival incision. BTA  was diluted to 100 IU/4 ml with 0.9% saline in a volume of 0.1 ml per muscle. Needle electrodes were inserted into the EOMs under EMG guidance. On emitting a high level acoustic signal, indicating that the neuromuscular junction had been reached, and the injection was performed. Depending on the severity of strabismus, patients received two different doses of treatment. 1.25 IU per muscle was injected in patients with strabismus ≥ − 15 PD and < − 20 PD, while 2.5 IU per muscle was injected in patients with strabismus ≥ − 20 PD and < − 50 PD  . For caSurgical group. General anesthesia was performed with sevoflurane inhalation combined with intravenous infusion, and local anesthesia was performed with proparacaine hydrochloride surface anesthesia combined with lidocaine  subconjunctival infiltration anesthesia around the surgical muscle. Patients were treated surgically for strabismus according to the preoperative measurement of strabismus deviation. Unilateral lateral rectus recession was performed in children with deviation ≤ − 20 PD, and bilateral lateral rectus recession or unilateral recess-resect was chosen when the deviation was > − 20 PD and ≤ − 50 PD  .Follow-up was set at 1 week, 1 month, 3 months, and 6 months postoperatively . Ophthalmic examination included measurement of eye position, eye movements, binocular visual function and complications. In indicating the angle of deviation, the symbols (positive and negative) represent only the direction of horizontal strabismus, independent of the preoperative and postoperative period. Therefore, we use the positive value of prism diopters for exotropia pre-operation and the negative values for consecutive esotropia post-operation.Primary outcome: Success rate at 6 months post-treatment was set as the primary outcome. Treatment success was defined as a final absolute value of horizontal deviation of 10 PD or less .Secondary outcomes. (1) angle of deviation  before and after operation; and (2) binocular visual function. The normal range of binocular vision included simultaneous vision between − 3° and + 3°, the presence of fusion and distance stereopsis, and near stereopsis less than or equal to 60 arcsec.Safety evaluation indicators: complications such as ptosis, diplopia, vertical strabismus, etc.p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 23.0 statistical software. Assignment of near stereoacuity: Log was taken for near stereoacuity in arcsecond. “None” was assigned a value of 1600, and 3.20 after taking Log. The prismatic degree and quantitative components of binocular vision before and after treatment were expressed as mean plus standard deviation range and compared by paired t-test. Independent samples t-test was used to compare the results of the injection and surgery groups, including age, angle of deviation, and quantitative components of binocular vision. The Fisher exact test was used to compare the sex ratio, the qualitative component of binocular vision, success rate, and complications between the two groups, described by frequency and percentage. Patient demographics and clinical characteristics are shown in Table P = 0.13).All patients received 2.5 IU injections based on preoperative strabismus deviation measurements. None received 1.25 IU injections. At 6 months after treatment, there was no significant difference in treatment success  between the injection group  and the surgical group  or surgery (P < 0.001) at 6 months, and the deviation after treatment was significantly lower than before treatment in both groups. There was no statistically significant difference in the angle of deviation between the two groups at 6 months after treatment (P = 0.09) (Table There was a significant difference in the angle of deviation before and after injection (9) Table . The chaP = 0.12) and distance stereopsis (P = 0.40) between the two groups at 6 months after treatment, however, the recovery of fusion was significantly higher in the surgical group compared with the injection group (P < 0.001). And there was no difference between the two groups in terms of good post-treatment near stereopsis (P = 0.09). At 6 months after treatment, the logarithmic arcsecond of near stereopsis was 2.0 ± 0.4 in the injection and 2.0 ± 0.5 in the surgical group. There was no significant difference in the log arcsec of near stereopsis before and after injection (P = 0.98)/surgery (P = 0.08). And for the log arcsec of near stereopsis after injection and surgery, there was also no significant difference between two groups (P = 0.99). After 6 months of treatment, the proportion of patients with good binocular vision increased in both groups compared with pre-treatment .As shown in Table All children had normal eye movements before treatment, and all children had an initial EOM force of 0. In the injection group, 47 children had reduced EOM force at the 1-week follow-up (EOM force ranged from − 3 to − 1), and 9 cases had mild limitation of eye movements for 1 month consecutively, with reduced injection muscle force to − 1. After 3 months of injection, all children’s EOM force returned to preoperative baseline. There was no significant oculomotor limitation in the surgery group.P < 0.001). In the injection group, 33 cases (45.8%) underwent local anesthesia without sedation and 39 subjects (54.2%) under general anesthesia, whereas only 1 case (1.4%) in the surgery group underwent surgery under local anesthesia.There was a statistically significant difference in the mode of anesthesia between injection and surgery , none of whom developed amblyopia. The ptosis disappeared after a median of 1 to 3 months. Subconjunctival hemorrhage was noted in 1 patient, and this symptom disappeared at 1-month follow-up. In the surgery group, some children also complained of diplopia after surgery, usually lasting a few days or weeks. Subconjunctival hemorrhage and conjunctival edema are unavoidable. The edema is usually absorbed within a few weeks, but the hemorrhage is absorbed over a longer period of time and can disappear completely within 3–6 months. In the meantime, due to the surgical wound and sutures, many children develop a foreign body sensation or even pain of the eye, which usually lasts 1–3 months .Fifteen children (20.8% of the injection group) received a second injection , and no further repeat injections were performed. Of the 15 IXT patients, 5 relapsed  at 3 months after injection and 10 at 6 months after injection Table . ChildreTwenty other patients in the injection group relapsed but did not undergo a second injection. Of these subjects, 8 patients relapsed at 3 months after injection and 12 patients relapsed at 6 months after injection. The mean deviation of relapse was −18.2 ± 6.3 PD (range − 12 to − 35 PD). Considering their relatively low relapse deviation, the physicians agreed to observe temporarily after communication with the parents and then decide whether to repeat the injection according to the changes.Intermittent exotropia is the most common type of strabismus in East Asian children –17. One P = 0.13), but lower than previous studies. A variety of reasons may be involved, including different evaluation metrics for surgical success, the preoperative angle of deviation in pediatric IXT patients, and the inclusion of repeat injections. Previous studies [In the present study, we enrolled 144 children with IXT with indications for surgery and divided them into a BTA injection group and a surgery group. The treatment success rate at the last follow-up at 6 months in the injection group, evaluated with eye position, was 52.8%, which was as effective as the surgery group  compared to traditional surgery. At the same time, the children’s binocular visual function can be maintained, although the recovery of fusion is not as good as surgery. BTA injection, as a novel alternative method for the treatment of IXT in children, still has a promising application because of its simplicity, reproducibility, minimally invasive, low requirements for anesthesia care, and reliable safety.Additional file 1.Additional file 2.


Recent studies have shown the presence of SARS‐CoV‐2 in the tissues of clinically recovered patients and persistent immune symptoms in discharged patients for up to several months. Pregnant patients were shown to be a high‐risk group for COVID‐19. Based on these findings, we assessed SARS‐CoV‐2 nucleic acid and protein retention in the placentas of pregnant women who had fully recovered from COVID‐19 and cytokine fluctuations in maternal and foetal tissues.Remnant SARS‐CoV‐2 in the term placenta was detected using nucleic acid amplification and immunohistochemical staining of the SARS‐CoV‐2 protein. The infiltration of CD14+ macrophages into the placental villi was detected by immunostaining. The cytokines in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens at delivery were profiled using the Luminex assay.Residual SARS‐CoV‐2 nucleic acid and protein were detected in the term placentas of recovered pregnant women. The infiltration of CD14+ macrophages into the placental villi of the recovered pregnant women was higher than that in the controls. Furthermore, the cytokine levels in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens fluctuated significantly.Our study showed that SARS‐CoV‐2 nucleic acid (in one patient) and protein (in five patients) were present in the placentas of clinically recovered pregnant patients for more than 3 months after diagnosis. The immune responses induced by the virus may lead to prolonged and persistent symptoms in the maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid. in vivoRemnant SARS‐CoV‐2 virus RNA and protein could be detected in the term placentas of recovered pregnant women. Immune responses of maternal plasma, placenta, umbilical cord, cord blood, and amniotic fluid were activated by the viruses may persist even longer  The major manifestations of COVID‐19 are observed in the respiratory organs.ACE2 expression, whereas the lung cells show moderate expression.SARS‐CoV‐2 is primarily transmitted through the respiratory tract and infects airway epithelial cells, vascular endothelial cells and macrophages.Following infection, an immune response is typically induced against the pathogen, and patients with severe COVID‐19 may suffer from lymphocytopenia and macrophage activation syndrome.Pregnant women are a high‐risk population for severe COVID‐19 and exhibit high mortality owing to their unique immune status.During pregnancy, the placenta acts as a transient endocrine organ that supports foetal growth by extracting nutrients from the maternal blood and serving as a barrier against pathogens or mediators of the maternal immune system. The placental villus is a functional unit of the placenta, composed of a layer of mononucleated cytotrophoblast cells and an outer multinucleated syncytiotrophoblast.In this study, we used the placentas, maternal plasma, neonatal umbilical cords, cord blood and amniotic fluid donated at delivery by pregnant patients who had completely recovered from COVID‐19 and confirmed the presence of residual SARS‐CoV‐2 nucleic acid and protein in the placentas of patients with COVID‐19 long after initial diagnosis and complete recovery. The longest interval between diagnosis and sampling was approximately 3 months, and we believe that the immune response induced by SARS‐CoV‐2 may persist even longer.22.1The biological specimens used in this study were collected from 11 pregnant women who had recovered from COVID‐19 in Wuhan, China, between March 30 and April 24, 2020. The women were diagnosed with COVID‐19 by the local Center for Disease Control or by a designated diagnostic laboratory in accordance with the novel coronavirus pneumonia diagnosis and treatment program (5th edition). Among the women, four  had an asymptomatic infections. At the time of delivery, the patients met the requirements for clinical discharge or release from quarantine, with approval provided by clinicians. However, owing to the limited information about the pathogen and the lack of sensitive testing methods at the onset of suspected symptoms, the time point of initial infection could not be determined in some patients. All the patients were treated in isolation. The diagnosis was based on SARS‐CoV‐2 nucleic acid detection from throat swabs, serum antibody testing and chest CT imaging before delivery (Table S2). Four patients  underwent RNA and antibody testing after delivery. Maternal plasma, placentas, neonatal umbilical cords, cord blood and amniotic fluid were collected under stringent conditions to eliminate the chances of accidental contamination, and all samples were stored in a biosafety level 3 containment laboratory. The experiments were performed after temperature inactivation, with liquid samples processed at 56 ºC for 45 min and solid samples processed at 95 ºC for 45 min. All researchers had requisite qualifications for biosafety and experimental operations. The experimental procedures complied with the standard instructions and requirements of the Institute of Zoology, Chinese Academy of Sciences.2.2The study was approved by the Research Ethics Committee (reference number: TJ‐IRB20200732) of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All participants (patients and uninfected pregnant women) who donated biological specimens provided written informed consent.2.3RNA extracted from maternal throat swabs, placental villi (10 mg) and neonatal throat swabs were used to test for SARS‐CoV‐2 S protein using qRT‐PCR. The RNA titre was measured using a fluorescent probe targeting the S protein RNA. The sequences of the probes were as follows: CoV‐F (5’‐TCCTGGTGATTCTTCTTCAGGT‐3’), CoV‐R (5’‐TCTGAGAGAGGGTCAAGTGC‐3’) and CoV‐P (5’‐FAM‐AGCTGCAGCACCAGCTGTCCA‐BHQ1‐3’). Samples were considered SARS‐CoV‐2‐positive when the cycle threshold values for S protein were ≤ 38.2.4ELISA was used to detect SARS‐CoV‐2‐specific IgG and IgM in maternal serum, neonatal serum, umbilical cord, cord blood and amniotic fluid specimens, as instructed.2.5Immunohistochemical staining was performed as described by Fu et al2.6To characterize the cytokine profiles of maternal plasma, placenta, neonatal umbilical cord, cord blood and amniotic fluid specimens, the Luminex assay was performed using the Bio‐Plex Pro Human Cytokine Screening Panel , which can detect 48 cytokines and chemokines. The data were collected on a Luminex 200 Instrument System and analysed using Luminex xPONENT . The mean cytokine level in uninfected controls was considered the baseline and the cytokine levels in the specimens were expressed relative to the baseline level and termed "relative secretion levels". For negative controls, we used maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid donated by three, four, two, two and three uninfected pregnant women at delivery respectively.2.7P <.05; **, P <.01; ***, P <.001.Results are expressed in terms of means ± standard error of the mean. Statistical analysis was performed using a paired‐sample t‐test with Statistical Package for Social Science . Significance was defined as follows: *, 33.1+4W, whereas the other infants were full‐term newborns (Table S3). Subsequent assessments were based on the biological samples obtained.The clinical details of the pregnant patients with COVID‐19 and their fetuses have been summarized in Tables 3.2To explore the existence of SARS‐CoV‐2 in the placenta of clinically recovered patients, we first performed qRT‐PCR on isolated total RNAs of the placentas from six patients . As shown in Figure To estimate the duration of SARS‐CoV‐2 retention in the placenta, we analysed the clinical course of patient #46, who showed the presence of both SARS‐CoV‐2 nucleic acid and protein in the placenta. As shown in Figure 3.3After SARS‐CoV‐2 infection, the immune response in the patient is activated, and the infiltration of macrophages and lymphocytes into the infected locus is a common mechanism for virus clearance.Immune activation in response to SARS‐CoV‐2 infection has been shown to induce cytokine secretion, which may be an important contributor to disease progression.To further explore the immune fluctuation in vivo, we profiled the cytokines in maternal plasma samples collected at delivery from nine recovered pregnant women  and three normal controls using the Luminex assay Figure . The relOf the nine pregnant women who donated maternal plasma, four also donated their placentas . Among them, the plasma IL‐2 and IL‐15 levels in patients #26, #42 and #49, who showed the presence of viral protein in the placenta, were elevated compared with those in normal controls Figure . Plasma 3.4To determine whether placental SARS‐CoV‐2 infection affected the pregnancy outcome and led to the intrauterine infection of the foetus, we further investigated the foetal and accessory abnormalities at delivery. Of all newborns, the monochorionic diamniotic twins weighed 2500 g and 2100 g, whereas the other full‐term infants had body weights ranging from 2810 g to 3900 g. All infants received 1‐minute and 5‐minute Apgar scores of 8 ‐ 10 (Table S3). Maternal infection‐associated abnormalities were not observed in the foetuses and appendages of any of the 11 pregnant women (Table S4).To further explore the possibility of intrauterine infection of the foetus, we performed a COVID‐19 test on the newborns, and no viral nucleic acid was detected in the nasopharyngeal swab samples. However, seven newborns  tested positive for IgG but negative for IgM. None of the newborns showed typical symptoms of COVID‐19 pneumonia. In addition, the umbilical cords from five patients  and amniotic fluid from three patients  were collected for SARS‐CoV‐2 testing. All umbilical cord and two amniotic fluid samples (#26 and #47) tested positive for IgG, whereas none of the specimens tested positive for IgM , the relative secretion levels of vascular endothelial growth factor (VEGF) were high in five samples, which were collected from the patients whose placental villi showed the presence of viral protein; the two normal controls were used for comparison Figure . In addi4SARS‐CoV‐2 infection in the placenta and the placental pathology in COVID‐19‐positive mothers has been reported in previous studies.Several lines of evidence have shown that the adaptive immune response induced by SARS‐CoV‐2 infection can last for nearly 6 months.Collectively, our findings showed persistent SARS‐CoV‐2 infection and macrophage infiltration in the term placentas of pregnant women who had recovered from COVID‐19, suggesting a high probability of SARS‐CoV‐2 infection in human placentas.The authors declare there are no competing interests and all authors consent to publish the data.H. M. W., S. L., D. D., Y. Z. and Z. X. conceived the project. H. M. W. and Z. X. supervised the project. S. L., Y. Z., D. D., X. L., J. W., R. W., D. L., D. W., M. H., B. H., R. L., J. P., H. Y., H. Y., Y. Z., W. Z., X. W., Z. H. and K. L. collected clinical specimens. H. W., Y. W. and M. G. conducted experiments and data analysis. H. W. and Z. X. wrote the manuscript.Supplementary MaterialClick here for additional data file.


The respiration could decrease the time synchronization between odor stimulation and data acquisition, consequently deteriorating the functional activation and hemodynamic response function (HRF) in olfactory functional magnetic resonance imaging (fMRI) with a conventional repetition time (TR). In this study, we aimed to investigate whether simultaneous multislice (SMS) technology with reduced TR could improve the blood oxygen level-dependent (BOLD) activation and optimize HRF modeling in olfactory fMRI. t-scores) and the HRF modeling (response height and time to peak) were calculated in the bilateral amygdalae, hippocampi, and insulae.  Sixteen young healthy subjects with normal olfaction underwent olfactory fMRI on a 3T MRI scanner using a 64 channel head coil. FMRI data were acquired using SMS acceleration at three different TRs: 3000 ms, 1000 ms, and 500 ms. Both metrics of BOLD activation . But the increase of these metrics in the hippocampi did not reach a statistical significance . No significant difference in any BOLD activation metrics between TR 500 ms and 1000 ms was observed in all regions of interest (ROIs) . The HRF curves showed that higher response height and shorter time to peak in all ROIs were obtained at 500 ms and 1000 ms TRs compared to 3000 ms TR. TR 500 ms had a more significant effect on response height than TR 1000 ms in the amygdalae (P = 0.017), and there was no significant difference in time to peak between TR 500 ms and 1000 ms in all ROIs .  The 500 ms and 1000 ms TRs both significantly improved the number of activated voxels, mean, and maximum  The fast image acquisition technique of SMS with reduced TR could significantly improve the functional activation and HRF curve in olfactory fMRI. Functional magnetic resonance imaging (fMRI) has been widely used in evaluating brain functions. However, the application of olfactory fMRI is still challenging due to many methodological and physiological factors, such as complex odor stimulation paradigm , magnetiOlfactory fMRI is conventionally performed using echo planar imaging (EPI) sequences with the standard repetition time (TR) of 2-3 s for whole brain mapping. The fast image acquisition technique of simultaneous multislice (SMS) employs multiband excitation pulses to simultaneously excite and acquire multiple slices . SMS accThe primary goal of the study was to investigate whether SMS technology with reduced TR could improve the BOLD activation and optimize HRF modeling in olfactory fMRI.Sixteen young healthy subjects  were recruited into the study. All subjects were right-handed. The inclusion criteria were the age range of 20-30 years and normal olfaction. Olfactory evaluation was performed by T&T olfactory test  prior to2, FOV = 220 mm, matrix = 220 × 220, flip angle = 70°, slice thickness = 2.5 mm, number of slices = 32, and scan time = 11 min 30 s. Based on fMRI scanning time of 11 min 30 s, the total number of frames/scans was set to 1380, 690, and 230 for TRs of 500 ms, 1000 ms, and 3000 ms, respectively. The SMS EPI images were employed with slice GRAPPA image reconstruction.All imaging examinations were performed on a 3T MRI scanner  using a 64-channel head coil. Structural images were acquired using a T1-weighted magnetization-prepared rapid gradient-echo (MPRAGE) sequence with following imaging parameters: sagittal orientation, TR = 1550 ms, echo time (TE) = 2.98 ms, field of view (FOV) = 256 mm, matrix = 256 × 256, flip angle = 9°, slice thickness = 1.00 mm, number of slices = 176, no gap, effective TI = 900 ms, Generalized Autocalibrating Partially Parallel Acquisition (GRAPPA) acceleration 3, and acquisition time = 2 min 35 s. To investigate the effects of TR on BOLD activation and HRF modeling, three sets of fMRI data were acquired during the olfactory task using three SMS echo planar imaging (SMS-EPI) sequences with different TRs . Three corresponding SMS factors were 8, 4, and 1, respectively. Other imaging parameters of the SMS-EPI sequences were kept identical: transverse orientation, TE = 30.0 ms, spatial resolution = 2.4 × 2.4 mmThe odor stimulation paradigm was programmed in an MRI-compatible olfactometer . The olfactory task was performed with an event-related design, as displayed in The olfactometer delivered constant airflow of 6 L/min at room temperature (22°C). Prior to the fMRI scanning session, all subjects followed the instruction and received the practical training to breathe steadily and regularly through their nose without sniffing. A respiratory belt was attached to the lower sternal angle to closely detect the subject's respiration. Respiration cycle and timing of odor delivery were recorded by the olfactometer.http://www.fil.ion.ucl.ac.uk/spm) on Matlab 2013a . The standard data preprocessing procedures were performed identically for three sets of fMRI data: image realignment, coregistration of functional and anatomical images, spatial normalization in the Montreal Neurological Institute (MNI) template space, and smoothing with a Gaussian kernel of 8 mm full width at half maximum (FWHM). A cutoff low-pass filter of 0.01~0.1 Hz was applied to remove high-frequency signal from the fMRI data. No subjects were excluded from data analysis due to the head motion (more than 2 mm of displacement or 1° of rotation in any direction).All fMRI data preprocessing was performed using Statistical Parametric Mapping . The templates of three ROIs were then applied to the statistical parametric map. The spatial distribution of activation extent (the number of activated voxels) and intensity (the mean and maximum t-score) in each ROI were calculated from the statistical parametric map at the individual level. The olfactory activation map at group level was generated using one-sample t-tests  across all subjects. The group activation maps were displayed on the high-resolution T1-weighted image using the software of Data processing & Analysis for Brain Imaging .For postprocessing, all smoothed fMRI data were analyzed with general linear model (GLM) and ROI analysis. GLM analysis and ROI analysis were repeatedly performed for each TR condition. In the traditional GLM analysis, a statistical parametric map at the individual level was generated by fitting the olfactory stimulation paradigm to the functional data. The odor stimulations by the three lavender concentrations were used as one stimulation condition . To meashttps://rfmri.org/dpabi) on Matlab 2013a. The BOLD signal time course represented the percentage changes of the BOLD signal by time [ROI analysis was performed for smoothed fMRI data to extract the event-related time course in three ROIs using the toolbox of Data processing & Analysis for Brain Imaging  were expressed as the mean ± standard deviation. The repeated measures analysis of variance (ANOVA) was performed to examine the effects of TR on each metric. Pairwise comparison testing with Bonferroni's correction was carried for multiple comparisons. The P value was defined as smaller than 0.05 to be statistically significant.Statistical analysis was performed using SPSS version 23 . All metrics  = 18.307, P < 0.001) and insulae  = 15.738, P = 0.001). As shown in P = 0.001) and 500 ms . In the insulae, the number of activated voxels was increased when decreasing TR 3000 ms to 1000 ms  and 500 ms . Pairwise comparison testing revealed there was no significant difference in the number of activated voxels between TR 500 ms and 1000 ms in the amygdalae and insulae . The more activated voxels in the hippocampus were at TR 500 ms and 1000 ms than TR 3000 ms but did not reach a statistic significance (P = 0.422).Group activation maps of three different TRs are shown in t-score in the amygdalae (P < 0.001) and insulae (P < 0.001), and no significant effect was observed in the hippocampi (P = 0.077), which are shown in TR had a statistically significant effect on the mean t-score was consistent with it on the mean t-score. The maximum t-score increased significantly when decreasing TR in the amygdalae and insulae  but did not significantly change across TRs in the hippocampi (P = 0.082), which are shown in The effect of TR on the maximum t-scores were both significantly increased at TR 500 ms and 1000 ms than that at TR 3000 ms in the amygdalae and insulae . The maximum and mean t-scores did not significantly differ between TR 500 ms and 1000 ms in all three ROIs  (P = 0.002) and 1000 ms (P = 0.017), and the response height did not significantly differ between TR 1000 ms and 3000 ms (P = 0.269). In the hippocampi, the response height was only higher at TR of 500 ms compared to TR of 3000 ms (P = 0.008) and the other pair comparisons of TRs did not reach statistical differences . In the insulae, the response height was higher at TRs of 500 ms (P = 0.019) and 1000 ms (P = 0.035), compared with TR of 3000 ms. There was no significant difference in response height between TR 500 ms and 1000 ms in insulae (P = 1.000).Response height was significantly enhanced when decreasing TR in all ROIs (insulae) . In the P ≤ 0.001). In the amygdalae, the averaged time to peak was, respectively, decreased to 8.00s and 7.75 s at TR 1000 ms (P = 0.003) and 500 ms (P = 0.001) from 10.13 s at TR 3000 ms. Pair comparison testing revealed that the averaged time to peak in the hippocampi was reduced from 10.00s to 6.88 s and 6.44 s when decreasing TR 3000 ms to 1000 ms (P = 0.001) and 500 ms (P < 0.001), respectively. The averaged time to peak in the insulae was reduced from 9.56 s to 7.81 s and 7.81 s when decreasing TR 3000 ms to 1000 ms (P = 0.039) and 500 ms (P = 0.013), respectively. The time to peak did not significantly differ between TR 1000 ms and 500 ms in all ROIs .As presented in t-scores) was obtained with reduced TRs (500 ms and 1000 ms), compared with TR of 3000 ms. More than that, higher response height and shorter time to peak in HRF curve were measured with reduced TRs (500 ms and 1000 ms).The present study investigated whether SMS technology with reduced TR could improve the BOLD activation and optimize HRF modeling in the olfactory fMRI. The results showed that an improved BOLD activation  in improving functional activation and statistical power [In the olfactory fMRI scan, other imaging parameters of three SMS-EPI sequences were equal except for TR. The effect of TR was elucidated. The difference in the number of samples was occurred across three different TR conditions. Shorter TR significantly increased the number of samples in a given time. The fast sampling rate at TR of 500 ms and 1000 ms increased, respectively, the number of images by 6 times and 3 times at the same scan duration, compared with the TR of 3000 ms. The increased amount of data acquisition consequently improved the detection of functional activation. This study result demonstrated that larger activated voxels were measured with TRs of 500 ms and 1000 ms. Georgiopoulos et.al  investigal power , 21.In this olfactory fMRI, TR 500 ms did not seem to have obvious advantages over TR 1000 ms for improving functional activation. The steady-state MR signal was reduced at shorter TRs. At the short TR acquisition, image signal was exponentially reduced due to decreased T1 recovery within the TR . TherefoThe sampling density of BOLD response was determined by the TR of the fMRI sequence. SMS technology provided the higher sampling rate at TRs of 500 ms and 1000 ms, compared with 3000 ms. A high data sampling of the BOLD response was advantageous for precisely characterizing the peak signal . The resThe optimum value of TR in the fMRI experiment was not easily calculated. Temporal correlations of the noise limited the very short-TR imaging . SeveralBilateral hippocampi were poorly activated for the olfactory stimulation in the present study. The activation in the hippocampi was not drastically increased when decreasing TR. The function and location of the hippocampi might explain something. The hippocampi were classified into the secondary olfactory cortex, involving in higher-order odor-related processing, such as behavior regulation and emotional response , 27. In The present study had certain limitations. The first limitation was the small size of subjects, and all the subjects were young healthy. Further studies on elder subjects or clinical patients were still needed. Second, we only tested three TRs ; more TRs within 500 ms-1000 ms should be added into the tested TRs to determine the optimal TR for the BOLD activation and HRF curve. Third, a simple odor stimulation paradigm without respiration triggering was employed in the present olfactory fMRI given that the respiration-triggering method prolonged olfactory stimulation paradigm and increased complexity of data processing. Odor molecules only be sensed when entering into nasal cavity and activating the olfactory epithelium at the beginning of the inspiration phase. The correction of actual odor stimulation onsets as the start time of first inhalation during the odor delivery time period would minimize the time synchronization error between odor delivery and respiration. Reduced TRs using SMS imaging and respiration correction method might further improve BOLD activation and HRF modeling and could be explored in the future work.SMS provides an excellent opportunity to investigate neuronal responses to brief stimuli. A better understanding of how to best choose TR in order to optimize experimental design is needed. The acceleration technology of SMS allows to significantly reducing TR. Our study suggested decreasing TRs were beneficial for the improvement of BOLD activation and estimation of HRF in olfactory fMRI.