Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. If host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Moreover, how the vital and characteristic cyst matrix and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma intravacuolar-network-associated dense granule (GRA) proteins during cyst development in vitro. Intravacuolar-network GRAs were present within the cyst matrix and at the cyst wall in developing cysts, and genetic deletion of intravacuolar-network-associated GRAs reduced the rate of accumulation of cyst wall material at the cyst periphery. Our results show that intravacuolar-network-associated GRAs, particularly GRA2 and GRA12, play dynamic and essential roles in the development and maturation of the cyst matrix and the cyst wall structures. Toxoplasma gondii cyst develops. Here, we investigated intravacuolar-network-associated dense granule (GRA) proteins GRA1, GRA2, GRA4, GRA6, GRA9, and GRA12 during cyst development in vitro after differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 postdifferentiation, GRA1, GRA4, GRA6, GRA9, and GRA12 colocalized with Dolichos biflorus agglutinin stain at the cyst periphery. In contrast, GRA2 remained in the cyst matrix. By day 2 postdifferentiation, coinciding with localization of GRA2 to the cyst periphery, GRA1, GRA4, GRA6, and GRA9 established a continuous matrix pattern in the cyst. In contrast, GRA2 and GRA12 were colocalized in prominent cyst matrix puncta throughout cyst development. While GRA2, GRA6, and GRA12 localized in outer and inner layers of the cyst wall, GRA1, GRA4, and GRA9 localized predominantly in the inner layers of the cyst wall. GRA2 and GRA12 were colocalized in the cyst wall by day 7 postdifferentiation. However, by day 10 postdifferentiation, GRA12 was relocalized from the cyst wall to puncta in the cyst matrix. Differentiation of Δgra2 parasites revealed a defect in the ability to establish a normal cyst matrix. In addition, the deletion of any intravacuolar-network-associated GRA protein, except GRA1, reduced the rate of accumulation of cyst wall proteins at the cyst periphery relative to the cyst interior. Our findings reveal dynamic patterns of GRA protein localization during cyst development and suggest that intravacuolar-network-associated GRA proteins regulate the formation and maturation of the cyst matrix and cyst wall structures.Little is known regarding how the chronic IMPORTANCEToxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. If host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Moreover, how the vital and characteristic cyst matrix and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma intravacuolar-network-associated dense granule (GRA) proteins during cyst development in vitro. Intravacuolar-network GRAs were present within the cyst matrix and at the cyst wall in developing cysts, and genetic deletion of intravacuolar-network-associated GRAs reduced the rate of accumulation of cyst wall material at the cyst periphery. Our results show that intravacuolar-network-associated GRAs, particularly GRA2 and GRA12, play dynamic and essential roles in the development and maturation of the cyst matrix and the cyst wall structures. Toxoplasma gondii   . Inside ole (PV) , 12. If ole (PV) , 14, an ole (PV) , 16. Braole (PV) , 18. Theole (PV) . The cysole (PV)  and is oole (PV) . The cysted mice . An estaigher pH , 23 trigin types . These is of age .Dolichos biflorus agglutinin (DBA) stain  stain . The cysA) stain –27. DBA moieties  that heamoieties , 28. CSTmoieties . A recenin vitro .Tachyzoites massively secrete the dense granules and their GRA proteins into the PV space soon after the PV initially forms. These GRAs modify the nascent PV and the host cell environment , 31. Sho34–Deletion of IVN-associated GRA2, GRA4, GRA6, GRA9, or GRA12 caused a major defect in the development of cyst burdens during chronic infection , 50. TheHere, we determined the locations of IVN-associated GRA proteins during cyst development. Our findings show that deletion of IVN-associated GRAs significantly reduced the rate of accumulation of cyst wall proteins at the cyst periphery relative to the cyst interior, suggesting that these IVN GRA proteins play an important role in the development of the cyst wall. GRA2 expression was essential for normal development of the cyst matrix and accumulation of GRA4 and GRA6 at the cyst periphery. IVN-associated GRA2 and GRA12 showed dynamic patterns of relocalization between the cyst matrix and the cyst wall. Collectively, our results suggest that IVN-associated GRAs play vital roles in the formation and maturation of the cyst matrix and cyst wall structures.ku80 genetic background  Δckground , 50. Herce assay . Since tce assay  52), we, weku80 ce assay .10.1128/mSphere.00487-19.1FIG S1HXGPRT at the deleted GRA1 gene locus by selection with mycophenolic acid (MPA) and xanthine (X). As indicated in the schematic, the GRA1 gene and the MAG1 gene are neighbors on the chromosome. Double crossovers occurred at the GRA gene of interest (GOI) locus when the PruΔku80Δhxgprt strain allowed the replacement of the targeted GRA coding sequence by that of the HXGPRT resistance marker, leading to the isolation of the PruΔku80Δgra1::HXGPRT strain. The locations of the genotype validation PCRs are shown above the schematized PruΔku80Δgra1::HXGPRT genome. The genotype of the GRA1 knockout was verified by PCR (not shown). (B and C) The PruΔgra1 strain lacks expression of GRA1 and retains expression of MAG1. Host HFF cells on coverslips were infected with an MOI of 0.5 tachyzoite of each strain, PruΔku80 or PruΔku80Δgra1 (Δgra1), permeabilized with 0.01% saponin, and stained with α-GRA1 (B) or α-MAG1 (C) (shown in red). DAPI (shown in blue) stained the host cell and parasite nuclei. PVs were located using DIC microscopy, and representative images are shown. Scale bars = 5 μm. (D) Complementation strategy at the original locus through selection with 6TX (6-thioxanthine). Design of genotype validation PCR is shown. The genotype of the GRA12 complementation was verified by PCR (not shown). Download FIG S1, TIF file, 1.2 MB.GRA1 knockout and GRA12 complementation strategy. (A) Knockout strategy to insert Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gra1, Δgra2, Δgra4, Δgra6, Δgra9, and Δgra12 cysts   .10.1128/mSphere.00487-19.2FIG S2in vitro cysts. Cysts were located using DIC microscopy and imaged by confocal microscopy. The presence of bradyzoites inside cysts was verified by locating parasite nuclei with DAPI staining (not shown) and verifying that each parasite nucleus was surrounded by expression of cytosolic GFP (GFP+ bradyzoites). Cysts were stained with DBA (shown in red), which highlights the cyst wall structure. Scale bars = 50 pixels. (C and D) The dotted lines (upper panels) highlight the region that is magnified below (lower panels). (C) The mask (shown in blue) is drawn using the fluorescence intensity of DBA (shown in red) (panel 1). The mask was duplicated to create a region between layers. Fourteen layers (L-2 to L-15) were measured from the mask outside the cyst to provide background readings. (D) Six layers (L-1 to L5) are the layers of the cyst wall region. (E) Representative layers of the cyst interior. Download FIG S2, TIF file, 0.6 MB.Defining the cyst wall and the locations of IVN-associated GRA proteins relative to the cyst wall strategy. (A to E) Schematic showing the locations of GRA proteins within the cyst wall. (A and B) Schematic showing the method of measurement. The mask (shown by the dotted blue line) was duplicated to form a region between two layers, approximately 1 pixel thick where the fluorescence intensity of that region was measured. (C to E) Infected HFFs on coverslips were treated with bradyzoite-inducing conditions for 7 days to induce Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00487-19.3FIG S3in vitro cysts. Cysts were located using DIC microscopy and imaged by confocal microscopy. The presence of bradyzoites inside cysts was verified by locating parasite nuclei with DAPI staining (not shown) and verifying that each parasite nucleus was surrounded by expressed cytosolic GFP (GFP+ bradyzoites). Cysts were stained with DBA (shown in red), which highlights the cyst wall structure. The layers (L) (shown by the blue line) are denoted with a minus sign, which indicates layers outside the mask, or no sign, which indicates layers inside the mask. The total fluorescence intensity inside the blue line is measured at each layer. (C) Layers that compose the cyst wall. (D) Layers inside the cyst wall. Layer 5 (for day 1) or layer 6 (for day 2 through day 10) was determined to be the border between the cyst periphery, which includes the cyst wall plus two layers in the cyst matrix to account for proteins at the cyst periphery that are not yet incorporated into the cyst wall and the cyst interior (which includes everything within the cyst but excludes the cyst periphery). Download FIG S3, TIF file, 0.6 MB.Locations of IVN-associated GRAs. (A to D) Schematic illustration of the method used for quantification of the fluorescence intensity of GRA proteins within the cyst wall relative to the cyst interior. (A) A mask (shown by the blue line) is drawn using the fluorescence intensity of DBA (shown in red) to define the boundary of the cyst, which is named layer zero (L0). Scale bars = 50 pixels. (B) Schematic showing the primary features of the cyst and their definitions. The total fluorescence intensity of the cyst is one layer away from the mask, layer negative one (L-1). The fluorescence intensity of the cyst interior is layer five (L5) for day 1 or layer six (L6) for day 2 through day 10. The fluorescence intensity of the cyst periphery was determined by subtracting the fluorescence intensity of the cyst interior from the fluorescence intensity of the total cyst. (C and D) Infected HFFs on coverslips were treated with bradyzoite-inducing conditions for 7 days to induce Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00487-19.9TEXT S1T. gondii proteins within the cyst. Download Text S1, TXT file, 0.01 MB.Macro for location of Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro cyst development using specific antibodies to locate GRA1, GRA2, GRA4, GRA6, and GRA9 and a Δgra12 mutant that was complemented with wild-type GRA12 tagged at the C terminus with hemagglutinin (HA) for visualization of GRA12  bradyzoites were visible within the developing cyst  and verifying each parasite nucleus was surrounded by expression of cytosolic GFP (GFP+ bradyzoites). (A) Cysts were stained with DBA and anti-GRA antibody (αGRA). For each GRA protein assessed, panels show GFP and DBA, αGRA, DBA and αGRA, and DIC. Scale bars = 5 μm. (B) GRA proteins were assessed for colocalization with GRA2. Cysts were stained with α-GRA2 (red) and α-GRA (green). Scale bars = 5 μm. (C) Cysts were stained with α-GRA9 (red) and α-GRA1 (green). Scale bars = 5 μm. Fluorescence intensity profiles of the cysts in FIG S4, TIF file, 1.9 MB.In 6-h cysts, IVN-associated GRAs are localized to the cyst periphery shortly after differentiation while GRA2 molecules are delayed to the cyst periphery. Infected HFFs on coverslips were treated with bradyzoite-inducing conditions for 6 hours to differentiate Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + bradyzoites were visible within a DBA-stained cyst  of GRA proteins in the cyst, we measured the cyst fluorescence intensity profiles for GRA proteins, DBA, and GFP using the Fiji macro  to measuDBA-stained puncta were observed in the cyst matrix, indicating that the cyst wall was actively being constructed . GRA1, GIn contrast to five layers of the cyst wall region observed in 1-day cysts, the cyst wall region in 2-day cysts occupied six layers  to J. ThSimilar to 1-day cysts  or 2-dayThe cyst wall region in 3-day cysts occupied six layers  to J. ThAll GRAs were localized to the cyst matrix; GRA1, GRA4, GRA6, and GRA9 occupied the spaces between the bradyzoites, while bright puncta of GRA2 and GRA12 were visible within the cyst matrix . All GRA10.1128/mSphere.00487-19.5FIG S5FIG S5, TIF file, 0.5 MB.GRA2 and GRA12 localize at the cyst wall in 7-day and 10-day cysts, and then GRA12 leaves the cyst wall in 10-day cysts. (A) Images from Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The cyst wall region in 7-day cysts occupied six layers  to J. ThAll GRAs localized to the cyst matrix; GRA1, GRA4, GRA6, and GRA9 occupied the spaces between the bradyzoites, while bright puncta of GRA2 and GRA12 were prominent within the cyst matrix . GRA12 wThe cyst wall region of 10-day cysts occupied six layers  to J. Thgra2 cysts. GFP+ bradyzoites were visible within DBA-stained Δgra2 cysts that lacked expression of GRA2   , suggestyst size , and thi10.1128/mSphere.00487-19.6FIG S6ku80 or PruΔku80Δgra2 (Δgra2) and treated with bradyzoite-inducing conditions to differentiate in vitro cysts for 1 day (A), 2 days (B), 3 days (C), (D) 7 days, or 10 days (E). Cysts were located using DIC microscopy and imaged by confocal microscopy. The presence of bradyzoites inside cysts was verified by locating parasite nuclei with DAPI staining (not shown) and verifying that each parasite nucleus was surrounded by expressed cytosolic GFP (GFP+ bradyzoites shown in the first panel of panels A to E). Cysts were stained with DBA (red) and α-GRA2 (green). Representative cysts are shown. Scale bars = 5 μm. (F) The fluorescence intensity of DBA was measured at the cyst periphery and within the cyst (cyst interior) for each time point. Data plotted are the mean ratio of fluorescence intensity at the cyst periphery to cyst interior ± SEM . P values were calculated with a Student’s t test and indicated as follows: *, P < 0.05; ***, P < 0.005; ****, P < 0.0001; NS, not significant. (G) Cyst area ± SEM (n = 24 cysts) was measured at the indicated times postdifferentiation. P values were calculated with a Student’s t test . Download FIG S6, TIF file, 2.0 MB.GRA2 controls DBA staining intensity at the cyst periphery relative to the cyst interior. (A to E) HFFs on coverslips were infected with PruΔCopyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gra2 cysts, we evaluated the localization of GRA4 and GRA6 in Δgra2 and PruΔku80 cysts. GRA4 and GRA6 were observed at the cyst wall  of GRA4 and GRA6  Fluorescence intensity profiles of the cysts in  FluorescCopyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gra2 cysts and the known role of GRA2 in tubulating the IVN membranes .GRA2 tubulates vesicles into highly curved membranes in vitro . Correspin vitro . GRA6 wain vitro . Our datin vitro , 19, andin vivo cysts, CST1 is localized with granular material uniformly distributed throughout the wall  or IVN-associated GRA proteins was recently shown to markedly reduce cyst burdens  monolayers (ATCC SCRS-1041.1) cultured in Eagle’s modified essential medium (EMEM) (Lonza) containing 1% fetal bovine serum (FBS) (Life Technologies), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 36°C in 95% air and 5% CO2. HFF cells were maintained in EMEM containing 10% FBS (HyClone), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 95% air and 5% CO2. The parental Pru strain PruΔku80 was previously made transgenic for green fluorescent protein (GFP) under the control of the LDH2 bradyzoite stage-specific promoter (ku80 knockout strain of the type II Pru strain using previously described methods (Type II Prugniaud (Pru) background promoter . Strains methods  . Kn. Knku80 database . All olidatabase . Linearishown in  to measu10.1128/mSphere.00487-19.8TABLE S1Table S1, DOCX file, 0.03 MB.DNA primers for construction of gene replacement plasmids. Download Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gra12 through targeted chromosomal integration and expression of wild-type GRA12 at the endogenous locus using previously described methods , 69gra12 methods . Oligonu methods  were use methods  to measu methods .2+ and Mg2+ (HyClone) and incubated for 1 h at room temperature (RT) with a 1:1,000 dilution of secondary goat anti-mouse IgG antibodies conjugated to Alexa Fluor 594. All samples were mounted in Slowfade Gold antifade with DAPI  (Life Technologies) and imaged at 100× with a Nikon A1R SI confocal microscope . PVs were located using differential interference contrast (DIC) microscopy. Confocal images as raw .nd2 files were imported and minimally processed for brightness in Fiji  and infected with parasites for 24 h. Samples were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.01% saponin (Sigma) for 10 min, and blocked with 10% FBS for 20 min. All samples were incubated with 1:500 dilution of primary mouse monoclonal anti-GRA1 or 1:50 dilution of anti-MAG1 antibody. Preparations were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) supplemented with Ca in Fiji .in vitro into bradyzoites within cysts essentially as previously and elegantly described by Tobin and colleagues (l-glutamine (HyClone), 20 mM HEPES (free acid) (IBI Scientific), 1% XL-glutamine (a long-lasting stable form of glutamine) (VWR), 1% FBS, and 1% penicillin-streptomycin. The pH of the differentiation medium was adjusted to 8.1 with sodium hydroxide and filter sterilized. HFF cells were cultured on circular micro cover glass until confluent (Electron Microscopy Sciences), and confluent monolayers were infected with type II Pru parasites at a multiplicity of infection (MOI) of ∼0.5. Three hours after infection, infected cells were washed once in DPBS supplemented with Ca2+ and Mg2+ and incubated in differentiation medium for 6 h, 1 day, 2 days, 3 days, 7 days, or 10 days at 37°C in ambient air. The medium was replaced with fresh medium on day 3 and day 7.Tachyzoites were differentiated lleagues . Differe2+ and Mg2+ and incubated for 1 h at RT with a 1:1,000 dilution of secondary goat anti-rabbit (H+L) (Thermofisher) and goat anti-mouse IgG (H+L) antibodies conjugated to Alexa Fluor 647 . All samples were incubated with a 1:250 dilution of rhodamine-labeled Dolichos biflorus agglutinin (Vector Laboratories) for 1 h at RT. Samples were mounted in Slowfade Gold antifade with DAPI (Life Technologies) and then imaged with a Nikon A1R SI confocal microscope  using an Apo TIRF 100× Oil DIC N20 objective. Cysts were randomly selected for analysis by locating cysts using DIC microscopy. Bradyzoite differentiation in cysts was confirmed by GFP+ bradyzoites. The focal plane (from a Z-stack) selected for quantification was from the middle of the cyst, where the cyst size is maximal  to stain GRA12, or 1:1,000 dilution of primary mouse monoclonal anti-GRA1, anti-GRA2 antibody , or 1:1, maximal . Raw .nd maximal . The num10.1128/mSphere.00487-19.10MOVIE S1Movie S1, MOV file, 0.04 MB.Movie of a 7-day cyst stained with DBA and GRA6 (Z-stack). Download Copyright © 2019 Guevara et al.2019Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the n = 10 to 34 cysts) and multiplied by 100 to be presented as a percentage of cysts that showed colocalization.Each imaged cyst was analyzed for spots of yellow, which indicated colocalization between GRA2 and GRA4/GRA6/GRA9/GRA12 or GRA9 and GRA1. The numbers of cysts that were positive for yellow spots were counted out of the total number of cysts  or GFP , respectively. The rationale for selecting these parameters is discussed in the Results section. This standardized analysis was performed for each cyst assessed for each time point on or after day 1. One-day cysts revealed five layers  that defined the cyst wall region, while 2-day-, 3-day-, 7-day-, and 10-day cysts revealed six layers  that defined the cyst wall region, denoted by dotted vertical black lines in the graphs in the figures. Consistently, the peak of DBA fluorescence was observed in layer L1 in 1-day cysts . P values were calculated with a Student’s t test.The surface area of each cyst was measured during quantification of total fluorescence intensity. Area values were measured at L-1 in differentiated 3-, 7-, and 10-day t tests were used to calculate P values. All calculations of averages, standard errors of the means (SEM), and P values were performed using GraphPad PRISM software version 5.0c.Unpaired


Newborns, especially preterm infants, have an immature immune system, which, in combination with the required medical interventions necessary for keeping the neonate alive may lead to an increased risk of infection. Even after reaching stability and adapting to the environment, preterm infants have adverse prognoses regarding infections and long-term outcomes compared to their full-term counterparts. The objective of this study was to research differences in the number and severity of infections between preterm and full-term infants during their first year of life. To answer this question, a monocentric prospective study was conducted in a pediatric practice in Vienna, including 71 full-term infants and 72 preterm infants who were observed during their first year of life regarding occurring infections. In respective samples, there was a significantly higher total number of infections in preterm (mean 6.01 ± 3.90) compared to full-term infants (3.85 ± 1.72) during the observation period of one year. Particularly the count of respiratory and severe infections was considerably higher in preterm infants. Otorhinolaryngeal infections were the most frequent of all types of infections in both groups. The pregnancy period, number of siblings, and length of the postnatal hospital stay, were observed as significantly influencing factors which affected the total number of infections. The group of early term infants (37+0 weeks to 38+6) was not significantly different to late term babies (>39+0). The acquired knowledge about the increased risk of infections should lead to a more extensive care for preterm infants, with the objective of reducing the rates of complications, morbidity and mortality in this vulnerable age group in the future. A premature birth is by definition a birth taking place before the 37th week of gestation (WOG), with further classification into late 32–37 WOG), very (28–32 WOG) and extremely preterm birth (<28 WOG), or with regard to birth weight, into low , very low VLBW; <1500g) and extremely low birth weight  [500g and –37 WOG, The combination of undeveloped organs and the high need for invasive care and life support measures is of great importance in the pathogenesis of many complications including infections . In partSeveral studies have shown that the risk of infection increases with decreasing gestational age and birth weight , 9. PretThe group of late preterms is an extremely important subgroup, accounting for almost 75% of all preterm infants and numerically the most fasting growing subgroup of newborns . AlthougThe overall aim of this monocentric prospective observational study was to investigate whether preterm infants were more likely to suffer from infections than full-term infants during their first year of life, what types of infections were present and whether associated factors were observable.Ethical approval for this monocentric prospective observational study was applied for and granted by the ethics committee of the Medical University of Vienna (EK-Nr: 1330/2016). Participants were recruited in a Viennese pediatric practice, the “First Vienna Pediatric Medical Center”. Exact case number planning was not required due to the exploratory approach of this study. Parents were informed about the study and gave written consent. Data of infants were pseudonymized for analysis.Inclusion criteria for the observation group of preterm infants was a birth before WOG<37+0, and for full-term group a birth after WOG >37+0. Gestational age was based on parental report and calculation according to a gynaecologist. In case of inconsistent results of sonography and calculated gestational age, adaptions were done by the gynaecologist. Children with chronic disease were excluded from the study. Those infants, who were primarily recruited, but did not appear in the practice during the observation period were finally excluded from the study analysis as drop out. Data were analysed only from those infants, who completed the observation period of one year. The general, demographic and birth-specific data were collected by the legal guardians using a questionnaire.other infections. Serious infections were thus defined as those that led to hospitalisation.Subsequently, study subjects were asked to attend the study center in case of infections. They were observed throughout the first year of life for infections and complications that occurred since birth, which was documented in the patient records. At the end of the first year of life, the total number of infections and the number of different types of infection were calculated for each child. The different types of infection included respiratory, otorhinolaryngeal (ENT-infections), urinary tract (UT- infections), gastrointestinal (GI-infections), dermal and severe infections. Infections that could not be assigned to any of these groups were assigned to the category of The raw data  was compr was used according to the Cohen classification  [2) or in cases of expected values <5 in more than 20% of the cells, Fishers Exact (cF) test were performed. The McNemar test was used to test relationships of dependent nominally scaled variables. For analysis of strength and direction of association of two ranked, non-parametric variables, Spearman correlation coefficient (rs) was calculated. Differences of two independent samples were tested either with t-test  or the Mann Whitney U-test as a parameter-free alternative. Kruskal-Wallis test (H) was performed for comparison of medians of more than 2 groups. Multiple linear regression was performed for analysing the influence of diverse predictors. Two-way ANOVA was used for testing for differences of infection frequency per month of age. Correction of multiple testing was done using the Holm-Bonferroni method and adjusted p-values were calculated.To estimate the practical relevance of statistically significant relations, the standardised effect size  effect) . The oddEighty preterm and eighty term infants were primarily recruited in this study. Due to loss of follow up, being originated in changing the paediatrician during the observation period, the original case count was reduced to 71 for the full-term and 72 for the preterm infants, which corresponds to a drop-out rate of 10.6%.As depicted in There was an even gender distribution with 40 (54.1%) male and 31 (44.9%) female full-term infants and 34 (45.9%) male and 38 (55.1%) female preterm infants.2(1) = 9.96 and p = .002) compared to term infants (28.2%) indicating a threefold higher rate of being born by caesarean section compared to full-term infants .With regard to the birth procedure, significantly more preterms (54.2%) were born by Caesarean section (X2(1) = 12.444, p < .001). Nuchal cord as most common complication in term infants (7%), leading to intrauterine asphyxia in two cases, was followed by gestational diabetes (GDM) with concomitant macrosomia, isolated GDM, polyhydramnios and premature rupture of the membranes (PROM) (1.4% each)). In the preterm infants group, the most frequent complications were pre-eclampsia (9.7%), preterm rupture of membranes (9.7%), maternal HELLP syndrome (5.5%) and GDM (5.5%). Severe preeclampsia in combination with placental insufficiency occurred with 2.8%, and placenta praevia and premature placental abruption with 1.4%.Concerning pregnancy and birth complications, 36.1% of preterm and 23.9% of full-term infants showed a complication significantly associated with preterm birth (X2(1) = 8.511 and p = .004) with about five times greater risk . In preterm infants, 28 (38.9%) had respiratory distress syndrome, 2.8% intraventricular hemorrhage, 1.4% necrotizing enterocolitis, 9.7% patent ductus arteriosus and 4.2% a retinopathy of prematurity. Twelve (16.7%) suffered from a congenital infection, 1.4% had neonatal sepsis 55.5% had neonatal jaundice. Thirty-three (46.5%) full-term infants had neonatal jaundice and one of them each had a congenital infection and neonatal sepsis (1.4%).Perinatal complications were observed in 81.9% of preterm infants and 47.9% of full-term infants with a significantly higher rate in preterm infants (XU = 2175.5 (-1.664), p = .096).As for the number of siblings, a trend could be assumed that preterm infants with a mean of 0.92  had a higher number of siblings than full-term infants with a mean value of 0.66   full term infants were looked after at home and seven (9.9%) in a nursery, whereas within the preterms 65 (90.3%) grew up at home and seven (9.7%) in a nursery. As for the housing situation, 19 (26.4%) preterms grew up in a house, 52 (72.2%) in an apartment and one (1.4%) in a smoking household. Regarding the full-term infants 17 (23.9%) grew up in a house, 52 (73.2%) in an apartment and two (2.8%) in a smoking household. As for the breastfeeding factor, preterm infants (72.7%) and full-term infants (74.6%) were breastfed equally frequently.U = 858.0 (z = -7.951), p < .001, r = .67), with an average duration of 5.18 days for full-term and 27.52 days for preterm infants.In the present sample, it could be shown that preterm infants had a significantly longer hospital stay after birth compared to full-term infants (p < .001). In full-term infants, 3.85 ±1.72 , and in preterm infants 6.01 ±3.90  infections were detected during the first year of life. The coincidence was tested by a bivariate plot graph (r(143) = -.394, p < .001).Preterm infants had a significantly higher total number of infections within the first year of life than full-term infants (ot graph  and show2(2) = 3.193, p = .203; birth weight: X2(3) = 1.766, p = .622)  .Extremely and very preterm infants did not differ regarding the number of infections, which is the reason for combining these groups for the next analysis. As gestational age might be only estimated within 1–2 weeks, the full-term babies were divided into early term infants (WOG 37+0 to 38+6) and late term (> = 39+0 WOG). The total frequency of infections within the first year of life was compared between very preterm babies , late preterm infants , early term (n = 21) and late term infants (n = 50) . No signWith regard to the different types of infection, it was found that respiratory  and severe  infections showed a significant difference in the comparison of the two observation groups. The most frequent severe infections leading to hospitalization were, except for connatal infections directly after birth, respiratory infections (42%), gastroenteritis (33%), fever of unknown origin (9%), urinary tract infection, influenza with emesis and meningitis (each 4%).For the group of UTI, the quantity tested  was significant, although only 3 preterm and no term infants suffered from UT-infections .p = .002) as well as the number of siblings (p = .038) had a significant influence. The individual predictors were also analysed separately for their influence on the risk of infection, which in turn showed that there was a weak positive relationship between number of siblings and total number of infections (rs(143).235, p = .002). A weak positive relationship between hospital stay after birth in days and the total number of infections during the first year of life were found (rs(142).17, p = .044). The analysis of the remaining predictors revealed insignificant effects.As possible predictors that can influence the total number of infections, the following additional factors were checked: gender, birth procedure , birth in the warm (March-September) vs. cold season (September-March), number of siblings, presence of pregnancy and childbirth complications, occurrence of perinatal complications, child care, living conditions and breastfeeding during the first year of life. The analysis showed that only the duration of pregnancy  in preterm and 0–5 (Md = 2.0) in full-term infants. The most frequent reasons for consultation were bronchitis, rhinopharyngitis, otitis and gastroenteritis, while in the group of preterm infants, more severe infections, febrile convulsions and breath-holding spells were observed [In the sample concerned, there was a significantly higher total number of infections in preterm compared to full-term infants. In the only comparable study, the total number of infectious diseases in the first 12 months of life were 0–13  could have falsified the results.Preterm infants represent a particularly significant and vulnerable group with increasing survival rates. Knowing the higher risk of complications and infections in preterm infants during their first year of life, more comprehensive care should be offered to these children. The care of preterm infants should not end after discharging them and, above all, late-preterm infants should not be mistakenly regarded as full-terms and treated as such. Parents should be informed about the increased risk of infection and the preventive measures urgently. Vaccination of all family members who come into contact with the infant, adequate hand disinfection and avoidance of environments that increase the microbial load are important prophylactic measures.S1 Table(SAV)Click here for additional data file.


Pulmonary hypertension (PAH) among children and adults has been linked to premature birth, even after adjustments for known risk factors such as congenital heart disease and chronic lung disease. The aim of this population-based registry study was to assess the risk of PAH following exposure to premature birth and other factors in the decades when modern neonatal care was introduced and survival rates increased.Data on pulmonary hypertension and perinatal factors were retrieved from population-based governmental and national quality registers. Cases were adults and children over five years of age with pulmonary hypertension born from 1973 to 2010 and individually matched to six controls by birth year and delivery hospital. Conditional multiple logistic regression was performed to assess the risk of pulmonary hypertension following premature birth and to adjust for known confounding factors for the total study population and for time of birth, grouped into five-year intervals.In total, 128 cases and 768 controls were included in the study group. Preterm birth was over three times more common among cases (21%) than among controls (6%). The overall adjusted risk of pulmonary hypertension was associated with premature birth, OR = 4.48 . Maternal hypertension, several neonatal risk factors and female gender were independently associated with PAH when potential confounders were taken into account. For each five-year period, the risk of PAH following premature birth increased several times for children born in the 2000s and later, OR = 17.08 (95% CI 5.60–52.14).Preterm birth, along with other factors, significantly contributes to PAH. PAH following premature birth has increased over the last few decades. Our study indicates that new, yet unknown factors may play a role in the risk of preterm-born infants developing PAH later in life. Preterm birth has previously been linked to pulmonary arterial hypertension (PAH) in children and adults . PAH is This population-based national case-control registry study assessed neonatal risk factors for children and young adults with pulmonary hypertension compared to healthy controls. The study is based on registry data, and individual informed consent from each participant is not required due to a waiver from the ethical committee and national guidelines.All children aged five years or older and adults who were born between 1973 and 2010 and who were registered in the population-based Swedish Medical Birth Register were included in this case-control study. Cases were all diagnosed with PAH; those who were born in 1973–1996 were retrieved from the Swedish Pulmonary Arterial Hypertension Registry (SPAHR), and those who were born in 1993–2010 were retrieved from the Swedish registry of Congenital heart disease (SWEDCON). All cases retrieved from SPAHR were diagnosed according to the Dana Point Classification using right heart catheterization, and the cases retrieved from SWEDCON were diagnosed using either right heart catheterization and/or transthoracic Doppler echocardiography. The registries and retrieval of cases and controls are described in previously published studies .Six controls without pulmonary hypertension were matched to each case by year of birth and hospital. Cases who were not born in Sweden were excluded.A national registration number is assigned at birth to every child born in Sweden.Maternal factors during pregnancy , neonatal data  were retrieved from the Swedish Medical Birth Register using the International Classification of Diseases ICD-9 or ICD-10 codes [Linkages between governmental and national quality-based registries was possible with national registration numbers, which were used for the retrieval of exposure information for both cases and controls  .The association between preterm birth prior to 37 weeks of gestation and PAH for the whole period (1973–2010) was calculated by conditional logistic regression and adjusted for confounding factors.To assess the association between preterm birth and PAH over time, we subgrouped the study population into five-year intervals based on the year of birth. We then calculated the risk of PAH following premature birth for each group and adjusted for confounding factors. Exposure to potential confounders is described in Table Maximum-likelihood estimates of the odds ratio (OR) and 95% confidence interval (CI) were obtained. SAS version 9.4  was used to fit the conditional logistic model to our 1:6 matched case-control data.The study was approved by the regional ethics committee of Umeå University (D2011–396-31 M).N = 768). The median birth year was 1994, with an interquartile range (IQR) of 26 years.Overall, 128 cases, children and adults with PAH, were included in the study and individually matched to six controls each (Preterm birth was over three times more common among cases (6%) than among controls 21%) for the total study group  Table . MaternaBeing born premature in 1983–87, 2003–07 and 2008–15 was significantly associated with PAH later in life, although with wide confidence intervals  and even more common when a pulmonary disease is related to the PAH diagnosis , 23. PreAngiogenesis has been shown to be necessary for alveolarization during normal lung development . The expWe speculate that the discontinuation of normal lung vascularization in premature birth has an adverse impact on the vascular development of the infant’s lungs and on future growth. This may induce stress on the myocardium, causing PAH to occur later in life as the individual is exposed to other factors that further impair heart function. Evidence of echocardiographic myocardial changes has recently been found in preterm children at one year of age, but further studies of pulmonary vascular maturation in relation to cardiac function are needed . MedicalMatching cases and controls by year and birth hospital reduced the risk of selection bias due to differences in medical care and survival rates. To increase power, we choose to match six controls to each case.Cardiovascular malformations, as well as chromosomal abnormalities, include heterogeneous conditions; they are more common among preterm infants than term-born infants and are also known risk factors for PAH , 40. By There is always the risk of the misclassification of diagnosis when using registers. We believe that the potential bias of cases is small in our study as all adult cases were retrieved from the SPAHR, which includes patients according to the Dana Point classification , 42, andPreterm birth, along with other factors, significantly contributes to the development of PAH. Previously, CHD, pulmonary diseases and other factors have been linked to PAH in children and young adults who were born preterm. By adjusting for previously known risk factors, our study indicates that new, yet undefined and unknown factors may play a role in the risk of PAH development in later life among those born preterm. In this paper, we discuss some hypotheses to be tested in future studies.


Antenatal maternal psychological distress is common in low and middle-income countries (LMIC), but there is a dearth of research on its effect on birth and developmental outcomes in these settings, particularly in Sub-Saharan Africa. This study set out to identify risk factors for antenatal maternal psychological distress and determine whether antenatal maternal psychological distress was associated with infant birth and developmental outcomes, using data from the Drakenstein Child Health Study (DCHS), a birth cohort study in South Africa.Pregnant women were enrolled in the DCHS from primary care antenatal clinics. Antenatal maternal psychological distress was measured using the Self-Reporting Questionnaire 20-item (SRQ-20). A range of psychosocial measures, including maternal childhood trauma, depression, and posttraumatic stress disorder (PTSD) were administered. Birth outcomes, including premature birth, weight-for-age z-score and head circumference-for-age z-score, were measured using revised Fenton growth charts. The Bayley III Scales of Infant and Toddler Development was administered at 6 months of age to assess infant development outcomes, including cognitive, language, and motor domains in a subset of n = 231. Associations of maternal antenatal psychological distress with psychosocial measures, and with infant birth and developmental outcomes were examined using linear regression models.961 women were included in this analysis, with 197 (21%) reporting scores indicating the presence of psychological distress. Antenatal psychological distress was associated with maternal childhood trauma, antenatal depression, and PTSD, and inversely associated with partner support. No association was observed between antenatal maternal psychological distress and preterm birth or early developmental outcomes, but antenatal maternal psychological distress was associated with a smaller head circumference at birth .Antenatal maternal psychological distress is common in LMIC settings and was found to be associated with key psychosocial measures during pregnancy, as well as with adverse birth outcomes, in our study population. These associations highlight the potential value of screening for antenatal maternal psychological distress as well as of developing targeted interventions. When A systematic review of maternal well-being in Africa  reportedRisk factors that have previously been found to predict maternal depression and anxiety in HIC include poor marital relationships, history of psychological disorders, poor social support and stressful life events . In the The impact of antenatal maternal psychological distress on infant birth outcomes has been researched in HIC. A number of studies have found no relation between antenatal maternal psychological distress and infant birth outcomes ; howeverThere is concern that antenatal maternal psychological distress may also have an impact on infant developmental outcomes that impact later health and education potential. Many studies have reported an association between antenatal maternal psychological distress and cognitive and behavioural development ; howeverWhile there is research on the effects of antenatal maternal psychological distress on the infant in HIC, data are lacking in a LMIC context, particularly in Sub-Saharan Africa where increased prevalence of antenatal maternal psychological distress ,42 and aThe aim of this study was to explore the risk factors for antenatal maternal psychological distress and determine any associations between antenatal maternal psychological distress and infant birth and developmental outcomes in a South African birth cohort.22.1Pregnant women, 18 years or older, between 20 and 28 weeks of gestation and attending one of two primary health care clinics in the Drakenstein peri-urban sub-district of the Western Cape, South Africa were recruited in the Drakenstein Child Health Study (DCHS) ,45. The 2.22.2.1Sociodemographic information was collected from the mother at enrolment, and many of these measures were adapted from items used in the South African Stress and Health Study . This in2.2.2) , and SMK is funded by a MQ fellows award and EDCTP early-career fellowship.The Drakenstein Child Health Study (DCHS) is funded by the None.


Fetal brain diffusion tensor imaging (DTI) offers quantitative analysis of the developing brain. The objective was to 1) quantify DTI measures across gestation in a cohort of fetuses without brain abnormalities using full retrospective correction for fetal head motion 2) compare results obtained in utero to those in preterm infants.Motion-corrected DTI analysis was performed on data sets obtained at 1.5T from 32 fetuses scanned between 21.29 and 37.57 (median 31.86) weeks. Results were compared to 32 preterm infants scanned at 3T between 27.43 and 37.14 (median 33.07) weeks. Apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were quantified by region of interest measurements and tractography was performed.p = 0.001; R2 = 0.469; slope = 0.011; splenium FA: p < 0.001; R2 = 0.597; slope = 0.019; thalamus ADC: p = 0.001; R2 = 0.420; slope = − 0.023); similar trends were found in preterm infants.Fetal DTI was successful in 84% of fetuses for whom there was sufficient data for DTI estimation, and at least one tract could be obtained in 25 cases. Fetal FA values increased and ADC values decreased with age at scan  contains supplementary material, which is available to authorized users. Diffusion tensor imaging (DTI) has proved valuable for assessing the developing brain, with studies conducted in both preterm and term age infants providing important information about its structure in early life –4. In utIn this study, motion-corrected methods were employed to quantify fetal FA and apparent diffusion coefficient (ADC) measures across gestation in a cohort of fetuses without brain abnormalities. The results were compared with data from premature neonates examined at similar post-menstrual ages providing validation that in utero measurements can provide robust data and a first exploration of whether DTI can detect developmental differences between in utero and ex utero brain maturation.DTI was conducted on a total of 36 fetal cases scanned for a variety of reasons: 10 were healthy volunteers, 2 were the surviving fetus from a monochorionic diamniotic twin pregnancy, 2 had atrioventricular septal defects, 4 had transposition of great arteries, 1 had left heart obstructive lesions, and the remaining cases were scanned for possible abnormalities suspected from an ultrasound scan. All cases were assessed by an experienced perinatal neuroradiologist (MAR) and found to have normal brain appearances on anatomical MRI. One subject had a slightly prominent fourth ventricle and three had mild enlargement of the cisterna magna, which were not considered to be of clinical significance. All fetuses subsequently had normal deliveries; no infants required resuscitation at birth and all had 1 and 5 min Apgar scores > 8 (1 case lost to follow up). The gestational age (GA) at scan of the fetuses ranged from 21.29 to 37.57 (median 29.71) weeks.1- and T2-weighted MRI scans. For both fetal and preterm MRI, all parents gave written consent prior to scanning .For comparison of fetal DTI with preterm data, 32 preterm neonate DTI maps were produced. Preterm neonates were chosen to match the ages of the fetal group as closely as possible. They were scanned between 27.43 and 37.14 (median 33.07) week post-menstrual age (PMA), and had a GA at birth ranging from 24.57 to 34.71 (median 29.86) weeks. Preterm neonates were only included for comparison if there was no evidence of focal lesions on conventional Tb value 0 (b0) and 500 s/mm2, 15 non-collinear directions, TE 121 ms, TR 8500 ms, FoV 290 × 290 × 128 mm3, voxel size 2.3 × 2.3 × 3.5 mm3, slice gap − 1.75 mm, and number of slices 62–66 , and acquisition order was set to: odd–even slice, ascending order. Overlapping slices allow for oversampling the fetal brain, which increases the likelihood of sampling enough data for reconstruction, even in cases of significant motion . Moreover, fetal FA values, for example, in the splenium of the corpus callosum (0.49 ± 0.09), is consistent with a mean FA value of 0.47 ± 0.01 previously studies of preterm infants imaged between 33 and 37.5 weeks . The ROIA specific hierarchical pattern of DTI measures was also found, with the majority of WM regions demonstrating significantly greater FA and ADC values than the grey matter. These same regional patterns have previously been found in prematurely born infants , 29.A few previous studies have provided normal fetal FA values across gestation –7, 30. SIn a recent repeatability study of fetal DTI, Jakab et al.  demonstrFetal DTI data presented in this study produced diffusion maps that provided FA values across gestation which are comparable to data obtained ex utero. The motion correction technique applied proved effective, provided that there were sufficient independent samples to reliably fit the six elements of the symmetric diffusion tensor. We studied changes in FA and ADC values with increasing age at scan in a cohort of fetuses without CNS abnormalities. This study demonstrated the potential of the technique to support tractography in the fetal brain; however, the scope of this kind of analysis is currently limited as compared to ex utero data. Fetal DTI has the potential to explore the relationship between in utero and ex utero brain development, and may be used in the future to study abnormalities of pregnancies in utero, and the impact of exposure to the ex utero environment on brain development. Slice-by-slice motion correction is a critical technique for the stable DTI results that have been achieved in the fetal brain, paving the way for reliable fetal DTI in both scientific and clinical applications.Supplementary material 1 (DOCX 20 kb)Below is the link to the electronic supplementary material.


Empagliflozin, a sodium glucose co-transporter 2 (SGLT2) inhibitor, ameliorates hyperglycemia in patients with type 2 diabetes (T2D) by inducing sustained glucosuria. Empagliflozin treatment was previously associated with a transient increase in 24-h urine volume in Caucasian patients with T2D, however comparable evidence in Japanese T2D individuals is scarce. We therefore assessed acute and chronic changes in 24-h urine volume and fluid intake with empagliflozin in Japanese patients with T2D.In this randomized, double-blind, placebo-controlled, parallel-group, multiple-dose, 4-week trial, 100 Japanese patients with T2D were randomized to receive either 1, 5, 10, or 25 mg empagliflozin or placebo once-daily. Changes from baseline in 24-h urine volume and fluid intake were assessed at days 1, 27, and 28 after the initiation of empagliflozin.p < 0.001 vs. placebo). However, 24-h urine volume levels in the empagliflozin groups were comparable to placebo at day 27 and 28 . The 24-h fluid intake was comparable across all study groups throughout the entire study period. No events consistent with dehydration were reported during empagliflozin treatment.The 24-h urine volume and fluid intake were comparable across all treatment groups at baseline. Patients treated with either 10 or 25 mg empagliflozin  showed a significant increase in 24-h urine volume compared to placebo at day 1 (mean change from baseline: + 0.83, + 1.08, and + 0.29 L/day in the empagliflozin 10 and 25 mg groups and the placebo group, respectively; both Treatment initiation with empagliflozin in Japanese patients with T2D was associated with transient diuresis; however, overall urine volume returned towards baseline levels within 4 weeks of treatment. These findings are consistent with a physiological, adaptive mechanism of the kidney to maintain overall body fluid balance in response to treatment initiation with a SGLT2 inhibitor.NCT00885118.Nippon Boehringer Ingelheim Co., Ltd. Sodium glucose co-transporter 2 (SGLT2) is abundantly expressed in proximal tubular cells and mediates approximately 90% of glucose reabsorption within the kidney. SGLT2 inhibitors are a new class of oral antidiabetic drugs (OADs) that specifically target glucose reabsorption in the kidney, thereby facilitating glucose excretion into urine. This removal of excess glucose from the body is associated with a reduction in blood glucose levels in patients with type 2 diabetes (T2D) . Moreove®; Boehringer Ingelheim), a highly selective SGLT2 inhibitor . These results are in accordance with evidence from a previous Caucasian study with empagliflozin [How could the transient effect of SGLT2 inhibitors on urine volume . In addition, 24-h urine collections were performed on day 1 and then again after 4 weeks of treatment. Hence, it is still expected to obtain a more detailed time course for the observed adaptations in daily urine volume, especially during the first 1–2 weeks of treatment with empagliflozin in Japanese patients with type 2 diabetes.Empagliflozin was associated with a transient increase in diuresis in Japanese patients with T2D within the first 24 hours of treatment. Thereafter, urine volume returned towards placebo levels after 4 weeks of therapy with empagliflozin. Neither an increased daily fluid intake nor events consistent with dehydration were observed with empagliflozin throughout the 4-week treatment period. Our findings support an early and effective compensatory mechanism of the kidney that  in order to maintain whole-body fluid balance, despite sustained glucosuria, in Japanese T2D patients treated with empagliflozin. Clinical evidence relating to empagliflozin supports safe and effective treatment initiation in appropriate Japanese patients with T2D without an excess risk of undesirable volume-related events.


Arachnoid cysts (ACs) account for a small proportion of all intracranial lesions. They are often incidental but can become symptomatic and even cause a threat to life. Symptoms are usually due to direct compression of neural elements and/or raised intracranial pressure.We report the case of an infant with an enlarging posterior fossa arachnoid cyst (PFAC) causing torticollis and gastro-oesophageal reflux (GOR), the combination of which had been previously unreported in this context. Endoscopic fenestration and cyst decompression were followed by complete resolution of the symptoms. We discuss the possible mechanisms of torticollis and GOR in this context. Arachnoid cysts (ACs) are congenital lesions –4 that aA 2-month-old male infant was referred to the emergency department with macrocephaly. He was born at term via a normal vaginal delivery. Antenatal screening was normal with no initial post-natal concerns. Two weeks prior to admission, the head circumference increased significantly, and he started to have difficulty feeding with severe GOR. On examination, the anterior fontanelle was bulging and tense with prominent scalp veins. Urgent CT followed by MRI Fig.  of the hAn endoscopic third ventriculostomy (ETV) was performed and a Rickham reservoir connected to an intraventricular catheter was inserted. The post-operative scan revealed decompression of the ventricular system and a stable PFAC. The infant was discharged home 3 days later.In the following weeks, he developed torticollis  and GOR refractory to medical treatment. A repeat MRI revealed an increase in the size of the PFAC such that it was extending into the spinal canal through the craniocervical junction and causing significant mass effect on the brainstem. The previous ETV was still functioning Fig. .Fig. 2T2We proceeded with endoscopic cyst fenestration. The cyst was entered and its wall was coagulated in places (to reduce its size) then fenestrated. The fenestrations were into the craniocervical junction, the fourth ventricle, out through the right foramen of Luschka and into the pre-pontine cistern. Choroid plexus was seen within the cyst Fig. c. NeuropHe was readmitted with a CSF leak 1 week later. A CT scan demonstrated that the cyst was smaller and ruled out hydrocephalus. A trans-fontanelle tap revealed a raised white cell count with no organisms detected. He returned to surgery where the reservoir and intraventricular catheter were removed and replaced by an external ventricular drain (EVD). He received 14 days of intrathecal (IT) vancomycin and 16 days of intravenous meropenem and vancomycin. He made a good recovery. The EVD was removed and he was discharged home 20 days after surgery.Despite reduction in the size of the cyst and a functioning ventriculostomy, the patient developed communicating hydrocephalus likely due to the infection. A left ventriculoperitoneal shunt was inserted after serial lumbar punctures. At 18-month follow-up he is fit and well, with no recurrence of symptoms.There are four previous reports of PFACs presenting with torticollis –10 (TablThe precise mechanism resulting in torticollis in the context of a PFAC is unclear. However, symptom resolution after cyst decompression implies a role of mass effect. Compression of structures such as the vermis and fastigial nucleus may explain this presentation due to their involvement in the control of head movement . Other hWe also observed GOR refractory to medical management resolve upon cyst fenestration. This association, to the best of our knowledge, has not been reported before. Vagal nerve impairment has been implicated in the pathogenesis of GOR , for exaA minority of PFACs require intervention ; at presWe report an unusual, and to the best of our knowledge, unique presentation with torticollis and GOR secondary to a PFAC in an infant which resolved upon fenestration. Whilst the exact mechanism causing such symptoms is unclear, resolution of mass effect treated the symptomatology and allowed normal development. This is the second report of a PFAC presenting with torticollis to be managed endoscopically and the first report of GOR in this context.


What are the prenatal risk factors and perinatal and postnatal outcomes associated with maternal opioid use during pregnancy?In this cohort study based on data from 8509 mother-child pairs in the Boston Birth Cohort, in utero opioid exposure was significantly associated with higher risks of fetal growth restriction, preterm birth, lack of expected physiological development, childhood conduct disorder or emotional disturbance in preschool-aged children, and attention-deficit/hyperactivity disorder in school-aged children.Prenatal opioid exposure was associated with higher risks of adverse perinatal and postnatal physical health and neurodevelopmental outcomes, suggesting that efforts to mitigate the health consequences of the opioid epidemic require more intergenerational research. This cohort study examines the prenatal risk factors and perinatal and postnatal outcomes associated with maternal opioid use during pregnancy. The opioid epidemic increasingly affects pregnant women and developing fetuses, resulting in high rates of neonatal abstinence syndrome. However, longitudinal studies that prospectively observe newborns with neonatal abstinence syndrome or with maternal opioid use and examine their long-term physical and neurodevelopmental outcomes are lacking.To examine prenatal risk factors associated with maternal opioid use during pregnancy and the short-term and long-term health consequences on their children.This cohort study analyzed data from the Boston Birth Cohort, an urban, low-income, multiethnic cohort that enrolled mother-newborn pairs at birth at Boston Medical Center  starting in 1998, and a subset of children were prospectively observed at Boston Medical Center pediatric primary care and subspecialty clinics from birth to age 21 years. Data analysis began in June 2018 and was completed in May 2019.In utero opioid exposure was defined as maternal self-reported opioid use and/or clinical diagnosis of neonatal abstinence syndrome.Pregnancy outcomes, postnatal child physical health, and major neurodevelopmental disabilities, documented in maternal and child medical records.This study included 8509 Boston Birth Cohort mother-newborn pairs for prenatal and perinatal analyses. Of those, 3153 children continued to receive pediatric care at Boston Medical Center and were included in assessing postnatal outcomes. Overall, 454 of the 8509 children (5.3%) in the Boston Birth Cohort had in utero opioid exposure. At birth, opioid exposure was associated with higher risks of fetal growth restriction  and preterm birth . Opioid exposure was associated with increased risks of lack of expected physiological development  and conduct disorder/emotional disturbance  among preschool-aged children. In school-aged children, opioid exposure was associated with a higher risk of attention-deficit/hyperactivity disorder .In this sample of urban, high-risk, low-income mother-child pairs, in utero opioid exposure was significantly associated with adverse short-term and long-term outcomes across developmental stages, including higher rates of physical and neurodevelopmental disorders in affected children. Efforts to prevent the opioid epidemic and mitigate its health consequences would benefit from more intergenerational research. Leading governmental and professional associations, including the US Department of Health and Human Services, the American College of Obstetricians and Gynecologists, and the American Academy of Pediatrics, are making concerted efforts to identify risk factors and improve prevention strategies to reduce the negative health effects of opioids on pregnant women and mitigate the developmental consequences of in utero opioid exposure.However, many questions related to in utero opioid exposure remain unanswered. Significant knowledge gaps exist regarding the long-term consequences of maternal opioid use on physical health and neurodevelopmental outcomes among children exposed to opioids. Foremost, there is a lack of large, prospective birth cohort studies to assess the long-term impact of in utero opioid exposure, in part owing to the need for a long duration of follow-up, the risk of attrition, complicated logistics, and the large expense associated with sustaining longitudinal studies. There is a recognized disparity in opioid-related data available on urban low-income minority populations and historically understudied populations in the United States.17 have reported an increased risk of chronic health conditions, prolonged hospitalizations, unintended pregnancies, and psychopathology among women exposed or addicted to opioids. Whiteman et al17 have also reported an increased risk of threatened preterm labor and early-onset delivery among these women. Meanwhile, among infants and children exposed to opioids in utero, studies24 report preterm birth and stillbirth, hypoxia, low mental and psychomotor development, language and reading deficits, low cognitive development and functioning, lower IQ score, neurodevelopmental impairment, and early marijuana use. Tolia et al25 found that NAS was associated with the increasing amount of resources related to neonatal intensive care units from 2004 to 2013.25 There is a trend in the incidence of NAS and its related health care expenditures between 2000 and 2009 in the United States26 and between 2000 and 2011 in Australia.27 Haight et al3 reported a 333% increase in opioid use disorder in terms of delivery hospitalizations in the United States.Prior studiesWhile each study offers important contributions, most are dated, used small sample sizes, were conducted in different social-cultural settings, or relied on maternal self-reports for identification of child perinatal and postnatal outcomes. To our knowledge, no longitudinal cohort studies of urban, low-income US residents to date have examined maternal sociodemographic and epidemiological characteristics associated with opioid use or perinatal and postnatal outcomes of children exposed to opioids. Understanding the characteristics of pregnant women and the factors that potentially put them at risk of opioid exposure or opioid use disorder and opioids’ long-term effect on their children is important for developing and implementing policies and programs to address the opioid epidemic burden among pregnant women and prevent adverse outcomes in their children.In this study, we conducted a longitudinal analysis of the risks and consequences of maternal opioid exposure, using a rich longitudinal database of mother-newborn pairs included in the Boston Birth Cohort (BBC), a large sample of an urban, low-income, multiethnic population in the United States. Specifically, we examined (1) demographic and epidemiological characteristics of mothers with opioid exposure compared with mothers without exposure, (2) prenatal risk factors of maternal opioid exposure, (3) pregnancy complications and birth outcomes of mother-newborn pairs with opioid exposure compared with their counterparts without exposure, and (4) the postnatal physical health and neurodevelopmental outcomes of children with opioid exposure compared with children without exposure.29 Briefly, eligible mothers were those who delivered singleton live births at Boston Medical Center (BMC), Boston, Massachusetts, starting in 1998. The enrollment targeted singleton, live-preterm-birth (<37 weeks’ gestation), low-birth-weight infants (<2500 g), and full-term, normal-weight infants, with oversampling of preterm and/or low-birth-weight babies. Multiple-gestation pregnancies , newborns with major birth defects, birth by in vitro fertilization, and infants with congenital chromosomal abnormalities were excluded from the cohort. Among those enrolled at birth, 3153 mother-newborn pairs who continued to receive pediatric primary care at BMC were enrolled in a postnatal follow-up study.31 The institutional review boards of Boston University Medical Center and the Johns Hopkins Bloomberg School of Public Health approved the study protocol. This report follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.A detailed description of the study cohort has been previously published.International Statistical Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM)  and Tenth Revision (ICD-10) .After obtaining written informed consent from the enrolled participants, trained research staff conducted face-to-face interviews 48 to 72 hours after delivery using a standardized questionnaire to determine sociodemographic variables, illicit drug use patterns, and smoking and drinking status. Pregnancy complications, birth outcomes, and diagnoses for NAS were obtained via maternal and infant medical record abstraction. Starting in 2003, postnatal growth and developmental outcomes were collected from electronic medical records (EMRs) as part of routine clinical data collection. For each primary care visit, EMRs contain codes for primary and secondary physician diagnoses corresponding to the Pertinent maternal sociodemographic information was collected using standard maternal questionnaires, including maternal age, self-reported height and prepregnancy body weight, number of live births, annual household income, race/ethnicity, marital status, and education status. Body mass index (BMI) before pregnancy was calculated as prepregnancy body weight in kilograms divided by height in meters squared. In addition, maternal questionnaires collected information on cigarette smoking status, alcohol use, and illicit drug use. Illicit drugs were categorized into 3 groups: (1) marijuana, (2) stimulants, and (3) opioids. Stimulants included cocaine, crack, ecstasy, amphetamines, and phencyclidine. Opioids included heroin, oxycodone, and methadone. The duration of ever smoking, drinking, and drug use was defined as the period from 6 months before pregnancy to delivery.ICD-9 code 779.5  or ICD-10 code P96.1 . Diagnoses were documented in medical records.Neonatal abstinence syndrome was defined for infants with a hospital diagnosis of 32; maternal diabetes was defined as having either gestational or pregestational diabetes33; and hypertensive disorder was classified yes if preeclampsia, eclampsia, chronic hypertension, and/or hemolysis, elevated liver enzymes, and low platelets syndrome were present during pregnancy.34 Birth outcomes, including birth weight, gestational age, and fetal growth pattern, were obtained from maternal and child medical records. Gestational age was assessed based on the first day of the last menstrual period and early prenatal ultrasonographic results, as described previously.29 Preterm birth was defined as gestational age less than 37 weeks. Low birth weight was defined as less than 2500 g.29 Fetal growth was defined by birth weight for gestational age and categorized into 3 groups: (1) small for gestational age (<10th percentile), (2), appropriate for gestational age (10th-90th percentile), and (3) large for gestational age (>90th percentile), according to an established local sex- and race-specific reference population living in the Boston area.35This study’s major outcomes were pregnancy complications, birth outcomes, and postnatal physical health and neurodevelopmental disabilities. The following pregnancy complications were defined based on maternal medical records: prepregnancy BMI categorized as underweight (<18.5), normal weight (18.5-25), or overweight (≥25) according to the Institute of Medicine BMI classificationICD-9 and ICD-10 codes from study children’s EMRs. They included being ever diagnosed with attention-deficit/hyperactivity disorder (ADHD) , conduct disorder/emotional disturbance , and lack of expected normal physiological development .Postnatal physical health and neurodevelopmental disabilities were defined according to 2 tests  were used; for continuous data, t tests were used. Baseline characteristics of mother-infant pairs in the BBC follow-up were compared with those of mother-infant pairs not in the follow-up using univariate analysis. Pregnancy complications, birth, and postnatal physical health and neurodevelopmental outcomes between the 2 groups were compared using crude and multiple logistic regression analyses. The following sociodemographic variables were adjusted for pregnancy complications and birth outcomes in multiple logistic regression: maternal age, household income, race/ethnicity, marital status, and maternal education. For children’s postnatal physical health and neurodevelopmental outcomes, preterm birth and low-birth-weight conditions were also adjusted in the multiple logistic regression. The postnatal physical health and neurodevelopmental outcomes were further examined by child age groups (<6 years vs ≥6 years). Categorical outcomes were analyzed using multinomial logistic regression. The following sensitivity analyses were conducted in this study: (1) log-linear regression for pregnancy complications, birth, and postnatal physical health and neurodevelopmental outcomes, (2) Cox regression model for risk of ADHD diagnosis, (3) logistic regression for pregnancy complications, birth, and postnatal physical health and neurodevelopmental outcomes after excluding infants born before 2003, and (4) logistic regression for pregnancy complications and birth outcomes further adjusting birth year period (1998-2007 vs 2008-2014 and later). The pooled estimates of 10 imputed data sets were used for all analytical results using multiple imputed data according to Rubin rule.36 All analyses were performed using R version 3.4.3 (The R Foundation). Statistical significance was set at P < .05, and all tests were 2-tailed.The incidence rates for NAS from 2003 to 2016 were calculated and plotted as a bar chart using each year’s enrolled newborns and NAS diagnoses from the EMRs  were prenatally exposed to opioids . There wPregnancy complications and birth outcomes between the opioid-exposed group and the nonexposed group are presented in 9 A US Centers for Disease Control and Prevention Morbidity and Mortality Weekly Report article shows that NAS incidence by US state in 2013 ranged from 0.7 cases per 1000 hospital births (Hawaii) to 33.4 cases per 1000 hospital births (West Virginia).10 Because our sample was a predominantly urban, low-income, minority cohort, the higher incidence rates in our study underscore the potential disparities in NAS experienced by urban low-income populations living in the inner cities of the United States. Our higher rates may partly be attributed to the implementation of Project Recovery, Empowerment, Social Services, Prenatal Care, Education, Community, and Treatment (RESPECT) at BMC in 2006.37 This program identified and treated pregnant women with substance use disorder and their children, who were subsequently delivered at BMC.To our knowledge, this is the first study to describe the risk factors and perinatal and postnatal outcomes of maternal opioid exposure. To do this, we used a rich longitudinal database of mother-newborn pairs included in the BBC, a large sample of an urban, low-income, multiethnic population in the United States. We found that NAS incidence in the BBC increased drastically from 12.1 per 1000 hospital births to more than 30 per 1000 hospital births over the past 15 years. This rising trend is consistent with the national trend, although the BBC rate was much higher compared with national data from 2009 to 2012.17 They also lend further credence to the previous finding that opioid use is associated with multiple deleterious exposures, such as low socioeconomic status, poor nutrition, and other substance use disorders, which were highlighted in an executive summary of a joint workshop by the major federal and national public health agencies and assocaitons.13In addition to a higher rate of opioid use, our study demonstrates that mothers exposed to opioids had a higher likelihood of polysubstance use, including alcohol consumption, cigarette smoking, and stimulant use. They were also more likely to be multiparous, be non-Hispanic white, be unmarried, and have less than a college education. These findings regarding the risk factors of opioid use and consequences of NAS are consistent with previous literature.38 More importantly, our study fills a major gap in the knowledge base regarding the long-term postnatal outcomes associated with maternal opioid exposure. Our examination of the association of maternal exposure to opioids with major physical health and neurodevelopmental outcomes for children younger and older than 6 years is noteworthy. We found that within the first 6 years, opioid exposure was associated with a higher risk of diagnosis of conduct disorder/emotional disturbance and lack of expected normal physiological development. After 6 years, opioid exposure was associated with higher likelihood of a diagnosis of ADHD. Taken together, our data suggests that prenatal opioid exposure was associated with higher risk of different yet interrelated adverse perinatal and postnatal physical health and neurodevelopmental outcomes unfolding over the life course. This differential risk over the life course may explain the inconsistent postnatal outcome findings from a previous prenatal drug exposure study.39Furthermore, we found that infants’ exposure to opioids was associated with lower gestational age, lower birth weight, and higher rate of being small for gestational age, consistent with previous literature on NAS and birth outcomes.If confirmed, the findings of this study could have multiple clinical and public health implications. First, most mothers with opioid exposure have polysubstance use in addition to opioids. Thus, opioid addiction is only part of the problem. The root causes and other substance use should be addressed simultaneously. Second, children born from mothers exposed to opioids had a higher risk of physical and mental health problems along the life course, including poor fetal growth, low birth weight, preterm birth, conduct disorder/emotional disturbance, lack of expected normal physiological development, and ADHD. Additionally, maternal exposure to opioids together with poor birth outcomes may further increase the risk of adverse childhood physical health and neurodevelopmental outcomes. Thus, efforts to prevent maternal opioid use and mitigate its health consequences require a longitudinal approach that addresses physical and mental health sequelae for mothers and children. Finally, from a health economics perspective, the opioid epidemic is an intergenerational problem. Successfully addressing it will not only benefit current and future generations but also mitigate exorbitant health care expenditures.23 This type of data is more reliable than maternal self-reported outcomes, which were used by many prior studies. Third, the study sample is mainly from a low-income, urban, minority population in the Boston area. This is a high-risk yet understudied population. We showed that this sample had a NAS rate that was almost 10-fold that of the general population, which further underscores the well-noted need for expanded research and programmatic interventions within these populations.This study has several strengths. First, the prospective birth cohort design of the study made it possible to investigate early life factors before the onset of opioid-associated NAS and the long-term postnatal outcomes of maternal opioid exposure. This longitudinal approach helped us to better understand the temporal associations of opioids exposure, NAS, and their consequences later in life. Additionally, to our knowledge, BBC is among the largest contemporary prospective US birth cohorts, which allowed us to investigate multiple potential postnatal outcomes. Second, this study used physician diagnoses extracted from EMRs to identify a wide range of postnatal physical and neurodevelopmental outcomes, which is rarely found in the literature.This study has some limitations. First, it may be subject to exposure misclassification. We defined exposure heterogeneously to include illicit, therapeutic, and unknown opioid exposures leading to NAS. This heterogeneity in exposure could lead to differential effects on long-term child outcomes, which are difficult to disentangle in this study. Second, our analyses of long-term associations might be confounded by other pregnancy exposures listed in In this sample of an urban, low-income, multiethnic US population, the prevalence of NAS was almost 10-fold the national rate. Most mothers who were exposed to opioids used other substances, including marijuana, stimulants, cigarettes, and alcohol. Maternal opioid exposure was associated with a higher risk of pregnancy complications and poor birth outcomes as well as adverse postnatal child physical health and neurodevelopmental outcomes. However, the effect of opioids is still difficult to disentangle from effects of other childhood exposures. Policy and programmatic efforts to prevent NAS and mitigate its health consequences require more comprehensive longitudinal and intergenerational research.


The purpose of this study was to evaluate the association of influenza vaccine during pregnancy and adverse fetal outcomes. Preventing fetal death, low birth weight, small for gestational age birth and preterm birth are important potential effects of antenatal maternal influenza immunization for which there are conflicting data.A double-blind, randomized, placebo-controlled clinical trial of trivalent inactivated influenza vaccine was conducted in South Africa from March 2011 until after the 2012 influenza season when the infants born had reached the age of 24 weeks. Mothers were administered the vaccine or placebo during pregnancy at a gestation of 20 to 36 weeks. A comparison of rates of fetal death, low birth weight, small for gestational age birth, and preterm birth, between vaccinated and placebo groups was made. Fetal outcome differences between the groups were measured using Student’s t-tests, vaccine efficacy with 95% confidence intervals, and Poisson regression for incidence rates. All analyses except fetal death excluded mothers who were administered vaccine or placebo after 34 weeks gestational age.There were 2116 HIV-uninfected pregnant women age 18 to 38 years in the trial; 2005 infants were born to mothers where vaccine or placebo had been administered ≥ 14 days prior to delivery, and there were 6 miscarriages and 23 stillbirths. There was no significant vaccine efficacy  on fetal death , low birth weight , small for gestational age birth , or preterm birth . Neither was vaccine efficacy demonstrated when the analysis was restricted to infants of mothers who were exposed to an influenza season (1832 outcomes available).We did not find a beneficial effect of trivalent inactivated influenza vaccine during pregnancy on adverse fetal outcomes. Enrollment started on March 3, 2011, for the first cohort and on March 6, 2012, in the second year. Eligibility criteria included maternal ages 18 to 38 years, estimated gestational age between 20 and 36 weeks, and absence of certain medical conditions. Complete inclusion and exclusion criteria, vaccine characteristics and subject demographics are described elsewhere Methods for estimating vaccine efficacy of IIV3 versus placebo in protecting mothers and their infants from contracting influenza illness, comparison of seroconversion rates, and safety outcomes are discussed elsewhere The study  was approved by the Human Research Ethics Committee of the University of the Witwatersrand (HREC number: 101106) and conducted in accordance with Good Clinical Practice guidelines. Signed, written informed consent was obtained from all participants.2.2Full cohort. All Mothers Enrolled Before or During an Influenza Season with Delivery Before, During or After an Influenza Season. This analysis included mothers who were enrolled in the study, whose fetal outcomes were known, and who had been administered vaccine or placebo a minimum of 14 days prior to delivery. The time between mother’s enrollment and delivery was used to compute the person-years (PY) of enrollment for incidence rate analysis and was counted whether or not that time period fell within an influenza season. All subjects who fit these criteria were included in the fetal death analyses. All other analyses included only live-born infants. This analysis is a modified intention-to-treat vaccine efficacy analysis. Further, live-born infants of mothers who were administered vaccine or placebo after 34 weeks gestational age (wGA) were excluded from the live-born analyses.and who had been administered vaccine or placebo a minimum of 14 days prior to delivery, were included in this subgroup. For incidence rate analysis, only the PY of enrollment until delivery that fell within an influenza season were counted towards the mother’s PY. This subgroup only included the outcomes of fetuses gestating during the influenza season and therefore were potentially at risk for maternal influenza virus infection associated outcomes. As per the previous cohort, live-born infants of mothers who were administered vaccine or placebo after 34 wGA were excluded from the live-born analyses.Influenza season gestation Subgroup. Mothers at Risk During Influenza Season Only. The South African influenza seasons were defined using the National Institute for Communicable Diseases surveillance data.  2.3Study subjects were randomized shortly after they consented to participate in the study and eligibility had been confirmed. Except for the statistician and pharmacist, all study personnel and participants were blinded to the computer-generated, randomly assigned [1:1 ratio] designation of mothers who received either IIV3 or placebo. Participants received either 0·5 ml of the influenza vaccine with the southern hemisphere composition for 2011 and 2012  in the active arm or an identical-looking placebo of 0·5 ml of 0·9% normal saline, administered into the deltoid muscle by study nurses.2.4a priori for the RCT study regarding vaccine efficacy versus contracting influenza t-test.Sample size requirement was computed 2.56/7 weeks) births. For term births (>376/7 weeks), the GA was computed as days elapsed between the enrollment visit and the birth, plus GA (in days) that was determined at enrollment. Gestational age of the mother at enrollment was determined using a hierarchy of methods which included, by order of priority, fetal ultrasound when available, the last menstrual period of the mother, and physical examination by palpation of fundal height Fetal outcomes were classified as: fetal death from miscarriages, spontaneous abortion of pregnancy occurring after 20 wGA, and stillbirths defined as a fetal death after 28 weeks gestation Study data were collected and managed using REDCap 2.6t-test.All statistical analyses were pre-specified and were performed using SAS version 9·4 . Graphs were created using SPSS v.22 . Data were analysed from the viewpoints of efficacy and safety, as these outcomes could be viewed from both viewpoints. We defined efficacy using simple vaccine efficacy (VE) proportions, however granting that safety examinations require a person time denominator for comparison across studies, we have also analyzed data  using a person time denominator. Comparisons between means were performed using a two-tailed, two-sample Student’s v/Ip), where Iv = incidence rate in the vaccinated-group and Ip = incidence rate in placebo-group; 95% confidence intervals (95% CI) were constructed Vaccine efficacy was calculated using the formula 100 × (1 − Iv/Ip). Incidence rates were computed per 1000 PY for these analyses. The associated 95% CI and P-values were computed using PROC GENMOD, using an exact approximation to the Poisson distribution.For the safety analyses, Incidence Rate Ratios (IRR) were computed using the formula .3A flow diagram  depicts Sample Size Requirements. After excluding mothers who were administered vaccine or placebo after 34 wGA, we were left with 1884 (cohort 1) and 1737 (cohort 2) liveborn infants with a known birth weight. Given these N, we are able to detect a difference in birth weight of 39 g with 69% power (cohort 1) and 65% power (cohort 2), using a 2-tailed t-test analysis. Using a 1-tailed test, we are able to detect a difference of 40 g with 79% power (cohort 1) and 76% power (cohort 2).3.1P = 0.025), with a concomitant non-significant 39 g difference in birth weight . VE fetal outcome measures  and safety for the full cohort are presented in . There were 29 fetal deaths distributed among the two study groups with VE of −21·2% : −150·8, 41·4) . The meaP = 0.048). Mean birth weight was lower in the IIV3 group  but only approached significance; birth weight differences are graphically illustrated . There were 24 fetal deaths and 1808 live births. GA was statistically greater in the placebo versus the vaccinated group .The birth weight of infants born prior to an influenza season did not differ by mother’s intervention status , panel A4Our study, which is one of 3 recent placebo-controlled trials of IIV3 given to pregnant women in utero , overall or restricted to infants of mothers who were potentially exposed to influenza in utero B, as welmeta-analysis Our results differ substantially from the first randomized controlled trial from Bangladesh Given the global importance of preventing LBW, prematurity and small size for gestational age with a simple intervention, we urge caution in generalizing our findings, in light of the results of the other randomized controlled trials in Mali meta-analysis.Of note, none of the three trials showed an impact on premature birth. It is possible that this was because all three trials were underpowered to show an impact as suggested by Hutcheon et al. Given the CIs, this neutral study can rule out a vaccine efficacy of 12% for LBW, 8% for preterm birth, 11% for SGA, and 41% for fetal death . From th5Despite a significant reduction in maternal influenza infections Author declares that there is no conflicts of interest.


A pseudoaneurysm occurs as the result of a contained rupture of an arterial wall, yielding a perfused sac that communicates with the arterial lumen. Pseudoaneurysm of an intercostal artery is an extremely rare event but it carries with it a significant risk of rupture and subsequent hemothorax. It must be considered as a potential complication of thoracentesis.Here, we report a rare case of an intercostal artery pseudoaneurysm following thoracentesis in an 82-year old male. The patient presented with respiratory distress 1 day after a therapeutic thoracentesis had been performed. Computed tomography (CT) with contrast revealed a left intercostal pseudoaneurysm with hemothorax and adjacent compressive atelectasis. Doppler ultrasound revealed bidirectional blood flow in the pseudoaneurysm sac. An intercostal arteriogram and thoracic aortogram aided in confirmation of the pseudoaneurysm and successful treatment with coil embolization.An intercostal pseudoaneurysm complication following thoracentesis is very rare but important to rule out as a possible cause of hemothorax after the procedure. Capturing this finding with the aid of multiple imaging modalities allowed for diagnostic certainty and rapid treatment with coil embolization, leading to a successful patient recovery. Thoracentesis is a commonly performed procedure in the diagnosis and possible treatment of pleural effusion. Typical complications of the procedure include pneumothorax, bleeding including hemothorax, and re-expansion pulmonary edema . A pseudAn 82-year-old male was admitted to the emergency department for worsening shortness of breath and hypoxia. He was admitted a week after he was diagnosed with a left ninth rib fracture secondary to a fall. He had long-standing history of chronic obstructive pulmonary disease, coronary artery disease, and peripheral vascular disease. Chest radiographs revealed a left pleural effusion and possible infiltrate. The patient was initially treated with a nebulizer, prednisone, and empiric antibiotic coverage with ceftriaxone and azithromycin. The patient failed to improve with the medical interventions and a therapeutic thoracentesis was performed. The thoracentesis was completed with ultrasound guidance, with the puncture made above the 11th rib at mid chest on the left. The pleural effusion was found to be frank blood. No immediate complications were noted, and the patient was taken to recovery. The next day the patient was found to be in respiratory distress. A chest x-ray revealed an opaque left hemithorax that was likely rapid accumulation of pleural fluid Fig.  directlyFollowing identification of the left intercostal pseudoaneurysm, the patient underwent a thoracic aortogram and multiple-level left intercostal angiogram Fig.  under IVThoracentesis is a very common procedure used to remove pleural fluid for diagnostic or therapeutic purposes. The most common complications of the procedure include pneumothorax, bleeding includinghemothorax, and re-expansion pulmonary edema . FollowiAn intercostal pseudoaneurysm is an extremely rare phenomenon, seldomly described in literature. Etiology of previously described intercostal artery pseudoaneurysms includes blunt thoracic trauma , 8, 12, Our patient’s intercostal pseudoaneurysm was captured with the aid of three different imaging modalities prior to treatment intervention. The pseudoaneurysm was initially detected on the contrast-enhanced CT. The procedure was performed during arterial phase and this played a pivotal role in the diagnostic process. Doppler ultrasound identifying the possible pseudoaneurysm revealed a “yin-yang sign” which can be characteristic of the complication. This radiological sign has been described to represent the bidirectional flow of blood in the pseudoaneurysm sac and is helpful in the diagnostic confirmation , 13.The gold standard for diagnosing pseudoaneurysms on imaging is conventional angiography, however, less invasive modalities are increasing in use including ultrasonography, computed tomographic angiography (CTA), and magnetic resonance angiography (MRA) . CTA andn-butyl cyanoacrylate or re-embolization at a different location [Early treatment of the pseudoaneurysm before rupture is critical in the success of the patient’s recovery. If rupture did occur, the patient would be at risk of a possible life-threatening hemorrhage . Currentlocation , 16. OurAn intercostal artery pseudoaneurysm as a complication of thoracentesis is an extremely unusual condition but it is important to rule out as a possible cause of hemothorax after the procedure. Capturing this finding with the aid of multiple imaging modalities allowed for diagnostic certainty and rapid treatment with coil embolization, leading to a successful patient recovery.